Extreme environments and the origins of biodiversity...
Transcript of Extreme environments and the origins of biodiversity...
I N V I T E D R E V I EW S AND S YN TH E S E S
Extreme environments and the origins of biodiversityAdaptation and speciation in sulphide spring fishes
Michael Tobler1 | Joanna L Kelley2 | Martin Plath3 | Reuroudiger Riesch4
1Division of Biology Kansas State
University Manhattan KS USA
2School of Biological Sciences Washington
State University Pullman WA USA
3Shaanxi Key Laboratory of Molecular
Biology for Agriculture College of Animal
Science and Technology Northwest AampF
University Yangling Shaanxi China
4School of Biological Sciences Centre for
Ecology Evolution and Behaviour Royal
Holloway University of London Egham
Surrey UK
Correspondence
Michael Tobler Division of Biology Kansas
State University Manhattan KS USA
Email toblerksuedu
Funding information
Army Research Office GrantAward
Number W911NF-15-1-0175 National
Science Foundation Division of Integrative
Organismal Systems GrantAward Numbers
IOS-1557795 IOS-1557860
Abstract
Organisms adapted to physiochemical stressors provide ideal systems to study evo-
lutionary mechanisms that drive adaptation and speciation This review study
focuses on livebearing fishes of the Poecilia mexicana species complex (Poeciliidae)
members of which have repeatedly colonized hydrogen sulphide (H2S)-rich springs
H2S is a potent respiratory toxicant that creates extreme environmental conditions
in aquatic ecosystems There is also a rich history of research on H2S in toxicology
and biomedicine which has facilitated the generation of a priori hypotheses about
the proximate mechanisms of adaptation Testing these hypotheses through the
application of high-throughput genomic and transcriptomic analyses has led to the
identification of the physiological underpinnings mediating adaptation to H2S-rich
environments In addition systematic natural history studies have provided a
nuanced understanding of how the presence of a physiochemical stressor interacts
with other sources of selection to drive evolutionary change in a variety of organis-
mal traits including physiology morphology behaviour and life history Adaptation
to extreme environments in P mexicana also coincides with ecological speciation
and evolutionarily independent lineages span almost the full range of the speciation
continuum from panmixia to complete reproductive isolation Multiple mechanisms
of reproductive isolation are involved in reducing gene flow between adjacent popu-
lations that are adapted to contrasting environmental conditions Comparative stud-
ies among evolutionarily independent lineages within the P mexicana species
complex and more recently other members of the family Poeciliidae that have colo-
nized H2S-rich environments will provide insights into the factors facilitating or
impeding convergent evolution providing tangible links between micro-evolutionary
processes and macro-evolutionary patterns
K E YWORD S
ecological genomics extremophile fishes hydrogen sulphide local adaptation Poeciliidae
speciation
1 | INTRODUCTION
Organisms adapted to extreme environmental conditions provide
excellent systems to investigate basic questions about the origins of
biodiversity Extreme environments are characterized by the pres-
ence of physicochemical stressors that are lethal to most organisms
(Bell 2012 Riesch Tobler amp Plath 2015 Waterman 1999) Most
extreme environments harbour relatively simple biological communi-
ties with low levels of diversity both because there are constraints
for adaptation to physiochemical stressors and because resources
are often scarce (Gibert amp Deharveng 2002 Wall amp Virginia 1999)
Comparing highly specialized extremophiles to ancestral lineages in
Received 25 July 2017 | Revised 8 January 2018 | Accepted 10 January 2018
DOI 101111mec14497
Molecular Ecology 201827843ndash859 wileyonlinelibrarycomjournalmec copy 2018 John Wiley amp Sons Ltd | 843
ldquonormalrdquo habitats provides unique opportunities to illuminate evolu-
tionary processes that give rise to diversity both in terms of pheno-
typic novelty that mediates organismal function under adverse
environmental conditions and the origin of species The clearly
defined selective regimes in extreme environments enable hypothe-
sis-driven tests of responses at all levels of biological organization
and facilitate integrative approaches to study the causes and conse-
quences of adaptive evolution (Nevo 2011 Tobler Riesch amp Plath
2015) In addition the availability of replicated lineages exposed to
the same physicochemical stressors allows for evolutionary analyses
in a comparative context spanning vast geographic and phylogenetic
distances (Riesch Plath Schlupp Tobler amp Langerhans 2014 Riesch
et al 2016) Here we review recent research on fishes inhabiting
hydrogen sulphide-rich springs that sheds light onto mechanisms of
adaptation speciation and convergent evolution
Hydrogen sulphide (H2S) is a noxious gas that profoundly shaped
the diversification of life on the planet It served as a substrate for
anaerobic energy production for early life forms that lived in the
oxygen-poor oceans of the Proterozoic (Olson amp Straub 2015) and
caused mass extinctions after aerobic organisms diversified during
the Cambrian explosion (Kump Pavlov amp Arthur 2005) In todayrsquos
well-oxygenated atmosphere H2S is relatively rare but it is pro-
duced through geochemical and biological processes in a variety of
aquatic ecosystems (Tobler Passow Greenway Kelley amp Shaw
2016) The presence of H2S creates extreme environments for meta-
zoans because it interferes with fundamental biochemical and physi-
ological processes that mediate the function of aerobic organisms
(Bagarinao 1992) Most importantly H2S binds to cytochrome c oxi-
dase (COX) thereby interrupting the oxidative phosphorylation path-
way (OXPHOS) in mitochondria and inhibiting the aerobic
production of ATP (Cooper amp Brown 2008) Consequently H2S is
highly toxic for most metazoans even in micromolar concentrations
(Beauchamp Bus Popp Boreiko amp Andjelkovich 1984 Reiffenstein
Hulbert amp Roth 1992)
In southern Mexico H2S occurs naturally in multiple freshwater
spring complexes that are fed by groundwater sources (Rosales
Lagarde 2012) These sulphide springs are distributed across multi-
ple river drainages (Rıos Tacotalpa Puyacatengo Ixtapangajoya and
Pichucalco) that are part of the Rıo Grijalva basin (Figure 1) and
they exhibit H2S concentrations orders of magnitude higher than
what is considered lethal for most metazoans (Tobler et al 2011)
All springs directly drain into adjacent streams or rivers where H2S
is immediately diluted Thus there are contrasting sulphidic and non-
sulphidic habitat types that lack physical separation Despite the high
levels of toxicity multiple lineages of small livebearing fish of the
Poecilia mexicana species complex (subgenus Mollienesia family Poe-
ciliidae) have independently colonized the H2S-rich springs in each
of the river drainages Members of this species complex inhabit a
variety of freshwater and brackish habitats from northern Mexico
south to Panama (Alda Reina Doadrio amp Bermingham 2013 Pala-
cios Voelker Arias-Rodriguez Mateos amp Tobler 2016) and the
nominal species P mexicana is common in nonsulphidic habitats sur-
rounding the sulphide springs In the eastern two drainages with
sulphide springs (Rıos Tacotalpa and Puyacatengo) colonization of
the H2S-rich habitats has occurred relatively recently
(lt100000 years ago Pfenninger et al 2014) and H2S-adapted pop-
ulations are closely related tomdashand taxonomically considered the
same species asmdashP mexicana in adjacent nonsulphidic habitats (Pala-
cios et al 2013) In the western two drainages (Rıos Ixtapangajoya
and Pichucalco) sulphide springs have been colonized earlier
(gt200000 years ago Pfenninger et al 2014) and sulphide spring
populations in these drainages are more closely related to Poecilia
limantouri which primarily occurs north of the Trans-Mexican Vol-
canic Belt in northeastern Mexico (Brown et al 2017 Palacios et al
2013 2016) These older extremophile lineages have been described
as distinct species that are endemic to sulphide springs Poecilia ther-
malis (Steindachner 1863) in the Ixtapangajoya and Poecilia sulphu-
raria (Alvarez del Villar 1948) in the Pichucalco river drainage All
sulphide spring populationsmdashirrespective of their taxonomic statusmdash
represent locally adapted ecotypes that diverged in a variety of phe-
notypic traits (Riesch et al 2016 Tobler et al 2011) Sulphide
spring ecotypes can tolerate high H2S concentrations whereas eco-
types from adjacent nonsulphidic sites succumb to the toxic effects
of H2S over short periods of time (Plath et al 2013 Tobler et al
2011) indicating strong survival selection that impacts fitness across
small spatial scales Consequently high tolerance to environmental
H2S in sulphide spring fish can be viewed as an evolutionary innova-
tion that facilitated the colonization of unoccupied ecological niches
The sulphide spring fish of the P mexicana species complex pro-
vide an ideal system for an integrative approach to studying adapta-
tion and speciation for several reasons H2S provides a strong source
of selection with predictable physiological effects and known bio-
chemical consequences (Bagarinao 1992 Li Rose amp Moore 2011
Tobler et al 2016) which facilitates hypothesis-driven approaches
to studying mechanisms underlying physiological adaptation In addi-
tion the environmental gradients are clearly defined and replicated
which allows for an assessment of convergent patterns of evolution
across independent lineages that have colonized the same habitat
type The ease of cultivation of livebearing fish in the laboratory also
permits experimental approaches to corroborate patterns docu-
mented in the field and establish causal relationships between speci-
fic sources of selection and the evolution of specific traits Finally
growing genomic resources for fish of the family Poeciliidae facilitate
genetic analyses (Kelley et al 2012 Keuroustner et al 2016 Schartl
et al 2013)
2 | UNCOVERING PROXIMATEMECHANISMS OF ADAPTATION
Recent technological and analytical breakthroughs have revolution-
ized the genomewide quantification of genetic variation and gene
expression (Metzker 2010 Wang Gerstein amp Snyder 2009) facili-
tating the identification of genetic biochemical and physiological
underpinnings of adaptation to physiochemical stressors in natural
systems The rapid generation of large data sets allows for inductive
844 | TOBLER ET AL
Pichucalco Ixtapangajoya Puyacatengo Tacotalpa
Pichucalco
Teapa
Tacotalpa
Tapijulapa
4 km
(a)
(b) (c)
(d) (e)
F IGURE 1 Overview of the distribution of sulphide springs and their inhabitants in southern Mexico (a) Map depicting the four river drainages(Pichucalco Ixtapangajoya Puyacatengo and Tacotalpa) that exhibit sulphide springs inhabited by members of the P mexicana species complexThe locations of sulphide springs are indicated with yellow triangles black triangles represent commonly used field sites for the collection ofecotypes from nonsulphidic waters Note that roads (black lines) and major towns (grey shaded areas) were added for orientation The insethighlights the location of the study region within Mexico (bndashd) Examples of H2S-rich habitats in the Pichucalco (b) Puyacatengo (c) and Tacotalpa(d) river drainages (e) Representative individuals from the nonsulphidic (top) and the sulphidic (bottom) ecotypes from the Pichucalco river drainage
TOBLER ET AL | 845
inference of putative mechanisms underlying adaptation even in sys-
tems without a priori predictions (Kell amp Oliver 2004) However
genetic and gene expression data alone are insufficient to infer
adaptation at specific loci because neutral evolutionary processes
demographic history and genetic hitchhiking can generate false posi-
tives in genomewide analyses of selection and gene expression (Bar-
ton 1998 Ramirez-Soriano Ramos-Onsins Rozas Calafell amp
Navarro 2008 Whitehead amp Crawford 2006) This underscores the
importance of generating a priori hypotheses about the molecular
underpinnings of adaptation and testing whether genetic and gene
expression variation at specific loci actually affects organismal per-
formance under different environmental conditions (Dalziel Rogers
amp Schulte 2009 Storz amp Wheat 2010)
21 | Developing and testing a priori hypotheses
There is a rich history of research in toxicology and biomedicine that
has investigated the molecular targets of H2S illuminating the mech-
anisms of H2S toxicity and detoxification in model organisms and
human cell cultures (see Li et al 2011 Olson Donald Dombkowski
amp Perry 2012 Wang 2012 Wallace amp Wang 2015 for recent
reviews) In a recent attempt of ldquoseparating hype and hoperdquo in
potential biomedical applications of H2S Olson (2011) lamented that
H2S ldquoseemingly affects all organ systems and biological processes in
which it has been investigatedrdquo While it often remains unclear
whether the documented effects are a direct consequence of H2S
exposure or a result of the disruption of cellular homeostasis this
body of work provides fruitful grounds for the development of
hypotheses about the molecular mechanisms underlying adaptation
to H2S-rich environments that can then be tested using genome
scans for selection and analyses of gene expression variation
between sulphide-adapted and nonadapted populations (see Table 1
for an overview) Most importantly candidates include direct toxicity
targets involved in OXPHOS (Cooper amp Brown 2008) and oxygen
transport (Pietri Roman-Morales amp Lopez-Garriga 2011) anaerobic
metabolism (Dubilier Giere amp Grieshaber 2005) and oxidative stress
response systems (Eghbal Pennefather amp OrsquoBrien 2004) that may
help mitigate adverse consequences of H2S exposure as well as
pathways involved in enzymatic H2S detoxification sulphur process-
ing and sulphur excretion (Hildebrandt amp Grieshaber 2008) It is
important to note that H2S is also produced endogenously by meta-
zoans in the cytosol and in mitochondria through enzymatic reac-
tions associated with the reverse trans-sulphuration pathway
(Stipanuk 2004 Stipanuk amp Ueki 2011) At concentrations well
below the toxicity threshold endogenously produced H2S serves as
a cellular signalling molecule that modulates physiological processes
including the function of ion channels that underlie smooth muscle
control and the function of neural cells (Peers Bauer Boyle Scragg
amp Dallas 2012) and it interacts with transcription factors that con-
trol inflammation cell survival and proliferation the expression of
cellular protective enzymes and hypoxia responses (Li et al 2011
Wallace amp Wang 2015 Wang 2012) Organisms may thus not strive
to merely eliminate H2S from their system and avoid effects of
toxicity Instead they are likely selected for maintaining optimal
endogenous concentrations that facilitate H2S-mediated signalling
and assure proper physiological functioning As a consequence
endogenous H2S production pathways and targets of H2S signalling
may also be modified during adaptation to environmental H2S
A series of studies in members of the P mexicana species com-
plex have tested hypotheses about the molecular underpinnings of
H2S adaptation by analysing patterns of molecular evolution in can-
didate genes (Barts et al 2018 Pfenninger et al 2014) deploying
genomewide scans for selection based on FST outliers (Pfenninger
et al 2015) and quantifying genomewide patterns of gene expres-
sion (Kelley et al 2016 Passow Henpita et al 2017) Collectively
the results of these studies have indicated a striking congruence
between candidate genes inferred from toxicological and biomedical
research and genes with empirical evidence for selection andor dif-
ferential expression between at least some of the adjacent H2S-
adapted and nonadapted populations (Table 1) (i) There was evi-
dence for selection on genes encoding the primary toxicity target of
H2S COX (complex IV in OXPHOS Pfenninger et al 2014) In addi-
tion genes encoding other components and assembly factors of
OXPHOS are either under selection or differentially expressed in sul-
phide spring populations (Kelley et al 2016 Pfenninger et al
2015) Evidence for selection and differential expression was also
uncovered for subunits of haemoglobin whose function is directly
impaired by H2S (Barts et al 2018) (ii) Genes involved in anaerobic
ATP production and in coping with oxidative stress exhibited evi-
dence for selection and differential expression suggesting a shift
towards alternative sources of ATP production and an increased abil-
ity to withstand reactive oxygen species that are produced as a by-
product of OXPHOS inhibition by H2S (Kelley et al 2016 Pfen-
ninger et al 2015) (iii) There is overwhelming evidence for selection
on and differential regulation of genes encoding components of the
sulphidequinone oxidoreductase pathway which mediates enzymatic
H2S detoxification in metazoans (Kelley et al 2016 Pfenninger
et al 2015) In addition there is evidence for upregulation of genes
encoding transporters of oxidized sulphur species which are puta-
tively involved in sulphur excretion (Kelley et al 2016)
These results suggest that adaptation to H2S-rich environments
in members of the P mexicana species complex involves both an
increased ability to cope with elevated endogenous H2S and an
increased ability to keep endogenous H2S concentrations low
through detoxification and excretion However there is a noticeable
absence of evidence for genes and physiological pathways associ-
ated with endogenous H2S production and H2S-mediated cell sig-
nalling being involved in adaptation (see Kelley et al 2016
Pfenninger et al 2015) There are three nonmutually exclusive
hypotheses that could explain this finding First the ability of popu-
lations from sulphidic habitats to regulate endogenous H2S through
detoxification may be sufficient to maintain normal H2S-mediated
signalling Second compared to the ability for detoxification and for
mitigation of direct toxic effects in terms of ATP production and
oxidative stress H2Srsquos influence on cell signalling may have relatively
low fitness consequences with concomitantly smaller evolutionary
846 | TOBLER ET AL
responses Last inferences from biomedical studies about the causal
relationship between endogenously produced H2S and various physi-
ological functions may be weak and some documented physiological
effects may instead be an indirect consequence of the disruption of
homeostasis that cascades through interconnected pathways operat-
ing in a cell In this case evolutionary studies on natural systems
exposed to H2S may provide important insights in terms of prioritiz-
ing research on biomedically relevant aspects of H2S biology Study-
ing ancestral populations susceptible to H2S will allow for
quantifying consequences of disruptions in H2S homeostasis at all
levels of biological organization (like in presently available animal
models) Adding derived H2S tolerant populations will further allow
for the detection of critical modifications in physiological processes
affected by H2S Consequently such a comparative approach may
provide a powerful tool to disentangle direct and indirect physiologi-
cal consequences of deviations in H2S homeostasis which is cur-
rently difficult in traditional biomedical models
Despite some of these discrepancies the studies exploring mech-
anisms underlying H2S tolerance in sulphide spring fishes highlight
the utility of research in toxicology and biomedicine in generating
testable hypotheses about evolutionary processes Biomedical stud-
ies on metabolic disorders (Aspiras Rohner Martineau Borowsky amp
Clifford 2015) stress responses (Rohner et al 2013) responses to
low oxygen (Eales Hollinshead amp Tennant 2016) and effects of
xenobiotics (Stockinger Di Meglio Gialitakis amp Duarte 2014) have
also facilitated our understanding of adaptive evolution in other nat-
ural systems Considering the increasingly specialized and frag-
mented research areas in the life sciences interdisciplinary
approaches and reciprocal exchange of ideas and methodologies
continue to be critical for facilitating breakthroughs in basic and
applied research on biomedical models and natural systems (Carroll
et al 2014 Nesse amp Stearns 2008 Schartl 2014)
22 | Convergent and nonconvergent evolution inindependent lineages
Our discussion of putative candidate genes that are under selection
andor differentially expressed in sulphide spring populations so far
focused on general patterns documented across evolutionarily inde-
pendent lineages in different river drainages Selection scans (Pfen-
ninger et al 2015) and analyses of differential gene expression
(Kelley et al 2016) have indicated high congruence of biological
processes associated with candidate genes across lineages in differ-
ent river drainages indicating that evolution has repeatedly and pre-
dictably modified the same physiological pathways during adaptation
to environmental H2S However the signal of evolutionary conver-
gence among sulphide spring lineages decreases with the level of
organismal organization (from physiological pathways down to geno-
mic regions genes and individual SNPs in selection scans Pfenninger
et al 2015 from physiological pathways down to genes and tran-
scripts in expression studies Passow Brown et al 2017) indicating
that different lineages have often modified the same physiological
pathways in different ways While knowledge about the biochemical
TABLE 1 Adaptation to H2S can occur through three principlemechanisms (1) Resistance involves direct toxicity targets whosefunctions are impaired by H2S Adaptive modifications of thesetoxicity targets are predicted to assure proper function despitethe presence of H2S (2) Mitigation involves alternativephysiological pathways that compensate for the inhibition ofdirect toxicity targets Adaptive modifications of these alternativephysiological pathways are predicted to allow for the maintenanceof organismal function by other means (3) Regulation involvesphysiological pathways associated with the maintenance ofendogenous H2S homeostasis Adaptive modifications ofregulatory pathways are predicted to maintain low endogenousconcentrations despite continuous flux of H2S from theenvironment into the body
Candidate pathways Evidence
(1) Resistance
OXPHOS bull Positive selection on COX in some lineages of
P mexicana (Puyacatengo and Pichucalco)a as
well as some other genera of sulphide spring
poeciliidsb COX amino acid substitutions in P
mexicana allow for the maintenance of function
in the presence of H2Sa
bull Upregulation of cytochrome c and some COX
subunits as well as downregulation of compo-
nents of complex I in all sulphide spring lineages
of P mexicana indicating complex changes in
aerobic ATP productionc
Oxygen transport
genesbull Positive selection on and upregulation of
several oxygen transport genes (including myo-
globin and multiple haemoglobin subunits) in
sulphide spring lineages of the genera
Gambusia Limia Poecilia and Pseudoxiphophorus
indicating modifications to putatively maintain
oxygen transport in the presence of H2Sd
(2) Mitigation
Anaerobic
metabolismbull Upregulation of genes associated with glycolysis
and gluconeogenesis in all sulphide spring lin-
eages of P mexicana indicating increased
capacity of anaerobic ATP productionc
Oxidative stress
responsesbull Upregulation of genes associated with
oxidative stress responses in all sulphide
spring lineages of P mexicana indicating
increased capacity to cope with H2S-induced
oxidative stressc
(3) Regulation
H2S detoxification bull Positive selection on and upregulation of sev-
eral genes associated with enzymatic H2S
detoxification in all sulphide spring lineages of
P mexicanace Upregulation of H2S detoxifica-
tion enzymes in other sulphide spring lineages
of the genera Gambusia Limia Poecilia Pseudox-
iphophorus and Xiphophorusb This indicates an
increased ability to maintain low endogenous
H2S concentrations during exposure
H2S production bull No evidence for modifications of genes associ-
ated with enzymatic production of H2Sc
aPfenninger et al (2014) bR Greenway J L Kelley amp M Tobler unpub-
lished data cKelley et al (2016) dBarts et al (2018) ePfenninger et al
(2015)
TOBLER ET AL | 847
and physiological consequences of H2S may thus enable us to pre-
dict the physiological pathways in which adaptive modifications
occur it may be impossible to predict specific modifications within
those pathways This contrasts with a common expectation in the
field that convergent evolution of molecular mechanisms underlying
phenotypic traits (ie parallel evolution) necessarily hinges on modi-
fication of the same loci across lineages as found in some of the
best-known examples of parallelism (Colosimo et al 2005 Feldman
Brodie Brodie amp Pfrender 2012 Grant Grant Markert Keller amp
Petren 2004) However modification of the same loci may only be
common under particular circumstances (Rosenblum Parent amp
Brandt 2014) for example when selection acts on standing genetic
variation (Colosimo et al 2005) when adaptive alleles are intro-
gressed across population boundaries through gene flow (Grant
et al 2004) or when there are strong genetic constraints (Feldman
et al 2012) Exploration of molecular mechanisms underlying con-
vergent evolution in many systems may therefore be better
approached from the perspective of interacting genes that are orga-
nized in pathways in which alternative genetic changes can have
equivalent functional consequences (ie functional redundancy)
Developing and testing hypotheses about parallel evolution at the
pathway level will hinge on improving genome annotations (Pavey
Bernatchez Aubin-Horth amp Landry 2012) and provide a more
nuanced understanding of the factors determining the predictability
of evolution at different levels of organismal organization (Rosen-
blum et al 2014)
Despite evidence for convergent evolution in some candidate
physiological pathways it also needs to be emphasized that this
does not apply to all P mexicana lineages or to all candidate physio-
logical pathways associated with H2S adaptation investigated to
date Nonconvergent evolutionmdashevidenced by unique lineage-speci-
fic signals of selection and differential expressionmdashmay be indicative
of unique molecular mechanisms to cope with H2S toxicity For
example signatures of positive selection on COXmdashH2Srsquos primary
toxicity targetmdashcan be found in sulphide spring lineages of the Puy-
acatengo and Pichucalco river drainages (a lineage with recent diver-
gence time and a lineage with older divergence time respectively)
but not in those of the Tacotalpa drainage (Pfenninger et al 2014)
The presence of nonconvergent genetic and gene expression differ-
ences between populations from sulphidic and non-sulphidic habitats
in different river drainages highlights the limitations of inferences
that can be drawn based on data sets generated with high-through-
put DNA and RNA sequencing The potential importance of modifi-
cations at candidate locimdashor the lack thereofmdashcan only be assessed
through functional assays that document how alternative alleles and
changes in gene expression actually affect organismal performance
The functional consequences of genetic and gene expression varia-
tion at most candidate loci remain to be investigated However
enzyme activity assays have indicated that COX maintains its activity
with increasing H2S concentrations in the two sulphide spring popu-
lations with evidence for positive selection on mitochondrially
encoded subunits (Puyacatengo and Pichucalco Pfenninger et al
2014) Key amino acid substitutions occur in the D-pathway channel
of COX1 which putatively narrow the channel width and reduce
H2Srsquos access to the binuclear centre of the protein Consequently
some lineages of P mexicana exhibit an H2S-resistant COX that can
maintain its activity and aerobic ATP production even if endogenous
H2S concentrations are elevated In contrast COX activity in sul-
phide spring fish of the Tacotalpa river drainage and in fish from
nonsulphidic habitats decreases when H2S is present which is con-
sistent with mechanisms of H2S toxicity (Pfenninger et al 2014) In
vitro assays on isolated mitochondria have nonetheless indicated
that an H2S-resistant COX is not necessary to maintain aerobic ATP
production during H2S exposure Unlike in populations from nonsul-
phidic habitats mitochondrial spare respiratory capacity is main-
tained in the presence of H2S even in the Tacotalpa sulphide spring
populations that lack an H2S-resistant COX (C Henpita M Tobler amp
JH Shaw unpublished data) This may indicate that modification of
detoxification pathways in sulphide spring fishes is sufficient to
maintain mitochondrial function and aerobic ATP production
23 | Ensuing challenges linking genotypes tofitness
Understanding the mechanistic basis of adaptation requires linking
genotypic variation to phenotypic traits that ultimately shape fitness
along selective gradients The evolution of H2S tolerance in sulphide
spring fishes appears to be a consequence of modifications in many
genes and many physiological pathways whose functions interact to
eliminate H2S from the system minimize effects of elevated endoge-
nous concentrations on direct toxicity targets and mitigate adverse
effects that cascade through cellular metabolism during disruption of
homeostasis (Table 1) Such complex genetic changes are likely com-
mon during adaptation and have been documented in other systems
that involve major evolutionary shifts including the emergence of
parasitic lifestyles (Werren et al 2010) eusociality (Simola et al
2013) asexual reproduction (Fradin et al 2017) and internal gesta-
tion with livebearing (Schartl et al 2013) Consequently comple-
mentary approaches will be required to uncover the molecular
mechanisms of adaptation to H2S in the future
1 The involvement of many genes and physiological pathways in
shaping tolerance emphasizes the importance of quantitative
genetic approaches that leverage experimental crosses between
H2S tolerant and nontolerant populations and test whether toler-
ancemdashand adaptive trait modifications that contribute to itmdashis
explained by a few major effects loci or whether it is a conse-
quence of a number of genes with small effects (Klerks Xie amp
Levinton 2011 Saltz Hessel amp Kelly 2017) Combining analyses
of phenotype-fitness associations and their underlying genetic
mechanisms via QTL mapping with the results of selection scans
from population genomic analyses (Pfenninger et al 2015) will
allow for an integrative understanding of how selection acting on
the genome results in phenotypic variation that mediates adapta-
tion to different environments (Stinchcombe amp Hoekstra 2008)
From a functional perspective breaking up correlations between
848 | TOBLER ET AL
different physiological traits (associated with resistance mitiga-
tion and regulation Table 1) in F2 crosses will further allow test-
ing what molecular mechanisms are necessary for tolerance
whether there are alternative strategies with equivalent fitness
benefits and whether there are complementary modifications
with additive effects on tolerance
2 Considering the number of genes and physiological pathways
likely involved in H2S tolerance assessing the functional conse-
quences of genetic and gene expression variation is a daunting
task At the same time such functional tests can significantly
contribute to closing the genotype-to-phenotype gap because
physiochemical stress responses can be assessed along the con-
tinuum of organismal organization (Gjuvsland Vik Beard Hunter
amp Omholt 2013) Standardized assays can be used to quantify
the biochemical makeup of organelles cells and tissues (eg pro-
tein abundance and enzyme activity) physiological performance
in vitro and in vivo (eg rates of H2S detoxification ATP produc-
tion and reactive oxygen production) and fitness-relevant proxies
of whole organism performance (eg H2S tolerance Figure 2)
This highlights the importance of methods development for the
quantification of complex phenotypic traits or even whole phe-
nomes that can keep pace with rapid data generation through
high-throughput sequencing (Houle Govindaraju amp Omholt
2010 Ritchie Holzinger Li Pendergrass amp Kim 2015) In addi-
tion genotypendashphenotype links established through such correla-
tive approaches will ideally be verified through targeted gene
editing While gene editing with CRISPRndashCas9 is now routinely
employed in model organisms (Doudna amp Charpentier 2014 Sha-
lem Sanjana amp Zhang 2015) applying this technology to non-
models still remains challenging especially in livebearing species
like poeciliid fishes where in vitro culture of embryos is not rou-
tinely employed (Martyn Weigel amp Dreyer 2006) Nonetheless
the effects of specific mutations can potentially be verified
through the establishment and manipulation of cells in culture
(Liu et al 2014) or by generating transgenics (Lin et al 2016)
3 Organismal responses to physiochemical stressors occur at multi-
ple hierarchical levels that can be plastically modulated in
response to endogenous and exogenous cues (Whitehead 2012)
Understanding adaptation to physiochemical stressors conse-
quently requires disentangling the effects of genetic variation
developmental plasticity and short-term acclimation in gene
expression patterns on the emerging physiological processes
Common garden experiments on sulphide spring fishes have
shown that many genes differentially expressed between popula-
tions from sulphidic and nonsulphidic habitats in nature remain
differentially expressed in laboratory-reared fish that have never
been exposed to H2S indicating evolved differences in constitu-
tive gene expression (Passow Henpita et al 2017) At the same
time expression patterns in the laboratory also exhibit evidence
for plasticity in response to short-term H2S exposure (Passow
Henpita et al 2017) Some of these plastic responses are shared
between populations from sulphidic and nonsulphidic habitats
(likely representing shared ancestral plasticity) while others have
been lost or gained during sulphide spring colonization A core
challenge for future research is to overcome logistical constraints
that have so far prevented long-term H2S exposures of fish in
the laboratory Exposing individuals throughout development will
be required to test how developmental plasticity and short-term
acclimation shape responses to H2S in individuals from adapted
and nonadapted populations Finally the next big scientific
advance in this context will also require the identification of reg-
ulatory mechanisms that shape expression differences during
adaptation including sequence changes in transcription factors
and their binding sites patterns of DNA methylation and chro-
matin accessibility as well as the production of microRNAs and
competing endogenous RNAs that can modulate transcript abun-
dance (Flores Wolschin amp Amdam 2013 Lopez-Maury Mar-
guerat amp Beuroahler 2008 Silva Bye el Azzouzi amp Wisloslashff 2017)
In addition the role of alternative splicing and other post-tran-
scriptional and post-translational modifications in mediating adap-
tation remains understudied by evolutionary biologists (Ast
2004 Bush Chen Tovar-Corona amp Urritia 2017) This is particu-
larly relevant because H2S has been hypothesized to play a role
in the post-translational modification of proteins with sulphur-
containing amino acids through sulfhydration (Mustafa et al
2009)
Bridging the genotypendashphenotype gap remains one of the major
challenges in biology (Gjuvsland et al 2013 Ritchie et al 2015) and
will provide opportunities to understand how organisms function in
the face of environmental stress and how evolution shapes these
functions during adaptation to different environmental conditions
Establishing the molecular mechanisms of adaptation across repli-
cated lineages exposed to the same sources of selection will also
provide insights into how evolutionary history demography and
genetics shape convergent evolutionary outcomes providing a better
understanding at what hierarchical levels of organismal organization
evolutionary change may actually be predictable (Rosenblum et al
2014)
3 | COMPLEX ORGANISMS IN A COMPLEXWORLD DISENTANGLING THE TANGLEDBANK
Mechanisms of adaptation can be studied through bottom-up
(screening for genetic modifications that allow for inferences about
relevant phenotypic traits) and top-down approaches (assessing fit-
ness-relevant phenotypic differences among populations without
knowledge about the genetic basis see Schluter 2009) Over the
years genomic and transcriptomic studies on sulphide spring popula-
tions of the P mexicana species complex have been complemented
with analyses of phenotypic variation Just as genetic analyses have
indicated that adaptation to H2S is mediated by the modification
and modulation of many genes associated with multiple physiological
pathways phenotypic studies uncovered a variety of differences
TOBLER ET AL | 849
between populations from sulphidic and nonsulphidic environments
Many of these phenotypic differences have evolved in convergence
across replicated sulphide spring lineages in different river drainages
Examples include modifications of morphological (body shape and
organ size Tobler et al 2011 Tobler Scharnweber et al 2015
Schulz-Mirbach et al 2016) physiological (tolerance and metabolic
rates Tobler et al 2011 Passow Arias-Rodriguez amp Tobler 2017)
behavioural (aggression and personality Riesch et al 2009 Bierbach
et al 2012 Sommer-Trembo et al 2016 Bierbach Arias Rodriguez
amp Plath 2018) and life history traits (fecundity and offspring size
Riesch Plath Garcia de Leon amp Schlupp 2010 Riesch et al 2014)
Hence proximate populations in different habitat types have
diverged in complex phenotypes that span many different suites of
traits and a key question is how divergence in multivariate pheno-
types arises In theory H2S could be a key source of selection driv-
ing multivariate phenotypic divergence either because it directly
exerts selection on a multitude of traits that are independently modi-
fied to reach new fitness peaks or because selection on a few fit-
ness-relevant traits causes simultaneous changes in correlated
characteristics Neither explanation seems sufficient because many
Abiotic environmentOxygen concentrations
Ionic compositionTemperature
Structure and flow
Biotic environmentTrophic resources
DemographyPredationParasitism
H2S
Genetic variation
Gene expression
Biochemistry
Physiologicalfunction
Organismal performance
Primary fitness components
Survivalgrowth
reproduction
Organismal function Environment
F IGURE 2 Understanding how organisms function in the context of their environment is a core question in evolutionary biology yet thecomplexities associated with organismal function and environmental variation provide significant challenges High-throughput sequencing hasallowed for the identification of candidate genes subject to selection and differential expression during adaptation to H2S The next step is tobridge the genotype-to-phenotype gap by quantifying the integrated responses to H2S across the continuum of organismal organization includingbiochemical phenotypes physiological functions whole organism performance and ultimately fitness In addition understanding adaptation toH2S-rich environments will also require acknowledging H2Srsquos relation to other abiotic and biotic environmental variables which can exert selectionand modulate aspects of organismal function This will allow for the establishment of cause-and-effect relationships between specific sources ofselection and evolutionary change in specific traits and contribute to our understanding of organismal function in natural systems
850 | TOBLER ET AL
of the divergent traits between populations from sulphidic and non-
sulphidic environments have no clearmdashor at best tenuousmdashlinks to
H2S toxicity and detoxification and there is only limited evidence
for within-population correlations between different traits groups
(Riesch et al 2016)
Evolutionary analyses frequently focus on seemingly simple
sources of selection juxtaposing populations that are assigned to dis-
crete habitat types to understand the evolution of specific traits
(Kaeuffer Peichel Bolnick amp Hendry 2012) But as Darwin (1859)
already observed in his ldquotangled bankrdquo allegory selection in natural
systems is rarely univariate but multifarious selective regimes con-
tribute to trait divergence even among populations that occur along
apparently simple environmental gradients Unquantified environmen-
tal variation that is subsumed in the categorization of habitat types
can profoundly affect trait variation in natural systems (Stuart et al
2017) highlighting the importance of considering and testing alterna-
tive hypotheses about the cause-and-effect relationship between
specific sources of selection and evolution of specific traits Thus
accurate natural history observations that consider organisms as holis-
tic entities consisting of interacting traits in the context of the abiotic
and biotic factors comprising their environment are critical to inform
the design and interpretation of empirical research in natural systems
(Greene 2005) Indeed adjacent sulphidic and nonsulphidic habitat
types do not merely differ in the presence or absence of H2S but also
in a variety of other abiotic and biotic factors (Figure 2) Unlike nonsul-
phidic habitats sulphide springs in southern Mexico are characterized
by extreme hypoxia elevated salinity and lower pH (Rosales Lagarde
2012 Tobler et al 2011) In addition the presence of physiochemical
stressors affects the biotic communities in these sulphide springs Spe-
cies richness of aquatic invertebrates and vertebrates is drastically
reduced as a consequence of H2S toxicity (Greenway Arias-Rodriguez
Diaz amp Tobler 2014) and primary production appears to be driven in
part by chemoautotrophic bacteria that use H2S as an energy source
rather than just by autochthonous and allochthonous photosynthetic
organisms (Roach Tobler amp Winemiller 2011) These changes in the
biotic environment affect the relative importance of intra-vs inter-
specific competition trophic resource use and exposure to predators
and parasites (Riesch Oranth et al 2010 Tobler et al 2014 Tobler
Scharnweber et al 2015)
31 | Alternative drivers of trait evolution
Acknowledging the complexity of selective regimes in sulphide
springs allows for a more nuanced understanding of trait evolution
and for the development of alternative hypotheses about the ulti-
mate mechanisms that cause divergence in specific genetic and phe-
notypic traits In some instances sources of selection other than H2S
can plausibly explain trait divergence between populations from sul-
phidic and nonsulphidic habitats For example hypoxia likely drives
increases in head size and gill surface area (Tobler et al 2011)
changes in the ionic composition of the water can explain differential
expression of genes associated with osmoregulation (Kelley et al
2016) and shifts in trophic resource use are associated with
modifications of the gastrointestinal system (Tobler Scharnweber
et al 2015) In other cases disentangling alternative hypotheses is
not trivial because selection mediated by H2S and other environmen-
tal factors are predicted to affect trait modifications in similar ways
Shifts towards anaerobic metabolism in sulphide spring fish could be
the consequence of inhibition of aerobic ATP production by H2S or
by constraints in oxygen availability Similarly reproductive life his-
tory shifts towards the production of larger but fewer offspring could
be driven by H2S (decreasing offspringrsquos surfacevolume ratio to mini-
mize the diffusion of H2S into the body Powell 1989) by habitat dif-
ferences in the quality and quantity of predation that can affect size-
specific rates of extrinsic mortality (Jorgensen Auer amp Reznick
2011) or by energetic constraints (see below) While comparisons
across different selective regimes can shed light into the ultimate dri-
vers of trait evolution (Moore Riesch amp Martin 2016) isolating the
effects of different sources of selection may not be possible because
they could act in concert and facilitate increases of organismal perfor-
mance in multiple contexts (Ghalambor Walker amp Reznick 2003)
When multifarious selection shapes multivariate phenotypic evo-
lution it is important to also consider the role of trade-offs both in
terms of trait expression (increased investment in one trait may
impose constraints on the expression of other traits) and in terms of
trait function (optimizing the trait for one function may impair the
traitrsquos utility for other functions) For example selection on large off-
spring size in sulphide spring fishes has inadvertent consequences
for fecundity (Riesch et al 2014) and selection for increased gill
surface areas due to hypoxia simultaneously increases exposure to
environmental H2S (Tobler et al 2011) Thus the outcome of phe-
notypic evolution is ultimately a result of balancing competing needs
in a manner that optimizes trait expression and organismal function
in multiple contexts to maximize fitness (Arnold 1983) and the pres-
ence of trade-offs can have cascading indirect effects with selection
on one or a few traits leading to the modification of a multitude of
apparently unrelated characteristics
In this context bioenergetics may represent a nexus in the evo-
lution of complex traits observed in sulphide spring fish and other
extreme environments Living in and adapting to sulphide springs
imposes constraints on organismal energy availability (Plath et al
2007) with according trade-offs for energy allocation Energy limita-
tion in sulphide springs does not arise from a scarcity of food
resources (chemoautotrophic bacteria eaten by the fish are abundant
in sulphide springs Tobler Scharnweber et al 2015) but from
behavioural and physiological adaptation A primary survival strategy
for fish in the toxic and hypoxic springs is to engage in aquatic sur-
face respiration where individuals breathe directly at the water sur-
face to maximize oxygen uptake and minimize H2S exposure This
behaviour mediates short-term survival (Plath et al 2007) but it
also constrains energy acquisition for benthic foragers like P mexi-
cana (Tobler Riesch Tobler amp Plath 2009) and increases susceptibil-
ity to bird predation (Riesch Oranth et al 2010) Costly investments
into H2S detoxification and tissue repair as well as a shift from aero-
bic to less efficient anaerobic metabolism further constrain energy
allocation to other functions (Passow Greenway Arias-Rodriguez
TOBLER ET AL | 851
Jeyasingh amp Tobler 2015) Theory predicts that energetic con-
straints ndash whether mediated by limitations in the rate of resource
acquisition or in the rate of energy allocation to reproduction (as
opposed to maintenance or growth) ndash should exert selection for a
reduction in organismal energy budgets which allows for the maxi-
mization of residual energy allocation for the production of offspring
(Brown Marquet amp Taper 1993) Several lines of evidence support
the hypothesis that energy limitation plays a critical role in pheno-
typic evolution of sulphide spring fish Compared to individuals in
nonsulphidic environments fish in sulphide springs have significantly
lower routine energy demands which is mediated both by reduc-
tions in body size and mass-specific metabolism (Passow Arias-
Rodriguez et al 2017) Reductions in other traits such as energeti-
cally costly behaviours (Bierbach et al 2012 Plath 2008) and the
size of energy-demanding organs (Schulz-Mirbach et al 2016) may
also contribute to the reduced energy demands and shifts in repro-
ductive life history traits (Riesch et al 2014) are consistent with
selection from energy limitation (Reznick amp Yang 1993)
32 | Ensuing challenges Disentanglingenvironmental and organismal complexities
Future research needs to empirically address how multifarious selec-
tive regimes shape the evolution of organisms with complex pheno-
types We have made great advances in our understanding of how
specific traits evolve and mediate adaptation (eg morphology Chan
et al 2010 coloration Dasmahapatra et al 2012 sensory organs
Cartwright Schwartz Merry amp Howell 2017) and we have a thor-
ough understanding of how single environmental factors can shape
organismal responses (eg climatic conditions Johnston amp Bennett
1996 predation Langerhans 2007 competition Pfennig amp Pfennig
2012) In contrast our understanding of phenotypic evolution in
response to multifarious selection is comparatively limited especially
because interactive effects of different environmental factors can lead
to organismal and evolutionary responses that are unpredictable based
on experiments that manipulate single environmental variables in iso-
lation (Gunderson Armstrong amp Stillman 2016 Todgham amp Stillman
2013) Future research thus needs to acknowledge the complexities of
environments and organismal design and address them explicitly in
empirical investigations to gain a robust understanding of biological
diversification (Culumber amp Tobler 2017 Stuart et al 2017)
4 | LINKING ADAPTATION TOSPECIATION
So far we primarily explored the causes of adaptive evolution but
what are its consequences Theory predicts that adaptation to local
environmental conditions can lead to speciation because reproduc-
tive isolation emerges incidentally as a by-product (ecological specia-
tion Nosil 2012) Population genetic analyses of Poecilia spp in
sulphide springs and adjacent populations in nonsulphidic environ-
ments have indicated strong genetic differentiation across small
spatial scales (in some instances lt100 m) and in the absence of
physical barriers that would prevent fish movement (Plath et al
2013) In addition population genetic structuring has been shown to
be stable even in the face of major environmental disturbance
BOX 1 Speciation in sulphide springs and conservation
biology
Adaptation to H2S-rich environments in poeciliids coincides
with ecological speciation giving rise to phenotypically dis-
tinct and reproductively isolated lineages In some
instances this has led to the evolution of highly distinct
micro-endemic species including Poecilia sulphuraria and
P thermalis Limia sulphurophila and Gambusia eurystoma
(Figure 3) Except for P sulphuraria which is found in two
spring complexes of the Rıo Pichucalco drainage of Mexico
(Tobler amp Plath 2009b) the distributions of these sulphide
spring endemics are restricted to single localities that
include only small areas of suitable habitat For example
the largest sulphide spring complex (the Ba~nos del Azufre
in southern Mexico which harbours P sulphuraria and G
eurystoma) includes ~18000 m2 of suitable habitat (equiva-
lent to lt35 football fields) while one of the smallest (La
Esperanza springs harbouring P thermalis) merely covers
~600 m2 (just over a tenth of a football field Brown et al
2017) Accordingly some sulphide spring endemics are
considered critically endangered (IUCN 2012) and pro-
tected by law (SEDESOL 1994)
Many threatened species with restricted ranges exhibit
low genetic diversity and high incidences of inbreeding
(Bouzat 2010 Willi Van Buskirk amp Hoffmann 2006) In
contrast at least P sulphuraria and P thermalismdashthe only
species investigated to datemdashactually exhibit relatively high
effective population sizes (Ne = 1200ndash3800 depending on
the population) and no evidence for inbreeding (FIS lt 0)
(Brown et al 2017) This is likely a consequence of the
long-term stability of sulphide springs and the high pro-
ductivity and low levels of interspecific competition also
support unusually high fish densities and thus population
census sizes (Brown et al 2017 Culumber et al 2016)
Nonetheless sulphide spring endemics are threatened by
habitat destruction which is mediated primarily by the
modification of springs for recreational purposes defor-
estation and conversion of land for agriculture and most
recently high-intensity palm oil plantations (Tobler amp Plath
2009ab) Despite the official protection of sulphide spring
endemics there have been no tangible conservation mea-
sures that foster the sustainable development of land
around sulphide springs with the well-being of the endemic
fishes in mind
852 | TOBLER ET AL
(catastrophic flood event Plath et al 2010) Population genetic dif-
ferentiation between adjacent sulphide-adapted and nonadapted
populations is indicative of ongoing ecological speciation (Box 1) but
different population pairs from sulphidic and nonsulphidic waters
have made variable progress along the speciation continuum towards
complete reproductive isolation (Plath et al 2013) This prompts
questions about the mechanisms involved in reducing gene flow
along the environmental gradient connecting the two habitat types
41 | Prezygotic mechanisms of reproductiveisolation
The simplest form of reproductive isolation between locally adapted
populations arises from selection against migrants between habitat
types (Nosil Vines amp Funk 2005) Translocation experiments in nat-
ural habitats have indicated strong survival selection against
migrants especially those translocated from nonsulphidic to sulphidic
habitats (Plath et al 2013) Fish translocated from nonsulphidic
environments face high mortality within short periods of time
(lt24 hr) which is likely caused by H2S toxicity and hypoxia in sul-
phide springs Hence low gene flow from populations inhabiting
nonsulphidic waters into populations inhabiting sulphidic habitats is
simply explained by a lack of adaptation to the extreme environmen-
tal conditions At least in some drainages there is also appreciable
mortality for sulphide spring fish translocated to nonsulphidic habi-
tats although mechanisms causing inviability are unclear and the
strength of selection appears to be lower (Plath et al 2013) If sur-
vival selection against immigrants was the sole source of reproduc-
tive isolation this should result in unidirectional gene flow from
H2S-adapted to nonadapted populations but population genetic
analyses provided no evidence for this implying that additional
mechanisms are at work (Plath et al 2013)
One such mechanism could be intersexual selection and mate
choice experiments have indicated that migrants from sulphidic to
nonsulphidic habitats are at a significant disadvantage with fish from
nonsulphidic habitats avoiding potential mating partners from sul-
phidic ones (Plath et al 2013 Sommer-Trembo et al 2016) Such
assortative mating has been found to be critical during ecological spe-
ciation because it counteracts the homogenizing effects of gene flow
(van Doorn Edelaar amp Weissing 2009) yet how assortative mating
patterns arise often remains unclear In theory assortative mating can
arise through a variety of processes (Nosil 2012) ldquoMagic traitrdquo mecha-
nisms where mating preferences evolve as a by-product of divergent
selection on ecological traits that serve as cues have recently garnered
much attention (Servedio van Doorn Kopp Frame amp Nosil 2011)
Consistent with magic trait mechanisms assortative mating in P mexi-
cana appears to be linked to adaptive population differences in body
shape which serve as cues to distinguish potential mating partners
from different habitats (Greenway Drexler Arias-Rodriguez amp Tobler
2016) However neither sulphide spring fish (which are unlikely to
encounter migrants from nonsulphidic habitats due to H2S toxicity)
nor allopatric P mexicana from nonsulphidic waters (which have never
experienced migrants from sulphide spring populations) exhibit a
preference for mating partners from their own ecotype suggesting
that assortative mating in this system is a product of reinforcement
(ie direct selection for assortative mating Greenway et al 2016)
This is different from classic magic traits mechanisms because assorta-
tive mating is not a mere by-product of adaptation but adaptive traits
have instead been co-opted as mating signals in response to selection
on mating preferences (Maan amp Seehausen 2012) Moreover males of
some H2S-adapted populations have reduced aggressive behaviour
(Bierbach et al 2012 2018) In staged contests of size-matched males
from different ecotypes intruders had a significantly lower likelihood
to establish dominance than resident males independent of whether
or not they showed reduced aggressiveness overall Hence intrasexual
selection may represent another mechanism mediating reproductive
isolation between locally adapted ecotypes Overall H2S-adapted poe-
ciliids allow disentangling the relative contributions of different forms
of ecological and sexual selectionmdashand their interdependencymdashduring
ecological speciation
42 | Ensuing challenges quantifying postzygoticmechanisms of reproductive isolation
In contrast to prezygotic mechanisms little is known about postzy-
gotic mechanisms of reproductive isolation that potentially act to
reduce gene flow between adjacent populations in sulphidic and
nonsulphidic habitats Putative hybrids between sulphidic and non-
sulphidic ecotypes have occasionally been observed in nonsulphidic
habitats immediately adjacent to confluences with sulphide springs
(R Riesch M Plath and M Tobler personal observations) but it
remains unclear whether and through what mechanisms such hybrids
exhibit reduced fitness compared to parentals
Direct selection of mitochondrially encoded genes in the pres-
ence of H2S (Pfenninger et al 2014) should be accompanied by co-
evolution at nuclear loci that directly interact with mitochondrial
gene products in the formation of protein complexes involved in the
OXPHOS pathway (Sunnucks Morales Lamb Pavlova amp Greening
2017 Willett amp Burton 2004) Disruption of co-evolved gene com-
plexes in hybrids should therefore lead to intrinsic fitness reductions
through mito-nuclear incompatibilities (Burton Pereira amp Barreto
2013 Wolff Ladoukakis Enrıquez amp Dowling 2014) which have
been hypothesized to be a key driver in speciation (Chou amp Leu
2010 Gershoni Templeton amp Mishmar 2009) Empirical tests of
these ideas remain to be conducted in sulphide spring fishes just
like studies on potential ecological and sexual selection against
hybrids that arise from interpopulation mating events
5 | PERSPECTIVES LIFE IN SULPHIDICENVIRONMENTS BEYOND P MEXICANA
Research on sulphide spring fishes of the P mexicana species com-
plex in Mexico has provided insights into mechanisms underlying
adaptation and the links between adaptation and speciation Sul-
phide springs do not only occur in Mexico however but are found
TOBLER ET AL | 853
worldwide and have been colonized by 24 evolutionarily indepen-
dent fish lineages in North America the Neotropics and the Middle
East (Greenway et al 2014) The vast majority of lineages belong to
the order Cyprinodontiformes (95) and livebearing members of the
family Poeciliidae in particular (80) This potentially indicates that
internal gestation of young has facilitated the colonization of
extreme environments because it shields developing embryos from
physiochemical stressors (Blackburn 1999) Among the Poeciliidae
sulphide spring fishes include members of the genera Brachyrhaphis
Gambusia Poeciliopsis Priapichthys Pseudoxiphophorus Xiphophorus
and multiple subgenera of Poecilia (Figure 3 Greenway et al 2014)
spanning over 40 million years of evolution (based on Reznick Fur-
ness Meredith amp Springer 2017)
A handful of studies have started to address molecular and phe-
notypic evolution across a broader taxon sampling of sulphide spring
fishes finding evidence for convergent evolution in morphology
(Riesch et al 2016 Tobler amp Hastings 2011) life history traits
(Riesch Plath et al 2010 Riesch et al 2014) and oxygen transport
genes (Barts et al 2018) In addition ecological speciation in
response to adaptation to H2S-rich environments has also been doc-
umented in sulphide spring poeciliids other than P mexicana (Riesch
et al 2016) These studies have laid a fruitful foundation for the
investigation of mechanisms of adaptation to H2S-rich springs from
genes to phenotypic traits linking micro-evolutionary processes to
macro-evolutionary patterns
The availability of broad-scale evolutionary replication of popula-
tions that have invaded habitats with the same physiochemical stressors
allows for addressing some of the ensuing challenges discussed above
and investigating the factors that give rise to predictable evolutionary
outcomes Convergent evolution is a commonmdashbut not omnipresentmdash
theme in adaptive evolution and multiple factors can determine the
degree of convergence among replicated lineages exposed to the same
sources of selection (Kaeuffer et al 2012 Landry amp Bernatchez 2010
Oke Rolshausen LeBlond amp Hendry 2017 Rosenblum amp Harmon
2011) Thus leveraging replicated lineages that have colonized sulphide
springs to assess molecular mechanisms underlying convergent pheno-
typic changes analyse how environmental heterogeneity among sul-
phide spring habitats causes lineage-specific patterns of trait evolution
and test whether and by what mechanisms adaptation to extreme envi-
ronments is linked to speciation will provide insights into how ecologi-
cal genetic and functional factors contribute to evolutionary
convergence at different levels of organismal organization (Rosenblum
et al 2014) An expanded taxon sampling with population pairs that
span almost the full range of the speciation continuum from panmixia to
complete reproductive isolation (Riesch et al 2016) will also help
addressing questions about temporal aspects of H2S adaptation In par-
ticular comparing younger to older lineages will provide insights about
the order at which genetic and phenotypic modifications accrue during
the adaptation process
Animals have also invaded H2S-rich habitats in other aquatic
ecosystems including cold seeps and deep-sea hydrothermal vents
(Tobler et al 2016) Adaptation to H2S in the P mexicana species com-
plex has involved modification of physiological pathways associated
with H2S toxicity (OXPHOS pathway) and detoxification (sulphide
F IGURE 3 Phylogeny of the family Poeciliidae (adopted from Culumber amp Tobler 2017) Various lineages in the genera Poecilia (subgeneraAcanthophacelus Limia and Mollienesia) Priapichthys Brachyrhaphis Poeciliopsis Xiphophorus Pseudoxiphophorus and Gambusia have colonizedH2S-rich springs in different parts of the Americas (see Greenway et al 2014) Lineages highlighted in yellow represent endemic speciesrestricted to H2S-rich springs while red lineages represent species in which some populations have invaded sulphidic habitats Pictures depict arepresentative of each focal genussubgenus
854 | TOBLER ET AL
quinone oxidoreductase pathway) which have been highly conserved
during the diversification of life on Earth (Saraste 1999 Shahak amp
Hauska 2008) The high replicability of H2S as an environmental factor
that shaped animal evolution across vastly different ecological contexts
consequently offers the opportunity to investigate whether selection
mediated by the presence of H2S has repeatedly modified these con-
served physiological pathways across animal phyla that have diverged
during the Cambrian explosion spanning over 500 million years of evo-
lution Such comparisons would provide unprecedented insights into
the effects of a single physiochemical stressor on evolutionary change
irrespective of ecological or phylogenetic contexts
6 | CONCLUSIONS
H2S-rich environments and their inhabitants represent useful models
for research in organismal biology ecology and evolution illustrating
both opportunities (eg strong and explicit source of selection evolu-
tionary replication) and challenges (eg complexity of environments
and organismal responses) in asking basic question about the origins
of biodiversity Understanding how organisms thrive in apparently
inhospitable environments will provide us with insights into lifersquos
capacities and limitations to adapt to novel environments (Waterman
1999) This is particularly relevant considering that human activities
substantially contribute to the exacerbation of physiochemical stres-
sors at local regional and global scales impacting ecological and evo-
lutionary dynamics (Alberti 2015 Norberg Urban Vellend
Klausmeier amp Loeuille 2012) Thus a focus on ecological and evolu-
tionary consequences of natural stressors that push organisms to the
brink of their physiological capacities may be instrumental to predict
consequences of anthropogenic environmental change
ACKNOWLEDGEMENTS
We would like to thank Lenin Arias-Rodriguez the Universidad
Juarez Autonoma de Tabasco and the Centro de Investigacion E
Innovacion Para la Ense~nanza y El Aprendizaje for their continued
collaboration enabling our work on the P mexicana system Funding
for this review article has been provided by the National Science
Foundation (IOS-1557860 IOS-1557795) and the US Army
Research Office (W911NF-15-1-0175)
AUTHOR CONTRIBUTIONS
MT wrote the first draft of the paper all authors contributed text
and comments
ORCID
Michael Tobler httporcidorg0000-0002-0326-0890
Joanna L Kelley httporcidorg0000-0002-7731-605X
Reuroudiger Riesch httporcidorg0000-0002-0223-1254
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Alvarez del Villar J (1948) Descripcion de una nueva especie de Mol-
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Arnold S J (1983) Morphology performance and fitness American Zool-
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Aspiras A C Rohner N Martineau B Borowsky R L amp Clifford C J
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Ast G (2004) How did alternative splicing evolve Nature Reviews
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Bagarinao T (1992) Sulfide as an environmental factor and toxicant
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Barton N H (1998) The effect of hitch-hiking on neutral genealogies
Genetic Research 72 123ndash133 httpsdoiorg101017
S0016672398003462
Barts N Greenway R Passow C N Arias Rodriguez L Kelley J L amp
Tobler M (2018) Expression and molecular evolution of oxygen
transport genes in livebearing fishes (Poeciliidae) from hydrogen sul-
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[Epub ahead of print]
Beauchamp R O Bus J S Popp C S Boreiko C J amp Andjelkovich
D A (1984) A critical review of the literature on hydrogen sulfide
CRC Critical Reviews in Toxicology 13 25ndash97 httpsdoiorg10
310910408448409029321
Bell E M (2012) Life at extremes Environments organisms and strategies
for survival Oxfordshire CABI httpsdoiorg101079
97818459381470000
Bierbach D Arias Rodriguez L amp Plath M (2018) Intrasexual competi-
tion enhances reproductive isolation between locally adapted popula-
tions Current Zoology httpsdoiorg101093czzox071 [Epub
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Bierbach D Klein M Sassmannshausen V Schlupp I Riesch R
Parzefall J amp Plath M (2012) Divergent evolution of male aggres-
sive behavior Another reproductive isolation barrier in extremophile
poeciliid fishes International Journal of Evolutionary Biology 2012
148745
Blackburn D G (1999) Viviparity and oviparity Evolution and reproduc-
tive strategies In T E Knobil amp J D Neill (Eds) Encyclopedia of
reproduction Vol 4 (pp 994ndash1003) New York NY Academic Press
Bouzat J L (2010) Conservation genetics of population bottlenecks
The role of chance selection and history Conservation Genetics 11
463ndash478
Brown A P Greenway R Morgan S Quackenbush C R Giordani
L Arias-Rodriguez L Kelley J L (2017) Genome-scale data
reveals that endemic Poecilia populations from small sulfide spring
display no evidence of inbreeding Molecular Ecology 26 4920ndash
4934
Brown J H Marquet P A amp Taper M L (1993) Evolution of body
size Consequences of an energetic definition of fitness American
Naturalist 142 573ndash584 httpsdoiorg101086285558
Burton R S Pereira R J amp Barreto F S (2013) Cytonuclear genomic
interactions and hybrid breakdown Annual Review of Ecology
TOBLER ET AL | 855
Evolution and Systematics 44 281ndash302 httpsdoiorg101146an
nurev-ecolsys-110512-135758
Bush S J Chen L Tovar-Corona J M amp Urritia A O (2017) Alterna-
tive splicing and the evolution of phenotypic novelty Philosophical
Transactions of the Royal Society B 372 20150474 httpsdoiorg
101098rstb20150474
Carroll S P Jorgensen P S Kinnison M T Bergstrom C T Denison
R F Gluckman P Tabashnik B E (2014) Applying evolutionary
biology to address global challenges Science 346 313 httpsdoi
org101364FIO2014JW3A13
Cartwright R A Schwartz R S Merry A L amp Howell M M (2017)
The importance of selection in the evolution of blindness in cavefish
Bmc Evolutionary Biology 17 45 httpsdoiorg101186s12862-
017-0876-4
Chan Y F Marks M E Jones F C Villarreal G Shapiro M D Brady
S D Kingsley D M (2010) Adaptive evolution of pelvic reduc-
tion in sticklebacks by recurrent deletion of Pitx1 enhancer Science
327 302ndash305 httpsdoiorg101126science1182213
Chou J Y amp Leu J Y (2010) Speciation through cytonuclear incompat-
ibility Insights from yeast and implications for higher eukaryotes
BioEssays 32 401ndash411 httpsdoiorg101002bies200900162
Colosimo P F Hosemann K E Balabhadra S Villarreal G Dickson
M Grimwood J Kingsley D M (2005) Widespread parallel evo-
lution in sticklebacks by repeated fixation of ectodysplasin alleles
Science 307 1928ndash1933 httpsdoiorg101126science1107239
Cooper C E amp Brown G C (2008) The inhibition of mitochondrial
cytochrome oxidase by the gases carbon monoxide nitric oxide
hydrogen cyanide and hydrogen sulfide Chemical mechanism and
physiological significance Journal of Bioenergy and Biomembranes 40
533ndash539 httpsdoiorg101007s10863-008-9166-6
Culumber Z W Hopper G W Barts N Passow C N Morgan S Brown
A Tobler M (2016) Habitat use of two extremophile highly ende-
mic and critically endangered fish species (Gambusia eurystoma and Poe-
cilia sulphurophila Poeciliidae) Aquatic Conservation-Marine and
Freshwater Ecosystems 26 1155ndash1167 httpsdoiorg101002aqc
2640
Culumber Z W amp Tobler M (2017) Sex-specific evolution during the
diversification of live-bearing fishes Nature Ecology amp Evolution 1
1185ndash1191
Dalziel A C Rogers S M amp Schulte P M (2009) Linking genotypes
to phenotypes and fitness How mechanistic biology can inform
molecular ecology Molecular Ecology 18 4997ndash5017 httpsdoi
org101111j1365-294X200904427x
Darwin C (1859) On the origin of species based on natural selection or
the preservation of favoured races in the struggle of life 6th ed Lon-
don John Murray
Dasmahapatra K K Walters J R Briscoe A D Davey J W Whibley
A Nadeau N J Jiggins C D (2012) Butterfly genome reveals
promiscuous exchange of mimicry adaptations among species Nature
487 94ndash98 httpsdoiorg101038nature11041
Doudna J A amp Charpentier E (2014) The new frontier of genome edit-
ing with CRISPR-Cas9 Science 346 1258096 httpsdoiorg10
1126science1258096
Dubilier N Giere O amp Grieshaber M (2005) Concomitant effects of sul-
fide and hypoxia on the aerobic metabolism of the marine oligochaete
Tubificoides benedii Journal of Experimental Zoology 269 287ndash298
Eales K L Hollinshead K E R amp Tennant D A (2016) Hypoxia and
metabolic adaptation of cancer cells Oncogenesis 5 e190 httpsd
oiorg101038oncsis201550
Eghbal M A Pennefather P S amp OrsquoBrien P J (2004) H2S cytotoxicity
mechanism involves reactive oxygen species formation and mito-
chondrial depolarisation Toxicology 203 69ndash76 httpsdoiorg10
1016jtox200405020
Feldman C R Brodie Jr E D Brodie III E D amp Pfrender M E (2012)
Constraint shapes convergence in tetrodotoxin-resistant sodium
channels of snakes Proceedings of the National Academy of Sciences
USA 109 4556ndash4561 httpsdoiorg101073pnas1113468109
Flores K B Wolschin F amp Amdam G V (2013) The role of methyla-
tion of DNA in environmental adaptation Integrative and Comparative
Biology 53 359ndash372 httpsdoiorg101093icbict019
Fradin H Kiontke K Zegar C Gutwein M Lucas J Kovtun M
Piano F (2017) Genome architecture and evolution of a unichromo-
somal asexual nematode Current Biology 27(2928ndash2939) e2926
Gershoni M Templeton A amp Mishmar D (2009) Mitochondrial bioen-
ergetics as a major motive force of speciation BioEssays 31 642ndash
650 httpsdoiorg101002bies200800139
Ghalambor C K Walker J A amp Reznick D N (2003) Multi-trait selec-
tion adaptation and constraints on the evolution of burst swimming
performance Integrative and Comparative Biology 43 431ndash438
httpsdoiorg101093icb433431
Gibert J amp Deharveng L (2002) Subterranean ecosystems A truncated
functional biodiversity BioScience 52 473ndash481 httpsdoiorg10
16410006-3568(2002)052[0473SEATFB]20CO2
Gjuvsland A B Vik J O Beard D A Hunter P J amp Omholt S W
(2013) Bridging the genotype-phenotype gap What does it take
Journal of Physiology 591 2055ndash2066 httpsdoiorg101113jphys
iol2012248864
Grant P R Grant B R Markert J A Keller L F amp Petren K (2004)
Convergent evolution of Darwinrsquos finches caused by introgressive
hybridization and selection Evolution 58 1588ndash1599 httpsdoi
org101111j0014-38202004tb01738x
Greene H W (2005) Organisms in nature as a central focus for biology
Trends in Ecology and Evolution 20 23ndash27 httpsdoiorg101016j
tree200411005
Greenway R Arias-Rodriguez L Diaz P amp Tobler M (2014) Patterns
of macroinvertebrate and fish diversity in freshwater sulphide springs
Diversity 6 597ndash632 httpsdoiorg103390d6030597
Greenway R Drexler S Arias-Rodriguez L amp Tobler M (2016) Adap-
tive but not condition-dependent body shape differences contribute
to assortative mating preferences during ecological speciation Evolu-
tion 70 2809ndash2822 httpsdoiorg101111evo13087
Gunderson A R Armstrong E J amp Stillman J H (2016) Multiple stres-
sors in a changing world The need for an improved perspective on
physiological responses to the dynamic marine environment Annual
Review of Marine Science 8 357ndash378 httpsdoiorg101146an
nurev-marine-122414-033953
Hildebrandt T M amp Grieshaber M (2008) Three enzymatic activities
catalyze the oxidation of sulfide to thiosulfate in mammalian and
invertebrate mitochondria FEBS Journal 275 3352ndash3361 httpsd
oiorg101111j1742-4658200806482x
Houle D Govindaraju D R amp Omholt S (2010) Phenomics The next
challenge Nature Reviews Genetics 11 855ndash866 httpsdoiorg10
1038nrg2897
IUCN (2012) Red List of threatened species version 20132 www
iucnredlistorg
Johnston I A amp Bennett A F (1996) Animals and temperature Pheno-
typic and evolutionary adaptation Cambridge Cambridge University
Press httpsdoiorg101017CBO9780511721854
Jorgensen C Auer S K amp Reznick D N (2011) A model for optimal
offspring size in fish including live-bearing and parental effects Amer-
ican Naturalist 177 E119ndashE135 httpsdoiorg101086659622
Kaeuffer R Peichel C L Bolnick D I amp Hendry A P (2012) Parallel
and nonparallel aspects of ecological phenotypic and genetic diver-
gence across replicate population pairs of lake and stream stickle-
back Evolution 66 402ndash418 httpsdoiorg101111j1558-5646
201101440x
Kell D B amp Oliver S G (2004) Here is the evidence now what is the
hypothesis The complementary roles of inductive and hypothesis-
driven science in the post-genomic era BioEssays 26 99ndash105
httpsdoiorg101002(ISSN)1521-1878
856 | TOBLER ET AL
Kelley J L Arias-Rodriguez L Patacsil Martin D Yee M C Busta-
mante C amp Tobler M (2016) Mechanisms underlying adaptation to
life in hydrogen sulfide rich environments Molecular Biology and Evo-
lution 33 1419ndash1434 httpsdoiorg101093molbevmsw020
Kelley J L Passow C Plath M Arias-Rodriguez L Yee M C amp
Tobler M (2012) Genomic resources for a model in adaptation and
speciation research The characterization of the Poecilia mexicana
transcriptome BMC Genomics 13 652 httpsdoiorg101186
1471-2164-13-652
Klerks P L Xie L amp Levinton J S (2011) Quantitative genetics
approaches to study evolutionary processes in ecotoxicology a per-
spective from research on the evolution of resistance Ecotoxicology
20 513ndash523 httpsdoiorg101007s10646-011-0640-2
Kump L R Pavlov A amp Arthur M A (2005) Massive release of hydrogen
sulfide to the surface ocean and atmosphere during intervals of oceanic
anoxia Geology 33 397ndash400 httpsdoiorg101130G212951
Keuroustner A Hoffmann M Fraser B A Kottler V A Sharma E Wei-
gel D amp Dreyer C (2016) The genome of the Trinidadian guppy
Poecilia reticulata and variation in the guanapo population PLoS ONE
11 e0169087 httpsdoiorg101371journalpone0169087
Landry L amp Bernatchez L (2010) Role of epibenthic resource opportu-
nities in the parallel evolution of lake whitefish species pairs (Core-
gonus sp) Journal of Evolutionary Biology 23 2602ndash2613 httpsdoi
org101111j1420-9101201002121x
Langerhans R B (2007) Evolutionary consequences of predation Avoid-
ance escape reproduction and diversification In A M T Elewa
(Ed) Predation in organisms Berlin Springer
Li L Rose P amp Moore P K (2011) Hydrogen sulfide and cell signaling
Annual Review of Pharmacology and Toxicology 51 169ndash187
httpsdoiorg101146annurev-pharmtox-010510-100505
Lin Q Fan S Zhang Y Xu M Zhang H Yang Y Gunter H M
(2016) The seahorse genome and the evolution of its specialized mor-
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Liu Y Ma S Wang X Chang J Gao J Shi R Zhao P (2014)
Highly efficient multiplex targeted mutagenesis and genomic struc-
ture variation in Bombyx mori cells using CRISPRCas9 Insect bio-
chemistry and molecular biology 49 35ndash42 httpsdoiorg101016j
ibmb201403010
Lopez-Maury L Marguerat S amp Beuroahler J (2008) Tuning gene expres-
sion to changing environments From rapid responses to evolutionary
adaptation Nature Reviews Genetics 9 583ndash593 httpsdoiorg10
1038nrg2398
Maan M E amp Seehausen O (2012) Magic traits versus magic prefer-
ences in speciation Evolutionary Ecology Research 14 779ndash785
Martyn U Weigel D amp Dreyer C (2006) In vitro culture of embryos
of the guppy Poecilia reticulata Developmental Dynamics 235 617ndash
622 httpsdoiorg101002(ISSN)1097-0177
Metzker M L (2010) Sequencing technologies The next generation
Nature Reviews Genetics 11 31ndash46 httpsdoiorg101038nrg2626
Moore M P Riesch R amp Martin R A (2016) The predictability and
magnitude of life-history divergence to ecological agents of selection
A meta-analysis in livebearing fishes Ecology Letters 19 435ndash442
httpsdoiorg101111ele12576
Mustafa A K Gadalla M M Sen N Kim S Mu W Gazi S K
Snyder S H (2009) H2S signals through protein S-sulfhydration
Science Signaling 2 ra72
Nesse R M amp Stearns S C (2008) The great opportunity Evolutionary
applications to medicine and public health Evolutionary Applications
1 28ndash48 httpsdoiorg101111j1752-4571200700006x
Nevo E (2011) Evolution under environmental stress at macro and
microscales Genome Biology and Evolution 3 1039ndash1052 httpsdoi
org101093gbeevr052
Norberg J Urban M C Vellend M Klausmeier C A amp Loeuille N (2012)
Eco-evolutionary responses of biodiversity to climate change Nature Cli-
mate Change 2 747ndash751 httpsdoiorg101038nclimate1588
Nosil P (2012) Ecological speciation Oxford UK Oxford University
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0001
Nosil P Vines T H amp Funk D J (2005) Perspective Reproductive
isolation caused by natural selection against immigrants from diver-
gent habitats Evolution 59 705ndash719
Oke K B Rolshausen G LeBlond C amp Hendry A P (2017) How par-
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Olson K R (2011) The therapeutic potential of hydrogen sulfide
Separating hype from hope American Journal of Physiology-Regulatory
Integrative and Comparative Physiology 301 R297ndashR312 httpsdoi
org101152ajpregu000452011
Olson K R Donald J A Dombkowski R A amp Perry S F (2012) Evo-
lutionary and comparative aspects of nitric oxide carbon monoxide
and hydrogen sulfide Respiratory Physiology amp Neurobiology 184
117ndash129 httpsdoiorg101016jresp201204004
Olson K R amp Straub K D (2015) The role of hydrogen sulfide in evo-
lution and the evolution of hydrogen sulfide in metabolism and sig-
naling Physiology 31 60ndash72
Palacios M Arias-Rodriguez L Plath M Eifert C Lerp H Lamboj A
Tobler M (2013) The rediscovery of a long described species
reveals additional complexity in speciation patterns of poeciliid fishes
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nalpone0071069
Palacios M Voelker G Arias-Rodriguez L Mateos M amp Tobler M
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Poeciliidae Teleostei) reveal taxonomic inconsistencies cryptic biodi-
versity and spatio-temporal aspects of diversification in Middle
America Molecular Phylogenetics and Evolution 103 230ndash244
httpsdoiorg101016jympev201607025
Passow C Arias-Rodriguez L amp Tobler M (2017) Convergent evolu-
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e0186935 httpsdoiorg101371journalpone0186935
Passow C N Brown A Arias-Rodriguez L Yee M-C Sockell A
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patterns in natural populations of an extremophile fish (Poecilia mexi-
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101111mec14198
Passow C Greenway R Arias-Rodriguez L Jeyasingh P D amp Tobler
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body size and metabolic rates in locally adapted extremophile fishes
Physiological and Biochemical Zoology 88 371ndash383 httpsdoiorg
101086681053
Passow C N Henpita C Shaw J H Quakenbush C Warren W C
Schartl M Tobler M (2017) The roles of plasticity and evolu-
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lations of extremophile fish Molecular Ecology 26 6384ndash6399
Pavey S A Bernatchez L Aubin-Horth N amp Landry C R (2012)
What is needed for next-generation ecological and evolutionary
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org101016jtree201207014
Peers C Bauer C Boyle J P Scragg J L amp Dallas M L (2012)
Modulation of ion channels by hydrogen sulfide Antioxidants amp Redox
Signaling 17 95ndash105 httpsdoiorg101089ars20114359
Pfennig D W amp Pfennig K S (2012) Evolutionrsquos wedge competition and
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httpsdoiorg101525california97805202741810010001
Pfenninger M Lerp H Tobler M Passow C Kelley J L Funke E
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Pfenninger M Patel S Arias-Rodriguez L Feldmeyer B Riesch R amp
Plath M (2015) Unique evolutionary trajectories in repeated adapta-
tion to hydrogen sulphide-toxic habitats of a neotropical fish (Poecilia
TOBLER ET AL | 857
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1111mec13397
Pietri R Roman-Morales E amp Lopez-Garriga J (2011) Hydrogen sul-
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Plath M (2008) Male mating behavior and costs of sexual harassment
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Plath M Hermann C Schreurooder R Riesch R Tobler M Garcia de
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Plath M Pfenninger M Lerp H Riesch R Eschenbrenner C Slattery
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migrants in evolutionarily replicated extreme environments Evolution
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Plath M Tobler M Riesch R Garcia de Leon F J Giere O amp
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httpsdoiorg101007s00114-007-0279-2
Powell E (1989) Oxygen sulfide and diffusion Why thiobiotic meiofauna
must be sulfide-insensitive first-order respirers Journal of Marine
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Ramirez-Soriano A Ramos-Onsins S E Rozas J Calafell F amp Navarro A
(2008) Statistical power analysis of neutrality tests under demographic
expansions contractions and bottlenecks with recombination Genetics
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Reiffenstein R Hulbert W amp Roth S (1992) Toxicology of hydrogen
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134 httpsdoiorg101146annurevpa32040192000545
Reznick D N Furness A I Meredith R W amp Springer M S (2017)
The origin and biogeographic diversification of fishes in the family
Poeciliidae PLoS ONE 12 e0172546 httpsdoiorg101371journa
lpone0172546
Reznick D amp Yang A P (1993) The influence of fluctuating resources
on life history Patterns of allocation and plasticity in female guppies
Ecology 74 2011ndash2019 httpsdoiorg1023071940844
Riesch R Duwe V Herrmann N Padur L Ramm A Scharnweber K
Plath M (2009) Variation along the shy-bold continuum in extre-
mophile fishes (Poecilia mexicana Poecilia sulphuraria) Behavioral Ecol-
ogy and Sociobiology 63 1515ndash1526 httpsdoiorg101007
s00265-009-0780-z
Riesch R Oranth A Dzienko J Karau N Schiessl A Stadler S
Plath M (2010) Extreme habitats are not refuges Poeciliids suffer
from increased aerial predation risk in sulphidic southern Mexican
habitats Biological Journal of the Linnean Society 101 417ndash426
httpsdoiorg101111j1095-8312201001522x
Riesch R Plath M Garcia de Leon F J amp Schlupp I (2010) Conver-
gent life-history shifts Toxic environments result in big babies in two
clades of poeciliids Naturwissenschaften 97 133ndash141 httpsdoi
org101007s00114-009-0613-y
Riesch R Plath M Schlupp I Tobler M amp Langerhans R B (2014)
Colonization of toxic springs drives predictable life-history shift in
livebearing fishes (Poeciliidae) Ecology Letters 17 65ndash71 httpsdoi
org101111ele12209
Riesch R Tobler M Lerp H Jourdan J Doumas L T Nosil P Plath
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plexity of speciation along replicated ecological gradients BMC Evolu-
tionary Biology 16 136 httpsdoiorg101186s12862-016-0705-1
Riesch R Tobler M amp Plath M (2015) Extremophile fishes Ecology
evolution and physiology of teleosts in extreme environments p 290
Heidelberg Germany Springer httpsdoiorg101007978-3-319-
13362-1
Ritchie M E Holzinger E R Li R Pendergrass S A amp Kim S (2015)
Methods of integrating data to uncover genotype-phenotype interac-
tions Nature Reviews Genetics 16 85ndash97 httpsdoiorg101038
nrg3868
Roach K Tobler M amp Winemiller K O (2011) Hydrogen sulfide bac-
teria and fish A unique subterranean food chain Ecology 92 2056ndash
2062 httpsdoiorg10189011-02761
Rohner N Jarosz D F Kowalko J E Yoshizawa M Jeffery W R
Borowsky R L Tabin C J (2013) Cryptic variation in morpho-
logical evolution HSP90 as a capacitor for loss of eyes in cavefish
Science 342 1372ndash1375 httpsdoiorg101126science1240276
Rosales Lagarde L (2012) Investigation of karst brackish-sulfidic springs
and their role in the hydrogeology subsurface water-rock interactions
and speleogenesis at northern Sierra de Chiapas New Mexico Institute
of Mining and Technology Mexico
Rosenblum E B amp Harmon L J (2011) ldquoSame but differentrdquo Repli-
cated ecological speciation at White Sands Evolution 65 946ndash960
httpsdoiorg101111j1558-5646201001190x
Rosenblum E B Parent C E amp Brandt E E (2014) The molecular
basis of convergent evolution Annual Review of Ecology Evolution and
Systematics 45 203ndash226 httpsdoiorg101146annurev-ecolsys-
120213-091851
Saltz J B Hessel F C amp Kelly M W (2017) Trait correlations in the
genomics era Trends in Ecology amp Evolution 32 279ndash290 httpsdoi
org101016jtree201612008
Saraste M (1999) Oxidative phosphorylation at the fin de siecle Science
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Schartl M (2014) Beyond the zebrafish Diverse fish species for model-
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httpsdoiorg101242dmm012245
Schartl M Walter R B Shen Y Garcia T Catchen J Amores A
Warren W C (2013) The genome of the platyfish Xiphophorus mac-
ulatus provides insights into evolutionary adaptation and several
complex traits Nature Genetics 45 567ndash572 httpsdoiorg10
1038ng2604
Schluter D (2009) Evidence for ecological speciation and its alternative
Science 323 737ndash741 httpsdoiorg101126science1160006
Schulz-Mirbach T Eifert C Riesch R Farnworth M S Zimmer C
Bierbach D Plath M (2016) Toxic hydrogen sulphide shapes
brain anatomy A comparative study of sulphide-adapted ecotypes in
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determina las especies y subspecies de flora y fauna silvestres ter-
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sujetas a proteccion especial y que establece especificaciones para
su proteccion Diario Oficial 16 May 1994 488 2ndash60
Servedio M R van Doorn G S Kopp M Frame A M amp Nosil P
(2011) Magic traits in speciation lsquomagicrsquo but not rare Trends in Ecology
amp Evolution 26 389ndash397 httpsdoiorg101016jtree201104005
Shahak Y amp Hauska G (2008) Sulfide oxidation from cyanobacteria to
humans Sulfide-quinone oxidoreductase (SQR) In R Hell C Dahl D
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Shalem O Sanjana N E amp Zhang F (2015) High-throughput func-
tional genomics using CRISPR-Cas9 Nature Reviews Genetics 16
299ndash311 httpsdoiorg101038nrg3899
Silva G J J Bye A el Azzouzi H amp Wisloslashff U (2017) MicroRNAs as
important regulators of exercise adaptation Progress in Cardiovascular
Diseases 60 130ndash151 httpsdoiorg101016jpcad201706003
Simola D F Wissler L Donahue G Waterhouse R M Helmkampf
M Roux J Viljakainen L (2013) Social insect genomes exhibit
dramatic evolution in gene composition and regulation while preserv-
ing regulatory features linked to sociality Genome Research 23
1235ndash1247 httpsdoiorg101101gr155408113
858 | TOBLER ET AL
Sommer-Trembo C Bierbach D Arias Rodriguez L Verel Y Jourdan
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Steindachner F (1863) Beitreuroage zur Kentniss der Sciaenoiden Brasiliens
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Naturwissenschaftlichen Classe der Kaiserlichen Akademie der Wis-
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Stinchcombe J R amp Hoekstra H E (2008) Combining population geno-
mics and quantitative genetics Finding genes underlying ecologically
important traits Heredity 100 158ndash170 httpsdoiorg101038sj
hdy6800937
Stipanuk M H (2004) Sulfur amino acid metabolism Pathways for pro-
duction and removal of homocysteine and cysteine Annual Review of
Nutrition 24 539ndash577 httpsdoiorg101146annurevnutr24
012003132418
Stipanuk M H amp Ueki I (2011) Dealing with methioninehomocysteine
sulfur Cysteine metabolism to taurine and inorganic sulfur Journal of
Inherited Metabolic Disease 34 17ndash32 httpsdoiorg101007
s10545-009-9006-9
Stockinger B Di Meglio P Gialitakis M amp Duarte J H (2014) The
aryl hydrocarbon receptor Multitasking in the immune system
Annual Review of Immunology 32 403ndash432 httpsdoiorg101146
annurev-immunol-032713-120245
Storz J F amp Wheat C W (2010) Integrating evolutionary and
functional approaches to infer adaptation at specific loci Evolution
64 2489ndash2509 httpsdoiorg101111j1558-5646201001044x
Stuart Y E Veen T Weber J N Hanson D Ravinet M Lohman B
K Bolnick D I (2017) Contrasting effects of environment and
genetics generate a continuum of parallel evolution Nature Ecology amp
Evolution 1 0158 httpsdoiorg101038s41559-017-0158
Sunnucks P Morales H E Lamb A M Pavlova A amp Greening C
(2017) Integrative approaches for studying mitochondrial and nuclear
genome co-evolution in oxidative phosphorylation Frontiers in Genet-
ics 8 25
Tobler M amp Hastings L (2011) Convergent patterns of body shape dif-
ferentiation in four different clades of poeciliid fishes inhabiting sul-
fide springs Evolutionary Biology 38 412ndash421 httpsdoiorg10
1007s11692-011-9129-4
Tobler M Palacios M Chapman L J Mitrofanov I Bierbach D
Plath M Mateos M (2011) Evolution in extreme environments
Replicated phenotypic differentiation in livebearing fish inhabiting
sulfidic springs Evolution 65 2213ndash2228 httpsdoiorg101111j
1558-5646201101298x
Tobler M Passow C N Greenway R Kelley J L amp Shaw J H
(2016) The evolutionary ecology of animals inhabiting hydrogen sul-
fide-rich environments Annual Review of Ecology Evolution and Sys-
tematics 47 239ndash262 httpsdoiorg101146annurev-ecolsys-
121415-032418
Tobler M amp Plath M (2009a) Threatened fishes of the world Gambu-
sia eurystoma Miller 1975 (Poeciliidae) Environmental Biology of
Fishes 85 251 httpsdoiorg101007s10641-009-9481-8
Tobler M amp Plath M (2009b) Threatened fishes of the world Poecilia
sulphuraria (Alvarez 1948) (Poeciliidae) Environmental Biology of
Fishes 85 333ndash334 httpsdoiorg101007s10641-009-9506-3
Tobler M Plath M Riesch R Schlupp I Grasse A Munimanda G
Moodley Y (2014) Selection from parasites favors immunogenic diver-
sity but not divergence among locally adapted host populations Journal
of Evolutionary Biology 27 960ndash974 httpsdoiorg101111jeb12370
Tobler M Riesch R amp Plath M (2015) Extremophile fishes An inte-
grative synthesis In R Riesch M Tobler amp M Plath (Eds) Extremo-
phile fishes Ecology evolution and physiology of teleosts in extreme
environments (pp 279ndash296) Heidelberg Germany Springer
Tobler M Riesch R Tobler C M amp Plath M (2009) Compensatory
behaviour in response to sulfide-induced hypoxia affects time
budgets feeding efficiency and predation risk Evolutionary Ecology
Research 11 935ndash948
Tobler M Scharnweber K Greenway R Passow C Arias-Rodriguez
L amp Garcia-De-Leon F J (2015) Convergent changes in the
trophic ecology if extremophile fish along replicated environmental
gradients Freshwater Biology 60 768ndash780 httpsdoiorg10
1111fwb12530
Todgham A E amp Stillman J H (2013) Physiological responses to shifts
in multiple environmental stressors Relevance in a changing worlds
Integrative and Comparative Biology 53 539ndash544 httpsdoiorg10
1093icbict086
van Doorn G S Edelaar P amp Weissing F J (2009) On the origin of
species by natural selection and sexual selection Science 326 1704ndash
1707 httpsdoiorg101126science1181661
Wall D H amp Virginia R A (1999) Controls on soil biodiversity Insights
from extreme environments Applied Soil Ecology 13 137ndash150
httpsdoiorg101016S0929-1393(99)00029-3
Wallace J L amp Wang R (2015) Hydrogen-sulfide based therapeutics
Exploiting a unique but ubiquitous gasotransmitter Nature Reviews
Drug Discovery 14 329ndash345 httpsdoiorg101038nrd4433
Wang R (2012) Physiological implications of hydrogen sulfide A whiff
exploration that blossomed Physiological Reviews 92 791ndash896
httpsdoiorg101152physrev000172011
Wang Z Gerstein M amp Snyder M (2009) RNA-seq A revolutionary
tool for transcriptomics Nature Reviews Genetics 10 57ndash63
httpsdoiorg101038nrg2484
Waterman T H (1999) The evolutionary challenges of extreme environ-
ments (part 1) Journal of Experimental Zoology 285 326ndash359
httpsdoiorg101002(SICI)1097-010X(19991215)2854amplt326
AID-JEZ3ampgt30CO2-T
Werren J H Richards S Desjardins C A Niehuis O Gadau J Col-
bourne J K amp Group N G W (2010) Functional and evolutionary
insights from the genomes of three parasitoid Nasonia species
Science 327 343ndash348 httpsdoiorg101126science1178028
Whitehead A (2012) Comparative genomics in ecological physiology
Toward a more nuanced understanding of acclimation and adapta-
tion Journal of Experimental Biology 215 884ndash891 httpsdoiorg
101242jeb058735
Whitehead A amp Crawford D L (2006) Neutral and adaptive variation
in gene expression Proceedings of the National Academy of Sciences
USA 103 5425ndash5430 httpsdoiorg101073pnas0507648103
Willett C S amp Burton R S (2004) Evolution of interacting proteins in
the mitochondrial electron transport system in a marine copepod
Molecular Biology and Evolution 21 443ndash453 httpsdoiorg10
1093molbevmsh031
Willi Y Van Buskirk J amp Hoffmann A (2006) Limits to the adaptive
potential of small populations Annual Review of Ecology Evolution
and Systematics 37 433ndash458 httpsdoiorg101146annurevecol
sys37091305110145
Wolff J N Ladoukakis E D Enrıquez J A amp Dowling D K (2014)
Mitonuclear interactions Evolutionary consequences over multiple
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20130443 httpsdoiorg101098rstb20130443
How to cite this article Tobler M Kelley JL Plath M Riesch
R Extreme environments and the origins of biodiversity
Adaptation and speciation in sulphide spring fishes Mol Ecol
201827843ndash859 httpsdoiorg101111mec14497
TOBLER ET AL | 859
ldquonormalrdquo habitats provides unique opportunities to illuminate evolu-
tionary processes that give rise to diversity both in terms of pheno-
typic novelty that mediates organismal function under adverse
environmental conditions and the origin of species The clearly
defined selective regimes in extreme environments enable hypothe-
sis-driven tests of responses at all levels of biological organization
and facilitate integrative approaches to study the causes and conse-
quences of adaptive evolution (Nevo 2011 Tobler Riesch amp Plath
2015) In addition the availability of replicated lineages exposed to
the same physicochemical stressors allows for evolutionary analyses
in a comparative context spanning vast geographic and phylogenetic
distances (Riesch Plath Schlupp Tobler amp Langerhans 2014 Riesch
et al 2016) Here we review recent research on fishes inhabiting
hydrogen sulphide-rich springs that sheds light onto mechanisms of
adaptation speciation and convergent evolution
Hydrogen sulphide (H2S) is a noxious gas that profoundly shaped
the diversification of life on the planet It served as a substrate for
anaerobic energy production for early life forms that lived in the
oxygen-poor oceans of the Proterozoic (Olson amp Straub 2015) and
caused mass extinctions after aerobic organisms diversified during
the Cambrian explosion (Kump Pavlov amp Arthur 2005) In todayrsquos
well-oxygenated atmosphere H2S is relatively rare but it is pro-
duced through geochemical and biological processes in a variety of
aquatic ecosystems (Tobler Passow Greenway Kelley amp Shaw
2016) The presence of H2S creates extreme environments for meta-
zoans because it interferes with fundamental biochemical and physi-
ological processes that mediate the function of aerobic organisms
(Bagarinao 1992) Most importantly H2S binds to cytochrome c oxi-
dase (COX) thereby interrupting the oxidative phosphorylation path-
way (OXPHOS) in mitochondria and inhibiting the aerobic
production of ATP (Cooper amp Brown 2008) Consequently H2S is
highly toxic for most metazoans even in micromolar concentrations
(Beauchamp Bus Popp Boreiko amp Andjelkovich 1984 Reiffenstein
Hulbert amp Roth 1992)
In southern Mexico H2S occurs naturally in multiple freshwater
spring complexes that are fed by groundwater sources (Rosales
Lagarde 2012) These sulphide springs are distributed across multi-
ple river drainages (Rıos Tacotalpa Puyacatengo Ixtapangajoya and
Pichucalco) that are part of the Rıo Grijalva basin (Figure 1) and
they exhibit H2S concentrations orders of magnitude higher than
what is considered lethal for most metazoans (Tobler et al 2011)
All springs directly drain into adjacent streams or rivers where H2S
is immediately diluted Thus there are contrasting sulphidic and non-
sulphidic habitat types that lack physical separation Despite the high
levels of toxicity multiple lineages of small livebearing fish of the
Poecilia mexicana species complex (subgenus Mollienesia family Poe-
ciliidae) have independently colonized the H2S-rich springs in each
of the river drainages Members of this species complex inhabit a
variety of freshwater and brackish habitats from northern Mexico
south to Panama (Alda Reina Doadrio amp Bermingham 2013 Pala-
cios Voelker Arias-Rodriguez Mateos amp Tobler 2016) and the
nominal species P mexicana is common in nonsulphidic habitats sur-
rounding the sulphide springs In the eastern two drainages with
sulphide springs (Rıos Tacotalpa and Puyacatengo) colonization of
the H2S-rich habitats has occurred relatively recently
(lt100000 years ago Pfenninger et al 2014) and H2S-adapted pop-
ulations are closely related tomdashand taxonomically considered the
same species asmdashP mexicana in adjacent nonsulphidic habitats (Pala-
cios et al 2013) In the western two drainages (Rıos Ixtapangajoya
and Pichucalco) sulphide springs have been colonized earlier
(gt200000 years ago Pfenninger et al 2014) and sulphide spring
populations in these drainages are more closely related to Poecilia
limantouri which primarily occurs north of the Trans-Mexican Vol-
canic Belt in northeastern Mexico (Brown et al 2017 Palacios et al
2013 2016) These older extremophile lineages have been described
as distinct species that are endemic to sulphide springs Poecilia ther-
malis (Steindachner 1863) in the Ixtapangajoya and Poecilia sulphu-
raria (Alvarez del Villar 1948) in the Pichucalco river drainage All
sulphide spring populationsmdashirrespective of their taxonomic statusmdash
represent locally adapted ecotypes that diverged in a variety of phe-
notypic traits (Riesch et al 2016 Tobler et al 2011) Sulphide
spring ecotypes can tolerate high H2S concentrations whereas eco-
types from adjacent nonsulphidic sites succumb to the toxic effects
of H2S over short periods of time (Plath et al 2013 Tobler et al
2011) indicating strong survival selection that impacts fitness across
small spatial scales Consequently high tolerance to environmental
H2S in sulphide spring fish can be viewed as an evolutionary innova-
tion that facilitated the colonization of unoccupied ecological niches
The sulphide spring fish of the P mexicana species complex pro-
vide an ideal system for an integrative approach to studying adapta-
tion and speciation for several reasons H2S provides a strong source
of selection with predictable physiological effects and known bio-
chemical consequences (Bagarinao 1992 Li Rose amp Moore 2011
Tobler et al 2016) which facilitates hypothesis-driven approaches
to studying mechanisms underlying physiological adaptation In addi-
tion the environmental gradients are clearly defined and replicated
which allows for an assessment of convergent patterns of evolution
across independent lineages that have colonized the same habitat
type The ease of cultivation of livebearing fish in the laboratory also
permits experimental approaches to corroborate patterns docu-
mented in the field and establish causal relationships between speci-
fic sources of selection and the evolution of specific traits Finally
growing genomic resources for fish of the family Poeciliidae facilitate
genetic analyses (Kelley et al 2012 Keuroustner et al 2016 Schartl
et al 2013)
2 | UNCOVERING PROXIMATEMECHANISMS OF ADAPTATION
Recent technological and analytical breakthroughs have revolution-
ized the genomewide quantification of genetic variation and gene
expression (Metzker 2010 Wang Gerstein amp Snyder 2009) facili-
tating the identification of genetic biochemical and physiological
underpinnings of adaptation to physiochemical stressors in natural
systems The rapid generation of large data sets allows for inductive
844 | TOBLER ET AL
Pichucalco Ixtapangajoya Puyacatengo Tacotalpa
Pichucalco
Teapa
Tacotalpa
Tapijulapa
4 km
(a)
(b) (c)
(d) (e)
F IGURE 1 Overview of the distribution of sulphide springs and their inhabitants in southern Mexico (a) Map depicting the four river drainages(Pichucalco Ixtapangajoya Puyacatengo and Tacotalpa) that exhibit sulphide springs inhabited by members of the P mexicana species complexThe locations of sulphide springs are indicated with yellow triangles black triangles represent commonly used field sites for the collection ofecotypes from nonsulphidic waters Note that roads (black lines) and major towns (grey shaded areas) were added for orientation The insethighlights the location of the study region within Mexico (bndashd) Examples of H2S-rich habitats in the Pichucalco (b) Puyacatengo (c) and Tacotalpa(d) river drainages (e) Representative individuals from the nonsulphidic (top) and the sulphidic (bottom) ecotypes from the Pichucalco river drainage
TOBLER ET AL | 845
inference of putative mechanisms underlying adaptation even in sys-
tems without a priori predictions (Kell amp Oliver 2004) However
genetic and gene expression data alone are insufficient to infer
adaptation at specific loci because neutral evolutionary processes
demographic history and genetic hitchhiking can generate false posi-
tives in genomewide analyses of selection and gene expression (Bar-
ton 1998 Ramirez-Soriano Ramos-Onsins Rozas Calafell amp
Navarro 2008 Whitehead amp Crawford 2006) This underscores the
importance of generating a priori hypotheses about the molecular
underpinnings of adaptation and testing whether genetic and gene
expression variation at specific loci actually affects organismal per-
formance under different environmental conditions (Dalziel Rogers
amp Schulte 2009 Storz amp Wheat 2010)
21 | Developing and testing a priori hypotheses
There is a rich history of research in toxicology and biomedicine that
has investigated the molecular targets of H2S illuminating the mech-
anisms of H2S toxicity and detoxification in model organisms and
human cell cultures (see Li et al 2011 Olson Donald Dombkowski
amp Perry 2012 Wang 2012 Wallace amp Wang 2015 for recent
reviews) In a recent attempt of ldquoseparating hype and hoperdquo in
potential biomedical applications of H2S Olson (2011) lamented that
H2S ldquoseemingly affects all organ systems and biological processes in
which it has been investigatedrdquo While it often remains unclear
whether the documented effects are a direct consequence of H2S
exposure or a result of the disruption of cellular homeostasis this
body of work provides fruitful grounds for the development of
hypotheses about the molecular mechanisms underlying adaptation
to H2S-rich environments that can then be tested using genome
scans for selection and analyses of gene expression variation
between sulphide-adapted and nonadapted populations (see Table 1
for an overview) Most importantly candidates include direct toxicity
targets involved in OXPHOS (Cooper amp Brown 2008) and oxygen
transport (Pietri Roman-Morales amp Lopez-Garriga 2011) anaerobic
metabolism (Dubilier Giere amp Grieshaber 2005) and oxidative stress
response systems (Eghbal Pennefather amp OrsquoBrien 2004) that may
help mitigate adverse consequences of H2S exposure as well as
pathways involved in enzymatic H2S detoxification sulphur process-
ing and sulphur excretion (Hildebrandt amp Grieshaber 2008) It is
important to note that H2S is also produced endogenously by meta-
zoans in the cytosol and in mitochondria through enzymatic reac-
tions associated with the reverse trans-sulphuration pathway
(Stipanuk 2004 Stipanuk amp Ueki 2011) At concentrations well
below the toxicity threshold endogenously produced H2S serves as
a cellular signalling molecule that modulates physiological processes
including the function of ion channels that underlie smooth muscle
control and the function of neural cells (Peers Bauer Boyle Scragg
amp Dallas 2012) and it interacts with transcription factors that con-
trol inflammation cell survival and proliferation the expression of
cellular protective enzymes and hypoxia responses (Li et al 2011
Wallace amp Wang 2015 Wang 2012) Organisms may thus not strive
to merely eliminate H2S from their system and avoid effects of
toxicity Instead they are likely selected for maintaining optimal
endogenous concentrations that facilitate H2S-mediated signalling
and assure proper physiological functioning As a consequence
endogenous H2S production pathways and targets of H2S signalling
may also be modified during adaptation to environmental H2S
A series of studies in members of the P mexicana species com-
plex have tested hypotheses about the molecular underpinnings of
H2S adaptation by analysing patterns of molecular evolution in can-
didate genes (Barts et al 2018 Pfenninger et al 2014) deploying
genomewide scans for selection based on FST outliers (Pfenninger
et al 2015) and quantifying genomewide patterns of gene expres-
sion (Kelley et al 2016 Passow Henpita et al 2017) Collectively
the results of these studies have indicated a striking congruence
between candidate genes inferred from toxicological and biomedical
research and genes with empirical evidence for selection andor dif-
ferential expression between at least some of the adjacent H2S-
adapted and nonadapted populations (Table 1) (i) There was evi-
dence for selection on genes encoding the primary toxicity target of
H2S COX (complex IV in OXPHOS Pfenninger et al 2014) In addi-
tion genes encoding other components and assembly factors of
OXPHOS are either under selection or differentially expressed in sul-
phide spring populations (Kelley et al 2016 Pfenninger et al
2015) Evidence for selection and differential expression was also
uncovered for subunits of haemoglobin whose function is directly
impaired by H2S (Barts et al 2018) (ii) Genes involved in anaerobic
ATP production and in coping with oxidative stress exhibited evi-
dence for selection and differential expression suggesting a shift
towards alternative sources of ATP production and an increased abil-
ity to withstand reactive oxygen species that are produced as a by-
product of OXPHOS inhibition by H2S (Kelley et al 2016 Pfen-
ninger et al 2015) (iii) There is overwhelming evidence for selection
on and differential regulation of genes encoding components of the
sulphidequinone oxidoreductase pathway which mediates enzymatic
H2S detoxification in metazoans (Kelley et al 2016 Pfenninger
et al 2015) In addition there is evidence for upregulation of genes
encoding transporters of oxidized sulphur species which are puta-
tively involved in sulphur excretion (Kelley et al 2016)
These results suggest that adaptation to H2S-rich environments
in members of the P mexicana species complex involves both an
increased ability to cope with elevated endogenous H2S and an
increased ability to keep endogenous H2S concentrations low
through detoxification and excretion However there is a noticeable
absence of evidence for genes and physiological pathways associ-
ated with endogenous H2S production and H2S-mediated cell sig-
nalling being involved in adaptation (see Kelley et al 2016
Pfenninger et al 2015) There are three nonmutually exclusive
hypotheses that could explain this finding First the ability of popu-
lations from sulphidic habitats to regulate endogenous H2S through
detoxification may be sufficient to maintain normal H2S-mediated
signalling Second compared to the ability for detoxification and for
mitigation of direct toxic effects in terms of ATP production and
oxidative stress H2Srsquos influence on cell signalling may have relatively
low fitness consequences with concomitantly smaller evolutionary
846 | TOBLER ET AL
responses Last inferences from biomedical studies about the causal
relationship between endogenously produced H2S and various physi-
ological functions may be weak and some documented physiological
effects may instead be an indirect consequence of the disruption of
homeostasis that cascades through interconnected pathways operat-
ing in a cell In this case evolutionary studies on natural systems
exposed to H2S may provide important insights in terms of prioritiz-
ing research on biomedically relevant aspects of H2S biology Study-
ing ancestral populations susceptible to H2S will allow for
quantifying consequences of disruptions in H2S homeostasis at all
levels of biological organization (like in presently available animal
models) Adding derived H2S tolerant populations will further allow
for the detection of critical modifications in physiological processes
affected by H2S Consequently such a comparative approach may
provide a powerful tool to disentangle direct and indirect physiologi-
cal consequences of deviations in H2S homeostasis which is cur-
rently difficult in traditional biomedical models
Despite some of these discrepancies the studies exploring mech-
anisms underlying H2S tolerance in sulphide spring fishes highlight
the utility of research in toxicology and biomedicine in generating
testable hypotheses about evolutionary processes Biomedical stud-
ies on metabolic disorders (Aspiras Rohner Martineau Borowsky amp
Clifford 2015) stress responses (Rohner et al 2013) responses to
low oxygen (Eales Hollinshead amp Tennant 2016) and effects of
xenobiotics (Stockinger Di Meglio Gialitakis amp Duarte 2014) have
also facilitated our understanding of adaptive evolution in other nat-
ural systems Considering the increasingly specialized and frag-
mented research areas in the life sciences interdisciplinary
approaches and reciprocal exchange of ideas and methodologies
continue to be critical for facilitating breakthroughs in basic and
applied research on biomedical models and natural systems (Carroll
et al 2014 Nesse amp Stearns 2008 Schartl 2014)
22 | Convergent and nonconvergent evolution inindependent lineages
Our discussion of putative candidate genes that are under selection
andor differentially expressed in sulphide spring populations so far
focused on general patterns documented across evolutionarily inde-
pendent lineages in different river drainages Selection scans (Pfen-
ninger et al 2015) and analyses of differential gene expression
(Kelley et al 2016) have indicated high congruence of biological
processes associated with candidate genes across lineages in differ-
ent river drainages indicating that evolution has repeatedly and pre-
dictably modified the same physiological pathways during adaptation
to environmental H2S However the signal of evolutionary conver-
gence among sulphide spring lineages decreases with the level of
organismal organization (from physiological pathways down to geno-
mic regions genes and individual SNPs in selection scans Pfenninger
et al 2015 from physiological pathways down to genes and tran-
scripts in expression studies Passow Brown et al 2017) indicating
that different lineages have often modified the same physiological
pathways in different ways While knowledge about the biochemical
TABLE 1 Adaptation to H2S can occur through three principlemechanisms (1) Resistance involves direct toxicity targets whosefunctions are impaired by H2S Adaptive modifications of thesetoxicity targets are predicted to assure proper function despitethe presence of H2S (2) Mitigation involves alternativephysiological pathways that compensate for the inhibition ofdirect toxicity targets Adaptive modifications of these alternativephysiological pathways are predicted to allow for the maintenanceof organismal function by other means (3) Regulation involvesphysiological pathways associated with the maintenance ofendogenous H2S homeostasis Adaptive modifications ofregulatory pathways are predicted to maintain low endogenousconcentrations despite continuous flux of H2S from theenvironment into the body
Candidate pathways Evidence
(1) Resistance
OXPHOS bull Positive selection on COX in some lineages of
P mexicana (Puyacatengo and Pichucalco)a as
well as some other genera of sulphide spring
poeciliidsb COX amino acid substitutions in P
mexicana allow for the maintenance of function
in the presence of H2Sa
bull Upregulation of cytochrome c and some COX
subunits as well as downregulation of compo-
nents of complex I in all sulphide spring lineages
of P mexicana indicating complex changes in
aerobic ATP productionc
Oxygen transport
genesbull Positive selection on and upregulation of
several oxygen transport genes (including myo-
globin and multiple haemoglobin subunits) in
sulphide spring lineages of the genera
Gambusia Limia Poecilia and Pseudoxiphophorus
indicating modifications to putatively maintain
oxygen transport in the presence of H2Sd
(2) Mitigation
Anaerobic
metabolismbull Upregulation of genes associated with glycolysis
and gluconeogenesis in all sulphide spring lin-
eages of P mexicana indicating increased
capacity of anaerobic ATP productionc
Oxidative stress
responsesbull Upregulation of genes associated with
oxidative stress responses in all sulphide
spring lineages of P mexicana indicating
increased capacity to cope with H2S-induced
oxidative stressc
(3) Regulation
H2S detoxification bull Positive selection on and upregulation of sev-
eral genes associated with enzymatic H2S
detoxification in all sulphide spring lineages of
P mexicanace Upregulation of H2S detoxifica-
tion enzymes in other sulphide spring lineages
of the genera Gambusia Limia Poecilia Pseudox-
iphophorus and Xiphophorusb This indicates an
increased ability to maintain low endogenous
H2S concentrations during exposure
H2S production bull No evidence for modifications of genes associ-
ated with enzymatic production of H2Sc
aPfenninger et al (2014) bR Greenway J L Kelley amp M Tobler unpub-
lished data cKelley et al (2016) dBarts et al (2018) ePfenninger et al
(2015)
TOBLER ET AL | 847
and physiological consequences of H2S may thus enable us to pre-
dict the physiological pathways in which adaptive modifications
occur it may be impossible to predict specific modifications within
those pathways This contrasts with a common expectation in the
field that convergent evolution of molecular mechanisms underlying
phenotypic traits (ie parallel evolution) necessarily hinges on modi-
fication of the same loci across lineages as found in some of the
best-known examples of parallelism (Colosimo et al 2005 Feldman
Brodie Brodie amp Pfrender 2012 Grant Grant Markert Keller amp
Petren 2004) However modification of the same loci may only be
common under particular circumstances (Rosenblum Parent amp
Brandt 2014) for example when selection acts on standing genetic
variation (Colosimo et al 2005) when adaptive alleles are intro-
gressed across population boundaries through gene flow (Grant
et al 2004) or when there are strong genetic constraints (Feldman
et al 2012) Exploration of molecular mechanisms underlying con-
vergent evolution in many systems may therefore be better
approached from the perspective of interacting genes that are orga-
nized in pathways in which alternative genetic changes can have
equivalent functional consequences (ie functional redundancy)
Developing and testing hypotheses about parallel evolution at the
pathway level will hinge on improving genome annotations (Pavey
Bernatchez Aubin-Horth amp Landry 2012) and provide a more
nuanced understanding of the factors determining the predictability
of evolution at different levels of organismal organization (Rosen-
blum et al 2014)
Despite evidence for convergent evolution in some candidate
physiological pathways it also needs to be emphasized that this
does not apply to all P mexicana lineages or to all candidate physio-
logical pathways associated with H2S adaptation investigated to
date Nonconvergent evolutionmdashevidenced by unique lineage-speci-
fic signals of selection and differential expressionmdashmay be indicative
of unique molecular mechanisms to cope with H2S toxicity For
example signatures of positive selection on COXmdashH2Srsquos primary
toxicity targetmdashcan be found in sulphide spring lineages of the Puy-
acatengo and Pichucalco river drainages (a lineage with recent diver-
gence time and a lineage with older divergence time respectively)
but not in those of the Tacotalpa drainage (Pfenninger et al 2014)
The presence of nonconvergent genetic and gene expression differ-
ences between populations from sulphidic and non-sulphidic habitats
in different river drainages highlights the limitations of inferences
that can be drawn based on data sets generated with high-through-
put DNA and RNA sequencing The potential importance of modifi-
cations at candidate locimdashor the lack thereofmdashcan only be assessed
through functional assays that document how alternative alleles and
changes in gene expression actually affect organismal performance
The functional consequences of genetic and gene expression varia-
tion at most candidate loci remain to be investigated However
enzyme activity assays have indicated that COX maintains its activity
with increasing H2S concentrations in the two sulphide spring popu-
lations with evidence for positive selection on mitochondrially
encoded subunits (Puyacatengo and Pichucalco Pfenninger et al
2014) Key amino acid substitutions occur in the D-pathway channel
of COX1 which putatively narrow the channel width and reduce
H2Srsquos access to the binuclear centre of the protein Consequently
some lineages of P mexicana exhibit an H2S-resistant COX that can
maintain its activity and aerobic ATP production even if endogenous
H2S concentrations are elevated In contrast COX activity in sul-
phide spring fish of the Tacotalpa river drainage and in fish from
nonsulphidic habitats decreases when H2S is present which is con-
sistent with mechanisms of H2S toxicity (Pfenninger et al 2014) In
vitro assays on isolated mitochondria have nonetheless indicated
that an H2S-resistant COX is not necessary to maintain aerobic ATP
production during H2S exposure Unlike in populations from nonsul-
phidic habitats mitochondrial spare respiratory capacity is main-
tained in the presence of H2S even in the Tacotalpa sulphide spring
populations that lack an H2S-resistant COX (C Henpita M Tobler amp
JH Shaw unpublished data) This may indicate that modification of
detoxification pathways in sulphide spring fishes is sufficient to
maintain mitochondrial function and aerobic ATP production
23 | Ensuing challenges linking genotypes tofitness
Understanding the mechanistic basis of adaptation requires linking
genotypic variation to phenotypic traits that ultimately shape fitness
along selective gradients The evolution of H2S tolerance in sulphide
spring fishes appears to be a consequence of modifications in many
genes and many physiological pathways whose functions interact to
eliminate H2S from the system minimize effects of elevated endoge-
nous concentrations on direct toxicity targets and mitigate adverse
effects that cascade through cellular metabolism during disruption of
homeostasis (Table 1) Such complex genetic changes are likely com-
mon during adaptation and have been documented in other systems
that involve major evolutionary shifts including the emergence of
parasitic lifestyles (Werren et al 2010) eusociality (Simola et al
2013) asexual reproduction (Fradin et al 2017) and internal gesta-
tion with livebearing (Schartl et al 2013) Consequently comple-
mentary approaches will be required to uncover the molecular
mechanisms of adaptation to H2S in the future
1 The involvement of many genes and physiological pathways in
shaping tolerance emphasizes the importance of quantitative
genetic approaches that leverage experimental crosses between
H2S tolerant and nontolerant populations and test whether toler-
ancemdashand adaptive trait modifications that contribute to itmdashis
explained by a few major effects loci or whether it is a conse-
quence of a number of genes with small effects (Klerks Xie amp
Levinton 2011 Saltz Hessel amp Kelly 2017) Combining analyses
of phenotype-fitness associations and their underlying genetic
mechanisms via QTL mapping with the results of selection scans
from population genomic analyses (Pfenninger et al 2015) will
allow for an integrative understanding of how selection acting on
the genome results in phenotypic variation that mediates adapta-
tion to different environments (Stinchcombe amp Hoekstra 2008)
From a functional perspective breaking up correlations between
848 | TOBLER ET AL
different physiological traits (associated with resistance mitiga-
tion and regulation Table 1) in F2 crosses will further allow test-
ing what molecular mechanisms are necessary for tolerance
whether there are alternative strategies with equivalent fitness
benefits and whether there are complementary modifications
with additive effects on tolerance
2 Considering the number of genes and physiological pathways
likely involved in H2S tolerance assessing the functional conse-
quences of genetic and gene expression variation is a daunting
task At the same time such functional tests can significantly
contribute to closing the genotype-to-phenotype gap because
physiochemical stress responses can be assessed along the con-
tinuum of organismal organization (Gjuvsland Vik Beard Hunter
amp Omholt 2013) Standardized assays can be used to quantify
the biochemical makeup of organelles cells and tissues (eg pro-
tein abundance and enzyme activity) physiological performance
in vitro and in vivo (eg rates of H2S detoxification ATP produc-
tion and reactive oxygen production) and fitness-relevant proxies
of whole organism performance (eg H2S tolerance Figure 2)
This highlights the importance of methods development for the
quantification of complex phenotypic traits or even whole phe-
nomes that can keep pace with rapid data generation through
high-throughput sequencing (Houle Govindaraju amp Omholt
2010 Ritchie Holzinger Li Pendergrass amp Kim 2015) In addi-
tion genotypendashphenotype links established through such correla-
tive approaches will ideally be verified through targeted gene
editing While gene editing with CRISPRndashCas9 is now routinely
employed in model organisms (Doudna amp Charpentier 2014 Sha-
lem Sanjana amp Zhang 2015) applying this technology to non-
models still remains challenging especially in livebearing species
like poeciliid fishes where in vitro culture of embryos is not rou-
tinely employed (Martyn Weigel amp Dreyer 2006) Nonetheless
the effects of specific mutations can potentially be verified
through the establishment and manipulation of cells in culture
(Liu et al 2014) or by generating transgenics (Lin et al 2016)
3 Organismal responses to physiochemical stressors occur at multi-
ple hierarchical levels that can be plastically modulated in
response to endogenous and exogenous cues (Whitehead 2012)
Understanding adaptation to physiochemical stressors conse-
quently requires disentangling the effects of genetic variation
developmental plasticity and short-term acclimation in gene
expression patterns on the emerging physiological processes
Common garden experiments on sulphide spring fishes have
shown that many genes differentially expressed between popula-
tions from sulphidic and nonsulphidic habitats in nature remain
differentially expressed in laboratory-reared fish that have never
been exposed to H2S indicating evolved differences in constitu-
tive gene expression (Passow Henpita et al 2017) At the same
time expression patterns in the laboratory also exhibit evidence
for plasticity in response to short-term H2S exposure (Passow
Henpita et al 2017) Some of these plastic responses are shared
between populations from sulphidic and nonsulphidic habitats
(likely representing shared ancestral plasticity) while others have
been lost or gained during sulphide spring colonization A core
challenge for future research is to overcome logistical constraints
that have so far prevented long-term H2S exposures of fish in
the laboratory Exposing individuals throughout development will
be required to test how developmental plasticity and short-term
acclimation shape responses to H2S in individuals from adapted
and nonadapted populations Finally the next big scientific
advance in this context will also require the identification of reg-
ulatory mechanisms that shape expression differences during
adaptation including sequence changes in transcription factors
and their binding sites patterns of DNA methylation and chro-
matin accessibility as well as the production of microRNAs and
competing endogenous RNAs that can modulate transcript abun-
dance (Flores Wolschin amp Amdam 2013 Lopez-Maury Mar-
guerat amp Beuroahler 2008 Silva Bye el Azzouzi amp Wisloslashff 2017)
In addition the role of alternative splicing and other post-tran-
scriptional and post-translational modifications in mediating adap-
tation remains understudied by evolutionary biologists (Ast
2004 Bush Chen Tovar-Corona amp Urritia 2017) This is particu-
larly relevant because H2S has been hypothesized to play a role
in the post-translational modification of proteins with sulphur-
containing amino acids through sulfhydration (Mustafa et al
2009)
Bridging the genotypendashphenotype gap remains one of the major
challenges in biology (Gjuvsland et al 2013 Ritchie et al 2015) and
will provide opportunities to understand how organisms function in
the face of environmental stress and how evolution shapes these
functions during adaptation to different environmental conditions
Establishing the molecular mechanisms of adaptation across repli-
cated lineages exposed to the same sources of selection will also
provide insights into how evolutionary history demography and
genetics shape convergent evolutionary outcomes providing a better
understanding at what hierarchical levels of organismal organization
evolutionary change may actually be predictable (Rosenblum et al
2014)
3 | COMPLEX ORGANISMS IN A COMPLEXWORLD DISENTANGLING THE TANGLEDBANK
Mechanisms of adaptation can be studied through bottom-up
(screening for genetic modifications that allow for inferences about
relevant phenotypic traits) and top-down approaches (assessing fit-
ness-relevant phenotypic differences among populations without
knowledge about the genetic basis see Schluter 2009) Over the
years genomic and transcriptomic studies on sulphide spring popula-
tions of the P mexicana species complex have been complemented
with analyses of phenotypic variation Just as genetic analyses have
indicated that adaptation to H2S is mediated by the modification
and modulation of many genes associated with multiple physiological
pathways phenotypic studies uncovered a variety of differences
TOBLER ET AL | 849
between populations from sulphidic and nonsulphidic environments
Many of these phenotypic differences have evolved in convergence
across replicated sulphide spring lineages in different river drainages
Examples include modifications of morphological (body shape and
organ size Tobler et al 2011 Tobler Scharnweber et al 2015
Schulz-Mirbach et al 2016) physiological (tolerance and metabolic
rates Tobler et al 2011 Passow Arias-Rodriguez amp Tobler 2017)
behavioural (aggression and personality Riesch et al 2009 Bierbach
et al 2012 Sommer-Trembo et al 2016 Bierbach Arias Rodriguez
amp Plath 2018) and life history traits (fecundity and offspring size
Riesch Plath Garcia de Leon amp Schlupp 2010 Riesch et al 2014)
Hence proximate populations in different habitat types have
diverged in complex phenotypes that span many different suites of
traits and a key question is how divergence in multivariate pheno-
types arises In theory H2S could be a key source of selection driv-
ing multivariate phenotypic divergence either because it directly
exerts selection on a multitude of traits that are independently modi-
fied to reach new fitness peaks or because selection on a few fit-
ness-relevant traits causes simultaneous changes in correlated
characteristics Neither explanation seems sufficient because many
Abiotic environmentOxygen concentrations
Ionic compositionTemperature
Structure and flow
Biotic environmentTrophic resources
DemographyPredationParasitism
H2S
Genetic variation
Gene expression
Biochemistry
Physiologicalfunction
Organismal performance
Primary fitness components
Survivalgrowth
reproduction
Organismal function Environment
F IGURE 2 Understanding how organisms function in the context of their environment is a core question in evolutionary biology yet thecomplexities associated with organismal function and environmental variation provide significant challenges High-throughput sequencing hasallowed for the identification of candidate genes subject to selection and differential expression during adaptation to H2S The next step is tobridge the genotype-to-phenotype gap by quantifying the integrated responses to H2S across the continuum of organismal organization includingbiochemical phenotypes physiological functions whole organism performance and ultimately fitness In addition understanding adaptation toH2S-rich environments will also require acknowledging H2Srsquos relation to other abiotic and biotic environmental variables which can exert selectionand modulate aspects of organismal function This will allow for the establishment of cause-and-effect relationships between specific sources ofselection and evolutionary change in specific traits and contribute to our understanding of organismal function in natural systems
850 | TOBLER ET AL
of the divergent traits between populations from sulphidic and non-
sulphidic environments have no clearmdashor at best tenuousmdashlinks to
H2S toxicity and detoxification and there is only limited evidence
for within-population correlations between different traits groups
(Riesch et al 2016)
Evolutionary analyses frequently focus on seemingly simple
sources of selection juxtaposing populations that are assigned to dis-
crete habitat types to understand the evolution of specific traits
(Kaeuffer Peichel Bolnick amp Hendry 2012) But as Darwin (1859)
already observed in his ldquotangled bankrdquo allegory selection in natural
systems is rarely univariate but multifarious selective regimes con-
tribute to trait divergence even among populations that occur along
apparently simple environmental gradients Unquantified environmen-
tal variation that is subsumed in the categorization of habitat types
can profoundly affect trait variation in natural systems (Stuart et al
2017) highlighting the importance of considering and testing alterna-
tive hypotheses about the cause-and-effect relationship between
specific sources of selection and evolution of specific traits Thus
accurate natural history observations that consider organisms as holis-
tic entities consisting of interacting traits in the context of the abiotic
and biotic factors comprising their environment are critical to inform
the design and interpretation of empirical research in natural systems
(Greene 2005) Indeed adjacent sulphidic and nonsulphidic habitat
types do not merely differ in the presence or absence of H2S but also
in a variety of other abiotic and biotic factors (Figure 2) Unlike nonsul-
phidic habitats sulphide springs in southern Mexico are characterized
by extreme hypoxia elevated salinity and lower pH (Rosales Lagarde
2012 Tobler et al 2011) In addition the presence of physiochemical
stressors affects the biotic communities in these sulphide springs Spe-
cies richness of aquatic invertebrates and vertebrates is drastically
reduced as a consequence of H2S toxicity (Greenway Arias-Rodriguez
Diaz amp Tobler 2014) and primary production appears to be driven in
part by chemoautotrophic bacteria that use H2S as an energy source
rather than just by autochthonous and allochthonous photosynthetic
organisms (Roach Tobler amp Winemiller 2011) These changes in the
biotic environment affect the relative importance of intra-vs inter-
specific competition trophic resource use and exposure to predators
and parasites (Riesch Oranth et al 2010 Tobler et al 2014 Tobler
Scharnweber et al 2015)
31 | Alternative drivers of trait evolution
Acknowledging the complexity of selective regimes in sulphide
springs allows for a more nuanced understanding of trait evolution
and for the development of alternative hypotheses about the ulti-
mate mechanisms that cause divergence in specific genetic and phe-
notypic traits In some instances sources of selection other than H2S
can plausibly explain trait divergence between populations from sul-
phidic and nonsulphidic habitats For example hypoxia likely drives
increases in head size and gill surface area (Tobler et al 2011)
changes in the ionic composition of the water can explain differential
expression of genes associated with osmoregulation (Kelley et al
2016) and shifts in trophic resource use are associated with
modifications of the gastrointestinal system (Tobler Scharnweber
et al 2015) In other cases disentangling alternative hypotheses is
not trivial because selection mediated by H2S and other environmen-
tal factors are predicted to affect trait modifications in similar ways
Shifts towards anaerobic metabolism in sulphide spring fish could be
the consequence of inhibition of aerobic ATP production by H2S or
by constraints in oxygen availability Similarly reproductive life his-
tory shifts towards the production of larger but fewer offspring could
be driven by H2S (decreasing offspringrsquos surfacevolume ratio to mini-
mize the diffusion of H2S into the body Powell 1989) by habitat dif-
ferences in the quality and quantity of predation that can affect size-
specific rates of extrinsic mortality (Jorgensen Auer amp Reznick
2011) or by energetic constraints (see below) While comparisons
across different selective regimes can shed light into the ultimate dri-
vers of trait evolution (Moore Riesch amp Martin 2016) isolating the
effects of different sources of selection may not be possible because
they could act in concert and facilitate increases of organismal perfor-
mance in multiple contexts (Ghalambor Walker amp Reznick 2003)
When multifarious selection shapes multivariate phenotypic evo-
lution it is important to also consider the role of trade-offs both in
terms of trait expression (increased investment in one trait may
impose constraints on the expression of other traits) and in terms of
trait function (optimizing the trait for one function may impair the
traitrsquos utility for other functions) For example selection on large off-
spring size in sulphide spring fishes has inadvertent consequences
for fecundity (Riesch et al 2014) and selection for increased gill
surface areas due to hypoxia simultaneously increases exposure to
environmental H2S (Tobler et al 2011) Thus the outcome of phe-
notypic evolution is ultimately a result of balancing competing needs
in a manner that optimizes trait expression and organismal function
in multiple contexts to maximize fitness (Arnold 1983) and the pres-
ence of trade-offs can have cascading indirect effects with selection
on one or a few traits leading to the modification of a multitude of
apparently unrelated characteristics
In this context bioenergetics may represent a nexus in the evo-
lution of complex traits observed in sulphide spring fish and other
extreme environments Living in and adapting to sulphide springs
imposes constraints on organismal energy availability (Plath et al
2007) with according trade-offs for energy allocation Energy limita-
tion in sulphide springs does not arise from a scarcity of food
resources (chemoautotrophic bacteria eaten by the fish are abundant
in sulphide springs Tobler Scharnweber et al 2015) but from
behavioural and physiological adaptation A primary survival strategy
for fish in the toxic and hypoxic springs is to engage in aquatic sur-
face respiration where individuals breathe directly at the water sur-
face to maximize oxygen uptake and minimize H2S exposure This
behaviour mediates short-term survival (Plath et al 2007) but it
also constrains energy acquisition for benthic foragers like P mexi-
cana (Tobler Riesch Tobler amp Plath 2009) and increases susceptibil-
ity to bird predation (Riesch Oranth et al 2010) Costly investments
into H2S detoxification and tissue repair as well as a shift from aero-
bic to less efficient anaerobic metabolism further constrain energy
allocation to other functions (Passow Greenway Arias-Rodriguez
TOBLER ET AL | 851
Jeyasingh amp Tobler 2015) Theory predicts that energetic con-
straints ndash whether mediated by limitations in the rate of resource
acquisition or in the rate of energy allocation to reproduction (as
opposed to maintenance or growth) ndash should exert selection for a
reduction in organismal energy budgets which allows for the maxi-
mization of residual energy allocation for the production of offspring
(Brown Marquet amp Taper 1993) Several lines of evidence support
the hypothesis that energy limitation plays a critical role in pheno-
typic evolution of sulphide spring fish Compared to individuals in
nonsulphidic environments fish in sulphide springs have significantly
lower routine energy demands which is mediated both by reduc-
tions in body size and mass-specific metabolism (Passow Arias-
Rodriguez et al 2017) Reductions in other traits such as energeti-
cally costly behaviours (Bierbach et al 2012 Plath 2008) and the
size of energy-demanding organs (Schulz-Mirbach et al 2016) may
also contribute to the reduced energy demands and shifts in repro-
ductive life history traits (Riesch et al 2014) are consistent with
selection from energy limitation (Reznick amp Yang 1993)
32 | Ensuing challenges Disentanglingenvironmental and organismal complexities
Future research needs to empirically address how multifarious selec-
tive regimes shape the evolution of organisms with complex pheno-
types We have made great advances in our understanding of how
specific traits evolve and mediate adaptation (eg morphology Chan
et al 2010 coloration Dasmahapatra et al 2012 sensory organs
Cartwright Schwartz Merry amp Howell 2017) and we have a thor-
ough understanding of how single environmental factors can shape
organismal responses (eg climatic conditions Johnston amp Bennett
1996 predation Langerhans 2007 competition Pfennig amp Pfennig
2012) In contrast our understanding of phenotypic evolution in
response to multifarious selection is comparatively limited especially
because interactive effects of different environmental factors can lead
to organismal and evolutionary responses that are unpredictable based
on experiments that manipulate single environmental variables in iso-
lation (Gunderson Armstrong amp Stillman 2016 Todgham amp Stillman
2013) Future research thus needs to acknowledge the complexities of
environments and organismal design and address them explicitly in
empirical investigations to gain a robust understanding of biological
diversification (Culumber amp Tobler 2017 Stuart et al 2017)
4 | LINKING ADAPTATION TOSPECIATION
So far we primarily explored the causes of adaptive evolution but
what are its consequences Theory predicts that adaptation to local
environmental conditions can lead to speciation because reproduc-
tive isolation emerges incidentally as a by-product (ecological specia-
tion Nosil 2012) Population genetic analyses of Poecilia spp in
sulphide springs and adjacent populations in nonsulphidic environ-
ments have indicated strong genetic differentiation across small
spatial scales (in some instances lt100 m) and in the absence of
physical barriers that would prevent fish movement (Plath et al
2013) In addition population genetic structuring has been shown to
be stable even in the face of major environmental disturbance
BOX 1 Speciation in sulphide springs and conservation
biology
Adaptation to H2S-rich environments in poeciliids coincides
with ecological speciation giving rise to phenotypically dis-
tinct and reproductively isolated lineages In some
instances this has led to the evolution of highly distinct
micro-endemic species including Poecilia sulphuraria and
P thermalis Limia sulphurophila and Gambusia eurystoma
(Figure 3) Except for P sulphuraria which is found in two
spring complexes of the Rıo Pichucalco drainage of Mexico
(Tobler amp Plath 2009b) the distributions of these sulphide
spring endemics are restricted to single localities that
include only small areas of suitable habitat For example
the largest sulphide spring complex (the Ba~nos del Azufre
in southern Mexico which harbours P sulphuraria and G
eurystoma) includes ~18000 m2 of suitable habitat (equiva-
lent to lt35 football fields) while one of the smallest (La
Esperanza springs harbouring P thermalis) merely covers
~600 m2 (just over a tenth of a football field Brown et al
2017) Accordingly some sulphide spring endemics are
considered critically endangered (IUCN 2012) and pro-
tected by law (SEDESOL 1994)
Many threatened species with restricted ranges exhibit
low genetic diversity and high incidences of inbreeding
(Bouzat 2010 Willi Van Buskirk amp Hoffmann 2006) In
contrast at least P sulphuraria and P thermalismdashthe only
species investigated to datemdashactually exhibit relatively high
effective population sizes (Ne = 1200ndash3800 depending on
the population) and no evidence for inbreeding (FIS lt 0)
(Brown et al 2017) This is likely a consequence of the
long-term stability of sulphide springs and the high pro-
ductivity and low levels of interspecific competition also
support unusually high fish densities and thus population
census sizes (Brown et al 2017 Culumber et al 2016)
Nonetheless sulphide spring endemics are threatened by
habitat destruction which is mediated primarily by the
modification of springs for recreational purposes defor-
estation and conversion of land for agriculture and most
recently high-intensity palm oil plantations (Tobler amp Plath
2009ab) Despite the official protection of sulphide spring
endemics there have been no tangible conservation mea-
sures that foster the sustainable development of land
around sulphide springs with the well-being of the endemic
fishes in mind
852 | TOBLER ET AL
(catastrophic flood event Plath et al 2010) Population genetic dif-
ferentiation between adjacent sulphide-adapted and nonadapted
populations is indicative of ongoing ecological speciation (Box 1) but
different population pairs from sulphidic and nonsulphidic waters
have made variable progress along the speciation continuum towards
complete reproductive isolation (Plath et al 2013) This prompts
questions about the mechanisms involved in reducing gene flow
along the environmental gradient connecting the two habitat types
41 | Prezygotic mechanisms of reproductiveisolation
The simplest form of reproductive isolation between locally adapted
populations arises from selection against migrants between habitat
types (Nosil Vines amp Funk 2005) Translocation experiments in nat-
ural habitats have indicated strong survival selection against
migrants especially those translocated from nonsulphidic to sulphidic
habitats (Plath et al 2013) Fish translocated from nonsulphidic
environments face high mortality within short periods of time
(lt24 hr) which is likely caused by H2S toxicity and hypoxia in sul-
phide springs Hence low gene flow from populations inhabiting
nonsulphidic waters into populations inhabiting sulphidic habitats is
simply explained by a lack of adaptation to the extreme environmen-
tal conditions At least in some drainages there is also appreciable
mortality for sulphide spring fish translocated to nonsulphidic habi-
tats although mechanisms causing inviability are unclear and the
strength of selection appears to be lower (Plath et al 2013) If sur-
vival selection against immigrants was the sole source of reproduc-
tive isolation this should result in unidirectional gene flow from
H2S-adapted to nonadapted populations but population genetic
analyses provided no evidence for this implying that additional
mechanisms are at work (Plath et al 2013)
One such mechanism could be intersexual selection and mate
choice experiments have indicated that migrants from sulphidic to
nonsulphidic habitats are at a significant disadvantage with fish from
nonsulphidic habitats avoiding potential mating partners from sul-
phidic ones (Plath et al 2013 Sommer-Trembo et al 2016) Such
assortative mating has been found to be critical during ecological spe-
ciation because it counteracts the homogenizing effects of gene flow
(van Doorn Edelaar amp Weissing 2009) yet how assortative mating
patterns arise often remains unclear In theory assortative mating can
arise through a variety of processes (Nosil 2012) ldquoMagic traitrdquo mecha-
nisms where mating preferences evolve as a by-product of divergent
selection on ecological traits that serve as cues have recently garnered
much attention (Servedio van Doorn Kopp Frame amp Nosil 2011)
Consistent with magic trait mechanisms assortative mating in P mexi-
cana appears to be linked to adaptive population differences in body
shape which serve as cues to distinguish potential mating partners
from different habitats (Greenway Drexler Arias-Rodriguez amp Tobler
2016) However neither sulphide spring fish (which are unlikely to
encounter migrants from nonsulphidic habitats due to H2S toxicity)
nor allopatric P mexicana from nonsulphidic waters (which have never
experienced migrants from sulphide spring populations) exhibit a
preference for mating partners from their own ecotype suggesting
that assortative mating in this system is a product of reinforcement
(ie direct selection for assortative mating Greenway et al 2016)
This is different from classic magic traits mechanisms because assorta-
tive mating is not a mere by-product of adaptation but adaptive traits
have instead been co-opted as mating signals in response to selection
on mating preferences (Maan amp Seehausen 2012) Moreover males of
some H2S-adapted populations have reduced aggressive behaviour
(Bierbach et al 2012 2018) In staged contests of size-matched males
from different ecotypes intruders had a significantly lower likelihood
to establish dominance than resident males independent of whether
or not they showed reduced aggressiveness overall Hence intrasexual
selection may represent another mechanism mediating reproductive
isolation between locally adapted ecotypes Overall H2S-adapted poe-
ciliids allow disentangling the relative contributions of different forms
of ecological and sexual selectionmdashand their interdependencymdashduring
ecological speciation
42 | Ensuing challenges quantifying postzygoticmechanisms of reproductive isolation
In contrast to prezygotic mechanisms little is known about postzy-
gotic mechanisms of reproductive isolation that potentially act to
reduce gene flow between adjacent populations in sulphidic and
nonsulphidic habitats Putative hybrids between sulphidic and non-
sulphidic ecotypes have occasionally been observed in nonsulphidic
habitats immediately adjacent to confluences with sulphide springs
(R Riesch M Plath and M Tobler personal observations) but it
remains unclear whether and through what mechanisms such hybrids
exhibit reduced fitness compared to parentals
Direct selection of mitochondrially encoded genes in the pres-
ence of H2S (Pfenninger et al 2014) should be accompanied by co-
evolution at nuclear loci that directly interact with mitochondrial
gene products in the formation of protein complexes involved in the
OXPHOS pathway (Sunnucks Morales Lamb Pavlova amp Greening
2017 Willett amp Burton 2004) Disruption of co-evolved gene com-
plexes in hybrids should therefore lead to intrinsic fitness reductions
through mito-nuclear incompatibilities (Burton Pereira amp Barreto
2013 Wolff Ladoukakis Enrıquez amp Dowling 2014) which have
been hypothesized to be a key driver in speciation (Chou amp Leu
2010 Gershoni Templeton amp Mishmar 2009) Empirical tests of
these ideas remain to be conducted in sulphide spring fishes just
like studies on potential ecological and sexual selection against
hybrids that arise from interpopulation mating events
5 | PERSPECTIVES LIFE IN SULPHIDICENVIRONMENTS BEYOND P MEXICANA
Research on sulphide spring fishes of the P mexicana species com-
plex in Mexico has provided insights into mechanisms underlying
adaptation and the links between adaptation and speciation Sul-
phide springs do not only occur in Mexico however but are found
TOBLER ET AL | 853
worldwide and have been colonized by 24 evolutionarily indepen-
dent fish lineages in North America the Neotropics and the Middle
East (Greenway et al 2014) The vast majority of lineages belong to
the order Cyprinodontiformes (95) and livebearing members of the
family Poeciliidae in particular (80) This potentially indicates that
internal gestation of young has facilitated the colonization of
extreme environments because it shields developing embryos from
physiochemical stressors (Blackburn 1999) Among the Poeciliidae
sulphide spring fishes include members of the genera Brachyrhaphis
Gambusia Poeciliopsis Priapichthys Pseudoxiphophorus Xiphophorus
and multiple subgenera of Poecilia (Figure 3 Greenway et al 2014)
spanning over 40 million years of evolution (based on Reznick Fur-
ness Meredith amp Springer 2017)
A handful of studies have started to address molecular and phe-
notypic evolution across a broader taxon sampling of sulphide spring
fishes finding evidence for convergent evolution in morphology
(Riesch et al 2016 Tobler amp Hastings 2011) life history traits
(Riesch Plath et al 2010 Riesch et al 2014) and oxygen transport
genes (Barts et al 2018) In addition ecological speciation in
response to adaptation to H2S-rich environments has also been doc-
umented in sulphide spring poeciliids other than P mexicana (Riesch
et al 2016) These studies have laid a fruitful foundation for the
investigation of mechanisms of adaptation to H2S-rich springs from
genes to phenotypic traits linking micro-evolutionary processes to
macro-evolutionary patterns
The availability of broad-scale evolutionary replication of popula-
tions that have invaded habitats with the same physiochemical stressors
allows for addressing some of the ensuing challenges discussed above
and investigating the factors that give rise to predictable evolutionary
outcomes Convergent evolution is a commonmdashbut not omnipresentmdash
theme in adaptive evolution and multiple factors can determine the
degree of convergence among replicated lineages exposed to the same
sources of selection (Kaeuffer et al 2012 Landry amp Bernatchez 2010
Oke Rolshausen LeBlond amp Hendry 2017 Rosenblum amp Harmon
2011) Thus leveraging replicated lineages that have colonized sulphide
springs to assess molecular mechanisms underlying convergent pheno-
typic changes analyse how environmental heterogeneity among sul-
phide spring habitats causes lineage-specific patterns of trait evolution
and test whether and by what mechanisms adaptation to extreme envi-
ronments is linked to speciation will provide insights into how ecologi-
cal genetic and functional factors contribute to evolutionary
convergence at different levels of organismal organization (Rosenblum
et al 2014) An expanded taxon sampling with population pairs that
span almost the full range of the speciation continuum from panmixia to
complete reproductive isolation (Riesch et al 2016) will also help
addressing questions about temporal aspects of H2S adaptation In par-
ticular comparing younger to older lineages will provide insights about
the order at which genetic and phenotypic modifications accrue during
the adaptation process
Animals have also invaded H2S-rich habitats in other aquatic
ecosystems including cold seeps and deep-sea hydrothermal vents
(Tobler et al 2016) Adaptation to H2S in the P mexicana species com-
plex has involved modification of physiological pathways associated
with H2S toxicity (OXPHOS pathway) and detoxification (sulphide
F IGURE 3 Phylogeny of the family Poeciliidae (adopted from Culumber amp Tobler 2017) Various lineages in the genera Poecilia (subgeneraAcanthophacelus Limia and Mollienesia) Priapichthys Brachyrhaphis Poeciliopsis Xiphophorus Pseudoxiphophorus and Gambusia have colonizedH2S-rich springs in different parts of the Americas (see Greenway et al 2014) Lineages highlighted in yellow represent endemic speciesrestricted to H2S-rich springs while red lineages represent species in which some populations have invaded sulphidic habitats Pictures depict arepresentative of each focal genussubgenus
854 | TOBLER ET AL
quinone oxidoreductase pathway) which have been highly conserved
during the diversification of life on Earth (Saraste 1999 Shahak amp
Hauska 2008) The high replicability of H2S as an environmental factor
that shaped animal evolution across vastly different ecological contexts
consequently offers the opportunity to investigate whether selection
mediated by the presence of H2S has repeatedly modified these con-
served physiological pathways across animal phyla that have diverged
during the Cambrian explosion spanning over 500 million years of evo-
lution Such comparisons would provide unprecedented insights into
the effects of a single physiochemical stressor on evolutionary change
irrespective of ecological or phylogenetic contexts
6 | CONCLUSIONS
H2S-rich environments and their inhabitants represent useful models
for research in organismal biology ecology and evolution illustrating
both opportunities (eg strong and explicit source of selection evolu-
tionary replication) and challenges (eg complexity of environments
and organismal responses) in asking basic question about the origins
of biodiversity Understanding how organisms thrive in apparently
inhospitable environments will provide us with insights into lifersquos
capacities and limitations to adapt to novel environments (Waterman
1999) This is particularly relevant considering that human activities
substantially contribute to the exacerbation of physiochemical stres-
sors at local regional and global scales impacting ecological and evo-
lutionary dynamics (Alberti 2015 Norberg Urban Vellend
Klausmeier amp Loeuille 2012) Thus a focus on ecological and evolu-
tionary consequences of natural stressors that push organisms to the
brink of their physiological capacities may be instrumental to predict
consequences of anthropogenic environmental change
ACKNOWLEDGEMENTS
We would like to thank Lenin Arias-Rodriguez the Universidad
Juarez Autonoma de Tabasco and the Centro de Investigacion E
Innovacion Para la Ense~nanza y El Aprendizaje for their continued
collaboration enabling our work on the P mexicana system Funding
for this review article has been provided by the National Science
Foundation (IOS-1557860 IOS-1557795) and the US Army
Research Office (W911NF-15-1-0175)
AUTHOR CONTRIBUTIONS
MT wrote the first draft of the paper all authors contributed text
and comments
ORCID
Michael Tobler httporcidorg0000-0002-0326-0890
Joanna L Kelley httporcidorg0000-0002-7731-605X
Reuroudiger Riesch httporcidorg0000-0002-0223-1254
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Alda F Reina R G Doadrio I amp Bermingham E (2013) Phylogeny
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Alvarez del Villar J (1948) Descripcion de una nueva especie de Mol-
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Arnold S J (1983) Morphology performance and fitness American Zool-
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Aspiras A C Rohner N Martineau B Borowsky R L amp Clifford C J
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Ast G (2004) How did alternative splicing evolve Nature Reviews
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Bagarinao T (1992) Sulfide as an environmental factor and toxicant
Tolerance and adaptations in aquatic organisms Aquatic Toxicology
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Barton N H (1998) The effect of hitch-hiking on neutral genealogies
Genetic Research 72 123ndash133 httpsdoiorg101017
S0016672398003462
Barts N Greenway R Passow C N Arias Rodriguez L Kelley J L amp
Tobler M (2018) Expression and molecular evolution of oxygen
transport genes in livebearing fishes (Poeciliidae) from hydrogen sul-
fide rich springs Genome httpsdoiorg101139gen-2017-0051
[Epub ahead of print]
Beauchamp R O Bus J S Popp C S Boreiko C J amp Andjelkovich
D A (1984) A critical review of the literature on hydrogen sulfide
CRC Critical Reviews in Toxicology 13 25ndash97 httpsdoiorg10
310910408448409029321
Bell E M (2012) Life at extremes Environments organisms and strategies
for survival Oxfordshire CABI httpsdoiorg101079
97818459381470000
Bierbach D Arias Rodriguez L amp Plath M (2018) Intrasexual competi-
tion enhances reproductive isolation between locally adapted popula-
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ahead of print]
Bierbach D Klein M Sassmannshausen V Schlupp I Riesch R
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sive behavior Another reproductive isolation barrier in extremophile
poeciliid fishes International Journal of Evolutionary Biology 2012
148745
Blackburn D G (1999) Viviparity and oviparity Evolution and reproduc-
tive strategies In T E Knobil amp J D Neill (Eds) Encyclopedia of
reproduction Vol 4 (pp 994ndash1003) New York NY Academic Press
Bouzat J L (2010) Conservation genetics of population bottlenecks
The role of chance selection and history Conservation Genetics 11
463ndash478
Brown A P Greenway R Morgan S Quackenbush C R Giordani
L Arias-Rodriguez L Kelley J L (2017) Genome-scale data
reveals that endemic Poecilia populations from small sulfide spring
display no evidence of inbreeding Molecular Ecology 26 4920ndash
4934
Brown J H Marquet P A amp Taper M L (1993) Evolution of body
size Consequences of an energetic definition of fitness American
Naturalist 142 573ndash584 httpsdoiorg101086285558
Burton R S Pereira R J amp Barreto F S (2013) Cytonuclear genomic
interactions and hybrid breakdown Annual Review of Ecology
TOBLER ET AL | 855
Evolution and Systematics 44 281ndash302 httpsdoiorg101146an
nurev-ecolsys-110512-135758
Bush S J Chen L Tovar-Corona J M amp Urritia A O (2017) Alterna-
tive splicing and the evolution of phenotypic novelty Philosophical
Transactions of the Royal Society B 372 20150474 httpsdoiorg
101098rstb20150474
Carroll S P Jorgensen P S Kinnison M T Bergstrom C T Denison
R F Gluckman P Tabashnik B E (2014) Applying evolutionary
biology to address global challenges Science 346 313 httpsdoi
org101364FIO2014JW3A13
Cartwright R A Schwartz R S Merry A L amp Howell M M (2017)
The importance of selection in the evolution of blindness in cavefish
Bmc Evolutionary Biology 17 45 httpsdoiorg101186s12862-
017-0876-4
Chan Y F Marks M E Jones F C Villarreal G Shapiro M D Brady
S D Kingsley D M (2010) Adaptive evolution of pelvic reduc-
tion in sticklebacks by recurrent deletion of Pitx1 enhancer Science
327 302ndash305 httpsdoiorg101126science1182213
Chou J Y amp Leu J Y (2010) Speciation through cytonuclear incompat-
ibility Insights from yeast and implications for higher eukaryotes
BioEssays 32 401ndash411 httpsdoiorg101002bies200900162
Colosimo P F Hosemann K E Balabhadra S Villarreal G Dickson
M Grimwood J Kingsley D M (2005) Widespread parallel evo-
lution in sticklebacks by repeated fixation of ectodysplasin alleles
Science 307 1928ndash1933 httpsdoiorg101126science1107239
Cooper C E amp Brown G C (2008) The inhibition of mitochondrial
cytochrome oxidase by the gases carbon monoxide nitric oxide
hydrogen cyanide and hydrogen sulfide Chemical mechanism and
physiological significance Journal of Bioenergy and Biomembranes 40
533ndash539 httpsdoiorg101007s10863-008-9166-6
Culumber Z W Hopper G W Barts N Passow C N Morgan S Brown
A Tobler M (2016) Habitat use of two extremophile highly ende-
mic and critically endangered fish species (Gambusia eurystoma and Poe-
cilia sulphurophila Poeciliidae) Aquatic Conservation-Marine and
Freshwater Ecosystems 26 1155ndash1167 httpsdoiorg101002aqc
2640
Culumber Z W amp Tobler M (2017) Sex-specific evolution during the
diversification of live-bearing fishes Nature Ecology amp Evolution 1
1185ndash1191
Dalziel A C Rogers S M amp Schulte P M (2009) Linking genotypes
to phenotypes and fitness How mechanistic biology can inform
molecular ecology Molecular Ecology 18 4997ndash5017 httpsdoi
org101111j1365-294X200904427x
Darwin C (1859) On the origin of species based on natural selection or
the preservation of favoured races in the struggle of life 6th ed Lon-
don John Murray
Dasmahapatra K K Walters J R Briscoe A D Davey J W Whibley
A Nadeau N J Jiggins C D (2012) Butterfly genome reveals
promiscuous exchange of mimicry adaptations among species Nature
487 94ndash98 httpsdoiorg101038nature11041
Doudna J A amp Charpentier E (2014) The new frontier of genome edit-
ing with CRISPR-Cas9 Science 346 1258096 httpsdoiorg10
1126science1258096
Dubilier N Giere O amp Grieshaber M (2005) Concomitant effects of sul-
fide and hypoxia on the aerobic metabolism of the marine oligochaete
Tubificoides benedii Journal of Experimental Zoology 269 287ndash298
Eales K L Hollinshead K E R amp Tennant D A (2016) Hypoxia and
metabolic adaptation of cancer cells Oncogenesis 5 e190 httpsd
oiorg101038oncsis201550
Eghbal M A Pennefather P S amp OrsquoBrien P J (2004) H2S cytotoxicity
mechanism involves reactive oxygen species formation and mito-
chondrial depolarisation Toxicology 203 69ndash76 httpsdoiorg10
1016jtox200405020
Feldman C R Brodie Jr E D Brodie III E D amp Pfrender M E (2012)
Constraint shapes convergence in tetrodotoxin-resistant sodium
channels of snakes Proceedings of the National Academy of Sciences
USA 109 4556ndash4561 httpsdoiorg101073pnas1113468109
Flores K B Wolschin F amp Amdam G V (2013) The role of methyla-
tion of DNA in environmental adaptation Integrative and Comparative
Biology 53 359ndash372 httpsdoiorg101093icbict019
Fradin H Kiontke K Zegar C Gutwein M Lucas J Kovtun M
Piano F (2017) Genome architecture and evolution of a unichromo-
somal asexual nematode Current Biology 27(2928ndash2939) e2926
Gershoni M Templeton A amp Mishmar D (2009) Mitochondrial bioen-
ergetics as a major motive force of speciation BioEssays 31 642ndash
650 httpsdoiorg101002bies200800139
Ghalambor C K Walker J A amp Reznick D N (2003) Multi-trait selec-
tion adaptation and constraints on the evolution of burst swimming
performance Integrative and Comparative Biology 43 431ndash438
httpsdoiorg101093icb433431
Gibert J amp Deharveng L (2002) Subterranean ecosystems A truncated
functional biodiversity BioScience 52 473ndash481 httpsdoiorg10
16410006-3568(2002)052[0473SEATFB]20CO2
Gjuvsland A B Vik J O Beard D A Hunter P J amp Omholt S W
(2013) Bridging the genotype-phenotype gap What does it take
Journal of Physiology 591 2055ndash2066 httpsdoiorg101113jphys
iol2012248864
Grant P R Grant B R Markert J A Keller L F amp Petren K (2004)
Convergent evolution of Darwinrsquos finches caused by introgressive
hybridization and selection Evolution 58 1588ndash1599 httpsdoi
org101111j0014-38202004tb01738x
Greene H W (2005) Organisms in nature as a central focus for biology
Trends in Ecology and Evolution 20 23ndash27 httpsdoiorg101016j
tree200411005
Greenway R Arias-Rodriguez L Diaz P amp Tobler M (2014) Patterns
of macroinvertebrate and fish diversity in freshwater sulphide springs
Diversity 6 597ndash632 httpsdoiorg103390d6030597
Greenway R Drexler S Arias-Rodriguez L amp Tobler M (2016) Adap-
tive but not condition-dependent body shape differences contribute
to assortative mating preferences during ecological speciation Evolu-
tion 70 2809ndash2822 httpsdoiorg101111evo13087
Gunderson A R Armstrong E J amp Stillman J H (2016) Multiple stres-
sors in a changing world The need for an improved perspective on
physiological responses to the dynamic marine environment Annual
Review of Marine Science 8 357ndash378 httpsdoiorg101146an
nurev-marine-122414-033953
Hildebrandt T M amp Grieshaber M (2008) Three enzymatic activities
catalyze the oxidation of sulfide to thiosulfate in mammalian and
invertebrate mitochondria FEBS Journal 275 3352ndash3361 httpsd
oiorg101111j1742-4658200806482x
Houle D Govindaraju D R amp Omholt S (2010) Phenomics The next
challenge Nature Reviews Genetics 11 855ndash866 httpsdoiorg10
1038nrg2897
IUCN (2012) Red List of threatened species version 20132 www
iucnredlistorg
Johnston I A amp Bennett A F (1996) Animals and temperature Pheno-
typic and evolutionary adaptation Cambridge Cambridge University
Press httpsdoiorg101017CBO9780511721854
Jorgensen C Auer S K amp Reznick D N (2011) A model for optimal
offspring size in fish including live-bearing and parental effects Amer-
ican Naturalist 177 E119ndashE135 httpsdoiorg101086659622
Kaeuffer R Peichel C L Bolnick D I amp Hendry A P (2012) Parallel
and nonparallel aspects of ecological phenotypic and genetic diver-
gence across replicate population pairs of lake and stream stickle-
back Evolution 66 402ndash418 httpsdoiorg101111j1558-5646
201101440x
Kell D B amp Oliver S G (2004) Here is the evidence now what is the
hypothesis The complementary roles of inductive and hypothesis-
driven science in the post-genomic era BioEssays 26 99ndash105
httpsdoiorg101002(ISSN)1521-1878
856 | TOBLER ET AL
Kelley J L Arias-Rodriguez L Patacsil Martin D Yee M C Busta-
mante C amp Tobler M (2016) Mechanisms underlying adaptation to
life in hydrogen sulfide rich environments Molecular Biology and Evo-
lution 33 1419ndash1434 httpsdoiorg101093molbevmsw020
Kelley J L Passow C Plath M Arias-Rodriguez L Yee M C amp
Tobler M (2012) Genomic resources for a model in adaptation and
speciation research The characterization of the Poecilia mexicana
transcriptome BMC Genomics 13 652 httpsdoiorg101186
1471-2164-13-652
Klerks P L Xie L amp Levinton J S (2011) Quantitative genetics
approaches to study evolutionary processes in ecotoxicology a per-
spective from research on the evolution of resistance Ecotoxicology
20 513ndash523 httpsdoiorg101007s10646-011-0640-2
Kump L R Pavlov A amp Arthur M A (2005) Massive release of hydrogen
sulfide to the surface ocean and atmosphere during intervals of oceanic
anoxia Geology 33 397ndash400 httpsdoiorg101130G212951
Keuroustner A Hoffmann M Fraser B A Kottler V A Sharma E Wei-
gel D amp Dreyer C (2016) The genome of the Trinidadian guppy
Poecilia reticulata and variation in the guanapo population PLoS ONE
11 e0169087 httpsdoiorg101371journalpone0169087
Landry L amp Bernatchez L (2010) Role of epibenthic resource opportu-
nities in the parallel evolution of lake whitefish species pairs (Core-
gonus sp) Journal of Evolutionary Biology 23 2602ndash2613 httpsdoi
org101111j1420-9101201002121x
Langerhans R B (2007) Evolutionary consequences of predation Avoid-
ance escape reproduction and diversification In A M T Elewa
(Ed) Predation in organisms Berlin Springer
Li L Rose P amp Moore P K (2011) Hydrogen sulfide and cell signaling
Annual Review of Pharmacology and Toxicology 51 169ndash187
httpsdoiorg101146annurev-pharmtox-010510-100505
Lin Q Fan S Zhang Y Xu M Zhang H Yang Y Gunter H M
(2016) The seahorse genome and the evolution of its specialized mor-
phology Nature 540 395ndash399 httpsdoiorg101038nature20595
Liu Y Ma S Wang X Chang J Gao J Shi R Zhao P (2014)
Highly efficient multiplex targeted mutagenesis and genomic struc-
ture variation in Bombyx mori cells using CRISPRCas9 Insect bio-
chemistry and molecular biology 49 35ndash42 httpsdoiorg101016j
ibmb201403010
Lopez-Maury L Marguerat S amp Beuroahler J (2008) Tuning gene expres-
sion to changing environments From rapid responses to evolutionary
adaptation Nature Reviews Genetics 9 583ndash593 httpsdoiorg10
1038nrg2398
Maan M E amp Seehausen O (2012) Magic traits versus magic prefer-
ences in speciation Evolutionary Ecology Research 14 779ndash785
Martyn U Weigel D amp Dreyer C (2006) In vitro culture of embryos
of the guppy Poecilia reticulata Developmental Dynamics 235 617ndash
622 httpsdoiorg101002(ISSN)1097-0177
Metzker M L (2010) Sequencing technologies The next generation
Nature Reviews Genetics 11 31ndash46 httpsdoiorg101038nrg2626
Moore M P Riesch R amp Martin R A (2016) The predictability and
magnitude of life-history divergence to ecological agents of selection
A meta-analysis in livebearing fishes Ecology Letters 19 435ndash442
httpsdoiorg101111ele12576
Mustafa A K Gadalla M M Sen N Kim S Mu W Gazi S K
Snyder S H (2009) H2S signals through protein S-sulfhydration
Science Signaling 2 ra72
Nesse R M amp Stearns S C (2008) The great opportunity Evolutionary
applications to medicine and public health Evolutionary Applications
1 28ndash48 httpsdoiorg101111j1752-4571200700006x
Nevo E (2011) Evolution under environmental stress at macro and
microscales Genome Biology and Evolution 3 1039ndash1052 httpsdoi
org101093gbeevr052
Norberg J Urban M C Vellend M Klausmeier C A amp Loeuille N (2012)
Eco-evolutionary responses of biodiversity to climate change Nature Cli-
mate Change 2 747ndash751 httpsdoiorg101038nclimate1588
Nosil P (2012) Ecological speciation Oxford UK Oxford University
Press httpsdoiorg101093acprofosobl9780199587100001
0001
Nosil P Vines T H amp Funk D J (2005) Perspective Reproductive
isolation caused by natural selection against immigrants from diver-
gent habitats Evolution 59 705ndash719
Oke K B Rolshausen G LeBlond C amp Hendry A P (2017) How par-
allel is parallel evolution A comparative analysis in fishes American
Naturalist 190 1ndash16 httpsdoiorg101086691989
Olson K R (2011) The therapeutic potential of hydrogen sulfide
Separating hype from hope American Journal of Physiology-Regulatory
Integrative and Comparative Physiology 301 R297ndashR312 httpsdoi
org101152ajpregu000452011
Olson K R Donald J A Dombkowski R A amp Perry S F (2012) Evo-
lutionary and comparative aspects of nitric oxide carbon monoxide
and hydrogen sulfide Respiratory Physiology amp Neurobiology 184
117ndash129 httpsdoiorg101016jresp201204004
Olson K R amp Straub K D (2015) The role of hydrogen sulfide in evo-
lution and the evolution of hydrogen sulfide in metabolism and sig-
naling Physiology 31 60ndash72
Palacios M Arias-Rodriguez L Plath M Eifert C Lerp H Lamboj A
Tobler M (2013) The rediscovery of a long described species
reveals additional complexity in speciation patterns of poeciliid fishes
in sulfide springs PLoS ONE 8 e71069 httpsdoiorg101371jour
nalpone0071069
Palacios M Voelker G Arias-Rodriguez L Mateos M amp Tobler M
(2016) Phylogenetic analyses of the subgenus Mollienesia (Poecilia
Poeciliidae Teleostei) reveal taxonomic inconsistencies cryptic biodi-
versity and spatio-temporal aspects of diversification in Middle
America Molecular Phylogenetics and Evolution 103 230ndash244
httpsdoiorg101016jympev201607025
Passow C Arias-Rodriguez L amp Tobler M (2017) Convergent evolu-
tion of reduced energy demands in extremophile fish PLoS ONE 12
e0186935 httpsdoiorg101371journalpone0186935
Passow C N Brown A Arias-Rodriguez L Yee M-C Sockell A
Schartl M Tobler M (2017) Complexities of gene expression
patterns in natural populations of an extremophile fish (Poecilia mexi-
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101111mec14198
Passow C Greenway R Arias-Rodriguez L Jeyasingh P D amp Tobler
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body size and metabolic rates in locally adapted extremophile fishes
Physiological and Biochemical Zoology 88 371ndash383 httpsdoiorg
101086681053
Passow C N Henpita C Shaw J H Quakenbush C Warren W C
Schartl M Tobler M (2017) The roles of plasticity and evolu-
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lations of extremophile fish Molecular Ecology 26 6384ndash6399
Pavey S A Bernatchez L Aubin-Horth N amp Landry C R (2012)
What is needed for next-generation ecological and evolutionary
genomics Trends in Ecology amp Evolution 27 673ndash678 httpsdoi
org101016jtree201207014
Peers C Bauer C Boyle J P Scragg J L amp Dallas M L (2012)
Modulation of ion channels by hydrogen sulfide Antioxidants amp Redox
Signaling 17 95ndash105 httpsdoiorg101089ars20114359
Pfennig D W amp Pfennig K S (2012) Evolutionrsquos wedge competition and
the origins of diversity Berkeley CA University of California Press
httpsdoiorg101525california97805202741810010001
Pfenninger M Lerp H Tobler M Passow C Kelley J L Funke E
Plath M (2014) Parallel evolution of cox genes in H2S-tolerant
fish as key adaptation to a toxic environment Nature Communica-
tions 5 3873
Pfenninger M Patel S Arias-Rodriguez L Feldmeyer B Riesch R amp
Plath M (2015) Unique evolutionary trajectories in repeated adapta-
tion to hydrogen sulphide-toxic habitats of a neotropical fish (Poecilia
TOBLER ET AL | 857
mexicana) Molecular Ecology 24 5446ndash5459 httpsdoiorg10
1111mec13397
Pietri R Roman-Morales E amp Lopez-Garriga J (2011) Hydrogen sul-
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Redox Signaling 15 393ndash404 httpsdoiorg101089ars20103698
Plath M (2008) Male mating behavior and costs of sexual harassment
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1163156853908782687241
Plath M Hermann C Schreurooder R Riesch R Tobler M Garcia de
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maintain small-scale genetic differentiation despite perturbation by a
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oiorg1011861471-2148-10-256
Plath M Pfenninger M Lerp H Riesch R Eschenbrenner C Slattery
P Tobler M (2013) Genetic differentiation and selection against
migrants in evolutionarily replicated extreme environments Evolution
67 2647ndash2661 httpsdoiorg101111evo12133
Plath M Tobler M Riesch R Garcia de Leon F J Giere O amp
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httpsdoiorg101007s00114-007-0279-2
Powell E (1989) Oxygen sulfide and diffusion Why thiobiotic meiofauna
must be sulfide-insensitive first-order respirers Journal of Marine
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Ramirez-Soriano A Ramos-Onsins S E Rozas J Calafell F amp Navarro A
(2008) Statistical power analysis of neutrality tests under demographic
expansions contractions and bottlenecks with recombination Genetics
179 555ndash567 httpsdoiorg101534genetics107083006
Reiffenstein R Hulbert W amp Roth S (1992) Toxicology of hydrogen
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134 httpsdoiorg101146annurevpa32040192000545
Reznick D N Furness A I Meredith R W amp Springer M S (2017)
The origin and biogeographic diversification of fishes in the family
Poeciliidae PLoS ONE 12 e0172546 httpsdoiorg101371journa
lpone0172546
Reznick D amp Yang A P (1993) The influence of fluctuating resources
on life history Patterns of allocation and plasticity in female guppies
Ecology 74 2011ndash2019 httpsdoiorg1023071940844
Riesch R Duwe V Herrmann N Padur L Ramm A Scharnweber K
Plath M (2009) Variation along the shy-bold continuum in extre-
mophile fishes (Poecilia mexicana Poecilia sulphuraria) Behavioral Ecol-
ogy and Sociobiology 63 1515ndash1526 httpsdoiorg101007
s00265-009-0780-z
Riesch R Oranth A Dzienko J Karau N Schiessl A Stadler S
Plath M (2010) Extreme habitats are not refuges Poeciliids suffer
from increased aerial predation risk in sulphidic southern Mexican
habitats Biological Journal of the Linnean Society 101 417ndash426
httpsdoiorg101111j1095-8312201001522x
Riesch R Plath M Garcia de Leon F J amp Schlupp I (2010) Conver-
gent life-history shifts Toxic environments result in big babies in two
clades of poeciliids Naturwissenschaften 97 133ndash141 httpsdoi
org101007s00114-009-0613-y
Riesch R Plath M Schlupp I Tobler M amp Langerhans R B (2014)
Colonization of toxic springs drives predictable life-history shift in
livebearing fishes (Poeciliidae) Ecology Letters 17 65ndash71 httpsdoi
org101111ele12209
Riesch R Tobler M Lerp H Jourdan J Doumas L T Nosil P Plath
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plexity of speciation along replicated ecological gradients BMC Evolu-
tionary Biology 16 136 httpsdoiorg101186s12862-016-0705-1
Riesch R Tobler M amp Plath M (2015) Extremophile fishes Ecology
evolution and physiology of teleosts in extreme environments p 290
Heidelberg Germany Springer httpsdoiorg101007978-3-319-
13362-1
Ritchie M E Holzinger E R Li R Pendergrass S A amp Kim S (2015)
Methods of integrating data to uncover genotype-phenotype interac-
tions Nature Reviews Genetics 16 85ndash97 httpsdoiorg101038
nrg3868
Roach K Tobler M amp Winemiller K O (2011) Hydrogen sulfide bac-
teria and fish A unique subterranean food chain Ecology 92 2056ndash
2062 httpsdoiorg10189011-02761
Rohner N Jarosz D F Kowalko J E Yoshizawa M Jeffery W R
Borowsky R L Tabin C J (2013) Cryptic variation in morpho-
logical evolution HSP90 as a capacitor for loss of eyes in cavefish
Science 342 1372ndash1375 httpsdoiorg101126science1240276
Rosales Lagarde L (2012) Investigation of karst brackish-sulfidic springs
and their role in the hydrogeology subsurface water-rock interactions
and speleogenesis at northern Sierra de Chiapas New Mexico Institute
of Mining and Technology Mexico
Rosenblum E B amp Harmon L J (2011) ldquoSame but differentrdquo Repli-
cated ecological speciation at White Sands Evolution 65 946ndash960
httpsdoiorg101111j1558-5646201001190x
Rosenblum E B Parent C E amp Brandt E E (2014) The molecular
basis of convergent evolution Annual Review of Ecology Evolution and
Systematics 45 203ndash226 httpsdoiorg101146annurev-ecolsys-
120213-091851
Saltz J B Hessel F C amp Kelly M W (2017) Trait correlations in the
genomics era Trends in Ecology amp Evolution 32 279ndash290 httpsdoi
org101016jtree201612008
Saraste M (1999) Oxidative phosphorylation at the fin de siecle Science
283 1488ndash1493 httpsdoiorg101126science28354071488
Schartl M (2014) Beyond the zebrafish Diverse fish species for model-
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httpsdoiorg101242dmm012245
Schartl M Walter R B Shen Y Garcia T Catchen J Amores A
Warren W C (2013) The genome of the platyfish Xiphophorus mac-
ulatus provides insights into evolutionary adaptation and several
complex traits Nature Genetics 45 567ndash572 httpsdoiorg10
1038ng2604
Schluter D (2009) Evidence for ecological speciation and its alternative
Science 323 737ndash741 httpsdoiorg101126science1160006
Schulz-Mirbach T Eifert C Riesch R Farnworth M S Zimmer C
Bierbach D Plath M (2016) Toxic hydrogen sulphide shapes
brain anatomy A comparative study of sulphide-adapted ecotypes in
the Poecilia mexicana complex Journal of Zoology 300 163ndash176
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determina las especies y subspecies de flora y fauna silvestres ter-
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sujetas a proteccion especial y que establece especificaciones para
su proteccion Diario Oficial 16 May 1994 488 2ndash60
Servedio M R van Doorn G S Kopp M Frame A M amp Nosil P
(2011) Magic traits in speciation lsquomagicrsquo but not rare Trends in Ecology
amp Evolution 26 389ndash397 httpsdoiorg101016jtree201104005
Shahak Y amp Hauska G (2008) Sulfide oxidation from cyanobacteria to
humans Sulfide-quinone oxidoreductase (SQR) In R Hell C Dahl D
B Knaff amp Th Leustek (Eds) Advances in photosynthesis and respira-
tion (pp 319ndash335) Heidelberg Springer
Shalem O Sanjana N E amp Zhang F (2015) High-throughput func-
tional genomics using CRISPR-Cas9 Nature Reviews Genetics 16
299ndash311 httpsdoiorg101038nrg3899
Silva G J J Bye A el Azzouzi H amp Wisloslashff U (2017) MicroRNAs as
important regulators of exercise adaptation Progress in Cardiovascular
Diseases 60 130ndash151 httpsdoiorg101016jpcad201706003
Simola D F Wissler L Donahue G Waterhouse R M Helmkampf
M Roux J Viljakainen L (2013) Social insect genomes exhibit
dramatic evolution in gene composition and regulation while preserv-
ing regulatory features linked to sociality Genome Research 23
1235ndash1247 httpsdoiorg101101gr155408113
858 | TOBLER ET AL
Sommer-Trembo C Bierbach D Arias Rodriguez L Verel Y Jourdan
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Steindachner F (1863) Beitreuroage zur Kentniss der Sciaenoiden Brasiliens
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Naturwissenschaftlichen Classe der Kaiserlichen Akademie der Wis-
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Stinchcombe J R amp Hoekstra H E (2008) Combining population geno-
mics and quantitative genetics Finding genes underlying ecologically
important traits Heredity 100 158ndash170 httpsdoiorg101038sj
hdy6800937
Stipanuk M H (2004) Sulfur amino acid metabolism Pathways for pro-
duction and removal of homocysteine and cysteine Annual Review of
Nutrition 24 539ndash577 httpsdoiorg101146annurevnutr24
012003132418
Stipanuk M H amp Ueki I (2011) Dealing with methioninehomocysteine
sulfur Cysteine metabolism to taurine and inorganic sulfur Journal of
Inherited Metabolic Disease 34 17ndash32 httpsdoiorg101007
s10545-009-9006-9
Stockinger B Di Meglio P Gialitakis M amp Duarte J H (2014) The
aryl hydrocarbon receptor Multitasking in the immune system
Annual Review of Immunology 32 403ndash432 httpsdoiorg101146
annurev-immunol-032713-120245
Storz J F amp Wheat C W (2010) Integrating evolutionary and
functional approaches to infer adaptation at specific loci Evolution
64 2489ndash2509 httpsdoiorg101111j1558-5646201001044x
Stuart Y E Veen T Weber J N Hanson D Ravinet M Lohman B
K Bolnick D I (2017) Contrasting effects of environment and
genetics generate a continuum of parallel evolution Nature Ecology amp
Evolution 1 0158 httpsdoiorg101038s41559-017-0158
Sunnucks P Morales H E Lamb A M Pavlova A amp Greening C
(2017) Integrative approaches for studying mitochondrial and nuclear
genome co-evolution in oxidative phosphorylation Frontiers in Genet-
ics 8 25
Tobler M amp Hastings L (2011) Convergent patterns of body shape dif-
ferentiation in four different clades of poeciliid fishes inhabiting sul-
fide springs Evolutionary Biology 38 412ndash421 httpsdoiorg10
1007s11692-011-9129-4
Tobler M Palacios M Chapman L J Mitrofanov I Bierbach D
Plath M Mateos M (2011) Evolution in extreme environments
Replicated phenotypic differentiation in livebearing fish inhabiting
sulfidic springs Evolution 65 2213ndash2228 httpsdoiorg101111j
1558-5646201101298x
Tobler M Passow C N Greenway R Kelley J L amp Shaw J H
(2016) The evolutionary ecology of animals inhabiting hydrogen sul-
fide-rich environments Annual Review of Ecology Evolution and Sys-
tematics 47 239ndash262 httpsdoiorg101146annurev-ecolsys-
121415-032418
Tobler M amp Plath M (2009a) Threatened fishes of the world Gambu-
sia eurystoma Miller 1975 (Poeciliidae) Environmental Biology of
Fishes 85 251 httpsdoiorg101007s10641-009-9481-8
Tobler M amp Plath M (2009b) Threatened fishes of the world Poecilia
sulphuraria (Alvarez 1948) (Poeciliidae) Environmental Biology of
Fishes 85 333ndash334 httpsdoiorg101007s10641-009-9506-3
Tobler M Plath M Riesch R Schlupp I Grasse A Munimanda G
Moodley Y (2014) Selection from parasites favors immunogenic diver-
sity but not divergence among locally adapted host populations Journal
of Evolutionary Biology 27 960ndash974 httpsdoiorg101111jeb12370
Tobler M Riesch R amp Plath M (2015) Extremophile fishes An inte-
grative synthesis In R Riesch M Tobler amp M Plath (Eds) Extremo-
phile fishes Ecology evolution and physiology of teleosts in extreme
environments (pp 279ndash296) Heidelberg Germany Springer
Tobler M Riesch R Tobler C M amp Plath M (2009) Compensatory
behaviour in response to sulfide-induced hypoxia affects time
budgets feeding efficiency and predation risk Evolutionary Ecology
Research 11 935ndash948
Tobler M Scharnweber K Greenway R Passow C Arias-Rodriguez
L amp Garcia-De-Leon F J (2015) Convergent changes in the
trophic ecology if extremophile fish along replicated environmental
gradients Freshwater Biology 60 768ndash780 httpsdoiorg10
1111fwb12530
Todgham A E amp Stillman J H (2013) Physiological responses to shifts
in multiple environmental stressors Relevance in a changing worlds
Integrative and Comparative Biology 53 539ndash544 httpsdoiorg10
1093icbict086
van Doorn G S Edelaar P amp Weissing F J (2009) On the origin of
species by natural selection and sexual selection Science 326 1704ndash
1707 httpsdoiorg101126science1181661
Wall D H amp Virginia R A (1999) Controls on soil biodiversity Insights
from extreme environments Applied Soil Ecology 13 137ndash150
httpsdoiorg101016S0929-1393(99)00029-3
Wallace J L amp Wang R (2015) Hydrogen-sulfide based therapeutics
Exploiting a unique but ubiquitous gasotransmitter Nature Reviews
Drug Discovery 14 329ndash345 httpsdoiorg101038nrd4433
Wang R (2012) Physiological implications of hydrogen sulfide A whiff
exploration that blossomed Physiological Reviews 92 791ndash896
httpsdoiorg101152physrev000172011
Wang Z Gerstein M amp Snyder M (2009) RNA-seq A revolutionary
tool for transcriptomics Nature Reviews Genetics 10 57ndash63
httpsdoiorg101038nrg2484
Waterman T H (1999) The evolutionary challenges of extreme environ-
ments (part 1) Journal of Experimental Zoology 285 326ndash359
httpsdoiorg101002(SICI)1097-010X(19991215)2854amplt326
AID-JEZ3ampgt30CO2-T
Werren J H Richards S Desjardins C A Niehuis O Gadau J Col-
bourne J K amp Group N G W (2010) Functional and evolutionary
insights from the genomes of three parasitoid Nasonia species
Science 327 343ndash348 httpsdoiorg101126science1178028
Whitehead A (2012) Comparative genomics in ecological physiology
Toward a more nuanced understanding of acclimation and adapta-
tion Journal of Experimental Biology 215 884ndash891 httpsdoiorg
101242jeb058735
Whitehead A amp Crawford D L (2006) Neutral and adaptive variation
in gene expression Proceedings of the National Academy of Sciences
USA 103 5425ndash5430 httpsdoiorg101073pnas0507648103
Willett C S amp Burton R S (2004) Evolution of interacting proteins in
the mitochondrial electron transport system in a marine copepod
Molecular Biology and Evolution 21 443ndash453 httpsdoiorg10
1093molbevmsh031
Willi Y Van Buskirk J amp Hoffmann A (2006) Limits to the adaptive
potential of small populations Annual Review of Ecology Evolution
and Systematics 37 433ndash458 httpsdoiorg101146annurevecol
sys37091305110145
Wolff J N Ladoukakis E D Enrıquez J A amp Dowling D K (2014)
Mitonuclear interactions Evolutionary consequences over multiple
biological scales Philosophical Transactions of the Royal Society B 369
20130443 httpsdoiorg101098rstb20130443
How to cite this article Tobler M Kelley JL Plath M Riesch
R Extreme environments and the origins of biodiversity
Adaptation and speciation in sulphide spring fishes Mol Ecol
201827843ndash859 httpsdoiorg101111mec14497
TOBLER ET AL | 859
Pichucalco Ixtapangajoya Puyacatengo Tacotalpa
Pichucalco
Teapa
Tacotalpa
Tapijulapa
4 km
(a)
(b) (c)
(d) (e)
F IGURE 1 Overview of the distribution of sulphide springs and their inhabitants in southern Mexico (a) Map depicting the four river drainages(Pichucalco Ixtapangajoya Puyacatengo and Tacotalpa) that exhibit sulphide springs inhabited by members of the P mexicana species complexThe locations of sulphide springs are indicated with yellow triangles black triangles represent commonly used field sites for the collection ofecotypes from nonsulphidic waters Note that roads (black lines) and major towns (grey shaded areas) were added for orientation The insethighlights the location of the study region within Mexico (bndashd) Examples of H2S-rich habitats in the Pichucalco (b) Puyacatengo (c) and Tacotalpa(d) river drainages (e) Representative individuals from the nonsulphidic (top) and the sulphidic (bottom) ecotypes from the Pichucalco river drainage
TOBLER ET AL | 845
inference of putative mechanisms underlying adaptation even in sys-
tems without a priori predictions (Kell amp Oliver 2004) However
genetic and gene expression data alone are insufficient to infer
adaptation at specific loci because neutral evolutionary processes
demographic history and genetic hitchhiking can generate false posi-
tives in genomewide analyses of selection and gene expression (Bar-
ton 1998 Ramirez-Soriano Ramos-Onsins Rozas Calafell amp
Navarro 2008 Whitehead amp Crawford 2006) This underscores the
importance of generating a priori hypotheses about the molecular
underpinnings of adaptation and testing whether genetic and gene
expression variation at specific loci actually affects organismal per-
formance under different environmental conditions (Dalziel Rogers
amp Schulte 2009 Storz amp Wheat 2010)
21 | Developing and testing a priori hypotheses
There is a rich history of research in toxicology and biomedicine that
has investigated the molecular targets of H2S illuminating the mech-
anisms of H2S toxicity and detoxification in model organisms and
human cell cultures (see Li et al 2011 Olson Donald Dombkowski
amp Perry 2012 Wang 2012 Wallace amp Wang 2015 for recent
reviews) In a recent attempt of ldquoseparating hype and hoperdquo in
potential biomedical applications of H2S Olson (2011) lamented that
H2S ldquoseemingly affects all organ systems and biological processes in
which it has been investigatedrdquo While it often remains unclear
whether the documented effects are a direct consequence of H2S
exposure or a result of the disruption of cellular homeostasis this
body of work provides fruitful grounds for the development of
hypotheses about the molecular mechanisms underlying adaptation
to H2S-rich environments that can then be tested using genome
scans for selection and analyses of gene expression variation
between sulphide-adapted and nonadapted populations (see Table 1
for an overview) Most importantly candidates include direct toxicity
targets involved in OXPHOS (Cooper amp Brown 2008) and oxygen
transport (Pietri Roman-Morales amp Lopez-Garriga 2011) anaerobic
metabolism (Dubilier Giere amp Grieshaber 2005) and oxidative stress
response systems (Eghbal Pennefather amp OrsquoBrien 2004) that may
help mitigate adverse consequences of H2S exposure as well as
pathways involved in enzymatic H2S detoxification sulphur process-
ing and sulphur excretion (Hildebrandt amp Grieshaber 2008) It is
important to note that H2S is also produced endogenously by meta-
zoans in the cytosol and in mitochondria through enzymatic reac-
tions associated with the reverse trans-sulphuration pathway
(Stipanuk 2004 Stipanuk amp Ueki 2011) At concentrations well
below the toxicity threshold endogenously produced H2S serves as
a cellular signalling molecule that modulates physiological processes
including the function of ion channels that underlie smooth muscle
control and the function of neural cells (Peers Bauer Boyle Scragg
amp Dallas 2012) and it interacts with transcription factors that con-
trol inflammation cell survival and proliferation the expression of
cellular protective enzymes and hypoxia responses (Li et al 2011
Wallace amp Wang 2015 Wang 2012) Organisms may thus not strive
to merely eliminate H2S from their system and avoid effects of
toxicity Instead they are likely selected for maintaining optimal
endogenous concentrations that facilitate H2S-mediated signalling
and assure proper physiological functioning As a consequence
endogenous H2S production pathways and targets of H2S signalling
may also be modified during adaptation to environmental H2S
A series of studies in members of the P mexicana species com-
plex have tested hypotheses about the molecular underpinnings of
H2S adaptation by analysing patterns of molecular evolution in can-
didate genes (Barts et al 2018 Pfenninger et al 2014) deploying
genomewide scans for selection based on FST outliers (Pfenninger
et al 2015) and quantifying genomewide patterns of gene expres-
sion (Kelley et al 2016 Passow Henpita et al 2017) Collectively
the results of these studies have indicated a striking congruence
between candidate genes inferred from toxicological and biomedical
research and genes with empirical evidence for selection andor dif-
ferential expression between at least some of the adjacent H2S-
adapted and nonadapted populations (Table 1) (i) There was evi-
dence for selection on genes encoding the primary toxicity target of
H2S COX (complex IV in OXPHOS Pfenninger et al 2014) In addi-
tion genes encoding other components and assembly factors of
OXPHOS are either under selection or differentially expressed in sul-
phide spring populations (Kelley et al 2016 Pfenninger et al
2015) Evidence for selection and differential expression was also
uncovered for subunits of haemoglobin whose function is directly
impaired by H2S (Barts et al 2018) (ii) Genes involved in anaerobic
ATP production and in coping with oxidative stress exhibited evi-
dence for selection and differential expression suggesting a shift
towards alternative sources of ATP production and an increased abil-
ity to withstand reactive oxygen species that are produced as a by-
product of OXPHOS inhibition by H2S (Kelley et al 2016 Pfen-
ninger et al 2015) (iii) There is overwhelming evidence for selection
on and differential regulation of genes encoding components of the
sulphidequinone oxidoreductase pathway which mediates enzymatic
H2S detoxification in metazoans (Kelley et al 2016 Pfenninger
et al 2015) In addition there is evidence for upregulation of genes
encoding transporters of oxidized sulphur species which are puta-
tively involved in sulphur excretion (Kelley et al 2016)
These results suggest that adaptation to H2S-rich environments
in members of the P mexicana species complex involves both an
increased ability to cope with elevated endogenous H2S and an
increased ability to keep endogenous H2S concentrations low
through detoxification and excretion However there is a noticeable
absence of evidence for genes and physiological pathways associ-
ated with endogenous H2S production and H2S-mediated cell sig-
nalling being involved in adaptation (see Kelley et al 2016
Pfenninger et al 2015) There are three nonmutually exclusive
hypotheses that could explain this finding First the ability of popu-
lations from sulphidic habitats to regulate endogenous H2S through
detoxification may be sufficient to maintain normal H2S-mediated
signalling Second compared to the ability for detoxification and for
mitigation of direct toxic effects in terms of ATP production and
oxidative stress H2Srsquos influence on cell signalling may have relatively
low fitness consequences with concomitantly smaller evolutionary
846 | TOBLER ET AL
responses Last inferences from biomedical studies about the causal
relationship between endogenously produced H2S and various physi-
ological functions may be weak and some documented physiological
effects may instead be an indirect consequence of the disruption of
homeostasis that cascades through interconnected pathways operat-
ing in a cell In this case evolutionary studies on natural systems
exposed to H2S may provide important insights in terms of prioritiz-
ing research on biomedically relevant aspects of H2S biology Study-
ing ancestral populations susceptible to H2S will allow for
quantifying consequences of disruptions in H2S homeostasis at all
levels of biological organization (like in presently available animal
models) Adding derived H2S tolerant populations will further allow
for the detection of critical modifications in physiological processes
affected by H2S Consequently such a comparative approach may
provide a powerful tool to disentangle direct and indirect physiologi-
cal consequences of deviations in H2S homeostasis which is cur-
rently difficult in traditional biomedical models
Despite some of these discrepancies the studies exploring mech-
anisms underlying H2S tolerance in sulphide spring fishes highlight
the utility of research in toxicology and biomedicine in generating
testable hypotheses about evolutionary processes Biomedical stud-
ies on metabolic disorders (Aspiras Rohner Martineau Borowsky amp
Clifford 2015) stress responses (Rohner et al 2013) responses to
low oxygen (Eales Hollinshead amp Tennant 2016) and effects of
xenobiotics (Stockinger Di Meglio Gialitakis amp Duarte 2014) have
also facilitated our understanding of adaptive evolution in other nat-
ural systems Considering the increasingly specialized and frag-
mented research areas in the life sciences interdisciplinary
approaches and reciprocal exchange of ideas and methodologies
continue to be critical for facilitating breakthroughs in basic and
applied research on biomedical models and natural systems (Carroll
et al 2014 Nesse amp Stearns 2008 Schartl 2014)
22 | Convergent and nonconvergent evolution inindependent lineages
Our discussion of putative candidate genes that are under selection
andor differentially expressed in sulphide spring populations so far
focused on general patterns documented across evolutionarily inde-
pendent lineages in different river drainages Selection scans (Pfen-
ninger et al 2015) and analyses of differential gene expression
(Kelley et al 2016) have indicated high congruence of biological
processes associated with candidate genes across lineages in differ-
ent river drainages indicating that evolution has repeatedly and pre-
dictably modified the same physiological pathways during adaptation
to environmental H2S However the signal of evolutionary conver-
gence among sulphide spring lineages decreases with the level of
organismal organization (from physiological pathways down to geno-
mic regions genes and individual SNPs in selection scans Pfenninger
et al 2015 from physiological pathways down to genes and tran-
scripts in expression studies Passow Brown et al 2017) indicating
that different lineages have often modified the same physiological
pathways in different ways While knowledge about the biochemical
TABLE 1 Adaptation to H2S can occur through three principlemechanisms (1) Resistance involves direct toxicity targets whosefunctions are impaired by H2S Adaptive modifications of thesetoxicity targets are predicted to assure proper function despitethe presence of H2S (2) Mitigation involves alternativephysiological pathways that compensate for the inhibition ofdirect toxicity targets Adaptive modifications of these alternativephysiological pathways are predicted to allow for the maintenanceof organismal function by other means (3) Regulation involvesphysiological pathways associated with the maintenance ofendogenous H2S homeostasis Adaptive modifications ofregulatory pathways are predicted to maintain low endogenousconcentrations despite continuous flux of H2S from theenvironment into the body
Candidate pathways Evidence
(1) Resistance
OXPHOS bull Positive selection on COX in some lineages of
P mexicana (Puyacatengo and Pichucalco)a as
well as some other genera of sulphide spring
poeciliidsb COX amino acid substitutions in P
mexicana allow for the maintenance of function
in the presence of H2Sa
bull Upregulation of cytochrome c and some COX
subunits as well as downregulation of compo-
nents of complex I in all sulphide spring lineages
of P mexicana indicating complex changes in
aerobic ATP productionc
Oxygen transport
genesbull Positive selection on and upregulation of
several oxygen transport genes (including myo-
globin and multiple haemoglobin subunits) in
sulphide spring lineages of the genera
Gambusia Limia Poecilia and Pseudoxiphophorus
indicating modifications to putatively maintain
oxygen transport in the presence of H2Sd
(2) Mitigation
Anaerobic
metabolismbull Upregulation of genes associated with glycolysis
and gluconeogenesis in all sulphide spring lin-
eages of P mexicana indicating increased
capacity of anaerobic ATP productionc
Oxidative stress
responsesbull Upregulation of genes associated with
oxidative stress responses in all sulphide
spring lineages of P mexicana indicating
increased capacity to cope with H2S-induced
oxidative stressc
(3) Regulation
H2S detoxification bull Positive selection on and upregulation of sev-
eral genes associated with enzymatic H2S
detoxification in all sulphide spring lineages of
P mexicanace Upregulation of H2S detoxifica-
tion enzymes in other sulphide spring lineages
of the genera Gambusia Limia Poecilia Pseudox-
iphophorus and Xiphophorusb This indicates an
increased ability to maintain low endogenous
H2S concentrations during exposure
H2S production bull No evidence for modifications of genes associ-
ated with enzymatic production of H2Sc
aPfenninger et al (2014) bR Greenway J L Kelley amp M Tobler unpub-
lished data cKelley et al (2016) dBarts et al (2018) ePfenninger et al
(2015)
TOBLER ET AL | 847
and physiological consequences of H2S may thus enable us to pre-
dict the physiological pathways in which adaptive modifications
occur it may be impossible to predict specific modifications within
those pathways This contrasts with a common expectation in the
field that convergent evolution of molecular mechanisms underlying
phenotypic traits (ie parallel evolution) necessarily hinges on modi-
fication of the same loci across lineages as found in some of the
best-known examples of parallelism (Colosimo et al 2005 Feldman
Brodie Brodie amp Pfrender 2012 Grant Grant Markert Keller amp
Petren 2004) However modification of the same loci may only be
common under particular circumstances (Rosenblum Parent amp
Brandt 2014) for example when selection acts on standing genetic
variation (Colosimo et al 2005) when adaptive alleles are intro-
gressed across population boundaries through gene flow (Grant
et al 2004) or when there are strong genetic constraints (Feldman
et al 2012) Exploration of molecular mechanisms underlying con-
vergent evolution in many systems may therefore be better
approached from the perspective of interacting genes that are orga-
nized in pathways in which alternative genetic changes can have
equivalent functional consequences (ie functional redundancy)
Developing and testing hypotheses about parallel evolution at the
pathway level will hinge on improving genome annotations (Pavey
Bernatchez Aubin-Horth amp Landry 2012) and provide a more
nuanced understanding of the factors determining the predictability
of evolution at different levels of organismal organization (Rosen-
blum et al 2014)
Despite evidence for convergent evolution in some candidate
physiological pathways it also needs to be emphasized that this
does not apply to all P mexicana lineages or to all candidate physio-
logical pathways associated with H2S adaptation investigated to
date Nonconvergent evolutionmdashevidenced by unique lineage-speci-
fic signals of selection and differential expressionmdashmay be indicative
of unique molecular mechanisms to cope with H2S toxicity For
example signatures of positive selection on COXmdashH2Srsquos primary
toxicity targetmdashcan be found in sulphide spring lineages of the Puy-
acatengo and Pichucalco river drainages (a lineage with recent diver-
gence time and a lineage with older divergence time respectively)
but not in those of the Tacotalpa drainage (Pfenninger et al 2014)
The presence of nonconvergent genetic and gene expression differ-
ences between populations from sulphidic and non-sulphidic habitats
in different river drainages highlights the limitations of inferences
that can be drawn based on data sets generated with high-through-
put DNA and RNA sequencing The potential importance of modifi-
cations at candidate locimdashor the lack thereofmdashcan only be assessed
through functional assays that document how alternative alleles and
changes in gene expression actually affect organismal performance
The functional consequences of genetic and gene expression varia-
tion at most candidate loci remain to be investigated However
enzyme activity assays have indicated that COX maintains its activity
with increasing H2S concentrations in the two sulphide spring popu-
lations with evidence for positive selection on mitochondrially
encoded subunits (Puyacatengo and Pichucalco Pfenninger et al
2014) Key amino acid substitutions occur in the D-pathway channel
of COX1 which putatively narrow the channel width and reduce
H2Srsquos access to the binuclear centre of the protein Consequently
some lineages of P mexicana exhibit an H2S-resistant COX that can
maintain its activity and aerobic ATP production even if endogenous
H2S concentrations are elevated In contrast COX activity in sul-
phide spring fish of the Tacotalpa river drainage and in fish from
nonsulphidic habitats decreases when H2S is present which is con-
sistent with mechanisms of H2S toxicity (Pfenninger et al 2014) In
vitro assays on isolated mitochondria have nonetheless indicated
that an H2S-resistant COX is not necessary to maintain aerobic ATP
production during H2S exposure Unlike in populations from nonsul-
phidic habitats mitochondrial spare respiratory capacity is main-
tained in the presence of H2S even in the Tacotalpa sulphide spring
populations that lack an H2S-resistant COX (C Henpita M Tobler amp
JH Shaw unpublished data) This may indicate that modification of
detoxification pathways in sulphide spring fishes is sufficient to
maintain mitochondrial function and aerobic ATP production
23 | Ensuing challenges linking genotypes tofitness
Understanding the mechanistic basis of adaptation requires linking
genotypic variation to phenotypic traits that ultimately shape fitness
along selective gradients The evolution of H2S tolerance in sulphide
spring fishes appears to be a consequence of modifications in many
genes and many physiological pathways whose functions interact to
eliminate H2S from the system minimize effects of elevated endoge-
nous concentrations on direct toxicity targets and mitigate adverse
effects that cascade through cellular metabolism during disruption of
homeostasis (Table 1) Such complex genetic changes are likely com-
mon during adaptation and have been documented in other systems
that involve major evolutionary shifts including the emergence of
parasitic lifestyles (Werren et al 2010) eusociality (Simola et al
2013) asexual reproduction (Fradin et al 2017) and internal gesta-
tion with livebearing (Schartl et al 2013) Consequently comple-
mentary approaches will be required to uncover the molecular
mechanisms of adaptation to H2S in the future
1 The involvement of many genes and physiological pathways in
shaping tolerance emphasizes the importance of quantitative
genetic approaches that leverage experimental crosses between
H2S tolerant and nontolerant populations and test whether toler-
ancemdashand adaptive trait modifications that contribute to itmdashis
explained by a few major effects loci or whether it is a conse-
quence of a number of genes with small effects (Klerks Xie amp
Levinton 2011 Saltz Hessel amp Kelly 2017) Combining analyses
of phenotype-fitness associations and their underlying genetic
mechanisms via QTL mapping with the results of selection scans
from population genomic analyses (Pfenninger et al 2015) will
allow for an integrative understanding of how selection acting on
the genome results in phenotypic variation that mediates adapta-
tion to different environments (Stinchcombe amp Hoekstra 2008)
From a functional perspective breaking up correlations between
848 | TOBLER ET AL
different physiological traits (associated with resistance mitiga-
tion and regulation Table 1) in F2 crosses will further allow test-
ing what molecular mechanisms are necessary for tolerance
whether there are alternative strategies with equivalent fitness
benefits and whether there are complementary modifications
with additive effects on tolerance
2 Considering the number of genes and physiological pathways
likely involved in H2S tolerance assessing the functional conse-
quences of genetic and gene expression variation is a daunting
task At the same time such functional tests can significantly
contribute to closing the genotype-to-phenotype gap because
physiochemical stress responses can be assessed along the con-
tinuum of organismal organization (Gjuvsland Vik Beard Hunter
amp Omholt 2013) Standardized assays can be used to quantify
the biochemical makeup of organelles cells and tissues (eg pro-
tein abundance and enzyme activity) physiological performance
in vitro and in vivo (eg rates of H2S detoxification ATP produc-
tion and reactive oxygen production) and fitness-relevant proxies
of whole organism performance (eg H2S tolerance Figure 2)
This highlights the importance of methods development for the
quantification of complex phenotypic traits or even whole phe-
nomes that can keep pace with rapid data generation through
high-throughput sequencing (Houle Govindaraju amp Omholt
2010 Ritchie Holzinger Li Pendergrass amp Kim 2015) In addi-
tion genotypendashphenotype links established through such correla-
tive approaches will ideally be verified through targeted gene
editing While gene editing with CRISPRndashCas9 is now routinely
employed in model organisms (Doudna amp Charpentier 2014 Sha-
lem Sanjana amp Zhang 2015) applying this technology to non-
models still remains challenging especially in livebearing species
like poeciliid fishes where in vitro culture of embryos is not rou-
tinely employed (Martyn Weigel amp Dreyer 2006) Nonetheless
the effects of specific mutations can potentially be verified
through the establishment and manipulation of cells in culture
(Liu et al 2014) or by generating transgenics (Lin et al 2016)
3 Organismal responses to physiochemical stressors occur at multi-
ple hierarchical levels that can be plastically modulated in
response to endogenous and exogenous cues (Whitehead 2012)
Understanding adaptation to physiochemical stressors conse-
quently requires disentangling the effects of genetic variation
developmental plasticity and short-term acclimation in gene
expression patterns on the emerging physiological processes
Common garden experiments on sulphide spring fishes have
shown that many genes differentially expressed between popula-
tions from sulphidic and nonsulphidic habitats in nature remain
differentially expressed in laboratory-reared fish that have never
been exposed to H2S indicating evolved differences in constitu-
tive gene expression (Passow Henpita et al 2017) At the same
time expression patterns in the laboratory also exhibit evidence
for plasticity in response to short-term H2S exposure (Passow
Henpita et al 2017) Some of these plastic responses are shared
between populations from sulphidic and nonsulphidic habitats
(likely representing shared ancestral plasticity) while others have
been lost or gained during sulphide spring colonization A core
challenge for future research is to overcome logistical constraints
that have so far prevented long-term H2S exposures of fish in
the laboratory Exposing individuals throughout development will
be required to test how developmental plasticity and short-term
acclimation shape responses to H2S in individuals from adapted
and nonadapted populations Finally the next big scientific
advance in this context will also require the identification of reg-
ulatory mechanisms that shape expression differences during
adaptation including sequence changes in transcription factors
and their binding sites patterns of DNA methylation and chro-
matin accessibility as well as the production of microRNAs and
competing endogenous RNAs that can modulate transcript abun-
dance (Flores Wolschin amp Amdam 2013 Lopez-Maury Mar-
guerat amp Beuroahler 2008 Silva Bye el Azzouzi amp Wisloslashff 2017)
In addition the role of alternative splicing and other post-tran-
scriptional and post-translational modifications in mediating adap-
tation remains understudied by evolutionary biologists (Ast
2004 Bush Chen Tovar-Corona amp Urritia 2017) This is particu-
larly relevant because H2S has been hypothesized to play a role
in the post-translational modification of proteins with sulphur-
containing amino acids through sulfhydration (Mustafa et al
2009)
Bridging the genotypendashphenotype gap remains one of the major
challenges in biology (Gjuvsland et al 2013 Ritchie et al 2015) and
will provide opportunities to understand how organisms function in
the face of environmental stress and how evolution shapes these
functions during adaptation to different environmental conditions
Establishing the molecular mechanisms of adaptation across repli-
cated lineages exposed to the same sources of selection will also
provide insights into how evolutionary history demography and
genetics shape convergent evolutionary outcomes providing a better
understanding at what hierarchical levels of organismal organization
evolutionary change may actually be predictable (Rosenblum et al
2014)
3 | COMPLEX ORGANISMS IN A COMPLEXWORLD DISENTANGLING THE TANGLEDBANK
Mechanisms of adaptation can be studied through bottom-up
(screening for genetic modifications that allow for inferences about
relevant phenotypic traits) and top-down approaches (assessing fit-
ness-relevant phenotypic differences among populations without
knowledge about the genetic basis see Schluter 2009) Over the
years genomic and transcriptomic studies on sulphide spring popula-
tions of the P mexicana species complex have been complemented
with analyses of phenotypic variation Just as genetic analyses have
indicated that adaptation to H2S is mediated by the modification
and modulation of many genes associated with multiple physiological
pathways phenotypic studies uncovered a variety of differences
TOBLER ET AL | 849
between populations from sulphidic and nonsulphidic environments
Many of these phenotypic differences have evolved in convergence
across replicated sulphide spring lineages in different river drainages
Examples include modifications of morphological (body shape and
organ size Tobler et al 2011 Tobler Scharnweber et al 2015
Schulz-Mirbach et al 2016) physiological (tolerance and metabolic
rates Tobler et al 2011 Passow Arias-Rodriguez amp Tobler 2017)
behavioural (aggression and personality Riesch et al 2009 Bierbach
et al 2012 Sommer-Trembo et al 2016 Bierbach Arias Rodriguez
amp Plath 2018) and life history traits (fecundity and offspring size
Riesch Plath Garcia de Leon amp Schlupp 2010 Riesch et al 2014)
Hence proximate populations in different habitat types have
diverged in complex phenotypes that span many different suites of
traits and a key question is how divergence in multivariate pheno-
types arises In theory H2S could be a key source of selection driv-
ing multivariate phenotypic divergence either because it directly
exerts selection on a multitude of traits that are independently modi-
fied to reach new fitness peaks or because selection on a few fit-
ness-relevant traits causes simultaneous changes in correlated
characteristics Neither explanation seems sufficient because many
Abiotic environmentOxygen concentrations
Ionic compositionTemperature
Structure and flow
Biotic environmentTrophic resources
DemographyPredationParasitism
H2S
Genetic variation
Gene expression
Biochemistry
Physiologicalfunction
Organismal performance
Primary fitness components
Survivalgrowth
reproduction
Organismal function Environment
F IGURE 2 Understanding how organisms function in the context of their environment is a core question in evolutionary biology yet thecomplexities associated with organismal function and environmental variation provide significant challenges High-throughput sequencing hasallowed for the identification of candidate genes subject to selection and differential expression during adaptation to H2S The next step is tobridge the genotype-to-phenotype gap by quantifying the integrated responses to H2S across the continuum of organismal organization includingbiochemical phenotypes physiological functions whole organism performance and ultimately fitness In addition understanding adaptation toH2S-rich environments will also require acknowledging H2Srsquos relation to other abiotic and biotic environmental variables which can exert selectionand modulate aspects of organismal function This will allow for the establishment of cause-and-effect relationships between specific sources ofselection and evolutionary change in specific traits and contribute to our understanding of organismal function in natural systems
850 | TOBLER ET AL
of the divergent traits between populations from sulphidic and non-
sulphidic environments have no clearmdashor at best tenuousmdashlinks to
H2S toxicity and detoxification and there is only limited evidence
for within-population correlations between different traits groups
(Riesch et al 2016)
Evolutionary analyses frequently focus on seemingly simple
sources of selection juxtaposing populations that are assigned to dis-
crete habitat types to understand the evolution of specific traits
(Kaeuffer Peichel Bolnick amp Hendry 2012) But as Darwin (1859)
already observed in his ldquotangled bankrdquo allegory selection in natural
systems is rarely univariate but multifarious selective regimes con-
tribute to trait divergence even among populations that occur along
apparently simple environmental gradients Unquantified environmen-
tal variation that is subsumed in the categorization of habitat types
can profoundly affect trait variation in natural systems (Stuart et al
2017) highlighting the importance of considering and testing alterna-
tive hypotheses about the cause-and-effect relationship between
specific sources of selection and evolution of specific traits Thus
accurate natural history observations that consider organisms as holis-
tic entities consisting of interacting traits in the context of the abiotic
and biotic factors comprising their environment are critical to inform
the design and interpretation of empirical research in natural systems
(Greene 2005) Indeed adjacent sulphidic and nonsulphidic habitat
types do not merely differ in the presence or absence of H2S but also
in a variety of other abiotic and biotic factors (Figure 2) Unlike nonsul-
phidic habitats sulphide springs in southern Mexico are characterized
by extreme hypoxia elevated salinity and lower pH (Rosales Lagarde
2012 Tobler et al 2011) In addition the presence of physiochemical
stressors affects the biotic communities in these sulphide springs Spe-
cies richness of aquatic invertebrates and vertebrates is drastically
reduced as a consequence of H2S toxicity (Greenway Arias-Rodriguez
Diaz amp Tobler 2014) and primary production appears to be driven in
part by chemoautotrophic bacteria that use H2S as an energy source
rather than just by autochthonous and allochthonous photosynthetic
organisms (Roach Tobler amp Winemiller 2011) These changes in the
biotic environment affect the relative importance of intra-vs inter-
specific competition trophic resource use and exposure to predators
and parasites (Riesch Oranth et al 2010 Tobler et al 2014 Tobler
Scharnweber et al 2015)
31 | Alternative drivers of trait evolution
Acknowledging the complexity of selective regimes in sulphide
springs allows for a more nuanced understanding of trait evolution
and for the development of alternative hypotheses about the ulti-
mate mechanisms that cause divergence in specific genetic and phe-
notypic traits In some instances sources of selection other than H2S
can plausibly explain trait divergence between populations from sul-
phidic and nonsulphidic habitats For example hypoxia likely drives
increases in head size and gill surface area (Tobler et al 2011)
changes in the ionic composition of the water can explain differential
expression of genes associated with osmoregulation (Kelley et al
2016) and shifts in trophic resource use are associated with
modifications of the gastrointestinal system (Tobler Scharnweber
et al 2015) In other cases disentangling alternative hypotheses is
not trivial because selection mediated by H2S and other environmen-
tal factors are predicted to affect trait modifications in similar ways
Shifts towards anaerobic metabolism in sulphide spring fish could be
the consequence of inhibition of aerobic ATP production by H2S or
by constraints in oxygen availability Similarly reproductive life his-
tory shifts towards the production of larger but fewer offspring could
be driven by H2S (decreasing offspringrsquos surfacevolume ratio to mini-
mize the diffusion of H2S into the body Powell 1989) by habitat dif-
ferences in the quality and quantity of predation that can affect size-
specific rates of extrinsic mortality (Jorgensen Auer amp Reznick
2011) or by energetic constraints (see below) While comparisons
across different selective regimes can shed light into the ultimate dri-
vers of trait evolution (Moore Riesch amp Martin 2016) isolating the
effects of different sources of selection may not be possible because
they could act in concert and facilitate increases of organismal perfor-
mance in multiple contexts (Ghalambor Walker amp Reznick 2003)
When multifarious selection shapes multivariate phenotypic evo-
lution it is important to also consider the role of trade-offs both in
terms of trait expression (increased investment in one trait may
impose constraints on the expression of other traits) and in terms of
trait function (optimizing the trait for one function may impair the
traitrsquos utility for other functions) For example selection on large off-
spring size in sulphide spring fishes has inadvertent consequences
for fecundity (Riesch et al 2014) and selection for increased gill
surface areas due to hypoxia simultaneously increases exposure to
environmental H2S (Tobler et al 2011) Thus the outcome of phe-
notypic evolution is ultimately a result of balancing competing needs
in a manner that optimizes trait expression and organismal function
in multiple contexts to maximize fitness (Arnold 1983) and the pres-
ence of trade-offs can have cascading indirect effects with selection
on one or a few traits leading to the modification of a multitude of
apparently unrelated characteristics
In this context bioenergetics may represent a nexus in the evo-
lution of complex traits observed in sulphide spring fish and other
extreme environments Living in and adapting to sulphide springs
imposes constraints on organismal energy availability (Plath et al
2007) with according trade-offs for energy allocation Energy limita-
tion in sulphide springs does not arise from a scarcity of food
resources (chemoautotrophic bacteria eaten by the fish are abundant
in sulphide springs Tobler Scharnweber et al 2015) but from
behavioural and physiological adaptation A primary survival strategy
for fish in the toxic and hypoxic springs is to engage in aquatic sur-
face respiration where individuals breathe directly at the water sur-
face to maximize oxygen uptake and minimize H2S exposure This
behaviour mediates short-term survival (Plath et al 2007) but it
also constrains energy acquisition for benthic foragers like P mexi-
cana (Tobler Riesch Tobler amp Plath 2009) and increases susceptibil-
ity to bird predation (Riesch Oranth et al 2010) Costly investments
into H2S detoxification and tissue repair as well as a shift from aero-
bic to less efficient anaerobic metabolism further constrain energy
allocation to other functions (Passow Greenway Arias-Rodriguez
TOBLER ET AL | 851
Jeyasingh amp Tobler 2015) Theory predicts that energetic con-
straints ndash whether mediated by limitations in the rate of resource
acquisition or in the rate of energy allocation to reproduction (as
opposed to maintenance or growth) ndash should exert selection for a
reduction in organismal energy budgets which allows for the maxi-
mization of residual energy allocation for the production of offspring
(Brown Marquet amp Taper 1993) Several lines of evidence support
the hypothesis that energy limitation plays a critical role in pheno-
typic evolution of sulphide spring fish Compared to individuals in
nonsulphidic environments fish in sulphide springs have significantly
lower routine energy demands which is mediated both by reduc-
tions in body size and mass-specific metabolism (Passow Arias-
Rodriguez et al 2017) Reductions in other traits such as energeti-
cally costly behaviours (Bierbach et al 2012 Plath 2008) and the
size of energy-demanding organs (Schulz-Mirbach et al 2016) may
also contribute to the reduced energy demands and shifts in repro-
ductive life history traits (Riesch et al 2014) are consistent with
selection from energy limitation (Reznick amp Yang 1993)
32 | Ensuing challenges Disentanglingenvironmental and organismal complexities
Future research needs to empirically address how multifarious selec-
tive regimes shape the evolution of organisms with complex pheno-
types We have made great advances in our understanding of how
specific traits evolve and mediate adaptation (eg morphology Chan
et al 2010 coloration Dasmahapatra et al 2012 sensory organs
Cartwright Schwartz Merry amp Howell 2017) and we have a thor-
ough understanding of how single environmental factors can shape
organismal responses (eg climatic conditions Johnston amp Bennett
1996 predation Langerhans 2007 competition Pfennig amp Pfennig
2012) In contrast our understanding of phenotypic evolution in
response to multifarious selection is comparatively limited especially
because interactive effects of different environmental factors can lead
to organismal and evolutionary responses that are unpredictable based
on experiments that manipulate single environmental variables in iso-
lation (Gunderson Armstrong amp Stillman 2016 Todgham amp Stillman
2013) Future research thus needs to acknowledge the complexities of
environments and organismal design and address them explicitly in
empirical investigations to gain a robust understanding of biological
diversification (Culumber amp Tobler 2017 Stuart et al 2017)
4 | LINKING ADAPTATION TOSPECIATION
So far we primarily explored the causes of adaptive evolution but
what are its consequences Theory predicts that adaptation to local
environmental conditions can lead to speciation because reproduc-
tive isolation emerges incidentally as a by-product (ecological specia-
tion Nosil 2012) Population genetic analyses of Poecilia spp in
sulphide springs and adjacent populations in nonsulphidic environ-
ments have indicated strong genetic differentiation across small
spatial scales (in some instances lt100 m) and in the absence of
physical barriers that would prevent fish movement (Plath et al
2013) In addition population genetic structuring has been shown to
be stable even in the face of major environmental disturbance
BOX 1 Speciation in sulphide springs and conservation
biology
Adaptation to H2S-rich environments in poeciliids coincides
with ecological speciation giving rise to phenotypically dis-
tinct and reproductively isolated lineages In some
instances this has led to the evolution of highly distinct
micro-endemic species including Poecilia sulphuraria and
P thermalis Limia sulphurophila and Gambusia eurystoma
(Figure 3) Except for P sulphuraria which is found in two
spring complexes of the Rıo Pichucalco drainage of Mexico
(Tobler amp Plath 2009b) the distributions of these sulphide
spring endemics are restricted to single localities that
include only small areas of suitable habitat For example
the largest sulphide spring complex (the Ba~nos del Azufre
in southern Mexico which harbours P sulphuraria and G
eurystoma) includes ~18000 m2 of suitable habitat (equiva-
lent to lt35 football fields) while one of the smallest (La
Esperanza springs harbouring P thermalis) merely covers
~600 m2 (just over a tenth of a football field Brown et al
2017) Accordingly some sulphide spring endemics are
considered critically endangered (IUCN 2012) and pro-
tected by law (SEDESOL 1994)
Many threatened species with restricted ranges exhibit
low genetic diversity and high incidences of inbreeding
(Bouzat 2010 Willi Van Buskirk amp Hoffmann 2006) In
contrast at least P sulphuraria and P thermalismdashthe only
species investigated to datemdashactually exhibit relatively high
effective population sizes (Ne = 1200ndash3800 depending on
the population) and no evidence for inbreeding (FIS lt 0)
(Brown et al 2017) This is likely a consequence of the
long-term stability of sulphide springs and the high pro-
ductivity and low levels of interspecific competition also
support unusually high fish densities and thus population
census sizes (Brown et al 2017 Culumber et al 2016)
Nonetheless sulphide spring endemics are threatened by
habitat destruction which is mediated primarily by the
modification of springs for recreational purposes defor-
estation and conversion of land for agriculture and most
recently high-intensity palm oil plantations (Tobler amp Plath
2009ab) Despite the official protection of sulphide spring
endemics there have been no tangible conservation mea-
sures that foster the sustainable development of land
around sulphide springs with the well-being of the endemic
fishes in mind
852 | TOBLER ET AL
(catastrophic flood event Plath et al 2010) Population genetic dif-
ferentiation between adjacent sulphide-adapted and nonadapted
populations is indicative of ongoing ecological speciation (Box 1) but
different population pairs from sulphidic and nonsulphidic waters
have made variable progress along the speciation continuum towards
complete reproductive isolation (Plath et al 2013) This prompts
questions about the mechanisms involved in reducing gene flow
along the environmental gradient connecting the two habitat types
41 | Prezygotic mechanisms of reproductiveisolation
The simplest form of reproductive isolation between locally adapted
populations arises from selection against migrants between habitat
types (Nosil Vines amp Funk 2005) Translocation experiments in nat-
ural habitats have indicated strong survival selection against
migrants especially those translocated from nonsulphidic to sulphidic
habitats (Plath et al 2013) Fish translocated from nonsulphidic
environments face high mortality within short periods of time
(lt24 hr) which is likely caused by H2S toxicity and hypoxia in sul-
phide springs Hence low gene flow from populations inhabiting
nonsulphidic waters into populations inhabiting sulphidic habitats is
simply explained by a lack of adaptation to the extreme environmen-
tal conditions At least in some drainages there is also appreciable
mortality for sulphide spring fish translocated to nonsulphidic habi-
tats although mechanisms causing inviability are unclear and the
strength of selection appears to be lower (Plath et al 2013) If sur-
vival selection against immigrants was the sole source of reproduc-
tive isolation this should result in unidirectional gene flow from
H2S-adapted to nonadapted populations but population genetic
analyses provided no evidence for this implying that additional
mechanisms are at work (Plath et al 2013)
One such mechanism could be intersexual selection and mate
choice experiments have indicated that migrants from sulphidic to
nonsulphidic habitats are at a significant disadvantage with fish from
nonsulphidic habitats avoiding potential mating partners from sul-
phidic ones (Plath et al 2013 Sommer-Trembo et al 2016) Such
assortative mating has been found to be critical during ecological spe-
ciation because it counteracts the homogenizing effects of gene flow
(van Doorn Edelaar amp Weissing 2009) yet how assortative mating
patterns arise often remains unclear In theory assortative mating can
arise through a variety of processes (Nosil 2012) ldquoMagic traitrdquo mecha-
nisms where mating preferences evolve as a by-product of divergent
selection on ecological traits that serve as cues have recently garnered
much attention (Servedio van Doorn Kopp Frame amp Nosil 2011)
Consistent with magic trait mechanisms assortative mating in P mexi-
cana appears to be linked to adaptive population differences in body
shape which serve as cues to distinguish potential mating partners
from different habitats (Greenway Drexler Arias-Rodriguez amp Tobler
2016) However neither sulphide spring fish (which are unlikely to
encounter migrants from nonsulphidic habitats due to H2S toxicity)
nor allopatric P mexicana from nonsulphidic waters (which have never
experienced migrants from sulphide spring populations) exhibit a
preference for mating partners from their own ecotype suggesting
that assortative mating in this system is a product of reinforcement
(ie direct selection for assortative mating Greenway et al 2016)
This is different from classic magic traits mechanisms because assorta-
tive mating is not a mere by-product of adaptation but adaptive traits
have instead been co-opted as mating signals in response to selection
on mating preferences (Maan amp Seehausen 2012) Moreover males of
some H2S-adapted populations have reduced aggressive behaviour
(Bierbach et al 2012 2018) In staged contests of size-matched males
from different ecotypes intruders had a significantly lower likelihood
to establish dominance than resident males independent of whether
or not they showed reduced aggressiveness overall Hence intrasexual
selection may represent another mechanism mediating reproductive
isolation between locally adapted ecotypes Overall H2S-adapted poe-
ciliids allow disentangling the relative contributions of different forms
of ecological and sexual selectionmdashand their interdependencymdashduring
ecological speciation
42 | Ensuing challenges quantifying postzygoticmechanisms of reproductive isolation
In contrast to prezygotic mechanisms little is known about postzy-
gotic mechanisms of reproductive isolation that potentially act to
reduce gene flow between adjacent populations in sulphidic and
nonsulphidic habitats Putative hybrids between sulphidic and non-
sulphidic ecotypes have occasionally been observed in nonsulphidic
habitats immediately adjacent to confluences with sulphide springs
(R Riesch M Plath and M Tobler personal observations) but it
remains unclear whether and through what mechanisms such hybrids
exhibit reduced fitness compared to parentals
Direct selection of mitochondrially encoded genes in the pres-
ence of H2S (Pfenninger et al 2014) should be accompanied by co-
evolution at nuclear loci that directly interact with mitochondrial
gene products in the formation of protein complexes involved in the
OXPHOS pathway (Sunnucks Morales Lamb Pavlova amp Greening
2017 Willett amp Burton 2004) Disruption of co-evolved gene com-
plexes in hybrids should therefore lead to intrinsic fitness reductions
through mito-nuclear incompatibilities (Burton Pereira amp Barreto
2013 Wolff Ladoukakis Enrıquez amp Dowling 2014) which have
been hypothesized to be a key driver in speciation (Chou amp Leu
2010 Gershoni Templeton amp Mishmar 2009) Empirical tests of
these ideas remain to be conducted in sulphide spring fishes just
like studies on potential ecological and sexual selection against
hybrids that arise from interpopulation mating events
5 | PERSPECTIVES LIFE IN SULPHIDICENVIRONMENTS BEYOND P MEXICANA
Research on sulphide spring fishes of the P mexicana species com-
plex in Mexico has provided insights into mechanisms underlying
adaptation and the links between adaptation and speciation Sul-
phide springs do not only occur in Mexico however but are found
TOBLER ET AL | 853
worldwide and have been colonized by 24 evolutionarily indepen-
dent fish lineages in North America the Neotropics and the Middle
East (Greenway et al 2014) The vast majority of lineages belong to
the order Cyprinodontiformes (95) and livebearing members of the
family Poeciliidae in particular (80) This potentially indicates that
internal gestation of young has facilitated the colonization of
extreme environments because it shields developing embryos from
physiochemical stressors (Blackburn 1999) Among the Poeciliidae
sulphide spring fishes include members of the genera Brachyrhaphis
Gambusia Poeciliopsis Priapichthys Pseudoxiphophorus Xiphophorus
and multiple subgenera of Poecilia (Figure 3 Greenway et al 2014)
spanning over 40 million years of evolution (based on Reznick Fur-
ness Meredith amp Springer 2017)
A handful of studies have started to address molecular and phe-
notypic evolution across a broader taxon sampling of sulphide spring
fishes finding evidence for convergent evolution in morphology
(Riesch et al 2016 Tobler amp Hastings 2011) life history traits
(Riesch Plath et al 2010 Riesch et al 2014) and oxygen transport
genes (Barts et al 2018) In addition ecological speciation in
response to adaptation to H2S-rich environments has also been doc-
umented in sulphide spring poeciliids other than P mexicana (Riesch
et al 2016) These studies have laid a fruitful foundation for the
investigation of mechanisms of adaptation to H2S-rich springs from
genes to phenotypic traits linking micro-evolutionary processes to
macro-evolutionary patterns
The availability of broad-scale evolutionary replication of popula-
tions that have invaded habitats with the same physiochemical stressors
allows for addressing some of the ensuing challenges discussed above
and investigating the factors that give rise to predictable evolutionary
outcomes Convergent evolution is a commonmdashbut not omnipresentmdash
theme in adaptive evolution and multiple factors can determine the
degree of convergence among replicated lineages exposed to the same
sources of selection (Kaeuffer et al 2012 Landry amp Bernatchez 2010
Oke Rolshausen LeBlond amp Hendry 2017 Rosenblum amp Harmon
2011) Thus leveraging replicated lineages that have colonized sulphide
springs to assess molecular mechanisms underlying convergent pheno-
typic changes analyse how environmental heterogeneity among sul-
phide spring habitats causes lineage-specific patterns of trait evolution
and test whether and by what mechanisms adaptation to extreme envi-
ronments is linked to speciation will provide insights into how ecologi-
cal genetic and functional factors contribute to evolutionary
convergence at different levels of organismal organization (Rosenblum
et al 2014) An expanded taxon sampling with population pairs that
span almost the full range of the speciation continuum from panmixia to
complete reproductive isolation (Riesch et al 2016) will also help
addressing questions about temporal aspects of H2S adaptation In par-
ticular comparing younger to older lineages will provide insights about
the order at which genetic and phenotypic modifications accrue during
the adaptation process
Animals have also invaded H2S-rich habitats in other aquatic
ecosystems including cold seeps and deep-sea hydrothermal vents
(Tobler et al 2016) Adaptation to H2S in the P mexicana species com-
plex has involved modification of physiological pathways associated
with H2S toxicity (OXPHOS pathway) and detoxification (sulphide
F IGURE 3 Phylogeny of the family Poeciliidae (adopted from Culumber amp Tobler 2017) Various lineages in the genera Poecilia (subgeneraAcanthophacelus Limia and Mollienesia) Priapichthys Brachyrhaphis Poeciliopsis Xiphophorus Pseudoxiphophorus and Gambusia have colonizedH2S-rich springs in different parts of the Americas (see Greenway et al 2014) Lineages highlighted in yellow represent endemic speciesrestricted to H2S-rich springs while red lineages represent species in which some populations have invaded sulphidic habitats Pictures depict arepresentative of each focal genussubgenus
854 | TOBLER ET AL
quinone oxidoreductase pathway) which have been highly conserved
during the diversification of life on Earth (Saraste 1999 Shahak amp
Hauska 2008) The high replicability of H2S as an environmental factor
that shaped animal evolution across vastly different ecological contexts
consequently offers the opportunity to investigate whether selection
mediated by the presence of H2S has repeatedly modified these con-
served physiological pathways across animal phyla that have diverged
during the Cambrian explosion spanning over 500 million years of evo-
lution Such comparisons would provide unprecedented insights into
the effects of a single physiochemical stressor on evolutionary change
irrespective of ecological or phylogenetic contexts
6 | CONCLUSIONS
H2S-rich environments and their inhabitants represent useful models
for research in organismal biology ecology and evolution illustrating
both opportunities (eg strong and explicit source of selection evolu-
tionary replication) and challenges (eg complexity of environments
and organismal responses) in asking basic question about the origins
of biodiversity Understanding how organisms thrive in apparently
inhospitable environments will provide us with insights into lifersquos
capacities and limitations to adapt to novel environments (Waterman
1999) This is particularly relevant considering that human activities
substantially contribute to the exacerbation of physiochemical stres-
sors at local regional and global scales impacting ecological and evo-
lutionary dynamics (Alberti 2015 Norberg Urban Vellend
Klausmeier amp Loeuille 2012) Thus a focus on ecological and evolu-
tionary consequences of natural stressors that push organisms to the
brink of their physiological capacities may be instrumental to predict
consequences of anthropogenic environmental change
ACKNOWLEDGEMENTS
We would like to thank Lenin Arias-Rodriguez the Universidad
Juarez Autonoma de Tabasco and the Centro de Investigacion E
Innovacion Para la Ense~nanza y El Aprendizaje for their continued
collaboration enabling our work on the P mexicana system Funding
for this review article has been provided by the National Science
Foundation (IOS-1557860 IOS-1557795) and the US Army
Research Office (W911NF-15-1-0175)
AUTHOR CONTRIBUTIONS
MT wrote the first draft of the paper all authors contributed text
and comments
ORCID
Michael Tobler httporcidorg0000-0002-0326-0890
Joanna L Kelley httporcidorg0000-0002-7731-605X
Reuroudiger Riesch httporcidorg0000-0002-0223-1254
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Blackburn D G (1999) Viviparity and oviparity Evolution and reproduc-
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Brown A P Greenway R Morgan S Quackenbush C R Giordani
L Arias-Rodriguez L Kelley J L (2017) Genome-scale data
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Brown J H Marquet P A amp Taper M L (1993) Evolution of body
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nurev-ecolsys-110512-135758
Bush S J Chen L Tovar-Corona J M amp Urritia A O (2017) Alterna-
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Carroll S P Jorgensen P S Kinnison M T Bergstrom C T Denison
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Cartwright R A Schwartz R S Merry A L amp Howell M M (2017)
The importance of selection in the evolution of blindness in cavefish
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017-0876-4
Chan Y F Marks M E Jones F C Villarreal G Shapiro M D Brady
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Chou J Y amp Leu J Y (2010) Speciation through cytonuclear incompat-
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Colosimo P F Hosemann K E Balabhadra S Villarreal G Dickson
M Grimwood J Kingsley D M (2005) Widespread parallel evo-
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Cooper C E amp Brown G C (2008) The inhibition of mitochondrial
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hydrogen cyanide and hydrogen sulfide Chemical mechanism and
physiological significance Journal of Bioenergy and Biomembranes 40
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Culumber Z W Hopper G W Barts N Passow C N Morgan S Brown
A Tobler M (2016) Habitat use of two extremophile highly ende-
mic and critically endangered fish species (Gambusia eurystoma and Poe-
cilia sulphurophila Poeciliidae) Aquatic Conservation-Marine and
Freshwater Ecosystems 26 1155ndash1167 httpsdoiorg101002aqc
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Culumber Z W amp Tobler M (2017) Sex-specific evolution during the
diversification of live-bearing fishes Nature Ecology amp Evolution 1
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Dalziel A C Rogers S M amp Schulte P M (2009) Linking genotypes
to phenotypes and fitness How mechanistic biology can inform
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Darwin C (1859) On the origin of species based on natural selection or
the preservation of favoured races in the struggle of life 6th ed Lon-
don John Murray
Dasmahapatra K K Walters J R Briscoe A D Davey J W Whibley
A Nadeau N J Jiggins C D (2012) Butterfly genome reveals
promiscuous exchange of mimicry adaptations among species Nature
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Doudna J A amp Charpentier E (2014) The new frontier of genome edit-
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1126science1258096
Dubilier N Giere O amp Grieshaber M (2005) Concomitant effects of sul-
fide and hypoxia on the aerobic metabolism of the marine oligochaete
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Eales K L Hollinshead K E R amp Tennant D A (2016) Hypoxia and
metabolic adaptation of cancer cells Oncogenesis 5 e190 httpsd
oiorg101038oncsis201550
Eghbal M A Pennefather P S amp OrsquoBrien P J (2004) H2S cytotoxicity
mechanism involves reactive oxygen species formation and mito-
chondrial depolarisation Toxicology 203 69ndash76 httpsdoiorg10
1016jtox200405020
Feldman C R Brodie Jr E D Brodie III E D amp Pfrender M E (2012)
Constraint shapes convergence in tetrodotoxin-resistant sodium
channels of snakes Proceedings of the National Academy of Sciences
USA 109 4556ndash4561 httpsdoiorg101073pnas1113468109
Flores K B Wolschin F amp Amdam G V (2013) The role of methyla-
tion of DNA in environmental adaptation Integrative and Comparative
Biology 53 359ndash372 httpsdoiorg101093icbict019
Fradin H Kiontke K Zegar C Gutwein M Lucas J Kovtun M
Piano F (2017) Genome architecture and evolution of a unichromo-
somal asexual nematode Current Biology 27(2928ndash2939) e2926
Gershoni M Templeton A amp Mishmar D (2009) Mitochondrial bioen-
ergetics as a major motive force of speciation BioEssays 31 642ndash
650 httpsdoiorg101002bies200800139
Ghalambor C K Walker J A amp Reznick D N (2003) Multi-trait selec-
tion adaptation and constraints on the evolution of burst swimming
performance Integrative and Comparative Biology 43 431ndash438
httpsdoiorg101093icb433431
Gibert J amp Deharveng L (2002) Subterranean ecosystems A truncated
functional biodiversity BioScience 52 473ndash481 httpsdoiorg10
16410006-3568(2002)052[0473SEATFB]20CO2
Gjuvsland A B Vik J O Beard D A Hunter P J amp Omholt S W
(2013) Bridging the genotype-phenotype gap What does it take
Journal of Physiology 591 2055ndash2066 httpsdoiorg101113jphys
iol2012248864
Grant P R Grant B R Markert J A Keller L F amp Petren K (2004)
Convergent evolution of Darwinrsquos finches caused by introgressive
hybridization and selection Evolution 58 1588ndash1599 httpsdoi
org101111j0014-38202004tb01738x
Greene H W (2005) Organisms in nature as a central focus for biology
Trends in Ecology and Evolution 20 23ndash27 httpsdoiorg101016j
tree200411005
Greenway R Arias-Rodriguez L Diaz P amp Tobler M (2014) Patterns
of macroinvertebrate and fish diversity in freshwater sulphide springs
Diversity 6 597ndash632 httpsdoiorg103390d6030597
Greenway R Drexler S Arias-Rodriguez L amp Tobler M (2016) Adap-
tive but not condition-dependent body shape differences contribute
to assortative mating preferences during ecological speciation Evolu-
tion 70 2809ndash2822 httpsdoiorg101111evo13087
Gunderson A R Armstrong E J amp Stillman J H (2016) Multiple stres-
sors in a changing world The need for an improved perspective on
physiological responses to the dynamic marine environment Annual
Review of Marine Science 8 357ndash378 httpsdoiorg101146an
nurev-marine-122414-033953
Hildebrandt T M amp Grieshaber M (2008) Three enzymatic activities
catalyze the oxidation of sulfide to thiosulfate in mammalian and
invertebrate mitochondria FEBS Journal 275 3352ndash3361 httpsd
oiorg101111j1742-4658200806482x
Houle D Govindaraju D R amp Omholt S (2010) Phenomics The next
challenge Nature Reviews Genetics 11 855ndash866 httpsdoiorg10
1038nrg2897
IUCN (2012) Red List of threatened species version 20132 www
iucnredlistorg
Johnston I A amp Bennett A F (1996) Animals and temperature Pheno-
typic and evolutionary adaptation Cambridge Cambridge University
Press httpsdoiorg101017CBO9780511721854
Jorgensen C Auer S K amp Reznick D N (2011) A model for optimal
offspring size in fish including live-bearing and parental effects Amer-
ican Naturalist 177 E119ndashE135 httpsdoiorg101086659622
Kaeuffer R Peichel C L Bolnick D I amp Hendry A P (2012) Parallel
and nonparallel aspects of ecological phenotypic and genetic diver-
gence across replicate population pairs of lake and stream stickle-
back Evolution 66 402ndash418 httpsdoiorg101111j1558-5646
201101440x
Kell D B amp Oliver S G (2004) Here is the evidence now what is the
hypothesis The complementary roles of inductive and hypothesis-
driven science in the post-genomic era BioEssays 26 99ndash105
httpsdoiorg101002(ISSN)1521-1878
856 | TOBLER ET AL
Kelley J L Arias-Rodriguez L Patacsil Martin D Yee M C Busta-
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life in hydrogen sulfide rich environments Molecular Biology and Evo-
lution 33 1419ndash1434 httpsdoiorg101093molbevmsw020
Kelley J L Passow C Plath M Arias-Rodriguez L Yee M C amp
Tobler M (2012) Genomic resources for a model in adaptation and
speciation research The characterization of the Poecilia mexicana
transcriptome BMC Genomics 13 652 httpsdoiorg101186
1471-2164-13-652
Klerks P L Xie L amp Levinton J S (2011) Quantitative genetics
approaches to study evolutionary processes in ecotoxicology a per-
spective from research on the evolution of resistance Ecotoxicology
20 513ndash523 httpsdoiorg101007s10646-011-0640-2
Kump L R Pavlov A amp Arthur M A (2005) Massive release of hydrogen
sulfide to the surface ocean and atmosphere during intervals of oceanic
anoxia Geology 33 397ndash400 httpsdoiorg101130G212951
Keuroustner A Hoffmann M Fraser B A Kottler V A Sharma E Wei-
gel D amp Dreyer C (2016) The genome of the Trinidadian guppy
Poecilia reticulata and variation in the guanapo population PLoS ONE
11 e0169087 httpsdoiorg101371journalpone0169087
Landry L amp Bernatchez L (2010) Role of epibenthic resource opportu-
nities in the parallel evolution of lake whitefish species pairs (Core-
gonus sp) Journal of Evolutionary Biology 23 2602ndash2613 httpsdoi
org101111j1420-9101201002121x
Langerhans R B (2007) Evolutionary consequences of predation Avoid-
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(Ed) Predation in organisms Berlin Springer
Li L Rose P amp Moore P K (2011) Hydrogen sulfide and cell signaling
Annual Review of Pharmacology and Toxicology 51 169ndash187
httpsdoiorg101146annurev-pharmtox-010510-100505
Lin Q Fan S Zhang Y Xu M Zhang H Yang Y Gunter H M
(2016) The seahorse genome and the evolution of its specialized mor-
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Liu Y Ma S Wang X Chang J Gao J Shi R Zhao P (2014)
Highly efficient multiplex targeted mutagenesis and genomic struc-
ture variation in Bombyx mori cells using CRISPRCas9 Insect bio-
chemistry and molecular biology 49 35ndash42 httpsdoiorg101016j
ibmb201403010
Lopez-Maury L Marguerat S amp Beuroahler J (2008) Tuning gene expres-
sion to changing environments From rapid responses to evolutionary
adaptation Nature Reviews Genetics 9 583ndash593 httpsdoiorg10
1038nrg2398
Maan M E amp Seehausen O (2012) Magic traits versus magic prefer-
ences in speciation Evolutionary Ecology Research 14 779ndash785
Martyn U Weigel D amp Dreyer C (2006) In vitro culture of embryos
of the guppy Poecilia reticulata Developmental Dynamics 235 617ndash
622 httpsdoiorg101002(ISSN)1097-0177
Metzker M L (2010) Sequencing technologies The next generation
Nature Reviews Genetics 11 31ndash46 httpsdoiorg101038nrg2626
Moore M P Riesch R amp Martin R A (2016) The predictability and
magnitude of life-history divergence to ecological agents of selection
A meta-analysis in livebearing fishes Ecology Letters 19 435ndash442
httpsdoiorg101111ele12576
Mustafa A K Gadalla M M Sen N Kim S Mu W Gazi S K
Snyder S H (2009) H2S signals through protein S-sulfhydration
Science Signaling 2 ra72
Nesse R M amp Stearns S C (2008) The great opportunity Evolutionary
applications to medicine and public health Evolutionary Applications
1 28ndash48 httpsdoiorg101111j1752-4571200700006x
Nevo E (2011) Evolution under environmental stress at macro and
microscales Genome Biology and Evolution 3 1039ndash1052 httpsdoi
org101093gbeevr052
Norberg J Urban M C Vellend M Klausmeier C A amp Loeuille N (2012)
Eco-evolutionary responses of biodiversity to climate change Nature Cli-
mate Change 2 747ndash751 httpsdoiorg101038nclimate1588
Nosil P (2012) Ecological speciation Oxford UK Oxford University
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0001
Nosil P Vines T H amp Funk D J (2005) Perspective Reproductive
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gent habitats Evolution 59 705ndash719
Oke K B Rolshausen G LeBlond C amp Hendry A P (2017) How par-
allel is parallel evolution A comparative analysis in fishes American
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Olson K R (2011) The therapeutic potential of hydrogen sulfide
Separating hype from hope American Journal of Physiology-Regulatory
Integrative and Comparative Physiology 301 R297ndashR312 httpsdoi
org101152ajpregu000452011
Olson K R Donald J A Dombkowski R A amp Perry S F (2012) Evo-
lutionary and comparative aspects of nitric oxide carbon monoxide
and hydrogen sulfide Respiratory Physiology amp Neurobiology 184
117ndash129 httpsdoiorg101016jresp201204004
Olson K R amp Straub K D (2015) The role of hydrogen sulfide in evo-
lution and the evolution of hydrogen sulfide in metabolism and sig-
naling Physiology 31 60ndash72
Palacios M Arias-Rodriguez L Plath M Eifert C Lerp H Lamboj A
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nalpone0071069
Palacios M Voelker G Arias-Rodriguez L Mateos M amp Tobler M
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versity and spatio-temporal aspects of diversification in Middle
America Molecular Phylogenetics and Evolution 103 230ndash244
httpsdoiorg101016jympev201607025
Passow C Arias-Rodriguez L amp Tobler M (2017) Convergent evolu-
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Passow C N Brown A Arias-Rodriguez L Yee M-C Sockell A
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Passow C Greenway R Arias-Rodriguez L Jeyasingh P D amp Tobler
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body size and metabolic rates in locally adapted extremophile fishes
Physiological and Biochemical Zoology 88 371ndash383 httpsdoiorg
101086681053
Passow C N Henpita C Shaw J H Quakenbush C Warren W C
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Pavey S A Bernatchez L Aubin-Horth N amp Landry C R (2012)
What is needed for next-generation ecological and evolutionary
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Peers C Bauer C Boyle J P Scragg J L amp Dallas M L (2012)
Modulation of ion channels by hydrogen sulfide Antioxidants amp Redox
Signaling 17 95ndash105 httpsdoiorg101089ars20114359
Pfennig D W amp Pfennig K S (2012) Evolutionrsquos wedge competition and
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httpsdoiorg101525california97805202741810010001
Pfenninger M Lerp H Tobler M Passow C Kelley J L Funke E
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Pfenninger M Patel S Arias-Rodriguez L Feldmeyer B Riesch R amp
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TOBLER ET AL | 857
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1111mec13397
Pietri R Roman-Morales E amp Lopez-Garriga J (2011) Hydrogen sul-
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Plath M (2008) Male mating behavior and costs of sexual harassment
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Plath M Hermann C Schreurooder R Riesch R Tobler M Garcia de
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Plath M Pfenninger M Lerp H Riesch R Eschenbrenner C Slattery
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Plath M Tobler M Riesch R Garcia de Leon F J Giere O amp
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Powell E (1989) Oxygen sulfide and diffusion Why thiobiotic meiofauna
must be sulfide-insensitive first-order respirers Journal of Marine
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Ramirez-Soriano A Ramos-Onsins S E Rozas J Calafell F amp Navarro A
(2008) Statistical power analysis of neutrality tests under demographic
expansions contractions and bottlenecks with recombination Genetics
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Reiffenstein R Hulbert W amp Roth S (1992) Toxicology of hydrogen
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134 httpsdoiorg101146annurevpa32040192000545
Reznick D N Furness A I Meredith R W amp Springer M S (2017)
The origin and biogeographic diversification of fishes in the family
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lpone0172546
Reznick D amp Yang A P (1993) The influence of fluctuating resources
on life history Patterns of allocation and plasticity in female guppies
Ecology 74 2011ndash2019 httpsdoiorg1023071940844
Riesch R Duwe V Herrmann N Padur L Ramm A Scharnweber K
Plath M (2009) Variation along the shy-bold continuum in extre-
mophile fishes (Poecilia mexicana Poecilia sulphuraria) Behavioral Ecol-
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s00265-009-0780-z
Riesch R Oranth A Dzienko J Karau N Schiessl A Stadler S
Plath M (2010) Extreme habitats are not refuges Poeciliids suffer
from increased aerial predation risk in sulphidic southern Mexican
habitats Biological Journal of the Linnean Society 101 417ndash426
httpsdoiorg101111j1095-8312201001522x
Riesch R Plath M Garcia de Leon F J amp Schlupp I (2010) Conver-
gent life-history shifts Toxic environments result in big babies in two
clades of poeciliids Naturwissenschaften 97 133ndash141 httpsdoi
org101007s00114-009-0613-y
Riesch R Plath M Schlupp I Tobler M amp Langerhans R B (2014)
Colonization of toxic springs drives predictable life-history shift in
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org101111ele12209
Riesch R Tobler M Lerp H Jourdan J Doumas L T Nosil P Plath
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plexity of speciation along replicated ecological gradients BMC Evolu-
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Riesch R Tobler M amp Plath M (2015) Extremophile fishes Ecology
evolution and physiology of teleosts in extreme environments p 290
Heidelberg Germany Springer httpsdoiorg101007978-3-319-
13362-1
Ritchie M E Holzinger E R Li R Pendergrass S A amp Kim S (2015)
Methods of integrating data to uncover genotype-phenotype interac-
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nrg3868
Roach K Tobler M amp Winemiller K O (2011) Hydrogen sulfide bac-
teria and fish A unique subterranean food chain Ecology 92 2056ndash
2062 httpsdoiorg10189011-02761
Rohner N Jarosz D F Kowalko J E Yoshizawa M Jeffery W R
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Rosales Lagarde L (2012) Investigation of karst brackish-sulfidic springs
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of Mining and Technology Mexico
Rosenblum E B amp Harmon L J (2011) ldquoSame but differentrdquo Repli-
cated ecological speciation at White Sands Evolution 65 946ndash960
httpsdoiorg101111j1558-5646201001190x
Rosenblum E B Parent C E amp Brandt E E (2014) The molecular
basis of convergent evolution Annual Review of Ecology Evolution and
Systematics 45 203ndash226 httpsdoiorg101146annurev-ecolsys-
120213-091851
Saltz J B Hessel F C amp Kelly M W (2017) Trait correlations in the
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Saraste M (1999) Oxidative phosphorylation at the fin de siecle Science
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Schartl M (2014) Beyond the zebrafish Diverse fish species for model-
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Schartl M Walter R B Shen Y Garcia T Catchen J Amores A
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1038ng2604
Schluter D (2009) Evidence for ecological speciation and its alternative
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Schulz-Mirbach T Eifert C Riesch R Farnworth M S Zimmer C
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Servedio M R van Doorn G S Kopp M Frame A M amp Nosil P
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Shahak Y amp Hauska G (2008) Sulfide oxidation from cyanobacteria to
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Shalem O Sanjana N E amp Zhang F (2015) High-throughput func-
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Silva G J J Bye A el Azzouzi H amp Wisloslashff U (2017) MicroRNAs as
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Diseases 60 130ndash151 httpsdoiorg101016jpcad201706003
Simola D F Wissler L Donahue G Waterhouse R M Helmkampf
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858 | TOBLER ET AL
Sommer-Trembo C Bierbach D Arias Rodriguez L Verel Y Jourdan
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Naturwissenschaftlichen Classe der Kaiserlichen Akademie der Wis-
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Stinchcombe J R amp Hoekstra H E (2008) Combining population geno-
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hdy6800937
Stipanuk M H (2004) Sulfur amino acid metabolism Pathways for pro-
duction and removal of homocysteine and cysteine Annual Review of
Nutrition 24 539ndash577 httpsdoiorg101146annurevnutr24
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Stipanuk M H amp Ueki I (2011) Dealing with methioninehomocysteine
sulfur Cysteine metabolism to taurine and inorganic sulfur Journal of
Inherited Metabolic Disease 34 17ndash32 httpsdoiorg101007
s10545-009-9006-9
Stockinger B Di Meglio P Gialitakis M amp Duarte J H (2014) The
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Storz J F amp Wheat C W (2010) Integrating evolutionary and
functional approaches to infer adaptation at specific loci Evolution
64 2489ndash2509 httpsdoiorg101111j1558-5646201001044x
Stuart Y E Veen T Weber J N Hanson D Ravinet M Lohman B
K Bolnick D I (2017) Contrasting effects of environment and
genetics generate a continuum of parallel evolution Nature Ecology amp
Evolution 1 0158 httpsdoiorg101038s41559-017-0158
Sunnucks P Morales H E Lamb A M Pavlova A amp Greening C
(2017) Integrative approaches for studying mitochondrial and nuclear
genome co-evolution in oxidative phosphorylation Frontiers in Genet-
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Tobler M amp Hastings L (2011) Convergent patterns of body shape dif-
ferentiation in four different clades of poeciliid fishes inhabiting sul-
fide springs Evolutionary Biology 38 412ndash421 httpsdoiorg10
1007s11692-011-9129-4
Tobler M Palacios M Chapman L J Mitrofanov I Bierbach D
Plath M Mateos M (2011) Evolution in extreme environments
Replicated phenotypic differentiation in livebearing fish inhabiting
sulfidic springs Evolution 65 2213ndash2228 httpsdoiorg101111j
1558-5646201101298x
Tobler M Passow C N Greenway R Kelley J L amp Shaw J H
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fide-rich environments Annual Review of Ecology Evolution and Sys-
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Tobler M amp Plath M (2009a) Threatened fishes of the world Gambu-
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Fishes 85 251 httpsdoiorg101007s10641-009-9481-8
Tobler M amp Plath M (2009b) Threatened fishes of the world Poecilia
sulphuraria (Alvarez 1948) (Poeciliidae) Environmental Biology of
Fishes 85 333ndash334 httpsdoiorg101007s10641-009-9506-3
Tobler M Plath M Riesch R Schlupp I Grasse A Munimanda G
Moodley Y (2014) Selection from parasites favors immunogenic diver-
sity but not divergence among locally adapted host populations Journal
of Evolutionary Biology 27 960ndash974 httpsdoiorg101111jeb12370
Tobler M Riesch R amp Plath M (2015) Extremophile fishes An inte-
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environments (pp 279ndash296) Heidelberg Germany Springer
Tobler M Riesch R Tobler C M amp Plath M (2009) Compensatory
behaviour in response to sulfide-induced hypoxia affects time
budgets feeding efficiency and predation risk Evolutionary Ecology
Research 11 935ndash948
Tobler M Scharnweber K Greenway R Passow C Arias-Rodriguez
L amp Garcia-De-Leon F J (2015) Convergent changes in the
trophic ecology if extremophile fish along replicated environmental
gradients Freshwater Biology 60 768ndash780 httpsdoiorg10
1111fwb12530
Todgham A E amp Stillman J H (2013) Physiological responses to shifts
in multiple environmental stressors Relevance in a changing worlds
Integrative and Comparative Biology 53 539ndash544 httpsdoiorg10
1093icbict086
van Doorn G S Edelaar P amp Weissing F J (2009) On the origin of
species by natural selection and sexual selection Science 326 1704ndash
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Wall D H amp Virginia R A (1999) Controls on soil biodiversity Insights
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Wallace J L amp Wang R (2015) Hydrogen-sulfide based therapeutics
Exploiting a unique but ubiquitous gasotransmitter Nature Reviews
Drug Discovery 14 329ndash345 httpsdoiorg101038nrd4433
Wang R (2012) Physiological implications of hydrogen sulfide A whiff
exploration that blossomed Physiological Reviews 92 791ndash896
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Wang Z Gerstein M amp Snyder M (2009) RNA-seq A revolutionary
tool for transcriptomics Nature Reviews Genetics 10 57ndash63
httpsdoiorg101038nrg2484
Waterman T H (1999) The evolutionary challenges of extreme environ-
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AID-JEZ3ampgt30CO2-T
Werren J H Richards S Desjardins C A Niehuis O Gadau J Col-
bourne J K amp Group N G W (2010) Functional and evolutionary
insights from the genomes of three parasitoid Nasonia species
Science 327 343ndash348 httpsdoiorg101126science1178028
Whitehead A (2012) Comparative genomics in ecological physiology
Toward a more nuanced understanding of acclimation and adapta-
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Whitehead A amp Crawford D L (2006) Neutral and adaptive variation
in gene expression Proceedings of the National Academy of Sciences
USA 103 5425ndash5430 httpsdoiorg101073pnas0507648103
Willett C S amp Burton R S (2004) Evolution of interacting proteins in
the mitochondrial electron transport system in a marine copepod
Molecular Biology and Evolution 21 443ndash453 httpsdoiorg10
1093molbevmsh031
Willi Y Van Buskirk J amp Hoffmann A (2006) Limits to the adaptive
potential of small populations Annual Review of Ecology Evolution
and Systematics 37 433ndash458 httpsdoiorg101146annurevecol
sys37091305110145
Wolff J N Ladoukakis E D Enrıquez J A amp Dowling D K (2014)
Mitonuclear interactions Evolutionary consequences over multiple
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How to cite this article Tobler M Kelley JL Plath M Riesch
R Extreme environments and the origins of biodiversity
Adaptation and speciation in sulphide spring fishes Mol Ecol
201827843ndash859 httpsdoiorg101111mec14497
TOBLER ET AL | 859
inference of putative mechanisms underlying adaptation even in sys-
tems without a priori predictions (Kell amp Oliver 2004) However
genetic and gene expression data alone are insufficient to infer
adaptation at specific loci because neutral evolutionary processes
demographic history and genetic hitchhiking can generate false posi-
tives in genomewide analyses of selection and gene expression (Bar-
ton 1998 Ramirez-Soriano Ramos-Onsins Rozas Calafell amp
Navarro 2008 Whitehead amp Crawford 2006) This underscores the
importance of generating a priori hypotheses about the molecular
underpinnings of adaptation and testing whether genetic and gene
expression variation at specific loci actually affects organismal per-
formance under different environmental conditions (Dalziel Rogers
amp Schulte 2009 Storz amp Wheat 2010)
21 | Developing and testing a priori hypotheses
There is a rich history of research in toxicology and biomedicine that
has investigated the molecular targets of H2S illuminating the mech-
anisms of H2S toxicity and detoxification in model organisms and
human cell cultures (see Li et al 2011 Olson Donald Dombkowski
amp Perry 2012 Wang 2012 Wallace amp Wang 2015 for recent
reviews) In a recent attempt of ldquoseparating hype and hoperdquo in
potential biomedical applications of H2S Olson (2011) lamented that
H2S ldquoseemingly affects all organ systems and biological processes in
which it has been investigatedrdquo While it often remains unclear
whether the documented effects are a direct consequence of H2S
exposure or a result of the disruption of cellular homeostasis this
body of work provides fruitful grounds for the development of
hypotheses about the molecular mechanisms underlying adaptation
to H2S-rich environments that can then be tested using genome
scans for selection and analyses of gene expression variation
between sulphide-adapted and nonadapted populations (see Table 1
for an overview) Most importantly candidates include direct toxicity
targets involved in OXPHOS (Cooper amp Brown 2008) and oxygen
transport (Pietri Roman-Morales amp Lopez-Garriga 2011) anaerobic
metabolism (Dubilier Giere amp Grieshaber 2005) and oxidative stress
response systems (Eghbal Pennefather amp OrsquoBrien 2004) that may
help mitigate adverse consequences of H2S exposure as well as
pathways involved in enzymatic H2S detoxification sulphur process-
ing and sulphur excretion (Hildebrandt amp Grieshaber 2008) It is
important to note that H2S is also produced endogenously by meta-
zoans in the cytosol and in mitochondria through enzymatic reac-
tions associated with the reverse trans-sulphuration pathway
(Stipanuk 2004 Stipanuk amp Ueki 2011) At concentrations well
below the toxicity threshold endogenously produced H2S serves as
a cellular signalling molecule that modulates physiological processes
including the function of ion channels that underlie smooth muscle
control and the function of neural cells (Peers Bauer Boyle Scragg
amp Dallas 2012) and it interacts with transcription factors that con-
trol inflammation cell survival and proliferation the expression of
cellular protective enzymes and hypoxia responses (Li et al 2011
Wallace amp Wang 2015 Wang 2012) Organisms may thus not strive
to merely eliminate H2S from their system and avoid effects of
toxicity Instead they are likely selected for maintaining optimal
endogenous concentrations that facilitate H2S-mediated signalling
and assure proper physiological functioning As a consequence
endogenous H2S production pathways and targets of H2S signalling
may also be modified during adaptation to environmental H2S
A series of studies in members of the P mexicana species com-
plex have tested hypotheses about the molecular underpinnings of
H2S adaptation by analysing patterns of molecular evolution in can-
didate genes (Barts et al 2018 Pfenninger et al 2014) deploying
genomewide scans for selection based on FST outliers (Pfenninger
et al 2015) and quantifying genomewide patterns of gene expres-
sion (Kelley et al 2016 Passow Henpita et al 2017) Collectively
the results of these studies have indicated a striking congruence
between candidate genes inferred from toxicological and biomedical
research and genes with empirical evidence for selection andor dif-
ferential expression between at least some of the adjacent H2S-
adapted and nonadapted populations (Table 1) (i) There was evi-
dence for selection on genes encoding the primary toxicity target of
H2S COX (complex IV in OXPHOS Pfenninger et al 2014) In addi-
tion genes encoding other components and assembly factors of
OXPHOS are either under selection or differentially expressed in sul-
phide spring populations (Kelley et al 2016 Pfenninger et al
2015) Evidence for selection and differential expression was also
uncovered for subunits of haemoglobin whose function is directly
impaired by H2S (Barts et al 2018) (ii) Genes involved in anaerobic
ATP production and in coping with oxidative stress exhibited evi-
dence for selection and differential expression suggesting a shift
towards alternative sources of ATP production and an increased abil-
ity to withstand reactive oxygen species that are produced as a by-
product of OXPHOS inhibition by H2S (Kelley et al 2016 Pfen-
ninger et al 2015) (iii) There is overwhelming evidence for selection
on and differential regulation of genes encoding components of the
sulphidequinone oxidoreductase pathway which mediates enzymatic
H2S detoxification in metazoans (Kelley et al 2016 Pfenninger
et al 2015) In addition there is evidence for upregulation of genes
encoding transporters of oxidized sulphur species which are puta-
tively involved in sulphur excretion (Kelley et al 2016)
These results suggest that adaptation to H2S-rich environments
in members of the P mexicana species complex involves both an
increased ability to cope with elevated endogenous H2S and an
increased ability to keep endogenous H2S concentrations low
through detoxification and excretion However there is a noticeable
absence of evidence for genes and physiological pathways associ-
ated with endogenous H2S production and H2S-mediated cell sig-
nalling being involved in adaptation (see Kelley et al 2016
Pfenninger et al 2015) There are three nonmutually exclusive
hypotheses that could explain this finding First the ability of popu-
lations from sulphidic habitats to regulate endogenous H2S through
detoxification may be sufficient to maintain normal H2S-mediated
signalling Second compared to the ability for detoxification and for
mitigation of direct toxic effects in terms of ATP production and
oxidative stress H2Srsquos influence on cell signalling may have relatively
low fitness consequences with concomitantly smaller evolutionary
846 | TOBLER ET AL
responses Last inferences from biomedical studies about the causal
relationship between endogenously produced H2S and various physi-
ological functions may be weak and some documented physiological
effects may instead be an indirect consequence of the disruption of
homeostasis that cascades through interconnected pathways operat-
ing in a cell In this case evolutionary studies on natural systems
exposed to H2S may provide important insights in terms of prioritiz-
ing research on biomedically relevant aspects of H2S biology Study-
ing ancestral populations susceptible to H2S will allow for
quantifying consequences of disruptions in H2S homeostasis at all
levels of biological organization (like in presently available animal
models) Adding derived H2S tolerant populations will further allow
for the detection of critical modifications in physiological processes
affected by H2S Consequently such a comparative approach may
provide a powerful tool to disentangle direct and indirect physiologi-
cal consequences of deviations in H2S homeostasis which is cur-
rently difficult in traditional biomedical models
Despite some of these discrepancies the studies exploring mech-
anisms underlying H2S tolerance in sulphide spring fishes highlight
the utility of research in toxicology and biomedicine in generating
testable hypotheses about evolutionary processes Biomedical stud-
ies on metabolic disorders (Aspiras Rohner Martineau Borowsky amp
Clifford 2015) stress responses (Rohner et al 2013) responses to
low oxygen (Eales Hollinshead amp Tennant 2016) and effects of
xenobiotics (Stockinger Di Meglio Gialitakis amp Duarte 2014) have
also facilitated our understanding of adaptive evolution in other nat-
ural systems Considering the increasingly specialized and frag-
mented research areas in the life sciences interdisciplinary
approaches and reciprocal exchange of ideas and methodologies
continue to be critical for facilitating breakthroughs in basic and
applied research on biomedical models and natural systems (Carroll
et al 2014 Nesse amp Stearns 2008 Schartl 2014)
22 | Convergent and nonconvergent evolution inindependent lineages
Our discussion of putative candidate genes that are under selection
andor differentially expressed in sulphide spring populations so far
focused on general patterns documented across evolutionarily inde-
pendent lineages in different river drainages Selection scans (Pfen-
ninger et al 2015) and analyses of differential gene expression
(Kelley et al 2016) have indicated high congruence of biological
processes associated with candidate genes across lineages in differ-
ent river drainages indicating that evolution has repeatedly and pre-
dictably modified the same physiological pathways during adaptation
to environmental H2S However the signal of evolutionary conver-
gence among sulphide spring lineages decreases with the level of
organismal organization (from physiological pathways down to geno-
mic regions genes and individual SNPs in selection scans Pfenninger
et al 2015 from physiological pathways down to genes and tran-
scripts in expression studies Passow Brown et al 2017) indicating
that different lineages have often modified the same physiological
pathways in different ways While knowledge about the biochemical
TABLE 1 Adaptation to H2S can occur through three principlemechanisms (1) Resistance involves direct toxicity targets whosefunctions are impaired by H2S Adaptive modifications of thesetoxicity targets are predicted to assure proper function despitethe presence of H2S (2) Mitigation involves alternativephysiological pathways that compensate for the inhibition ofdirect toxicity targets Adaptive modifications of these alternativephysiological pathways are predicted to allow for the maintenanceof organismal function by other means (3) Regulation involvesphysiological pathways associated with the maintenance ofendogenous H2S homeostasis Adaptive modifications ofregulatory pathways are predicted to maintain low endogenousconcentrations despite continuous flux of H2S from theenvironment into the body
Candidate pathways Evidence
(1) Resistance
OXPHOS bull Positive selection on COX in some lineages of
P mexicana (Puyacatengo and Pichucalco)a as
well as some other genera of sulphide spring
poeciliidsb COX amino acid substitutions in P
mexicana allow for the maintenance of function
in the presence of H2Sa
bull Upregulation of cytochrome c and some COX
subunits as well as downregulation of compo-
nents of complex I in all sulphide spring lineages
of P mexicana indicating complex changes in
aerobic ATP productionc
Oxygen transport
genesbull Positive selection on and upregulation of
several oxygen transport genes (including myo-
globin and multiple haemoglobin subunits) in
sulphide spring lineages of the genera
Gambusia Limia Poecilia and Pseudoxiphophorus
indicating modifications to putatively maintain
oxygen transport in the presence of H2Sd
(2) Mitigation
Anaerobic
metabolismbull Upregulation of genes associated with glycolysis
and gluconeogenesis in all sulphide spring lin-
eages of P mexicana indicating increased
capacity of anaerobic ATP productionc
Oxidative stress
responsesbull Upregulation of genes associated with
oxidative stress responses in all sulphide
spring lineages of P mexicana indicating
increased capacity to cope with H2S-induced
oxidative stressc
(3) Regulation
H2S detoxification bull Positive selection on and upregulation of sev-
eral genes associated with enzymatic H2S
detoxification in all sulphide spring lineages of
P mexicanace Upregulation of H2S detoxifica-
tion enzymes in other sulphide spring lineages
of the genera Gambusia Limia Poecilia Pseudox-
iphophorus and Xiphophorusb This indicates an
increased ability to maintain low endogenous
H2S concentrations during exposure
H2S production bull No evidence for modifications of genes associ-
ated with enzymatic production of H2Sc
aPfenninger et al (2014) bR Greenway J L Kelley amp M Tobler unpub-
lished data cKelley et al (2016) dBarts et al (2018) ePfenninger et al
(2015)
TOBLER ET AL | 847
and physiological consequences of H2S may thus enable us to pre-
dict the physiological pathways in which adaptive modifications
occur it may be impossible to predict specific modifications within
those pathways This contrasts with a common expectation in the
field that convergent evolution of molecular mechanisms underlying
phenotypic traits (ie parallel evolution) necessarily hinges on modi-
fication of the same loci across lineages as found in some of the
best-known examples of parallelism (Colosimo et al 2005 Feldman
Brodie Brodie amp Pfrender 2012 Grant Grant Markert Keller amp
Petren 2004) However modification of the same loci may only be
common under particular circumstances (Rosenblum Parent amp
Brandt 2014) for example when selection acts on standing genetic
variation (Colosimo et al 2005) when adaptive alleles are intro-
gressed across population boundaries through gene flow (Grant
et al 2004) or when there are strong genetic constraints (Feldman
et al 2012) Exploration of molecular mechanisms underlying con-
vergent evolution in many systems may therefore be better
approached from the perspective of interacting genes that are orga-
nized in pathways in which alternative genetic changes can have
equivalent functional consequences (ie functional redundancy)
Developing and testing hypotheses about parallel evolution at the
pathway level will hinge on improving genome annotations (Pavey
Bernatchez Aubin-Horth amp Landry 2012) and provide a more
nuanced understanding of the factors determining the predictability
of evolution at different levels of organismal organization (Rosen-
blum et al 2014)
Despite evidence for convergent evolution in some candidate
physiological pathways it also needs to be emphasized that this
does not apply to all P mexicana lineages or to all candidate physio-
logical pathways associated with H2S adaptation investigated to
date Nonconvergent evolutionmdashevidenced by unique lineage-speci-
fic signals of selection and differential expressionmdashmay be indicative
of unique molecular mechanisms to cope with H2S toxicity For
example signatures of positive selection on COXmdashH2Srsquos primary
toxicity targetmdashcan be found in sulphide spring lineages of the Puy-
acatengo and Pichucalco river drainages (a lineage with recent diver-
gence time and a lineage with older divergence time respectively)
but not in those of the Tacotalpa drainage (Pfenninger et al 2014)
The presence of nonconvergent genetic and gene expression differ-
ences between populations from sulphidic and non-sulphidic habitats
in different river drainages highlights the limitations of inferences
that can be drawn based on data sets generated with high-through-
put DNA and RNA sequencing The potential importance of modifi-
cations at candidate locimdashor the lack thereofmdashcan only be assessed
through functional assays that document how alternative alleles and
changes in gene expression actually affect organismal performance
The functional consequences of genetic and gene expression varia-
tion at most candidate loci remain to be investigated However
enzyme activity assays have indicated that COX maintains its activity
with increasing H2S concentrations in the two sulphide spring popu-
lations with evidence for positive selection on mitochondrially
encoded subunits (Puyacatengo and Pichucalco Pfenninger et al
2014) Key amino acid substitutions occur in the D-pathway channel
of COX1 which putatively narrow the channel width and reduce
H2Srsquos access to the binuclear centre of the protein Consequently
some lineages of P mexicana exhibit an H2S-resistant COX that can
maintain its activity and aerobic ATP production even if endogenous
H2S concentrations are elevated In contrast COX activity in sul-
phide spring fish of the Tacotalpa river drainage and in fish from
nonsulphidic habitats decreases when H2S is present which is con-
sistent with mechanisms of H2S toxicity (Pfenninger et al 2014) In
vitro assays on isolated mitochondria have nonetheless indicated
that an H2S-resistant COX is not necessary to maintain aerobic ATP
production during H2S exposure Unlike in populations from nonsul-
phidic habitats mitochondrial spare respiratory capacity is main-
tained in the presence of H2S even in the Tacotalpa sulphide spring
populations that lack an H2S-resistant COX (C Henpita M Tobler amp
JH Shaw unpublished data) This may indicate that modification of
detoxification pathways in sulphide spring fishes is sufficient to
maintain mitochondrial function and aerobic ATP production
23 | Ensuing challenges linking genotypes tofitness
Understanding the mechanistic basis of adaptation requires linking
genotypic variation to phenotypic traits that ultimately shape fitness
along selective gradients The evolution of H2S tolerance in sulphide
spring fishes appears to be a consequence of modifications in many
genes and many physiological pathways whose functions interact to
eliminate H2S from the system minimize effects of elevated endoge-
nous concentrations on direct toxicity targets and mitigate adverse
effects that cascade through cellular metabolism during disruption of
homeostasis (Table 1) Such complex genetic changes are likely com-
mon during adaptation and have been documented in other systems
that involve major evolutionary shifts including the emergence of
parasitic lifestyles (Werren et al 2010) eusociality (Simola et al
2013) asexual reproduction (Fradin et al 2017) and internal gesta-
tion with livebearing (Schartl et al 2013) Consequently comple-
mentary approaches will be required to uncover the molecular
mechanisms of adaptation to H2S in the future
1 The involvement of many genes and physiological pathways in
shaping tolerance emphasizes the importance of quantitative
genetic approaches that leverage experimental crosses between
H2S tolerant and nontolerant populations and test whether toler-
ancemdashand adaptive trait modifications that contribute to itmdashis
explained by a few major effects loci or whether it is a conse-
quence of a number of genes with small effects (Klerks Xie amp
Levinton 2011 Saltz Hessel amp Kelly 2017) Combining analyses
of phenotype-fitness associations and their underlying genetic
mechanisms via QTL mapping with the results of selection scans
from population genomic analyses (Pfenninger et al 2015) will
allow for an integrative understanding of how selection acting on
the genome results in phenotypic variation that mediates adapta-
tion to different environments (Stinchcombe amp Hoekstra 2008)
From a functional perspective breaking up correlations between
848 | TOBLER ET AL
different physiological traits (associated with resistance mitiga-
tion and regulation Table 1) in F2 crosses will further allow test-
ing what molecular mechanisms are necessary for tolerance
whether there are alternative strategies with equivalent fitness
benefits and whether there are complementary modifications
with additive effects on tolerance
2 Considering the number of genes and physiological pathways
likely involved in H2S tolerance assessing the functional conse-
quences of genetic and gene expression variation is a daunting
task At the same time such functional tests can significantly
contribute to closing the genotype-to-phenotype gap because
physiochemical stress responses can be assessed along the con-
tinuum of organismal organization (Gjuvsland Vik Beard Hunter
amp Omholt 2013) Standardized assays can be used to quantify
the biochemical makeup of organelles cells and tissues (eg pro-
tein abundance and enzyme activity) physiological performance
in vitro and in vivo (eg rates of H2S detoxification ATP produc-
tion and reactive oxygen production) and fitness-relevant proxies
of whole organism performance (eg H2S tolerance Figure 2)
This highlights the importance of methods development for the
quantification of complex phenotypic traits or even whole phe-
nomes that can keep pace with rapid data generation through
high-throughput sequencing (Houle Govindaraju amp Omholt
2010 Ritchie Holzinger Li Pendergrass amp Kim 2015) In addi-
tion genotypendashphenotype links established through such correla-
tive approaches will ideally be verified through targeted gene
editing While gene editing with CRISPRndashCas9 is now routinely
employed in model organisms (Doudna amp Charpentier 2014 Sha-
lem Sanjana amp Zhang 2015) applying this technology to non-
models still remains challenging especially in livebearing species
like poeciliid fishes where in vitro culture of embryos is not rou-
tinely employed (Martyn Weigel amp Dreyer 2006) Nonetheless
the effects of specific mutations can potentially be verified
through the establishment and manipulation of cells in culture
(Liu et al 2014) or by generating transgenics (Lin et al 2016)
3 Organismal responses to physiochemical stressors occur at multi-
ple hierarchical levels that can be plastically modulated in
response to endogenous and exogenous cues (Whitehead 2012)
Understanding adaptation to physiochemical stressors conse-
quently requires disentangling the effects of genetic variation
developmental plasticity and short-term acclimation in gene
expression patterns on the emerging physiological processes
Common garden experiments on sulphide spring fishes have
shown that many genes differentially expressed between popula-
tions from sulphidic and nonsulphidic habitats in nature remain
differentially expressed in laboratory-reared fish that have never
been exposed to H2S indicating evolved differences in constitu-
tive gene expression (Passow Henpita et al 2017) At the same
time expression patterns in the laboratory also exhibit evidence
for plasticity in response to short-term H2S exposure (Passow
Henpita et al 2017) Some of these plastic responses are shared
between populations from sulphidic and nonsulphidic habitats
(likely representing shared ancestral plasticity) while others have
been lost or gained during sulphide spring colonization A core
challenge for future research is to overcome logistical constraints
that have so far prevented long-term H2S exposures of fish in
the laboratory Exposing individuals throughout development will
be required to test how developmental plasticity and short-term
acclimation shape responses to H2S in individuals from adapted
and nonadapted populations Finally the next big scientific
advance in this context will also require the identification of reg-
ulatory mechanisms that shape expression differences during
adaptation including sequence changes in transcription factors
and their binding sites patterns of DNA methylation and chro-
matin accessibility as well as the production of microRNAs and
competing endogenous RNAs that can modulate transcript abun-
dance (Flores Wolschin amp Amdam 2013 Lopez-Maury Mar-
guerat amp Beuroahler 2008 Silva Bye el Azzouzi amp Wisloslashff 2017)
In addition the role of alternative splicing and other post-tran-
scriptional and post-translational modifications in mediating adap-
tation remains understudied by evolutionary biologists (Ast
2004 Bush Chen Tovar-Corona amp Urritia 2017) This is particu-
larly relevant because H2S has been hypothesized to play a role
in the post-translational modification of proteins with sulphur-
containing amino acids through sulfhydration (Mustafa et al
2009)
Bridging the genotypendashphenotype gap remains one of the major
challenges in biology (Gjuvsland et al 2013 Ritchie et al 2015) and
will provide opportunities to understand how organisms function in
the face of environmental stress and how evolution shapes these
functions during adaptation to different environmental conditions
Establishing the molecular mechanisms of adaptation across repli-
cated lineages exposed to the same sources of selection will also
provide insights into how evolutionary history demography and
genetics shape convergent evolutionary outcomes providing a better
understanding at what hierarchical levels of organismal organization
evolutionary change may actually be predictable (Rosenblum et al
2014)
3 | COMPLEX ORGANISMS IN A COMPLEXWORLD DISENTANGLING THE TANGLEDBANK
Mechanisms of adaptation can be studied through bottom-up
(screening for genetic modifications that allow for inferences about
relevant phenotypic traits) and top-down approaches (assessing fit-
ness-relevant phenotypic differences among populations without
knowledge about the genetic basis see Schluter 2009) Over the
years genomic and transcriptomic studies on sulphide spring popula-
tions of the P mexicana species complex have been complemented
with analyses of phenotypic variation Just as genetic analyses have
indicated that adaptation to H2S is mediated by the modification
and modulation of many genes associated with multiple physiological
pathways phenotypic studies uncovered a variety of differences
TOBLER ET AL | 849
between populations from sulphidic and nonsulphidic environments
Many of these phenotypic differences have evolved in convergence
across replicated sulphide spring lineages in different river drainages
Examples include modifications of morphological (body shape and
organ size Tobler et al 2011 Tobler Scharnweber et al 2015
Schulz-Mirbach et al 2016) physiological (tolerance and metabolic
rates Tobler et al 2011 Passow Arias-Rodriguez amp Tobler 2017)
behavioural (aggression and personality Riesch et al 2009 Bierbach
et al 2012 Sommer-Trembo et al 2016 Bierbach Arias Rodriguez
amp Plath 2018) and life history traits (fecundity and offspring size
Riesch Plath Garcia de Leon amp Schlupp 2010 Riesch et al 2014)
Hence proximate populations in different habitat types have
diverged in complex phenotypes that span many different suites of
traits and a key question is how divergence in multivariate pheno-
types arises In theory H2S could be a key source of selection driv-
ing multivariate phenotypic divergence either because it directly
exerts selection on a multitude of traits that are independently modi-
fied to reach new fitness peaks or because selection on a few fit-
ness-relevant traits causes simultaneous changes in correlated
characteristics Neither explanation seems sufficient because many
Abiotic environmentOxygen concentrations
Ionic compositionTemperature
Structure and flow
Biotic environmentTrophic resources
DemographyPredationParasitism
H2S
Genetic variation
Gene expression
Biochemistry
Physiologicalfunction
Organismal performance
Primary fitness components
Survivalgrowth
reproduction
Organismal function Environment
F IGURE 2 Understanding how organisms function in the context of their environment is a core question in evolutionary biology yet thecomplexities associated with organismal function and environmental variation provide significant challenges High-throughput sequencing hasallowed for the identification of candidate genes subject to selection and differential expression during adaptation to H2S The next step is tobridge the genotype-to-phenotype gap by quantifying the integrated responses to H2S across the continuum of organismal organization includingbiochemical phenotypes physiological functions whole organism performance and ultimately fitness In addition understanding adaptation toH2S-rich environments will also require acknowledging H2Srsquos relation to other abiotic and biotic environmental variables which can exert selectionand modulate aspects of organismal function This will allow for the establishment of cause-and-effect relationships between specific sources ofselection and evolutionary change in specific traits and contribute to our understanding of organismal function in natural systems
850 | TOBLER ET AL
of the divergent traits between populations from sulphidic and non-
sulphidic environments have no clearmdashor at best tenuousmdashlinks to
H2S toxicity and detoxification and there is only limited evidence
for within-population correlations between different traits groups
(Riesch et al 2016)
Evolutionary analyses frequently focus on seemingly simple
sources of selection juxtaposing populations that are assigned to dis-
crete habitat types to understand the evolution of specific traits
(Kaeuffer Peichel Bolnick amp Hendry 2012) But as Darwin (1859)
already observed in his ldquotangled bankrdquo allegory selection in natural
systems is rarely univariate but multifarious selective regimes con-
tribute to trait divergence even among populations that occur along
apparently simple environmental gradients Unquantified environmen-
tal variation that is subsumed in the categorization of habitat types
can profoundly affect trait variation in natural systems (Stuart et al
2017) highlighting the importance of considering and testing alterna-
tive hypotheses about the cause-and-effect relationship between
specific sources of selection and evolution of specific traits Thus
accurate natural history observations that consider organisms as holis-
tic entities consisting of interacting traits in the context of the abiotic
and biotic factors comprising their environment are critical to inform
the design and interpretation of empirical research in natural systems
(Greene 2005) Indeed adjacent sulphidic and nonsulphidic habitat
types do not merely differ in the presence or absence of H2S but also
in a variety of other abiotic and biotic factors (Figure 2) Unlike nonsul-
phidic habitats sulphide springs in southern Mexico are characterized
by extreme hypoxia elevated salinity and lower pH (Rosales Lagarde
2012 Tobler et al 2011) In addition the presence of physiochemical
stressors affects the biotic communities in these sulphide springs Spe-
cies richness of aquatic invertebrates and vertebrates is drastically
reduced as a consequence of H2S toxicity (Greenway Arias-Rodriguez
Diaz amp Tobler 2014) and primary production appears to be driven in
part by chemoautotrophic bacteria that use H2S as an energy source
rather than just by autochthonous and allochthonous photosynthetic
organisms (Roach Tobler amp Winemiller 2011) These changes in the
biotic environment affect the relative importance of intra-vs inter-
specific competition trophic resource use and exposure to predators
and parasites (Riesch Oranth et al 2010 Tobler et al 2014 Tobler
Scharnweber et al 2015)
31 | Alternative drivers of trait evolution
Acknowledging the complexity of selective regimes in sulphide
springs allows for a more nuanced understanding of trait evolution
and for the development of alternative hypotheses about the ulti-
mate mechanisms that cause divergence in specific genetic and phe-
notypic traits In some instances sources of selection other than H2S
can plausibly explain trait divergence between populations from sul-
phidic and nonsulphidic habitats For example hypoxia likely drives
increases in head size and gill surface area (Tobler et al 2011)
changes in the ionic composition of the water can explain differential
expression of genes associated with osmoregulation (Kelley et al
2016) and shifts in trophic resource use are associated with
modifications of the gastrointestinal system (Tobler Scharnweber
et al 2015) In other cases disentangling alternative hypotheses is
not trivial because selection mediated by H2S and other environmen-
tal factors are predicted to affect trait modifications in similar ways
Shifts towards anaerobic metabolism in sulphide spring fish could be
the consequence of inhibition of aerobic ATP production by H2S or
by constraints in oxygen availability Similarly reproductive life his-
tory shifts towards the production of larger but fewer offspring could
be driven by H2S (decreasing offspringrsquos surfacevolume ratio to mini-
mize the diffusion of H2S into the body Powell 1989) by habitat dif-
ferences in the quality and quantity of predation that can affect size-
specific rates of extrinsic mortality (Jorgensen Auer amp Reznick
2011) or by energetic constraints (see below) While comparisons
across different selective regimes can shed light into the ultimate dri-
vers of trait evolution (Moore Riesch amp Martin 2016) isolating the
effects of different sources of selection may not be possible because
they could act in concert and facilitate increases of organismal perfor-
mance in multiple contexts (Ghalambor Walker amp Reznick 2003)
When multifarious selection shapes multivariate phenotypic evo-
lution it is important to also consider the role of trade-offs both in
terms of trait expression (increased investment in one trait may
impose constraints on the expression of other traits) and in terms of
trait function (optimizing the trait for one function may impair the
traitrsquos utility for other functions) For example selection on large off-
spring size in sulphide spring fishes has inadvertent consequences
for fecundity (Riesch et al 2014) and selection for increased gill
surface areas due to hypoxia simultaneously increases exposure to
environmental H2S (Tobler et al 2011) Thus the outcome of phe-
notypic evolution is ultimately a result of balancing competing needs
in a manner that optimizes trait expression and organismal function
in multiple contexts to maximize fitness (Arnold 1983) and the pres-
ence of trade-offs can have cascading indirect effects with selection
on one or a few traits leading to the modification of a multitude of
apparently unrelated characteristics
In this context bioenergetics may represent a nexus in the evo-
lution of complex traits observed in sulphide spring fish and other
extreme environments Living in and adapting to sulphide springs
imposes constraints on organismal energy availability (Plath et al
2007) with according trade-offs for energy allocation Energy limita-
tion in sulphide springs does not arise from a scarcity of food
resources (chemoautotrophic bacteria eaten by the fish are abundant
in sulphide springs Tobler Scharnweber et al 2015) but from
behavioural and physiological adaptation A primary survival strategy
for fish in the toxic and hypoxic springs is to engage in aquatic sur-
face respiration where individuals breathe directly at the water sur-
face to maximize oxygen uptake and minimize H2S exposure This
behaviour mediates short-term survival (Plath et al 2007) but it
also constrains energy acquisition for benthic foragers like P mexi-
cana (Tobler Riesch Tobler amp Plath 2009) and increases susceptibil-
ity to bird predation (Riesch Oranth et al 2010) Costly investments
into H2S detoxification and tissue repair as well as a shift from aero-
bic to less efficient anaerobic metabolism further constrain energy
allocation to other functions (Passow Greenway Arias-Rodriguez
TOBLER ET AL | 851
Jeyasingh amp Tobler 2015) Theory predicts that energetic con-
straints ndash whether mediated by limitations in the rate of resource
acquisition or in the rate of energy allocation to reproduction (as
opposed to maintenance or growth) ndash should exert selection for a
reduction in organismal energy budgets which allows for the maxi-
mization of residual energy allocation for the production of offspring
(Brown Marquet amp Taper 1993) Several lines of evidence support
the hypothesis that energy limitation plays a critical role in pheno-
typic evolution of sulphide spring fish Compared to individuals in
nonsulphidic environments fish in sulphide springs have significantly
lower routine energy demands which is mediated both by reduc-
tions in body size and mass-specific metabolism (Passow Arias-
Rodriguez et al 2017) Reductions in other traits such as energeti-
cally costly behaviours (Bierbach et al 2012 Plath 2008) and the
size of energy-demanding organs (Schulz-Mirbach et al 2016) may
also contribute to the reduced energy demands and shifts in repro-
ductive life history traits (Riesch et al 2014) are consistent with
selection from energy limitation (Reznick amp Yang 1993)
32 | Ensuing challenges Disentanglingenvironmental and organismal complexities
Future research needs to empirically address how multifarious selec-
tive regimes shape the evolution of organisms with complex pheno-
types We have made great advances in our understanding of how
specific traits evolve and mediate adaptation (eg morphology Chan
et al 2010 coloration Dasmahapatra et al 2012 sensory organs
Cartwright Schwartz Merry amp Howell 2017) and we have a thor-
ough understanding of how single environmental factors can shape
organismal responses (eg climatic conditions Johnston amp Bennett
1996 predation Langerhans 2007 competition Pfennig amp Pfennig
2012) In contrast our understanding of phenotypic evolution in
response to multifarious selection is comparatively limited especially
because interactive effects of different environmental factors can lead
to organismal and evolutionary responses that are unpredictable based
on experiments that manipulate single environmental variables in iso-
lation (Gunderson Armstrong amp Stillman 2016 Todgham amp Stillman
2013) Future research thus needs to acknowledge the complexities of
environments and organismal design and address them explicitly in
empirical investigations to gain a robust understanding of biological
diversification (Culumber amp Tobler 2017 Stuart et al 2017)
4 | LINKING ADAPTATION TOSPECIATION
So far we primarily explored the causes of adaptive evolution but
what are its consequences Theory predicts that adaptation to local
environmental conditions can lead to speciation because reproduc-
tive isolation emerges incidentally as a by-product (ecological specia-
tion Nosil 2012) Population genetic analyses of Poecilia spp in
sulphide springs and adjacent populations in nonsulphidic environ-
ments have indicated strong genetic differentiation across small
spatial scales (in some instances lt100 m) and in the absence of
physical barriers that would prevent fish movement (Plath et al
2013) In addition population genetic structuring has been shown to
be stable even in the face of major environmental disturbance
BOX 1 Speciation in sulphide springs and conservation
biology
Adaptation to H2S-rich environments in poeciliids coincides
with ecological speciation giving rise to phenotypically dis-
tinct and reproductively isolated lineages In some
instances this has led to the evolution of highly distinct
micro-endemic species including Poecilia sulphuraria and
P thermalis Limia sulphurophila and Gambusia eurystoma
(Figure 3) Except for P sulphuraria which is found in two
spring complexes of the Rıo Pichucalco drainage of Mexico
(Tobler amp Plath 2009b) the distributions of these sulphide
spring endemics are restricted to single localities that
include only small areas of suitable habitat For example
the largest sulphide spring complex (the Ba~nos del Azufre
in southern Mexico which harbours P sulphuraria and G
eurystoma) includes ~18000 m2 of suitable habitat (equiva-
lent to lt35 football fields) while one of the smallest (La
Esperanza springs harbouring P thermalis) merely covers
~600 m2 (just over a tenth of a football field Brown et al
2017) Accordingly some sulphide spring endemics are
considered critically endangered (IUCN 2012) and pro-
tected by law (SEDESOL 1994)
Many threatened species with restricted ranges exhibit
low genetic diversity and high incidences of inbreeding
(Bouzat 2010 Willi Van Buskirk amp Hoffmann 2006) In
contrast at least P sulphuraria and P thermalismdashthe only
species investigated to datemdashactually exhibit relatively high
effective population sizes (Ne = 1200ndash3800 depending on
the population) and no evidence for inbreeding (FIS lt 0)
(Brown et al 2017) This is likely a consequence of the
long-term stability of sulphide springs and the high pro-
ductivity and low levels of interspecific competition also
support unusually high fish densities and thus population
census sizes (Brown et al 2017 Culumber et al 2016)
Nonetheless sulphide spring endemics are threatened by
habitat destruction which is mediated primarily by the
modification of springs for recreational purposes defor-
estation and conversion of land for agriculture and most
recently high-intensity palm oil plantations (Tobler amp Plath
2009ab) Despite the official protection of sulphide spring
endemics there have been no tangible conservation mea-
sures that foster the sustainable development of land
around sulphide springs with the well-being of the endemic
fishes in mind
852 | TOBLER ET AL
(catastrophic flood event Plath et al 2010) Population genetic dif-
ferentiation between adjacent sulphide-adapted and nonadapted
populations is indicative of ongoing ecological speciation (Box 1) but
different population pairs from sulphidic and nonsulphidic waters
have made variable progress along the speciation continuum towards
complete reproductive isolation (Plath et al 2013) This prompts
questions about the mechanisms involved in reducing gene flow
along the environmental gradient connecting the two habitat types
41 | Prezygotic mechanisms of reproductiveisolation
The simplest form of reproductive isolation between locally adapted
populations arises from selection against migrants between habitat
types (Nosil Vines amp Funk 2005) Translocation experiments in nat-
ural habitats have indicated strong survival selection against
migrants especially those translocated from nonsulphidic to sulphidic
habitats (Plath et al 2013) Fish translocated from nonsulphidic
environments face high mortality within short periods of time
(lt24 hr) which is likely caused by H2S toxicity and hypoxia in sul-
phide springs Hence low gene flow from populations inhabiting
nonsulphidic waters into populations inhabiting sulphidic habitats is
simply explained by a lack of adaptation to the extreme environmen-
tal conditions At least in some drainages there is also appreciable
mortality for sulphide spring fish translocated to nonsulphidic habi-
tats although mechanisms causing inviability are unclear and the
strength of selection appears to be lower (Plath et al 2013) If sur-
vival selection against immigrants was the sole source of reproduc-
tive isolation this should result in unidirectional gene flow from
H2S-adapted to nonadapted populations but population genetic
analyses provided no evidence for this implying that additional
mechanisms are at work (Plath et al 2013)
One such mechanism could be intersexual selection and mate
choice experiments have indicated that migrants from sulphidic to
nonsulphidic habitats are at a significant disadvantage with fish from
nonsulphidic habitats avoiding potential mating partners from sul-
phidic ones (Plath et al 2013 Sommer-Trembo et al 2016) Such
assortative mating has been found to be critical during ecological spe-
ciation because it counteracts the homogenizing effects of gene flow
(van Doorn Edelaar amp Weissing 2009) yet how assortative mating
patterns arise often remains unclear In theory assortative mating can
arise through a variety of processes (Nosil 2012) ldquoMagic traitrdquo mecha-
nisms where mating preferences evolve as a by-product of divergent
selection on ecological traits that serve as cues have recently garnered
much attention (Servedio van Doorn Kopp Frame amp Nosil 2011)
Consistent with magic trait mechanisms assortative mating in P mexi-
cana appears to be linked to adaptive population differences in body
shape which serve as cues to distinguish potential mating partners
from different habitats (Greenway Drexler Arias-Rodriguez amp Tobler
2016) However neither sulphide spring fish (which are unlikely to
encounter migrants from nonsulphidic habitats due to H2S toxicity)
nor allopatric P mexicana from nonsulphidic waters (which have never
experienced migrants from sulphide spring populations) exhibit a
preference for mating partners from their own ecotype suggesting
that assortative mating in this system is a product of reinforcement
(ie direct selection for assortative mating Greenway et al 2016)
This is different from classic magic traits mechanisms because assorta-
tive mating is not a mere by-product of adaptation but adaptive traits
have instead been co-opted as mating signals in response to selection
on mating preferences (Maan amp Seehausen 2012) Moreover males of
some H2S-adapted populations have reduced aggressive behaviour
(Bierbach et al 2012 2018) In staged contests of size-matched males
from different ecotypes intruders had a significantly lower likelihood
to establish dominance than resident males independent of whether
or not they showed reduced aggressiveness overall Hence intrasexual
selection may represent another mechanism mediating reproductive
isolation between locally adapted ecotypes Overall H2S-adapted poe-
ciliids allow disentangling the relative contributions of different forms
of ecological and sexual selectionmdashand their interdependencymdashduring
ecological speciation
42 | Ensuing challenges quantifying postzygoticmechanisms of reproductive isolation
In contrast to prezygotic mechanisms little is known about postzy-
gotic mechanisms of reproductive isolation that potentially act to
reduce gene flow between adjacent populations in sulphidic and
nonsulphidic habitats Putative hybrids between sulphidic and non-
sulphidic ecotypes have occasionally been observed in nonsulphidic
habitats immediately adjacent to confluences with sulphide springs
(R Riesch M Plath and M Tobler personal observations) but it
remains unclear whether and through what mechanisms such hybrids
exhibit reduced fitness compared to parentals
Direct selection of mitochondrially encoded genes in the pres-
ence of H2S (Pfenninger et al 2014) should be accompanied by co-
evolution at nuclear loci that directly interact with mitochondrial
gene products in the formation of protein complexes involved in the
OXPHOS pathway (Sunnucks Morales Lamb Pavlova amp Greening
2017 Willett amp Burton 2004) Disruption of co-evolved gene com-
plexes in hybrids should therefore lead to intrinsic fitness reductions
through mito-nuclear incompatibilities (Burton Pereira amp Barreto
2013 Wolff Ladoukakis Enrıquez amp Dowling 2014) which have
been hypothesized to be a key driver in speciation (Chou amp Leu
2010 Gershoni Templeton amp Mishmar 2009) Empirical tests of
these ideas remain to be conducted in sulphide spring fishes just
like studies on potential ecological and sexual selection against
hybrids that arise from interpopulation mating events
5 | PERSPECTIVES LIFE IN SULPHIDICENVIRONMENTS BEYOND P MEXICANA
Research on sulphide spring fishes of the P mexicana species com-
plex in Mexico has provided insights into mechanisms underlying
adaptation and the links between adaptation and speciation Sul-
phide springs do not only occur in Mexico however but are found
TOBLER ET AL | 853
worldwide and have been colonized by 24 evolutionarily indepen-
dent fish lineages in North America the Neotropics and the Middle
East (Greenway et al 2014) The vast majority of lineages belong to
the order Cyprinodontiformes (95) and livebearing members of the
family Poeciliidae in particular (80) This potentially indicates that
internal gestation of young has facilitated the colonization of
extreme environments because it shields developing embryos from
physiochemical stressors (Blackburn 1999) Among the Poeciliidae
sulphide spring fishes include members of the genera Brachyrhaphis
Gambusia Poeciliopsis Priapichthys Pseudoxiphophorus Xiphophorus
and multiple subgenera of Poecilia (Figure 3 Greenway et al 2014)
spanning over 40 million years of evolution (based on Reznick Fur-
ness Meredith amp Springer 2017)
A handful of studies have started to address molecular and phe-
notypic evolution across a broader taxon sampling of sulphide spring
fishes finding evidence for convergent evolution in morphology
(Riesch et al 2016 Tobler amp Hastings 2011) life history traits
(Riesch Plath et al 2010 Riesch et al 2014) and oxygen transport
genes (Barts et al 2018) In addition ecological speciation in
response to adaptation to H2S-rich environments has also been doc-
umented in sulphide spring poeciliids other than P mexicana (Riesch
et al 2016) These studies have laid a fruitful foundation for the
investigation of mechanisms of adaptation to H2S-rich springs from
genes to phenotypic traits linking micro-evolutionary processes to
macro-evolutionary patterns
The availability of broad-scale evolutionary replication of popula-
tions that have invaded habitats with the same physiochemical stressors
allows for addressing some of the ensuing challenges discussed above
and investigating the factors that give rise to predictable evolutionary
outcomes Convergent evolution is a commonmdashbut not omnipresentmdash
theme in adaptive evolution and multiple factors can determine the
degree of convergence among replicated lineages exposed to the same
sources of selection (Kaeuffer et al 2012 Landry amp Bernatchez 2010
Oke Rolshausen LeBlond amp Hendry 2017 Rosenblum amp Harmon
2011) Thus leveraging replicated lineages that have colonized sulphide
springs to assess molecular mechanisms underlying convergent pheno-
typic changes analyse how environmental heterogeneity among sul-
phide spring habitats causes lineage-specific patterns of trait evolution
and test whether and by what mechanisms adaptation to extreme envi-
ronments is linked to speciation will provide insights into how ecologi-
cal genetic and functional factors contribute to evolutionary
convergence at different levels of organismal organization (Rosenblum
et al 2014) An expanded taxon sampling with population pairs that
span almost the full range of the speciation continuum from panmixia to
complete reproductive isolation (Riesch et al 2016) will also help
addressing questions about temporal aspects of H2S adaptation In par-
ticular comparing younger to older lineages will provide insights about
the order at which genetic and phenotypic modifications accrue during
the adaptation process
Animals have also invaded H2S-rich habitats in other aquatic
ecosystems including cold seeps and deep-sea hydrothermal vents
(Tobler et al 2016) Adaptation to H2S in the P mexicana species com-
plex has involved modification of physiological pathways associated
with H2S toxicity (OXPHOS pathway) and detoxification (sulphide
F IGURE 3 Phylogeny of the family Poeciliidae (adopted from Culumber amp Tobler 2017) Various lineages in the genera Poecilia (subgeneraAcanthophacelus Limia and Mollienesia) Priapichthys Brachyrhaphis Poeciliopsis Xiphophorus Pseudoxiphophorus and Gambusia have colonizedH2S-rich springs in different parts of the Americas (see Greenway et al 2014) Lineages highlighted in yellow represent endemic speciesrestricted to H2S-rich springs while red lineages represent species in which some populations have invaded sulphidic habitats Pictures depict arepresentative of each focal genussubgenus
854 | TOBLER ET AL
quinone oxidoreductase pathway) which have been highly conserved
during the diversification of life on Earth (Saraste 1999 Shahak amp
Hauska 2008) The high replicability of H2S as an environmental factor
that shaped animal evolution across vastly different ecological contexts
consequently offers the opportunity to investigate whether selection
mediated by the presence of H2S has repeatedly modified these con-
served physiological pathways across animal phyla that have diverged
during the Cambrian explosion spanning over 500 million years of evo-
lution Such comparisons would provide unprecedented insights into
the effects of a single physiochemical stressor on evolutionary change
irrespective of ecological or phylogenetic contexts
6 | CONCLUSIONS
H2S-rich environments and their inhabitants represent useful models
for research in organismal biology ecology and evolution illustrating
both opportunities (eg strong and explicit source of selection evolu-
tionary replication) and challenges (eg complexity of environments
and organismal responses) in asking basic question about the origins
of biodiversity Understanding how organisms thrive in apparently
inhospitable environments will provide us with insights into lifersquos
capacities and limitations to adapt to novel environments (Waterman
1999) This is particularly relevant considering that human activities
substantially contribute to the exacerbation of physiochemical stres-
sors at local regional and global scales impacting ecological and evo-
lutionary dynamics (Alberti 2015 Norberg Urban Vellend
Klausmeier amp Loeuille 2012) Thus a focus on ecological and evolu-
tionary consequences of natural stressors that push organisms to the
brink of their physiological capacities may be instrumental to predict
consequences of anthropogenic environmental change
ACKNOWLEDGEMENTS
We would like to thank Lenin Arias-Rodriguez the Universidad
Juarez Autonoma de Tabasco and the Centro de Investigacion E
Innovacion Para la Ense~nanza y El Aprendizaje for their continued
collaboration enabling our work on the P mexicana system Funding
for this review article has been provided by the National Science
Foundation (IOS-1557860 IOS-1557795) and the US Army
Research Office (W911NF-15-1-0175)
AUTHOR CONTRIBUTIONS
MT wrote the first draft of the paper all authors contributed text
and comments
ORCID
Michael Tobler httporcidorg0000-0002-0326-0890
Joanna L Kelley httporcidorg0000-0002-7731-605X
Reuroudiger Riesch httporcidorg0000-0002-0223-1254
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Ast G (2004) How did alternative splicing evolve Nature Reviews
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Bell E M (2012) Life at extremes Environments organisms and strategies
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Bierbach D Klein M Sassmannshausen V Schlupp I Riesch R
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Blackburn D G (1999) Viviparity and oviparity Evolution and reproduc-
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Bouzat J L (2010) Conservation genetics of population bottlenecks
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Brown A P Greenway R Morgan S Quackenbush C R Giordani
L Arias-Rodriguez L Kelley J L (2017) Genome-scale data
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Brown J H Marquet P A amp Taper M L (1993) Evolution of body
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nurev-ecolsys-110512-135758
Bush S J Chen L Tovar-Corona J M amp Urritia A O (2017) Alterna-
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Carroll S P Jorgensen P S Kinnison M T Bergstrom C T Denison
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Cartwright R A Schwartz R S Merry A L amp Howell M M (2017)
The importance of selection in the evolution of blindness in cavefish
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017-0876-4
Chan Y F Marks M E Jones F C Villarreal G Shapiro M D Brady
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Chou J Y amp Leu J Y (2010) Speciation through cytonuclear incompat-
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BioEssays 32 401ndash411 httpsdoiorg101002bies200900162
Colosimo P F Hosemann K E Balabhadra S Villarreal G Dickson
M Grimwood J Kingsley D M (2005) Widespread parallel evo-
lution in sticklebacks by repeated fixation of ectodysplasin alleles
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Cooper C E amp Brown G C (2008) The inhibition of mitochondrial
cytochrome oxidase by the gases carbon monoxide nitric oxide
hydrogen cyanide and hydrogen sulfide Chemical mechanism and
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533ndash539 httpsdoiorg101007s10863-008-9166-6
Culumber Z W Hopper G W Barts N Passow C N Morgan S Brown
A Tobler M (2016) Habitat use of two extremophile highly ende-
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Freshwater Ecosystems 26 1155ndash1167 httpsdoiorg101002aqc
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Culumber Z W amp Tobler M (2017) Sex-specific evolution during the
diversification of live-bearing fishes Nature Ecology amp Evolution 1
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Dalziel A C Rogers S M amp Schulte P M (2009) Linking genotypes
to phenotypes and fitness How mechanistic biology can inform
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Darwin C (1859) On the origin of species based on natural selection or
the preservation of favoured races in the struggle of life 6th ed Lon-
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Dasmahapatra K K Walters J R Briscoe A D Davey J W Whibley
A Nadeau N J Jiggins C D (2012) Butterfly genome reveals
promiscuous exchange of mimicry adaptations among species Nature
487 94ndash98 httpsdoiorg101038nature11041
Doudna J A amp Charpentier E (2014) The new frontier of genome edit-
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1126science1258096
Dubilier N Giere O amp Grieshaber M (2005) Concomitant effects of sul-
fide and hypoxia on the aerobic metabolism of the marine oligochaete
Tubificoides benedii Journal of Experimental Zoology 269 287ndash298
Eales K L Hollinshead K E R amp Tennant D A (2016) Hypoxia and
metabolic adaptation of cancer cells Oncogenesis 5 e190 httpsd
oiorg101038oncsis201550
Eghbal M A Pennefather P S amp OrsquoBrien P J (2004) H2S cytotoxicity
mechanism involves reactive oxygen species formation and mito-
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1016jtox200405020
Feldman C R Brodie Jr E D Brodie III E D amp Pfrender M E (2012)
Constraint shapes convergence in tetrodotoxin-resistant sodium
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Flores K B Wolschin F amp Amdam G V (2013) The role of methyla-
tion of DNA in environmental adaptation Integrative and Comparative
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Fradin H Kiontke K Zegar C Gutwein M Lucas J Kovtun M
Piano F (2017) Genome architecture and evolution of a unichromo-
somal asexual nematode Current Biology 27(2928ndash2939) e2926
Gershoni M Templeton A amp Mishmar D (2009) Mitochondrial bioen-
ergetics as a major motive force of speciation BioEssays 31 642ndash
650 httpsdoiorg101002bies200800139
Ghalambor C K Walker J A amp Reznick D N (2003) Multi-trait selec-
tion adaptation and constraints on the evolution of burst swimming
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Gibert J amp Deharveng L (2002) Subterranean ecosystems A truncated
functional biodiversity BioScience 52 473ndash481 httpsdoiorg10
16410006-3568(2002)052[0473SEATFB]20CO2
Gjuvsland A B Vik J O Beard D A Hunter P J amp Omholt S W
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Journal of Physiology 591 2055ndash2066 httpsdoiorg101113jphys
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Grant P R Grant B R Markert J A Keller L F amp Petren K (2004)
Convergent evolution of Darwinrsquos finches caused by introgressive
hybridization and selection Evolution 58 1588ndash1599 httpsdoi
org101111j0014-38202004tb01738x
Greene H W (2005) Organisms in nature as a central focus for biology
Trends in Ecology and Evolution 20 23ndash27 httpsdoiorg101016j
tree200411005
Greenway R Arias-Rodriguez L Diaz P amp Tobler M (2014) Patterns
of macroinvertebrate and fish diversity in freshwater sulphide springs
Diversity 6 597ndash632 httpsdoiorg103390d6030597
Greenway R Drexler S Arias-Rodriguez L amp Tobler M (2016) Adap-
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to assortative mating preferences during ecological speciation Evolu-
tion 70 2809ndash2822 httpsdoiorg101111evo13087
Gunderson A R Armstrong E J amp Stillman J H (2016) Multiple stres-
sors in a changing world The need for an improved perspective on
physiological responses to the dynamic marine environment Annual
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nurev-marine-122414-033953
Hildebrandt T M amp Grieshaber M (2008) Three enzymatic activities
catalyze the oxidation of sulfide to thiosulfate in mammalian and
invertebrate mitochondria FEBS Journal 275 3352ndash3361 httpsd
oiorg101111j1742-4658200806482x
Houle D Govindaraju D R amp Omholt S (2010) Phenomics The next
challenge Nature Reviews Genetics 11 855ndash866 httpsdoiorg10
1038nrg2897
IUCN (2012) Red List of threatened species version 20132 www
iucnredlistorg
Johnston I A amp Bennett A F (1996) Animals and temperature Pheno-
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Jorgensen C Auer S K amp Reznick D N (2011) A model for optimal
offspring size in fish including live-bearing and parental effects Amer-
ican Naturalist 177 E119ndashE135 httpsdoiorg101086659622
Kaeuffer R Peichel C L Bolnick D I amp Hendry A P (2012) Parallel
and nonparallel aspects of ecological phenotypic and genetic diver-
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back Evolution 66 402ndash418 httpsdoiorg101111j1558-5646
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Kell D B amp Oliver S G (2004) Here is the evidence now what is the
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856 | TOBLER ET AL
Kelley J L Arias-Rodriguez L Patacsil Martin D Yee M C Busta-
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life in hydrogen sulfide rich environments Molecular Biology and Evo-
lution 33 1419ndash1434 httpsdoiorg101093molbevmsw020
Kelley J L Passow C Plath M Arias-Rodriguez L Yee M C amp
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1471-2164-13-652
Klerks P L Xie L amp Levinton J S (2011) Quantitative genetics
approaches to study evolutionary processes in ecotoxicology a per-
spective from research on the evolution of resistance Ecotoxicology
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Kump L R Pavlov A amp Arthur M A (2005) Massive release of hydrogen
sulfide to the surface ocean and atmosphere during intervals of oceanic
anoxia Geology 33 397ndash400 httpsdoiorg101130G212951
Keuroustner A Hoffmann M Fraser B A Kottler V A Sharma E Wei-
gel D amp Dreyer C (2016) The genome of the Trinidadian guppy
Poecilia reticulata and variation in the guanapo population PLoS ONE
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Landry L amp Bernatchez L (2010) Role of epibenthic resource opportu-
nities in the parallel evolution of lake whitefish species pairs (Core-
gonus sp) Journal of Evolutionary Biology 23 2602ndash2613 httpsdoi
org101111j1420-9101201002121x
Langerhans R B (2007) Evolutionary consequences of predation Avoid-
ance escape reproduction and diversification In A M T Elewa
(Ed) Predation in organisms Berlin Springer
Li L Rose P amp Moore P K (2011) Hydrogen sulfide and cell signaling
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Lin Q Fan S Zhang Y Xu M Zhang H Yang Y Gunter H M
(2016) The seahorse genome and the evolution of its specialized mor-
phology Nature 540 395ndash399 httpsdoiorg101038nature20595
Liu Y Ma S Wang X Chang J Gao J Shi R Zhao P (2014)
Highly efficient multiplex targeted mutagenesis and genomic struc-
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ibmb201403010
Lopez-Maury L Marguerat S amp Beuroahler J (2008) Tuning gene expres-
sion to changing environments From rapid responses to evolutionary
adaptation Nature Reviews Genetics 9 583ndash593 httpsdoiorg10
1038nrg2398
Maan M E amp Seehausen O (2012) Magic traits versus magic prefer-
ences in speciation Evolutionary Ecology Research 14 779ndash785
Martyn U Weigel D amp Dreyer C (2006) In vitro culture of embryos
of the guppy Poecilia reticulata Developmental Dynamics 235 617ndash
622 httpsdoiorg101002(ISSN)1097-0177
Metzker M L (2010) Sequencing technologies The next generation
Nature Reviews Genetics 11 31ndash46 httpsdoiorg101038nrg2626
Moore M P Riesch R amp Martin R A (2016) The predictability and
magnitude of life-history divergence to ecological agents of selection
A meta-analysis in livebearing fishes Ecology Letters 19 435ndash442
httpsdoiorg101111ele12576
Mustafa A K Gadalla M M Sen N Kim S Mu W Gazi S K
Snyder S H (2009) H2S signals through protein S-sulfhydration
Science Signaling 2 ra72
Nesse R M amp Stearns S C (2008) The great opportunity Evolutionary
applications to medicine and public health Evolutionary Applications
1 28ndash48 httpsdoiorg101111j1752-4571200700006x
Nevo E (2011) Evolution under environmental stress at macro and
microscales Genome Biology and Evolution 3 1039ndash1052 httpsdoi
org101093gbeevr052
Norberg J Urban M C Vellend M Klausmeier C A amp Loeuille N (2012)
Eco-evolutionary responses of biodiversity to climate change Nature Cli-
mate Change 2 747ndash751 httpsdoiorg101038nclimate1588
Nosil P (2012) Ecological speciation Oxford UK Oxford University
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0001
Nosil P Vines T H amp Funk D J (2005) Perspective Reproductive
isolation caused by natural selection against immigrants from diver-
gent habitats Evolution 59 705ndash719
Oke K B Rolshausen G LeBlond C amp Hendry A P (2017) How par-
allel is parallel evolution A comparative analysis in fishes American
Naturalist 190 1ndash16 httpsdoiorg101086691989
Olson K R (2011) The therapeutic potential of hydrogen sulfide
Separating hype from hope American Journal of Physiology-Regulatory
Integrative and Comparative Physiology 301 R297ndashR312 httpsdoi
org101152ajpregu000452011
Olson K R Donald J A Dombkowski R A amp Perry S F (2012) Evo-
lutionary and comparative aspects of nitric oxide carbon monoxide
and hydrogen sulfide Respiratory Physiology amp Neurobiology 184
117ndash129 httpsdoiorg101016jresp201204004
Olson K R amp Straub K D (2015) The role of hydrogen sulfide in evo-
lution and the evolution of hydrogen sulfide in metabolism and sig-
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Palacios M Arias-Rodriguez L Plath M Eifert C Lerp H Lamboj A
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nalpone0071069
Palacios M Voelker G Arias-Rodriguez L Mateos M amp Tobler M
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Passow C Arias-Rodriguez L amp Tobler M (2017) Convergent evolu-
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e0186935 httpsdoiorg101371journalpone0186935
Passow C N Brown A Arias-Rodriguez L Yee M-C Sockell A
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patterns in natural populations of an extremophile fish (Poecilia mexi-
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101111mec14198
Passow C Greenway R Arias-Rodriguez L Jeyasingh P D amp Tobler
M (2015) Reduction of energetic demands through modification of
body size and metabolic rates in locally adapted extremophile fishes
Physiological and Biochemical Zoology 88 371ndash383 httpsdoiorg
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Passow C N Henpita C Shaw J H Quakenbush C Warren W C
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tionary change in shaping gene expression variation in natural popu-
lations of extremophile fish Molecular Ecology 26 6384ndash6399
Pavey S A Bernatchez L Aubin-Horth N amp Landry C R (2012)
What is needed for next-generation ecological and evolutionary
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org101016jtree201207014
Peers C Bauer C Boyle J P Scragg J L amp Dallas M L (2012)
Modulation of ion channels by hydrogen sulfide Antioxidants amp Redox
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Pfennig D W amp Pfennig K S (2012) Evolutionrsquos wedge competition and
the origins of diversity Berkeley CA University of California Press
httpsdoiorg101525california97805202741810010001
Pfenninger M Lerp H Tobler M Passow C Kelley J L Funke E
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fish as key adaptation to a toxic environment Nature Communica-
tions 5 3873
Pfenninger M Patel S Arias-Rodriguez L Feldmeyer B Riesch R amp
Plath M (2015) Unique evolutionary trajectories in repeated adapta-
tion to hydrogen sulphide-toxic habitats of a neotropical fish (Poecilia
TOBLER ET AL | 857
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1111mec13397
Pietri R Roman-Morales E amp Lopez-Garriga J (2011) Hydrogen sul-
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Redox Signaling 15 393ndash404 httpsdoiorg101089ars20103698
Plath M (2008) Male mating behavior and costs of sexual harassment
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Plath M Hermann C Schreurooder R Riesch R Tobler M Garcia de
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maintain small-scale genetic differentiation despite perturbation by a
catastrophic flood event BMC Evolutionary Biology 10 256 httpsd
oiorg1011861471-2148-10-256
Plath M Pfenninger M Lerp H Riesch R Eschenbrenner C Slattery
P Tobler M (2013) Genetic differentiation and selection against
migrants in evolutionarily replicated extreme environments Evolution
67 2647ndash2661 httpsdoiorg101111evo12133
Plath M Tobler M Riesch R Garcia de Leon F J Giere O amp
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httpsdoiorg101007s00114-007-0279-2
Powell E (1989) Oxygen sulfide and diffusion Why thiobiotic meiofauna
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Ramirez-Soriano A Ramos-Onsins S E Rozas J Calafell F amp Navarro A
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expansions contractions and bottlenecks with recombination Genetics
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Reiffenstein R Hulbert W amp Roth S (1992) Toxicology of hydrogen
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Reznick D N Furness A I Meredith R W amp Springer M S (2017)
The origin and biogeographic diversification of fishes in the family
Poeciliidae PLoS ONE 12 e0172546 httpsdoiorg101371journa
lpone0172546
Reznick D amp Yang A P (1993) The influence of fluctuating resources
on life history Patterns of allocation and plasticity in female guppies
Ecology 74 2011ndash2019 httpsdoiorg1023071940844
Riesch R Duwe V Herrmann N Padur L Ramm A Scharnweber K
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s00265-009-0780-z
Riesch R Oranth A Dzienko J Karau N Schiessl A Stadler S
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from increased aerial predation risk in sulphidic southern Mexican
habitats Biological Journal of the Linnean Society 101 417ndash426
httpsdoiorg101111j1095-8312201001522x
Riesch R Plath M Garcia de Leon F J amp Schlupp I (2010) Conver-
gent life-history shifts Toxic environments result in big babies in two
clades of poeciliids Naturwissenschaften 97 133ndash141 httpsdoi
org101007s00114-009-0613-y
Riesch R Plath M Schlupp I Tobler M amp Langerhans R B (2014)
Colonization of toxic springs drives predictable life-history shift in
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Riesch R Tobler M Lerp H Jourdan J Doumas L T Nosil P Plath
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Riesch R Tobler M amp Plath M (2015) Extremophile fishes Ecology
evolution and physiology of teleosts in extreme environments p 290
Heidelberg Germany Springer httpsdoiorg101007978-3-319-
13362-1
Ritchie M E Holzinger E R Li R Pendergrass S A amp Kim S (2015)
Methods of integrating data to uncover genotype-phenotype interac-
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Roach K Tobler M amp Winemiller K O (2011) Hydrogen sulfide bac-
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2062 httpsdoiorg10189011-02761
Rohner N Jarosz D F Kowalko J E Yoshizawa M Jeffery W R
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Science 342 1372ndash1375 httpsdoiorg101126science1240276
Rosales Lagarde L (2012) Investigation of karst brackish-sulfidic springs
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of Mining and Technology Mexico
Rosenblum E B amp Harmon L J (2011) ldquoSame but differentrdquo Repli-
cated ecological speciation at White Sands Evolution 65 946ndash960
httpsdoiorg101111j1558-5646201001190x
Rosenblum E B Parent C E amp Brandt E E (2014) The molecular
basis of convergent evolution Annual Review of Ecology Evolution and
Systematics 45 203ndash226 httpsdoiorg101146annurev-ecolsys-
120213-091851
Saltz J B Hessel F C amp Kelly M W (2017) Trait correlations in the
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Saraste M (1999) Oxidative phosphorylation at the fin de siecle Science
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Schartl M (2014) Beyond the zebrafish Diverse fish species for model-
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Schartl M Walter R B Shen Y Garcia T Catchen J Amores A
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1038ng2604
Schluter D (2009) Evidence for ecological speciation and its alternative
Science 323 737ndash741 httpsdoiorg101126science1160006
Schulz-Mirbach T Eifert C Riesch R Farnworth M S Zimmer C
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SEDESOL (1994) NORMA Oficial Mexicana NOM-059-ECOL-1994 que
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Servedio M R van Doorn G S Kopp M Frame A M amp Nosil P
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Shalem O Sanjana N E amp Zhang F (2015) High-throughput func-
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Silva G J J Bye A el Azzouzi H amp Wisloslashff U (2017) MicroRNAs as
important regulators of exercise adaptation Progress in Cardiovascular
Diseases 60 130ndash151 httpsdoiorg101016jpcad201706003
Simola D F Wissler L Donahue G Waterhouse R M Helmkampf
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858 | TOBLER ET AL
Sommer-Trembo C Bierbach D Arias Rodriguez L Verel Y Jourdan
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Stinchcombe J R amp Hoekstra H E (2008) Combining population geno-
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important traits Heredity 100 158ndash170 httpsdoiorg101038sj
hdy6800937
Stipanuk M H (2004) Sulfur amino acid metabolism Pathways for pro-
duction and removal of homocysteine and cysteine Annual Review of
Nutrition 24 539ndash577 httpsdoiorg101146annurevnutr24
012003132418
Stipanuk M H amp Ueki I (2011) Dealing with methioninehomocysteine
sulfur Cysteine metabolism to taurine and inorganic sulfur Journal of
Inherited Metabolic Disease 34 17ndash32 httpsdoiorg101007
s10545-009-9006-9
Stockinger B Di Meglio P Gialitakis M amp Duarte J H (2014) The
aryl hydrocarbon receptor Multitasking in the immune system
Annual Review of Immunology 32 403ndash432 httpsdoiorg101146
annurev-immunol-032713-120245
Storz J F amp Wheat C W (2010) Integrating evolutionary and
functional approaches to infer adaptation at specific loci Evolution
64 2489ndash2509 httpsdoiorg101111j1558-5646201001044x
Stuart Y E Veen T Weber J N Hanson D Ravinet M Lohman B
K Bolnick D I (2017) Contrasting effects of environment and
genetics generate a continuum of parallel evolution Nature Ecology amp
Evolution 1 0158 httpsdoiorg101038s41559-017-0158
Sunnucks P Morales H E Lamb A M Pavlova A amp Greening C
(2017) Integrative approaches for studying mitochondrial and nuclear
genome co-evolution in oxidative phosphorylation Frontiers in Genet-
ics 8 25
Tobler M amp Hastings L (2011) Convergent patterns of body shape dif-
ferentiation in four different clades of poeciliid fishes inhabiting sul-
fide springs Evolutionary Biology 38 412ndash421 httpsdoiorg10
1007s11692-011-9129-4
Tobler M Palacios M Chapman L J Mitrofanov I Bierbach D
Plath M Mateos M (2011) Evolution in extreme environments
Replicated phenotypic differentiation in livebearing fish inhabiting
sulfidic springs Evolution 65 2213ndash2228 httpsdoiorg101111j
1558-5646201101298x
Tobler M Passow C N Greenway R Kelley J L amp Shaw J H
(2016) The evolutionary ecology of animals inhabiting hydrogen sul-
fide-rich environments Annual Review of Ecology Evolution and Sys-
tematics 47 239ndash262 httpsdoiorg101146annurev-ecolsys-
121415-032418
Tobler M amp Plath M (2009a) Threatened fishes of the world Gambu-
sia eurystoma Miller 1975 (Poeciliidae) Environmental Biology of
Fishes 85 251 httpsdoiorg101007s10641-009-9481-8
Tobler M amp Plath M (2009b) Threatened fishes of the world Poecilia
sulphuraria (Alvarez 1948) (Poeciliidae) Environmental Biology of
Fishes 85 333ndash334 httpsdoiorg101007s10641-009-9506-3
Tobler M Plath M Riesch R Schlupp I Grasse A Munimanda G
Moodley Y (2014) Selection from parasites favors immunogenic diver-
sity but not divergence among locally adapted host populations Journal
of Evolutionary Biology 27 960ndash974 httpsdoiorg101111jeb12370
Tobler M Riesch R amp Plath M (2015) Extremophile fishes An inte-
grative synthesis In R Riesch M Tobler amp M Plath (Eds) Extremo-
phile fishes Ecology evolution and physiology of teleosts in extreme
environments (pp 279ndash296) Heidelberg Germany Springer
Tobler M Riesch R Tobler C M amp Plath M (2009) Compensatory
behaviour in response to sulfide-induced hypoxia affects time
budgets feeding efficiency and predation risk Evolutionary Ecology
Research 11 935ndash948
Tobler M Scharnweber K Greenway R Passow C Arias-Rodriguez
L amp Garcia-De-Leon F J (2015) Convergent changes in the
trophic ecology if extremophile fish along replicated environmental
gradients Freshwater Biology 60 768ndash780 httpsdoiorg10
1111fwb12530
Todgham A E amp Stillman J H (2013) Physiological responses to shifts
in multiple environmental stressors Relevance in a changing worlds
Integrative and Comparative Biology 53 539ndash544 httpsdoiorg10
1093icbict086
van Doorn G S Edelaar P amp Weissing F J (2009) On the origin of
species by natural selection and sexual selection Science 326 1704ndash
1707 httpsdoiorg101126science1181661
Wall D H amp Virginia R A (1999) Controls on soil biodiversity Insights
from extreme environments Applied Soil Ecology 13 137ndash150
httpsdoiorg101016S0929-1393(99)00029-3
Wallace J L amp Wang R (2015) Hydrogen-sulfide based therapeutics
Exploiting a unique but ubiquitous gasotransmitter Nature Reviews
Drug Discovery 14 329ndash345 httpsdoiorg101038nrd4433
Wang R (2012) Physiological implications of hydrogen sulfide A whiff
exploration that blossomed Physiological Reviews 92 791ndash896
httpsdoiorg101152physrev000172011
Wang Z Gerstein M amp Snyder M (2009) RNA-seq A revolutionary
tool for transcriptomics Nature Reviews Genetics 10 57ndash63
httpsdoiorg101038nrg2484
Waterman T H (1999) The evolutionary challenges of extreme environ-
ments (part 1) Journal of Experimental Zoology 285 326ndash359
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AID-JEZ3ampgt30CO2-T
Werren J H Richards S Desjardins C A Niehuis O Gadau J Col-
bourne J K amp Group N G W (2010) Functional and evolutionary
insights from the genomes of three parasitoid Nasonia species
Science 327 343ndash348 httpsdoiorg101126science1178028
Whitehead A (2012) Comparative genomics in ecological physiology
Toward a more nuanced understanding of acclimation and adapta-
tion Journal of Experimental Biology 215 884ndash891 httpsdoiorg
101242jeb058735
Whitehead A amp Crawford D L (2006) Neutral and adaptive variation
in gene expression Proceedings of the National Academy of Sciences
USA 103 5425ndash5430 httpsdoiorg101073pnas0507648103
Willett C S amp Burton R S (2004) Evolution of interacting proteins in
the mitochondrial electron transport system in a marine copepod
Molecular Biology and Evolution 21 443ndash453 httpsdoiorg10
1093molbevmsh031
Willi Y Van Buskirk J amp Hoffmann A (2006) Limits to the adaptive
potential of small populations Annual Review of Ecology Evolution
and Systematics 37 433ndash458 httpsdoiorg101146annurevecol
sys37091305110145
Wolff J N Ladoukakis E D Enrıquez J A amp Dowling D K (2014)
Mitonuclear interactions Evolutionary consequences over multiple
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How to cite this article Tobler M Kelley JL Plath M Riesch
R Extreme environments and the origins of biodiversity
Adaptation and speciation in sulphide spring fishes Mol Ecol
201827843ndash859 httpsdoiorg101111mec14497
TOBLER ET AL | 859
responses Last inferences from biomedical studies about the causal
relationship between endogenously produced H2S and various physi-
ological functions may be weak and some documented physiological
effects may instead be an indirect consequence of the disruption of
homeostasis that cascades through interconnected pathways operat-
ing in a cell In this case evolutionary studies on natural systems
exposed to H2S may provide important insights in terms of prioritiz-
ing research on biomedically relevant aspects of H2S biology Study-
ing ancestral populations susceptible to H2S will allow for
quantifying consequences of disruptions in H2S homeostasis at all
levels of biological organization (like in presently available animal
models) Adding derived H2S tolerant populations will further allow
for the detection of critical modifications in physiological processes
affected by H2S Consequently such a comparative approach may
provide a powerful tool to disentangle direct and indirect physiologi-
cal consequences of deviations in H2S homeostasis which is cur-
rently difficult in traditional biomedical models
Despite some of these discrepancies the studies exploring mech-
anisms underlying H2S tolerance in sulphide spring fishes highlight
the utility of research in toxicology and biomedicine in generating
testable hypotheses about evolutionary processes Biomedical stud-
ies on metabolic disorders (Aspiras Rohner Martineau Borowsky amp
Clifford 2015) stress responses (Rohner et al 2013) responses to
low oxygen (Eales Hollinshead amp Tennant 2016) and effects of
xenobiotics (Stockinger Di Meglio Gialitakis amp Duarte 2014) have
also facilitated our understanding of adaptive evolution in other nat-
ural systems Considering the increasingly specialized and frag-
mented research areas in the life sciences interdisciplinary
approaches and reciprocal exchange of ideas and methodologies
continue to be critical for facilitating breakthroughs in basic and
applied research on biomedical models and natural systems (Carroll
et al 2014 Nesse amp Stearns 2008 Schartl 2014)
22 | Convergent and nonconvergent evolution inindependent lineages
Our discussion of putative candidate genes that are under selection
andor differentially expressed in sulphide spring populations so far
focused on general patterns documented across evolutionarily inde-
pendent lineages in different river drainages Selection scans (Pfen-
ninger et al 2015) and analyses of differential gene expression
(Kelley et al 2016) have indicated high congruence of biological
processes associated with candidate genes across lineages in differ-
ent river drainages indicating that evolution has repeatedly and pre-
dictably modified the same physiological pathways during adaptation
to environmental H2S However the signal of evolutionary conver-
gence among sulphide spring lineages decreases with the level of
organismal organization (from physiological pathways down to geno-
mic regions genes and individual SNPs in selection scans Pfenninger
et al 2015 from physiological pathways down to genes and tran-
scripts in expression studies Passow Brown et al 2017) indicating
that different lineages have often modified the same physiological
pathways in different ways While knowledge about the biochemical
TABLE 1 Adaptation to H2S can occur through three principlemechanisms (1) Resistance involves direct toxicity targets whosefunctions are impaired by H2S Adaptive modifications of thesetoxicity targets are predicted to assure proper function despitethe presence of H2S (2) Mitigation involves alternativephysiological pathways that compensate for the inhibition ofdirect toxicity targets Adaptive modifications of these alternativephysiological pathways are predicted to allow for the maintenanceof organismal function by other means (3) Regulation involvesphysiological pathways associated with the maintenance ofendogenous H2S homeostasis Adaptive modifications ofregulatory pathways are predicted to maintain low endogenousconcentrations despite continuous flux of H2S from theenvironment into the body
Candidate pathways Evidence
(1) Resistance
OXPHOS bull Positive selection on COX in some lineages of
P mexicana (Puyacatengo and Pichucalco)a as
well as some other genera of sulphide spring
poeciliidsb COX amino acid substitutions in P
mexicana allow for the maintenance of function
in the presence of H2Sa
bull Upregulation of cytochrome c and some COX
subunits as well as downregulation of compo-
nents of complex I in all sulphide spring lineages
of P mexicana indicating complex changes in
aerobic ATP productionc
Oxygen transport
genesbull Positive selection on and upregulation of
several oxygen transport genes (including myo-
globin and multiple haemoglobin subunits) in
sulphide spring lineages of the genera
Gambusia Limia Poecilia and Pseudoxiphophorus
indicating modifications to putatively maintain
oxygen transport in the presence of H2Sd
(2) Mitigation
Anaerobic
metabolismbull Upregulation of genes associated with glycolysis
and gluconeogenesis in all sulphide spring lin-
eages of P mexicana indicating increased
capacity of anaerobic ATP productionc
Oxidative stress
responsesbull Upregulation of genes associated with
oxidative stress responses in all sulphide
spring lineages of P mexicana indicating
increased capacity to cope with H2S-induced
oxidative stressc
(3) Regulation
H2S detoxification bull Positive selection on and upregulation of sev-
eral genes associated with enzymatic H2S
detoxification in all sulphide spring lineages of
P mexicanace Upregulation of H2S detoxifica-
tion enzymes in other sulphide spring lineages
of the genera Gambusia Limia Poecilia Pseudox-
iphophorus and Xiphophorusb This indicates an
increased ability to maintain low endogenous
H2S concentrations during exposure
H2S production bull No evidence for modifications of genes associ-
ated with enzymatic production of H2Sc
aPfenninger et al (2014) bR Greenway J L Kelley amp M Tobler unpub-
lished data cKelley et al (2016) dBarts et al (2018) ePfenninger et al
(2015)
TOBLER ET AL | 847
and physiological consequences of H2S may thus enable us to pre-
dict the physiological pathways in which adaptive modifications
occur it may be impossible to predict specific modifications within
those pathways This contrasts with a common expectation in the
field that convergent evolution of molecular mechanisms underlying
phenotypic traits (ie parallel evolution) necessarily hinges on modi-
fication of the same loci across lineages as found in some of the
best-known examples of parallelism (Colosimo et al 2005 Feldman
Brodie Brodie amp Pfrender 2012 Grant Grant Markert Keller amp
Petren 2004) However modification of the same loci may only be
common under particular circumstances (Rosenblum Parent amp
Brandt 2014) for example when selection acts on standing genetic
variation (Colosimo et al 2005) when adaptive alleles are intro-
gressed across population boundaries through gene flow (Grant
et al 2004) or when there are strong genetic constraints (Feldman
et al 2012) Exploration of molecular mechanisms underlying con-
vergent evolution in many systems may therefore be better
approached from the perspective of interacting genes that are orga-
nized in pathways in which alternative genetic changes can have
equivalent functional consequences (ie functional redundancy)
Developing and testing hypotheses about parallel evolution at the
pathway level will hinge on improving genome annotations (Pavey
Bernatchez Aubin-Horth amp Landry 2012) and provide a more
nuanced understanding of the factors determining the predictability
of evolution at different levels of organismal organization (Rosen-
blum et al 2014)
Despite evidence for convergent evolution in some candidate
physiological pathways it also needs to be emphasized that this
does not apply to all P mexicana lineages or to all candidate physio-
logical pathways associated with H2S adaptation investigated to
date Nonconvergent evolutionmdashevidenced by unique lineage-speci-
fic signals of selection and differential expressionmdashmay be indicative
of unique molecular mechanisms to cope with H2S toxicity For
example signatures of positive selection on COXmdashH2Srsquos primary
toxicity targetmdashcan be found in sulphide spring lineages of the Puy-
acatengo and Pichucalco river drainages (a lineage with recent diver-
gence time and a lineage with older divergence time respectively)
but not in those of the Tacotalpa drainage (Pfenninger et al 2014)
The presence of nonconvergent genetic and gene expression differ-
ences between populations from sulphidic and non-sulphidic habitats
in different river drainages highlights the limitations of inferences
that can be drawn based on data sets generated with high-through-
put DNA and RNA sequencing The potential importance of modifi-
cations at candidate locimdashor the lack thereofmdashcan only be assessed
through functional assays that document how alternative alleles and
changes in gene expression actually affect organismal performance
The functional consequences of genetic and gene expression varia-
tion at most candidate loci remain to be investigated However
enzyme activity assays have indicated that COX maintains its activity
with increasing H2S concentrations in the two sulphide spring popu-
lations with evidence for positive selection on mitochondrially
encoded subunits (Puyacatengo and Pichucalco Pfenninger et al
2014) Key amino acid substitutions occur in the D-pathway channel
of COX1 which putatively narrow the channel width and reduce
H2Srsquos access to the binuclear centre of the protein Consequently
some lineages of P mexicana exhibit an H2S-resistant COX that can
maintain its activity and aerobic ATP production even if endogenous
H2S concentrations are elevated In contrast COX activity in sul-
phide spring fish of the Tacotalpa river drainage and in fish from
nonsulphidic habitats decreases when H2S is present which is con-
sistent with mechanisms of H2S toxicity (Pfenninger et al 2014) In
vitro assays on isolated mitochondria have nonetheless indicated
that an H2S-resistant COX is not necessary to maintain aerobic ATP
production during H2S exposure Unlike in populations from nonsul-
phidic habitats mitochondrial spare respiratory capacity is main-
tained in the presence of H2S even in the Tacotalpa sulphide spring
populations that lack an H2S-resistant COX (C Henpita M Tobler amp
JH Shaw unpublished data) This may indicate that modification of
detoxification pathways in sulphide spring fishes is sufficient to
maintain mitochondrial function and aerobic ATP production
23 | Ensuing challenges linking genotypes tofitness
Understanding the mechanistic basis of adaptation requires linking
genotypic variation to phenotypic traits that ultimately shape fitness
along selective gradients The evolution of H2S tolerance in sulphide
spring fishes appears to be a consequence of modifications in many
genes and many physiological pathways whose functions interact to
eliminate H2S from the system minimize effects of elevated endoge-
nous concentrations on direct toxicity targets and mitigate adverse
effects that cascade through cellular metabolism during disruption of
homeostasis (Table 1) Such complex genetic changes are likely com-
mon during adaptation and have been documented in other systems
that involve major evolutionary shifts including the emergence of
parasitic lifestyles (Werren et al 2010) eusociality (Simola et al
2013) asexual reproduction (Fradin et al 2017) and internal gesta-
tion with livebearing (Schartl et al 2013) Consequently comple-
mentary approaches will be required to uncover the molecular
mechanisms of adaptation to H2S in the future
1 The involvement of many genes and physiological pathways in
shaping tolerance emphasizes the importance of quantitative
genetic approaches that leverage experimental crosses between
H2S tolerant and nontolerant populations and test whether toler-
ancemdashand adaptive trait modifications that contribute to itmdashis
explained by a few major effects loci or whether it is a conse-
quence of a number of genes with small effects (Klerks Xie amp
Levinton 2011 Saltz Hessel amp Kelly 2017) Combining analyses
of phenotype-fitness associations and their underlying genetic
mechanisms via QTL mapping with the results of selection scans
from population genomic analyses (Pfenninger et al 2015) will
allow for an integrative understanding of how selection acting on
the genome results in phenotypic variation that mediates adapta-
tion to different environments (Stinchcombe amp Hoekstra 2008)
From a functional perspective breaking up correlations between
848 | TOBLER ET AL
different physiological traits (associated with resistance mitiga-
tion and regulation Table 1) in F2 crosses will further allow test-
ing what molecular mechanisms are necessary for tolerance
whether there are alternative strategies with equivalent fitness
benefits and whether there are complementary modifications
with additive effects on tolerance
2 Considering the number of genes and physiological pathways
likely involved in H2S tolerance assessing the functional conse-
quences of genetic and gene expression variation is a daunting
task At the same time such functional tests can significantly
contribute to closing the genotype-to-phenotype gap because
physiochemical stress responses can be assessed along the con-
tinuum of organismal organization (Gjuvsland Vik Beard Hunter
amp Omholt 2013) Standardized assays can be used to quantify
the biochemical makeup of organelles cells and tissues (eg pro-
tein abundance and enzyme activity) physiological performance
in vitro and in vivo (eg rates of H2S detoxification ATP produc-
tion and reactive oxygen production) and fitness-relevant proxies
of whole organism performance (eg H2S tolerance Figure 2)
This highlights the importance of methods development for the
quantification of complex phenotypic traits or even whole phe-
nomes that can keep pace with rapid data generation through
high-throughput sequencing (Houle Govindaraju amp Omholt
2010 Ritchie Holzinger Li Pendergrass amp Kim 2015) In addi-
tion genotypendashphenotype links established through such correla-
tive approaches will ideally be verified through targeted gene
editing While gene editing with CRISPRndashCas9 is now routinely
employed in model organisms (Doudna amp Charpentier 2014 Sha-
lem Sanjana amp Zhang 2015) applying this technology to non-
models still remains challenging especially in livebearing species
like poeciliid fishes where in vitro culture of embryos is not rou-
tinely employed (Martyn Weigel amp Dreyer 2006) Nonetheless
the effects of specific mutations can potentially be verified
through the establishment and manipulation of cells in culture
(Liu et al 2014) or by generating transgenics (Lin et al 2016)
3 Organismal responses to physiochemical stressors occur at multi-
ple hierarchical levels that can be plastically modulated in
response to endogenous and exogenous cues (Whitehead 2012)
Understanding adaptation to physiochemical stressors conse-
quently requires disentangling the effects of genetic variation
developmental plasticity and short-term acclimation in gene
expression patterns on the emerging physiological processes
Common garden experiments on sulphide spring fishes have
shown that many genes differentially expressed between popula-
tions from sulphidic and nonsulphidic habitats in nature remain
differentially expressed in laboratory-reared fish that have never
been exposed to H2S indicating evolved differences in constitu-
tive gene expression (Passow Henpita et al 2017) At the same
time expression patterns in the laboratory also exhibit evidence
for plasticity in response to short-term H2S exposure (Passow
Henpita et al 2017) Some of these plastic responses are shared
between populations from sulphidic and nonsulphidic habitats
(likely representing shared ancestral plasticity) while others have
been lost or gained during sulphide spring colonization A core
challenge for future research is to overcome logistical constraints
that have so far prevented long-term H2S exposures of fish in
the laboratory Exposing individuals throughout development will
be required to test how developmental plasticity and short-term
acclimation shape responses to H2S in individuals from adapted
and nonadapted populations Finally the next big scientific
advance in this context will also require the identification of reg-
ulatory mechanisms that shape expression differences during
adaptation including sequence changes in transcription factors
and their binding sites patterns of DNA methylation and chro-
matin accessibility as well as the production of microRNAs and
competing endogenous RNAs that can modulate transcript abun-
dance (Flores Wolschin amp Amdam 2013 Lopez-Maury Mar-
guerat amp Beuroahler 2008 Silva Bye el Azzouzi amp Wisloslashff 2017)
In addition the role of alternative splicing and other post-tran-
scriptional and post-translational modifications in mediating adap-
tation remains understudied by evolutionary biologists (Ast
2004 Bush Chen Tovar-Corona amp Urritia 2017) This is particu-
larly relevant because H2S has been hypothesized to play a role
in the post-translational modification of proteins with sulphur-
containing amino acids through sulfhydration (Mustafa et al
2009)
Bridging the genotypendashphenotype gap remains one of the major
challenges in biology (Gjuvsland et al 2013 Ritchie et al 2015) and
will provide opportunities to understand how organisms function in
the face of environmental stress and how evolution shapes these
functions during adaptation to different environmental conditions
Establishing the molecular mechanisms of adaptation across repli-
cated lineages exposed to the same sources of selection will also
provide insights into how evolutionary history demography and
genetics shape convergent evolutionary outcomes providing a better
understanding at what hierarchical levels of organismal organization
evolutionary change may actually be predictable (Rosenblum et al
2014)
3 | COMPLEX ORGANISMS IN A COMPLEXWORLD DISENTANGLING THE TANGLEDBANK
Mechanisms of adaptation can be studied through bottom-up
(screening for genetic modifications that allow for inferences about
relevant phenotypic traits) and top-down approaches (assessing fit-
ness-relevant phenotypic differences among populations without
knowledge about the genetic basis see Schluter 2009) Over the
years genomic and transcriptomic studies on sulphide spring popula-
tions of the P mexicana species complex have been complemented
with analyses of phenotypic variation Just as genetic analyses have
indicated that adaptation to H2S is mediated by the modification
and modulation of many genes associated with multiple physiological
pathways phenotypic studies uncovered a variety of differences
TOBLER ET AL | 849
between populations from sulphidic and nonsulphidic environments
Many of these phenotypic differences have evolved in convergence
across replicated sulphide spring lineages in different river drainages
Examples include modifications of morphological (body shape and
organ size Tobler et al 2011 Tobler Scharnweber et al 2015
Schulz-Mirbach et al 2016) physiological (tolerance and metabolic
rates Tobler et al 2011 Passow Arias-Rodriguez amp Tobler 2017)
behavioural (aggression and personality Riesch et al 2009 Bierbach
et al 2012 Sommer-Trembo et al 2016 Bierbach Arias Rodriguez
amp Plath 2018) and life history traits (fecundity and offspring size
Riesch Plath Garcia de Leon amp Schlupp 2010 Riesch et al 2014)
Hence proximate populations in different habitat types have
diverged in complex phenotypes that span many different suites of
traits and a key question is how divergence in multivariate pheno-
types arises In theory H2S could be a key source of selection driv-
ing multivariate phenotypic divergence either because it directly
exerts selection on a multitude of traits that are independently modi-
fied to reach new fitness peaks or because selection on a few fit-
ness-relevant traits causes simultaneous changes in correlated
characteristics Neither explanation seems sufficient because many
Abiotic environmentOxygen concentrations
Ionic compositionTemperature
Structure and flow
Biotic environmentTrophic resources
DemographyPredationParasitism
H2S
Genetic variation
Gene expression
Biochemistry
Physiologicalfunction
Organismal performance
Primary fitness components
Survivalgrowth
reproduction
Organismal function Environment
F IGURE 2 Understanding how organisms function in the context of their environment is a core question in evolutionary biology yet thecomplexities associated with organismal function and environmental variation provide significant challenges High-throughput sequencing hasallowed for the identification of candidate genes subject to selection and differential expression during adaptation to H2S The next step is tobridge the genotype-to-phenotype gap by quantifying the integrated responses to H2S across the continuum of organismal organization includingbiochemical phenotypes physiological functions whole organism performance and ultimately fitness In addition understanding adaptation toH2S-rich environments will also require acknowledging H2Srsquos relation to other abiotic and biotic environmental variables which can exert selectionand modulate aspects of organismal function This will allow for the establishment of cause-and-effect relationships between specific sources ofselection and evolutionary change in specific traits and contribute to our understanding of organismal function in natural systems
850 | TOBLER ET AL
of the divergent traits between populations from sulphidic and non-
sulphidic environments have no clearmdashor at best tenuousmdashlinks to
H2S toxicity and detoxification and there is only limited evidence
for within-population correlations between different traits groups
(Riesch et al 2016)
Evolutionary analyses frequently focus on seemingly simple
sources of selection juxtaposing populations that are assigned to dis-
crete habitat types to understand the evolution of specific traits
(Kaeuffer Peichel Bolnick amp Hendry 2012) But as Darwin (1859)
already observed in his ldquotangled bankrdquo allegory selection in natural
systems is rarely univariate but multifarious selective regimes con-
tribute to trait divergence even among populations that occur along
apparently simple environmental gradients Unquantified environmen-
tal variation that is subsumed in the categorization of habitat types
can profoundly affect trait variation in natural systems (Stuart et al
2017) highlighting the importance of considering and testing alterna-
tive hypotheses about the cause-and-effect relationship between
specific sources of selection and evolution of specific traits Thus
accurate natural history observations that consider organisms as holis-
tic entities consisting of interacting traits in the context of the abiotic
and biotic factors comprising their environment are critical to inform
the design and interpretation of empirical research in natural systems
(Greene 2005) Indeed adjacent sulphidic and nonsulphidic habitat
types do not merely differ in the presence or absence of H2S but also
in a variety of other abiotic and biotic factors (Figure 2) Unlike nonsul-
phidic habitats sulphide springs in southern Mexico are characterized
by extreme hypoxia elevated salinity and lower pH (Rosales Lagarde
2012 Tobler et al 2011) In addition the presence of physiochemical
stressors affects the biotic communities in these sulphide springs Spe-
cies richness of aquatic invertebrates and vertebrates is drastically
reduced as a consequence of H2S toxicity (Greenway Arias-Rodriguez
Diaz amp Tobler 2014) and primary production appears to be driven in
part by chemoautotrophic bacteria that use H2S as an energy source
rather than just by autochthonous and allochthonous photosynthetic
organisms (Roach Tobler amp Winemiller 2011) These changes in the
biotic environment affect the relative importance of intra-vs inter-
specific competition trophic resource use and exposure to predators
and parasites (Riesch Oranth et al 2010 Tobler et al 2014 Tobler
Scharnweber et al 2015)
31 | Alternative drivers of trait evolution
Acknowledging the complexity of selective regimes in sulphide
springs allows for a more nuanced understanding of trait evolution
and for the development of alternative hypotheses about the ulti-
mate mechanisms that cause divergence in specific genetic and phe-
notypic traits In some instances sources of selection other than H2S
can plausibly explain trait divergence between populations from sul-
phidic and nonsulphidic habitats For example hypoxia likely drives
increases in head size and gill surface area (Tobler et al 2011)
changes in the ionic composition of the water can explain differential
expression of genes associated with osmoregulation (Kelley et al
2016) and shifts in trophic resource use are associated with
modifications of the gastrointestinal system (Tobler Scharnweber
et al 2015) In other cases disentangling alternative hypotheses is
not trivial because selection mediated by H2S and other environmen-
tal factors are predicted to affect trait modifications in similar ways
Shifts towards anaerobic metabolism in sulphide spring fish could be
the consequence of inhibition of aerobic ATP production by H2S or
by constraints in oxygen availability Similarly reproductive life his-
tory shifts towards the production of larger but fewer offspring could
be driven by H2S (decreasing offspringrsquos surfacevolume ratio to mini-
mize the diffusion of H2S into the body Powell 1989) by habitat dif-
ferences in the quality and quantity of predation that can affect size-
specific rates of extrinsic mortality (Jorgensen Auer amp Reznick
2011) or by energetic constraints (see below) While comparisons
across different selective regimes can shed light into the ultimate dri-
vers of trait evolution (Moore Riesch amp Martin 2016) isolating the
effects of different sources of selection may not be possible because
they could act in concert and facilitate increases of organismal perfor-
mance in multiple contexts (Ghalambor Walker amp Reznick 2003)
When multifarious selection shapes multivariate phenotypic evo-
lution it is important to also consider the role of trade-offs both in
terms of trait expression (increased investment in one trait may
impose constraints on the expression of other traits) and in terms of
trait function (optimizing the trait for one function may impair the
traitrsquos utility for other functions) For example selection on large off-
spring size in sulphide spring fishes has inadvertent consequences
for fecundity (Riesch et al 2014) and selection for increased gill
surface areas due to hypoxia simultaneously increases exposure to
environmental H2S (Tobler et al 2011) Thus the outcome of phe-
notypic evolution is ultimately a result of balancing competing needs
in a manner that optimizes trait expression and organismal function
in multiple contexts to maximize fitness (Arnold 1983) and the pres-
ence of trade-offs can have cascading indirect effects with selection
on one or a few traits leading to the modification of a multitude of
apparently unrelated characteristics
In this context bioenergetics may represent a nexus in the evo-
lution of complex traits observed in sulphide spring fish and other
extreme environments Living in and adapting to sulphide springs
imposes constraints on organismal energy availability (Plath et al
2007) with according trade-offs for energy allocation Energy limita-
tion in sulphide springs does not arise from a scarcity of food
resources (chemoautotrophic bacteria eaten by the fish are abundant
in sulphide springs Tobler Scharnweber et al 2015) but from
behavioural and physiological adaptation A primary survival strategy
for fish in the toxic and hypoxic springs is to engage in aquatic sur-
face respiration where individuals breathe directly at the water sur-
face to maximize oxygen uptake and minimize H2S exposure This
behaviour mediates short-term survival (Plath et al 2007) but it
also constrains energy acquisition for benthic foragers like P mexi-
cana (Tobler Riesch Tobler amp Plath 2009) and increases susceptibil-
ity to bird predation (Riesch Oranth et al 2010) Costly investments
into H2S detoxification and tissue repair as well as a shift from aero-
bic to less efficient anaerobic metabolism further constrain energy
allocation to other functions (Passow Greenway Arias-Rodriguez
TOBLER ET AL | 851
Jeyasingh amp Tobler 2015) Theory predicts that energetic con-
straints ndash whether mediated by limitations in the rate of resource
acquisition or in the rate of energy allocation to reproduction (as
opposed to maintenance or growth) ndash should exert selection for a
reduction in organismal energy budgets which allows for the maxi-
mization of residual energy allocation for the production of offspring
(Brown Marquet amp Taper 1993) Several lines of evidence support
the hypothesis that energy limitation plays a critical role in pheno-
typic evolution of sulphide spring fish Compared to individuals in
nonsulphidic environments fish in sulphide springs have significantly
lower routine energy demands which is mediated both by reduc-
tions in body size and mass-specific metabolism (Passow Arias-
Rodriguez et al 2017) Reductions in other traits such as energeti-
cally costly behaviours (Bierbach et al 2012 Plath 2008) and the
size of energy-demanding organs (Schulz-Mirbach et al 2016) may
also contribute to the reduced energy demands and shifts in repro-
ductive life history traits (Riesch et al 2014) are consistent with
selection from energy limitation (Reznick amp Yang 1993)
32 | Ensuing challenges Disentanglingenvironmental and organismal complexities
Future research needs to empirically address how multifarious selec-
tive regimes shape the evolution of organisms with complex pheno-
types We have made great advances in our understanding of how
specific traits evolve and mediate adaptation (eg morphology Chan
et al 2010 coloration Dasmahapatra et al 2012 sensory organs
Cartwright Schwartz Merry amp Howell 2017) and we have a thor-
ough understanding of how single environmental factors can shape
organismal responses (eg climatic conditions Johnston amp Bennett
1996 predation Langerhans 2007 competition Pfennig amp Pfennig
2012) In contrast our understanding of phenotypic evolution in
response to multifarious selection is comparatively limited especially
because interactive effects of different environmental factors can lead
to organismal and evolutionary responses that are unpredictable based
on experiments that manipulate single environmental variables in iso-
lation (Gunderson Armstrong amp Stillman 2016 Todgham amp Stillman
2013) Future research thus needs to acknowledge the complexities of
environments and organismal design and address them explicitly in
empirical investigations to gain a robust understanding of biological
diversification (Culumber amp Tobler 2017 Stuart et al 2017)
4 | LINKING ADAPTATION TOSPECIATION
So far we primarily explored the causes of adaptive evolution but
what are its consequences Theory predicts that adaptation to local
environmental conditions can lead to speciation because reproduc-
tive isolation emerges incidentally as a by-product (ecological specia-
tion Nosil 2012) Population genetic analyses of Poecilia spp in
sulphide springs and adjacent populations in nonsulphidic environ-
ments have indicated strong genetic differentiation across small
spatial scales (in some instances lt100 m) and in the absence of
physical barriers that would prevent fish movement (Plath et al
2013) In addition population genetic structuring has been shown to
be stable even in the face of major environmental disturbance
BOX 1 Speciation in sulphide springs and conservation
biology
Adaptation to H2S-rich environments in poeciliids coincides
with ecological speciation giving rise to phenotypically dis-
tinct and reproductively isolated lineages In some
instances this has led to the evolution of highly distinct
micro-endemic species including Poecilia sulphuraria and
P thermalis Limia sulphurophila and Gambusia eurystoma
(Figure 3) Except for P sulphuraria which is found in two
spring complexes of the Rıo Pichucalco drainage of Mexico
(Tobler amp Plath 2009b) the distributions of these sulphide
spring endemics are restricted to single localities that
include only small areas of suitable habitat For example
the largest sulphide spring complex (the Ba~nos del Azufre
in southern Mexico which harbours P sulphuraria and G
eurystoma) includes ~18000 m2 of suitable habitat (equiva-
lent to lt35 football fields) while one of the smallest (La
Esperanza springs harbouring P thermalis) merely covers
~600 m2 (just over a tenth of a football field Brown et al
2017) Accordingly some sulphide spring endemics are
considered critically endangered (IUCN 2012) and pro-
tected by law (SEDESOL 1994)
Many threatened species with restricted ranges exhibit
low genetic diversity and high incidences of inbreeding
(Bouzat 2010 Willi Van Buskirk amp Hoffmann 2006) In
contrast at least P sulphuraria and P thermalismdashthe only
species investigated to datemdashactually exhibit relatively high
effective population sizes (Ne = 1200ndash3800 depending on
the population) and no evidence for inbreeding (FIS lt 0)
(Brown et al 2017) This is likely a consequence of the
long-term stability of sulphide springs and the high pro-
ductivity and low levels of interspecific competition also
support unusually high fish densities and thus population
census sizes (Brown et al 2017 Culumber et al 2016)
Nonetheless sulphide spring endemics are threatened by
habitat destruction which is mediated primarily by the
modification of springs for recreational purposes defor-
estation and conversion of land for agriculture and most
recently high-intensity palm oil plantations (Tobler amp Plath
2009ab) Despite the official protection of sulphide spring
endemics there have been no tangible conservation mea-
sures that foster the sustainable development of land
around sulphide springs with the well-being of the endemic
fishes in mind
852 | TOBLER ET AL
(catastrophic flood event Plath et al 2010) Population genetic dif-
ferentiation between adjacent sulphide-adapted and nonadapted
populations is indicative of ongoing ecological speciation (Box 1) but
different population pairs from sulphidic and nonsulphidic waters
have made variable progress along the speciation continuum towards
complete reproductive isolation (Plath et al 2013) This prompts
questions about the mechanisms involved in reducing gene flow
along the environmental gradient connecting the two habitat types
41 | Prezygotic mechanisms of reproductiveisolation
The simplest form of reproductive isolation between locally adapted
populations arises from selection against migrants between habitat
types (Nosil Vines amp Funk 2005) Translocation experiments in nat-
ural habitats have indicated strong survival selection against
migrants especially those translocated from nonsulphidic to sulphidic
habitats (Plath et al 2013) Fish translocated from nonsulphidic
environments face high mortality within short periods of time
(lt24 hr) which is likely caused by H2S toxicity and hypoxia in sul-
phide springs Hence low gene flow from populations inhabiting
nonsulphidic waters into populations inhabiting sulphidic habitats is
simply explained by a lack of adaptation to the extreme environmen-
tal conditions At least in some drainages there is also appreciable
mortality for sulphide spring fish translocated to nonsulphidic habi-
tats although mechanisms causing inviability are unclear and the
strength of selection appears to be lower (Plath et al 2013) If sur-
vival selection against immigrants was the sole source of reproduc-
tive isolation this should result in unidirectional gene flow from
H2S-adapted to nonadapted populations but population genetic
analyses provided no evidence for this implying that additional
mechanisms are at work (Plath et al 2013)
One such mechanism could be intersexual selection and mate
choice experiments have indicated that migrants from sulphidic to
nonsulphidic habitats are at a significant disadvantage with fish from
nonsulphidic habitats avoiding potential mating partners from sul-
phidic ones (Plath et al 2013 Sommer-Trembo et al 2016) Such
assortative mating has been found to be critical during ecological spe-
ciation because it counteracts the homogenizing effects of gene flow
(van Doorn Edelaar amp Weissing 2009) yet how assortative mating
patterns arise often remains unclear In theory assortative mating can
arise through a variety of processes (Nosil 2012) ldquoMagic traitrdquo mecha-
nisms where mating preferences evolve as a by-product of divergent
selection on ecological traits that serve as cues have recently garnered
much attention (Servedio van Doorn Kopp Frame amp Nosil 2011)
Consistent with magic trait mechanisms assortative mating in P mexi-
cana appears to be linked to adaptive population differences in body
shape which serve as cues to distinguish potential mating partners
from different habitats (Greenway Drexler Arias-Rodriguez amp Tobler
2016) However neither sulphide spring fish (which are unlikely to
encounter migrants from nonsulphidic habitats due to H2S toxicity)
nor allopatric P mexicana from nonsulphidic waters (which have never
experienced migrants from sulphide spring populations) exhibit a
preference for mating partners from their own ecotype suggesting
that assortative mating in this system is a product of reinforcement
(ie direct selection for assortative mating Greenway et al 2016)
This is different from classic magic traits mechanisms because assorta-
tive mating is not a mere by-product of adaptation but adaptive traits
have instead been co-opted as mating signals in response to selection
on mating preferences (Maan amp Seehausen 2012) Moreover males of
some H2S-adapted populations have reduced aggressive behaviour
(Bierbach et al 2012 2018) In staged contests of size-matched males
from different ecotypes intruders had a significantly lower likelihood
to establish dominance than resident males independent of whether
or not they showed reduced aggressiveness overall Hence intrasexual
selection may represent another mechanism mediating reproductive
isolation between locally adapted ecotypes Overall H2S-adapted poe-
ciliids allow disentangling the relative contributions of different forms
of ecological and sexual selectionmdashand their interdependencymdashduring
ecological speciation
42 | Ensuing challenges quantifying postzygoticmechanisms of reproductive isolation
In contrast to prezygotic mechanisms little is known about postzy-
gotic mechanisms of reproductive isolation that potentially act to
reduce gene flow between adjacent populations in sulphidic and
nonsulphidic habitats Putative hybrids between sulphidic and non-
sulphidic ecotypes have occasionally been observed in nonsulphidic
habitats immediately adjacent to confluences with sulphide springs
(R Riesch M Plath and M Tobler personal observations) but it
remains unclear whether and through what mechanisms such hybrids
exhibit reduced fitness compared to parentals
Direct selection of mitochondrially encoded genes in the pres-
ence of H2S (Pfenninger et al 2014) should be accompanied by co-
evolution at nuclear loci that directly interact with mitochondrial
gene products in the formation of protein complexes involved in the
OXPHOS pathway (Sunnucks Morales Lamb Pavlova amp Greening
2017 Willett amp Burton 2004) Disruption of co-evolved gene com-
plexes in hybrids should therefore lead to intrinsic fitness reductions
through mito-nuclear incompatibilities (Burton Pereira amp Barreto
2013 Wolff Ladoukakis Enrıquez amp Dowling 2014) which have
been hypothesized to be a key driver in speciation (Chou amp Leu
2010 Gershoni Templeton amp Mishmar 2009) Empirical tests of
these ideas remain to be conducted in sulphide spring fishes just
like studies on potential ecological and sexual selection against
hybrids that arise from interpopulation mating events
5 | PERSPECTIVES LIFE IN SULPHIDICENVIRONMENTS BEYOND P MEXICANA
Research on sulphide spring fishes of the P mexicana species com-
plex in Mexico has provided insights into mechanisms underlying
adaptation and the links between adaptation and speciation Sul-
phide springs do not only occur in Mexico however but are found
TOBLER ET AL | 853
worldwide and have been colonized by 24 evolutionarily indepen-
dent fish lineages in North America the Neotropics and the Middle
East (Greenway et al 2014) The vast majority of lineages belong to
the order Cyprinodontiformes (95) and livebearing members of the
family Poeciliidae in particular (80) This potentially indicates that
internal gestation of young has facilitated the colonization of
extreme environments because it shields developing embryos from
physiochemical stressors (Blackburn 1999) Among the Poeciliidae
sulphide spring fishes include members of the genera Brachyrhaphis
Gambusia Poeciliopsis Priapichthys Pseudoxiphophorus Xiphophorus
and multiple subgenera of Poecilia (Figure 3 Greenway et al 2014)
spanning over 40 million years of evolution (based on Reznick Fur-
ness Meredith amp Springer 2017)
A handful of studies have started to address molecular and phe-
notypic evolution across a broader taxon sampling of sulphide spring
fishes finding evidence for convergent evolution in morphology
(Riesch et al 2016 Tobler amp Hastings 2011) life history traits
(Riesch Plath et al 2010 Riesch et al 2014) and oxygen transport
genes (Barts et al 2018) In addition ecological speciation in
response to adaptation to H2S-rich environments has also been doc-
umented in sulphide spring poeciliids other than P mexicana (Riesch
et al 2016) These studies have laid a fruitful foundation for the
investigation of mechanisms of adaptation to H2S-rich springs from
genes to phenotypic traits linking micro-evolutionary processes to
macro-evolutionary patterns
The availability of broad-scale evolutionary replication of popula-
tions that have invaded habitats with the same physiochemical stressors
allows for addressing some of the ensuing challenges discussed above
and investigating the factors that give rise to predictable evolutionary
outcomes Convergent evolution is a commonmdashbut not omnipresentmdash
theme in adaptive evolution and multiple factors can determine the
degree of convergence among replicated lineages exposed to the same
sources of selection (Kaeuffer et al 2012 Landry amp Bernatchez 2010
Oke Rolshausen LeBlond amp Hendry 2017 Rosenblum amp Harmon
2011) Thus leveraging replicated lineages that have colonized sulphide
springs to assess molecular mechanisms underlying convergent pheno-
typic changes analyse how environmental heterogeneity among sul-
phide spring habitats causes lineage-specific patterns of trait evolution
and test whether and by what mechanisms adaptation to extreme envi-
ronments is linked to speciation will provide insights into how ecologi-
cal genetic and functional factors contribute to evolutionary
convergence at different levels of organismal organization (Rosenblum
et al 2014) An expanded taxon sampling with population pairs that
span almost the full range of the speciation continuum from panmixia to
complete reproductive isolation (Riesch et al 2016) will also help
addressing questions about temporal aspects of H2S adaptation In par-
ticular comparing younger to older lineages will provide insights about
the order at which genetic and phenotypic modifications accrue during
the adaptation process
Animals have also invaded H2S-rich habitats in other aquatic
ecosystems including cold seeps and deep-sea hydrothermal vents
(Tobler et al 2016) Adaptation to H2S in the P mexicana species com-
plex has involved modification of physiological pathways associated
with H2S toxicity (OXPHOS pathway) and detoxification (sulphide
F IGURE 3 Phylogeny of the family Poeciliidae (adopted from Culumber amp Tobler 2017) Various lineages in the genera Poecilia (subgeneraAcanthophacelus Limia and Mollienesia) Priapichthys Brachyrhaphis Poeciliopsis Xiphophorus Pseudoxiphophorus and Gambusia have colonizedH2S-rich springs in different parts of the Americas (see Greenway et al 2014) Lineages highlighted in yellow represent endemic speciesrestricted to H2S-rich springs while red lineages represent species in which some populations have invaded sulphidic habitats Pictures depict arepresentative of each focal genussubgenus
854 | TOBLER ET AL
quinone oxidoreductase pathway) which have been highly conserved
during the diversification of life on Earth (Saraste 1999 Shahak amp
Hauska 2008) The high replicability of H2S as an environmental factor
that shaped animal evolution across vastly different ecological contexts
consequently offers the opportunity to investigate whether selection
mediated by the presence of H2S has repeatedly modified these con-
served physiological pathways across animal phyla that have diverged
during the Cambrian explosion spanning over 500 million years of evo-
lution Such comparisons would provide unprecedented insights into
the effects of a single physiochemical stressor on evolutionary change
irrespective of ecological or phylogenetic contexts
6 | CONCLUSIONS
H2S-rich environments and their inhabitants represent useful models
for research in organismal biology ecology and evolution illustrating
both opportunities (eg strong and explicit source of selection evolu-
tionary replication) and challenges (eg complexity of environments
and organismal responses) in asking basic question about the origins
of biodiversity Understanding how organisms thrive in apparently
inhospitable environments will provide us with insights into lifersquos
capacities and limitations to adapt to novel environments (Waterman
1999) This is particularly relevant considering that human activities
substantially contribute to the exacerbation of physiochemical stres-
sors at local regional and global scales impacting ecological and evo-
lutionary dynamics (Alberti 2015 Norberg Urban Vellend
Klausmeier amp Loeuille 2012) Thus a focus on ecological and evolu-
tionary consequences of natural stressors that push organisms to the
brink of their physiological capacities may be instrumental to predict
consequences of anthropogenic environmental change
ACKNOWLEDGEMENTS
We would like to thank Lenin Arias-Rodriguez the Universidad
Juarez Autonoma de Tabasco and the Centro de Investigacion E
Innovacion Para la Ense~nanza y El Aprendizaje for their continued
collaboration enabling our work on the P mexicana system Funding
for this review article has been provided by the National Science
Foundation (IOS-1557860 IOS-1557795) and the US Army
Research Office (W911NF-15-1-0175)
AUTHOR CONTRIBUTIONS
MT wrote the first draft of the paper all authors contributed text
and comments
ORCID
Michael Tobler httporcidorg0000-0002-0326-0890
Joanna L Kelley httporcidorg0000-0002-7731-605X
Reuroudiger Riesch httporcidorg0000-0002-0223-1254
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httpsdoiorg101242dmm012245
Schartl M Walter R B Shen Y Garcia T Catchen J Amores A
Warren W C (2013) The genome of the platyfish Xiphophorus mac-
ulatus provides insights into evolutionary adaptation and several
complex traits Nature Genetics 45 567ndash572 httpsdoiorg10
1038ng2604
Schluter D (2009) Evidence for ecological speciation and its alternative
Science 323 737ndash741 httpsdoiorg101126science1160006
Schulz-Mirbach T Eifert C Riesch R Farnworth M S Zimmer C
Bierbach D Plath M (2016) Toxic hydrogen sulphide shapes
brain anatomy A comparative study of sulphide-adapted ecotypes in
the Poecilia mexicana complex Journal of Zoology 300 163ndash176
httpsdoiorg101111jzo12366
SEDESOL (1994) NORMA Oficial Mexicana NOM-059-ECOL-1994 que
determina las especies y subspecies de flora y fauna silvestres ter-
restres y acuaticas en peligro de extincion amenazadas raras y las
sujetas a proteccion especial y que establece especificaciones para
su proteccion Diario Oficial 16 May 1994 488 2ndash60
Servedio M R van Doorn G S Kopp M Frame A M amp Nosil P
(2011) Magic traits in speciation lsquomagicrsquo but not rare Trends in Ecology
amp Evolution 26 389ndash397 httpsdoiorg101016jtree201104005
Shahak Y amp Hauska G (2008) Sulfide oxidation from cyanobacteria to
humans Sulfide-quinone oxidoreductase (SQR) In R Hell C Dahl D
B Knaff amp Th Leustek (Eds) Advances in photosynthesis and respira-
tion (pp 319ndash335) Heidelberg Springer
Shalem O Sanjana N E amp Zhang F (2015) High-throughput func-
tional genomics using CRISPR-Cas9 Nature Reviews Genetics 16
299ndash311 httpsdoiorg101038nrg3899
Silva G J J Bye A el Azzouzi H amp Wisloslashff U (2017) MicroRNAs as
important regulators of exercise adaptation Progress in Cardiovascular
Diseases 60 130ndash151 httpsdoiorg101016jpcad201706003
Simola D F Wissler L Donahue G Waterhouse R M Helmkampf
M Roux J Viljakainen L (2013) Social insect genomes exhibit
dramatic evolution in gene composition and regulation while preserv-
ing regulatory features linked to sociality Genome Research 23
1235ndash1247 httpsdoiorg101101gr155408113
858 | TOBLER ET AL
Sommer-Trembo C Bierbach D Arias Rodriguez L Verel Y Jourdan
J Zimmer C Plath M (2016) Does personality affect premating
isolation between locally-adapted populations BMC Evolutionary Biol-
ogy 16 138 httpsdoiorg101186s12862-016-0712-2
Steindachner F (1863) Beitreuroage zur Kentniss der Sciaenoiden Brasiliens
und der Cyprinodonten Mejicos Sitzungsberichte der Mathematisch-
Naturwissenschaftlichen Classe der Kaiserlichen Akademie der Wis-
senschaften 48 162ndash185
Stinchcombe J R amp Hoekstra H E (2008) Combining population geno-
mics and quantitative genetics Finding genes underlying ecologically
important traits Heredity 100 158ndash170 httpsdoiorg101038sj
hdy6800937
Stipanuk M H (2004) Sulfur amino acid metabolism Pathways for pro-
duction and removal of homocysteine and cysteine Annual Review of
Nutrition 24 539ndash577 httpsdoiorg101146annurevnutr24
012003132418
Stipanuk M H amp Ueki I (2011) Dealing with methioninehomocysteine
sulfur Cysteine metabolism to taurine and inorganic sulfur Journal of
Inherited Metabolic Disease 34 17ndash32 httpsdoiorg101007
s10545-009-9006-9
Stockinger B Di Meglio P Gialitakis M amp Duarte J H (2014) The
aryl hydrocarbon receptor Multitasking in the immune system
Annual Review of Immunology 32 403ndash432 httpsdoiorg101146
annurev-immunol-032713-120245
Storz J F amp Wheat C W (2010) Integrating evolutionary and
functional approaches to infer adaptation at specific loci Evolution
64 2489ndash2509 httpsdoiorg101111j1558-5646201001044x
Stuart Y E Veen T Weber J N Hanson D Ravinet M Lohman B
K Bolnick D I (2017) Contrasting effects of environment and
genetics generate a continuum of parallel evolution Nature Ecology amp
Evolution 1 0158 httpsdoiorg101038s41559-017-0158
Sunnucks P Morales H E Lamb A M Pavlova A amp Greening C
(2017) Integrative approaches for studying mitochondrial and nuclear
genome co-evolution in oxidative phosphorylation Frontiers in Genet-
ics 8 25
Tobler M amp Hastings L (2011) Convergent patterns of body shape dif-
ferentiation in four different clades of poeciliid fishes inhabiting sul-
fide springs Evolutionary Biology 38 412ndash421 httpsdoiorg10
1007s11692-011-9129-4
Tobler M Palacios M Chapman L J Mitrofanov I Bierbach D
Plath M Mateos M (2011) Evolution in extreme environments
Replicated phenotypic differentiation in livebearing fish inhabiting
sulfidic springs Evolution 65 2213ndash2228 httpsdoiorg101111j
1558-5646201101298x
Tobler M Passow C N Greenway R Kelley J L amp Shaw J H
(2016) The evolutionary ecology of animals inhabiting hydrogen sul-
fide-rich environments Annual Review of Ecology Evolution and Sys-
tematics 47 239ndash262 httpsdoiorg101146annurev-ecolsys-
121415-032418
Tobler M amp Plath M (2009a) Threatened fishes of the world Gambu-
sia eurystoma Miller 1975 (Poeciliidae) Environmental Biology of
Fishes 85 251 httpsdoiorg101007s10641-009-9481-8
Tobler M amp Plath M (2009b) Threatened fishes of the world Poecilia
sulphuraria (Alvarez 1948) (Poeciliidae) Environmental Biology of
Fishes 85 333ndash334 httpsdoiorg101007s10641-009-9506-3
Tobler M Plath M Riesch R Schlupp I Grasse A Munimanda G
Moodley Y (2014) Selection from parasites favors immunogenic diver-
sity but not divergence among locally adapted host populations Journal
of Evolutionary Biology 27 960ndash974 httpsdoiorg101111jeb12370
Tobler M Riesch R amp Plath M (2015) Extremophile fishes An inte-
grative synthesis In R Riesch M Tobler amp M Plath (Eds) Extremo-
phile fishes Ecology evolution and physiology of teleosts in extreme
environments (pp 279ndash296) Heidelberg Germany Springer
Tobler M Riesch R Tobler C M amp Plath M (2009) Compensatory
behaviour in response to sulfide-induced hypoxia affects time
budgets feeding efficiency and predation risk Evolutionary Ecology
Research 11 935ndash948
Tobler M Scharnweber K Greenway R Passow C Arias-Rodriguez
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Waterman T H (1999) The evolutionary challenges of extreme environ-
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the mitochondrial electron transport system in a marine copepod
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How to cite this article Tobler M Kelley JL Plath M Riesch
R Extreme environments and the origins of biodiversity
Adaptation and speciation in sulphide spring fishes Mol Ecol
201827843ndash859 httpsdoiorg101111mec14497
TOBLER ET AL | 859
and physiological consequences of H2S may thus enable us to pre-
dict the physiological pathways in which adaptive modifications
occur it may be impossible to predict specific modifications within
those pathways This contrasts with a common expectation in the
field that convergent evolution of molecular mechanisms underlying
phenotypic traits (ie parallel evolution) necessarily hinges on modi-
fication of the same loci across lineages as found in some of the
best-known examples of parallelism (Colosimo et al 2005 Feldman
Brodie Brodie amp Pfrender 2012 Grant Grant Markert Keller amp
Petren 2004) However modification of the same loci may only be
common under particular circumstances (Rosenblum Parent amp
Brandt 2014) for example when selection acts on standing genetic
variation (Colosimo et al 2005) when adaptive alleles are intro-
gressed across population boundaries through gene flow (Grant
et al 2004) or when there are strong genetic constraints (Feldman
et al 2012) Exploration of molecular mechanisms underlying con-
vergent evolution in many systems may therefore be better
approached from the perspective of interacting genes that are orga-
nized in pathways in which alternative genetic changes can have
equivalent functional consequences (ie functional redundancy)
Developing and testing hypotheses about parallel evolution at the
pathway level will hinge on improving genome annotations (Pavey
Bernatchez Aubin-Horth amp Landry 2012) and provide a more
nuanced understanding of the factors determining the predictability
of evolution at different levels of organismal organization (Rosen-
blum et al 2014)
Despite evidence for convergent evolution in some candidate
physiological pathways it also needs to be emphasized that this
does not apply to all P mexicana lineages or to all candidate physio-
logical pathways associated with H2S adaptation investigated to
date Nonconvergent evolutionmdashevidenced by unique lineage-speci-
fic signals of selection and differential expressionmdashmay be indicative
of unique molecular mechanisms to cope with H2S toxicity For
example signatures of positive selection on COXmdashH2Srsquos primary
toxicity targetmdashcan be found in sulphide spring lineages of the Puy-
acatengo and Pichucalco river drainages (a lineage with recent diver-
gence time and a lineage with older divergence time respectively)
but not in those of the Tacotalpa drainage (Pfenninger et al 2014)
The presence of nonconvergent genetic and gene expression differ-
ences between populations from sulphidic and non-sulphidic habitats
in different river drainages highlights the limitations of inferences
that can be drawn based on data sets generated with high-through-
put DNA and RNA sequencing The potential importance of modifi-
cations at candidate locimdashor the lack thereofmdashcan only be assessed
through functional assays that document how alternative alleles and
changes in gene expression actually affect organismal performance
The functional consequences of genetic and gene expression varia-
tion at most candidate loci remain to be investigated However
enzyme activity assays have indicated that COX maintains its activity
with increasing H2S concentrations in the two sulphide spring popu-
lations with evidence for positive selection on mitochondrially
encoded subunits (Puyacatengo and Pichucalco Pfenninger et al
2014) Key amino acid substitutions occur in the D-pathway channel
of COX1 which putatively narrow the channel width and reduce
H2Srsquos access to the binuclear centre of the protein Consequently
some lineages of P mexicana exhibit an H2S-resistant COX that can
maintain its activity and aerobic ATP production even if endogenous
H2S concentrations are elevated In contrast COX activity in sul-
phide spring fish of the Tacotalpa river drainage and in fish from
nonsulphidic habitats decreases when H2S is present which is con-
sistent with mechanisms of H2S toxicity (Pfenninger et al 2014) In
vitro assays on isolated mitochondria have nonetheless indicated
that an H2S-resistant COX is not necessary to maintain aerobic ATP
production during H2S exposure Unlike in populations from nonsul-
phidic habitats mitochondrial spare respiratory capacity is main-
tained in the presence of H2S even in the Tacotalpa sulphide spring
populations that lack an H2S-resistant COX (C Henpita M Tobler amp
JH Shaw unpublished data) This may indicate that modification of
detoxification pathways in sulphide spring fishes is sufficient to
maintain mitochondrial function and aerobic ATP production
23 | Ensuing challenges linking genotypes tofitness
Understanding the mechanistic basis of adaptation requires linking
genotypic variation to phenotypic traits that ultimately shape fitness
along selective gradients The evolution of H2S tolerance in sulphide
spring fishes appears to be a consequence of modifications in many
genes and many physiological pathways whose functions interact to
eliminate H2S from the system minimize effects of elevated endoge-
nous concentrations on direct toxicity targets and mitigate adverse
effects that cascade through cellular metabolism during disruption of
homeostasis (Table 1) Such complex genetic changes are likely com-
mon during adaptation and have been documented in other systems
that involve major evolutionary shifts including the emergence of
parasitic lifestyles (Werren et al 2010) eusociality (Simola et al
2013) asexual reproduction (Fradin et al 2017) and internal gesta-
tion with livebearing (Schartl et al 2013) Consequently comple-
mentary approaches will be required to uncover the molecular
mechanisms of adaptation to H2S in the future
1 The involvement of many genes and physiological pathways in
shaping tolerance emphasizes the importance of quantitative
genetic approaches that leverage experimental crosses between
H2S tolerant and nontolerant populations and test whether toler-
ancemdashand adaptive trait modifications that contribute to itmdashis
explained by a few major effects loci or whether it is a conse-
quence of a number of genes with small effects (Klerks Xie amp
Levinton 2011 Saltz Hessel amp Kelly 2017) Combining analyses
of phenotype-fitness associations and their underlying genetic
mechanisms via QTL mapping with the results of selection scans
from population genomic analyses (Pfenninger et al 2015) will
allow for an integrative understanding of how selection acting on
the genome results in phenotypic variation that mediates adapta-
tion to different environments (Stinchcombe amp Hoekstra 2008)
From a functional perspective breaking up correlations between
848 | TOBLER ET AL
different physiological traits (associated with resistance mitiga-
tion and regulation Table 1) in F2 crosses will further allow test-
ing what molecular mechanisms are necessary for tolerance
whether there are alternative strategies with equivalent fitness
benefits and whether there are complementary modifications
with additive effects on tolerance
2 Considering the number of genes and physiological pathways
likely involved in H2S tolerance assessing the functional conse-
quences of genetic and gene expression variation is a daunting
task At the same time such functional tests can significantly
contribute to closing the genotype-to-phenotype gap because
physiochemical stress responses can be assessed along the con-
tinuum of organismal organization (Gjuvsland Vik Beard Hunter
amp Omholt 2013) Standardized assays can be used to quantify
the biochemical makeup of organelles cells and tissues (eg pro-
tein abundance and enzyme activity) physiological performance
in vitro and in vivo (eg rates of H2S detoxification ATP produc-
tion and reactive oxygen production) and fitness-relevant proxies
of whole organism performance (eg H2S tolerance Figure 2)
This highlights the importance of methods development for the
quantification of complex phenotypic traits or even whole phe-
nomes that can keep pace with rapid data generation through
high-throughput sequencing (Houle Govindaraju amp Omholt
2010 Ritchie Holzinger Li Pendergrass amp Kim 2015) In addi-
tion genotypendashphenotype links established through such correla-
tive approaches will ideally be verified through targeted gene
editing While gene editing with CRISPRndashCas9 is now routinely
employed in model organisms (Doudna amp Charpentier 2014 Sha-
lem Sanjana amp Zhang 2015) applying this technology to non-
models still remains challenging especially in livebearing species
like poeciliid fishes where in vitro culture of embryos is not rou-
tinely employed (Martyn Weigel amp Dreyer 2006) Nonetheless
the effects of specific mutations can potentially be verified
through the establishment and manipulation of cells in culture
(Liu et al 2014) or by generating transgenics (Lin et al 2016)
3 Organismal responses to physiochemical stressors occur at multi-
ple hierarchical levels that can be plastically modulated in
response to endogenous and exogenous cues (Whitehead 2012)
Understanding adaptation to physiochemical stressors conse-
quently requires disentangling the effects of genetic variation
developmental plasticity and short-term acclimation in gene
expression patterns on the emerging physiological processes
Common garden experiments on sulphide spring fishes have
shown that many genes differentially expressed between popula-
tions from sulphidic and nonsulphidic habitats in nature remain
differentially expressed in laboratory-reared fish that have never
been exposed to H2S indicating evolved differences in constitu-
tive gene expression (Passow Henpita et al 2017) At the same
time expression patterns in the laboratory also exhibit evidence
for plasticity in response to short-term H2S exposure (Passow
Henpita et al 2017) Some of these plastic responses are shared
between populations from sulphidic and nonsulphidic habitats
(likely representing shared ancestral plasticity) while others have
been lost or gained during sulphide spring colonization A core
challenge for future research is to overcome logistical constraints
that have so far prevented long-term H2S exposures of fish in
the laboratory Exposing individuals throughout development will
be required to test how developmental plasticity and short-term
acclimation shape responses to H2S in individuals from adapted
and nonadapted populations Finally the next big scientific
advance in this context will also require the identification of reg-
ulatory mechanisms that shape expression differences during
adaptation including sequence changes in transcription factors
and their binding sites patterns of DNA methylation and chro-
matin accessibility as well as the production of microRNAs and
competing endogenous RNAs that can modulate transcript abun-
dance (Flores Wolschin amp Amdam 2013 Lopez-Maury Mar-
guerat amp Beuroahler 2008 Silva Bye el Azzouzi amp Wisloslashff 2017)
In addition the role of alternative splicing and other post-tran-
scriptional and post-translational modifications in mediating adap-
tation remains understudied by evolutionary biologists (Ast
2004 Bush Chen Tovar-Corona amp Urritia 2017) This is particu-
larly relevant because H2S has been hypothesized to play a role
in the post-translational modification of proteins with sulphur-
containing amino acids through sulfhydration (Mustafa et al
2009)
Bridging the genotypendashphenotype gap remains one of the major
challenges in biology (Gjuvsland et al 2013 Ritchie et al 2015) and
will provide opportunities to understand how organisms function in
the face of environmental stress and how evolution shapes these
functions during adaptation to different environmental conditions
Establishing the molecular mechanisms of adaptation across repli-
cated lineages exposed to the same sources of selection will also
provide insights into how evolutionary history demography and
genetics shape convergent evolutionary outcomes providing a better
understanding at what hierarchical levels of organismal organization
evolutionary change may actually be predictable (Rosenblum et al
2014)
3 | COMPLEX ORGANISMS IN A COMPLEXWORLD DISENTANGLING THE TANGLEDBANK
Mechanisms of adaptation can be studied through bottom-up
(screening for genetic modifications that allow for inferences about
relevant phenotypic traits) and top-down approaches (assessing fit-
ness-relevant phenotypic differences among populations without
knowledge about the genetic basis see Schluter 2009) Over the
years genomic and transcriptomic studies on sulphide spring popula-
tions of the P mexicana species complex have been complemented
with analyses of phenotypic variation Just as genetic analyses have
indicated that adaptation to H2S is mediated by the modification
and modulation of many genes associated with multiple physiological
pathways phenotypic studies uncovered a variety of differences
TOBLER ET AL | 849
between populations from sulphidic and nonsulphidic environments
Many of these phenotypic differences have evolved in convergence
across replicated sulphide spring lineages in different river drainages
Examples include modifications of morphological (body shape and
organ size Tobler et al 2011 Tobler Scharnweber et al 2015
Schulz-Mirbach et al 2016) physiological (tolerance and metabolic
rates Tobler et al 2011 Passow Arias-Rodriguez amp Tobler 2017)
behavioural (aggression and personality Riesch et al 2009 Bierbach
et al 2012 Sommer-Trembo et al 2016 Bierbach Arias Rodriguez
amp Plath 2018) and life history traits (fecundity and offspring size
Riesch Plath Garcia de Leon amp Schlupp 2010 Riesch et al 2014)
Hence proximate populations in different habitat types have
diverged in complex phenotypes that span many different suites of
traits and a key question is how divergence in multivariate pheno-
types arises In theory H2S could be a key source of selection driv-
ing multivariate phenotypic divergence either because it directly
exerts selection on a multitude of traits that are independently modi-
fied to reach new fitness peaks or because selection on a few fit-
ness-relevant traits causes simultaneous changes in correlated
characteristics Neither explanation seems sufficient because many
Abiotic environmentOxygen concentrations
Ionic compositionTemperature
Structure and flow
Biotic environmentTrophic resources
DemographyPredationParasitism
H2S
Genetic variation
Gene expression
Biochemistry
Physiologicalfunction
Organismal performance
Primary fitness components
Survivalgrowth
reproduction
Organismal function Environment
F IGURE 2 Understanding how organisms function in the context of their environment is a core question in evolutionary biology yet thecomplexities associated with organismal function and environmental variation provide significant challenges High-throughput sequencing hasallowed for the identification of candidate genes subject to selection and differential expression during adaptation to H2S The next step is tobridge the genotype-to-phenotype gap by quantifying the integrated responses to H2S across the continuum of organismal organization includingbiochemical phenotypes physiological functions whole organism performance and ultimately fitness In addition understanding adaptation toH2S-rich environments will also require acknowledging H2Srsquos relation to other abiotic and biotic environmental variables which can exert selectionand modulate aspects of organismal function This will allow for the establishment of cause-and-effect relationships between specific sources ofselection and evolutionary change in specific traits and contribute to our understanding of organismal function in natural systems
850 | TOBLER ET AL
of the divergent traits between populations from sulphidic and non-
sulphidic environments have no clearmdashor at best tenuousmdashlinks to
H2S toxicity and detoxification and there is only limited evidence
for within-population correlations between different traits groups
(Riesch et al 2016)
Evolutionary analyses frequently focus on seemingly simple
sources of selection juxtaposing populations that are assigned to dis-
crete habitat types to understand the evolution of specific traits
(Kaeuffer Peichel Bolnick amp Hendry 2012) But as Darwin (1859)
already observed in his ldquotangled bankrdquo allegory selection in natural
systems is rarely univariate but multifarious selective regimes con-
tribute to trait divergence even among populations that occur along
apparently simple environmental gradients Unquantified environmen-
tal variation that is subsumed in the categorization of habitat types
can profoundly affect trait variation in natural systems (Stuart et al
2017) highlighting the importance of considering and testing alterna-
tive hypotheses about the cause-and-effect relationship between
specific sources of selection and evolution of specific traits Thus
accurate natural history observations that consider organisms as holis-
tic entities consisting of interacting traits in the context of the abiotic
and biotic factors comprising their environment are critical to inform
the design and interpretation of empirical research in natural systems
(Greene 2005) Indeed adjacent sulphidic and nonsulphidic habitat
types do not merely differ in the presence or absence of H2S but also
in a variety of other abiotic and biotic factors (Figure 2) Unlike nonsul-
phidic habitats sulphide springs in southern Mexico are characterized
by extreme hypoxia elevated salinity and lower pH (Rosales Lagarde
2012 Tobler et al 2011) In addition the presence of physiochemical
stressors affects the biotic communities in these sulphide springs Spe-
cies richness of aquatic invertebrates and vertebrates is drastically
reduced as a consequence of H2S toxicity (Greenway Arias-Rodriguez
Diaz amp Tobler 2014) and primary production appears to be driven in
part by chemoautotrophic bacteria that use H2S as an energy source
rather than just by autochthonous and allochthonous photosynthetic
organisms (Roach Tobler amp Winemiller 2011) These changes in the
biotic environment affect the relative importance of intra-vs inter-
specific competition trophic resource use and exposure to predators
and parasites (Riesch Oranth et al 2010 Tobler et al 2014 Tobler
Scharnweber et al 2015)
31 | Alternative drivers of trait evolution
Acknowledging the complexity of selective regimes in sulphide
springs allows for a more nuanced understanding of trait evolution
and for the development of alternative hypotheses about the ulti-
mate mechanisms that cause divergence in specific genetic and phe-
notypic traits In some instances sources of selection other than H2S
can plausibly explain trait divergence between populations from sul-
phidic and nonsulphidic habitats For example hypoxia likely drives
increases in head size and gill surface area (Tobler et al 2011)
changes in the ionic composition of the water can explain differential
expression of genes associated with osmoregulation (Kelley et al
2016) and shifts in trophic resource use are associated with
modifications of the gastrointestinal system (Tobler Scharnweber
et al 2015) In other cases disentangling alternative hypotheses is
not trivial because selection mediated by H2S and other environmen-
tal factors are predicted to affect trait modifications in similar ways
Shifts towards anaerobic metabolism in sulphide spring fish could be
the consequence of inhibition of aerobic ATP production by H2S or
by constraints in oxygen availability Similarly reproductive life his-
tory shifts towards the production of larger but fewer offspring could
be driven by H2S (decreasing offspringrsquos surfacevolume ratio to mini-
mize the diffusion of H2S into the body Powell 1989) by habitat dif-
ferences in the quality and quantity of predation that can affect size-
specific rates of extrinsic mortality (Jorgensen Auer amp Reznick
2011) or by energetic constraints (see below) While comparisons
across different selective regimes can shed light into the ultimate dri-
vers of trait evolution (Moore Riesch amp Martin 2016) isolating the
effects of different sources of selection may not be possible because
they could act in concert and facilitate increases of organismal perfor-
mance in multiple contexts (Ghalambor Walker amp Reznick 2003)
When multifarious selection shapes multivariate phenotypic evo-
lution it is important to also consider the role of trade-offs both in
terms of trait expression (increased investment in one trait may
impose constraints on the expression of other traits) and in terms of
trait function (optimizing the trait for one function may impair the
traitrsquos utility for other functions) For example selection on large off-
spring size in sulphide spring fishes has inadvertent consequences
for fecundity (Riesch et al 2014) and selection for increased gill
surface areas due to hypoxia simultaneously increases exposure to
environmental H2S (Tobler et al 2011) Thus the outcome of phe-
notypic evolution is ultimately a result of balancing competing needs
in a manner that optimizes trait expression and organismal function
in multiple contexts to maximize fitness (Arnold 1983) and the pres-
ence of trade-offs can have cascading indirect effects with selection
on one or a few traits leading to the modification of a multitude of
apparently unrelated characteristics
In this context bioenergetics may represent a nexus in the evo-
lution of complex traits observed in sulphide spring fish and other
extreme environments Living in and adapting to sulphide springs
imposes constraints on organismal energy availability (Plath et al
2007) with according trade-offs for energy allocation Energy limita-
tion in sulphide springs does not arise from a scarcity of food
resources (chemoautotrophic bacteria eaten by the fish are abundant
in sulphide springs Tobler Scharnweber et al 2015) but from
behavioural and physiological adaptation A primary survival strategy
for fish in the toxic and hypoxic springs is to engage in aquatic sur-
face respiration where individuals breathe directly at the water sur-
face to maximize oxygen uptake and minimize H2S exposure This
behaviour mediates short-term survival (Plath et al 2007) but it
also constrains energy acquisition for benthic foragers like P mexi-
cana (Tobler Riesch Tobler amp Plath 2009) and increases susceptibil-
ity to bird predation (Riesch Oranth et al 2010) Costly investments
into H2S detoxification and tissue repair as well as a shift from aero-
bic to less efficient anaerobic metabolism further constrain energy
allocation to other functions (Passow Greenway Arias-Rodriguez
TOBLER ET AL | 851
Jeyasingh amp Tobler 2015) Theory predicts that energetic con-
straints ndash whether mediated by limitations in the rate of resource
acquisition or in the rate of energy allocation to reproduction (as
opposed to maintenance or growth) ndash should exert selection for a
reduction in organismal energy budgets which allows for the maxi-
mization of residual energy allocation for the production of offspring
(Brown Marquet amp Taper 1993) Several lines of evidence support
the hypothesis that energy limitation plays a critical role in pheno-
typic evolution of sulphide spring fish Compared to individuals in
nonsulphidic environments fish in sulphide springs have significantly
lower routine energy demands which is mediated both by reduc-
tions in body size and mass-specific metabolism (Passow Arias-
Rodriguez et al 2017) Reductions in other traits such as energeti-
cally costly behaviours (Bierbach et al 2012 Plath 2008) and the
size of energy-demanding organs (Schulz-Mirbach et al 2016) may
also contribute to the reduced energy demands and shifts in repro-
ductive life history traits (Riesch et al 2014) are consistent with
selection from energy limitation (Reznick amp Yang 1993)
32 | Ensuing challenges Disentanglingenvironmental and organismal complexities
Future research needs to empirically address how multifarious selec-
tive regimes shape the evolution of organisms with complex pheno-
types We have made great advances in our understanding of how
specific traits evolve and mediate adaptation (eg morphology Chan
et al 2010 coloration Dasmahapatra et al 2012 sensory organs
Cartwright Schwartz Merry amp Howell 2017) and we have a thor-
ough understanding of how single environmental factors can shape
organismal responses (eg climatic conditions Johnston amp Bennett
1996 predation Langerhans 2007 competition Pfennig amp Pfennig
2012) In contrast our understanding of phenotypic evolution in
response to multifarious selection is comparatively limited especially
because interactive effects of different environmental factors can lead
to organismal and evolutionary responses that are unpredictable based
on experiments that manipulate single environmental variables in iso-
lation (Gunderson Armstrong amp Stillman 2016 Todgham amp Stillman
2013) Future research thus needs to acknowledge the complexities of
environments and organismal design and address them explicitly in
empirical investigations to gain a robust understanding of biological
diversification (Culumber amp Tobler 2017 Stuart et al 2017)
4 | LINKING ADAPTATION TOSPECIATION
So far we primarily explored the causes of adaptive evolution but
what are its consequences Theory predicts that adaptation to local
environmental conditions can lead to speciation because reproduc-
tive isolation emerges incidentally as a by-product (ecological specia-
tion Nosil 2012) Population genetic analyses of Poecilia spp in
sulphide springs and adjacent populations in nonsulphidic environ-
ments have indicated strong genetic differentiation across small
spatial scales (in some instances lt100 m) and in the absence of
physical barriers that would prevent fish movement (Plath et al
2013) In addition population genetic structuring has been shown to
be stable even in the face of major environmental disturbance
BOX 1 Speciation in sulphide springs and conservation
biology
Adaptation to H2S-rich environments in poeciliids coincides
with ecological speciation giving rise to phenotypically dis-
tinct and reproductively isolated lineages In some
instances this has led to the evolution of highly distinct
micro-endemic species including Poecilia sulphuraria and
P thermalis Limia sulphurophila and Gambusia eurystoma
(Figure 3) Except for P sulphuraria which is found in two
spring complexes of the Rıo Pichucalco drainage of Mexico
(Tobler amp Plath 2009b) the distributions of these sulphide
spring endemics are restricted to single localities that
include only small areas of suitable habitat For example
the largest sulphide spring complex (the Ba~nos del Azufre
in southern Mexico which harbours P sulphuraria and G
eurystoma) includes ~18000 m2 of suitable habitat (equiva-
lent to lt35 football fields) while one of the smallest (La
Esperanza springs harbouring P thermalis) merely covers
~600 m2 (just over a tenth of a football field Brown et al
2017) Accordingly some sulphide spring endemics are
considered critically endangered (IUCN 2012) and pro-
tected by law (SEDESOL 1994)
Many threatened species with restricted ranges exhibit
low genetic diversity and high incidences of inbreeding
(Bouzat 2010 Willi Van Buskirk amp Hoffmann 2006) In
contrast at least P sulphuraria and P thermalismdashthe only
species investigated to datemdashactually exhibit relatively high
effective population sizes (Ne = 1200ndash3800 depending on
the population) and no evidence for inbreeding (FIS lt 0)
(Brown et al 2017) This is likely a consequence of the
long-term stability of sulphide springs and the high pro-
ductivity and low levels of interspecific competition also
support unusually high fish densities and thus population
census sizes (Brown et al 2017 Culumber et al 2016)
Nonetheless sulphide spring endemics are threatened by
habitat destruction which is mediated primarily by the
modification of springs for recreational purposes defor-
estation and conversion of land for agriculture and most
recently high-intensity palm oil plantations (Tobler amp Plath
2009ab) Despite the official protection of sulphide spring
endemics there have been no tangible conservation mea-
sures that foster the sustainable development of land
around sulphide springs with the well-being of the endemic
fishes in mind
852 | TOBLER ET AL
(catastrophic flood event Plath et al 2010) Population genetic dif-
ferentiation between adjacent sulphide-adapted and nonadapted
populations is indicative of ongoing ecological speciation (Box 1) but
different population pairs from sulphidic and nonsulphidic waters
have made variable progress along the speciation continuum towards
complete reproductive isolation (Plath et al 2013) This prompts
questions about the mechanisms involved in reducing gene flow
along the environmental gradient connecting the two habitat types
41 | Prezygotic mechanisms of reproductiveisolation
The simplest form of reproductive isolation between locally adapted
populations arises from selection against migrants between habitat
types (Nosil Vines amp Funk 2005) Translocation experiments in nat-
ural habitats have indicated strong survival selection against
migrants especially those translocated from nonsulphidic to sulphidic
habitats (Plath et al 2013) Fish translocated from nonsulphidic
environments face high mortality within short periods of time
(lt24 hr) which is likely caused by H2S toxicity and hypoxia in sul-
phide springs Hence low gene flow from populations inhabiting
nonsulphidic waters into populations inhabiting sulphidic habitats is
simply explained by a lack of adaptation to the extreme environmen-
tal conditions At least in some drainages there is also appreciable
mortality for sulphide spring fish translocated to nonsulphidic habi-
tats although mechanisms causing inviability are unclear and the
strength of selection appears to be lower (Plath et al 2013) If sur-
vival selection against immigrants was the sole source of reproduc-
tive isolation this should result in unidirectional gene flow from
H2S-adapted to nonadapted populations but population genetic
analyses provided no evidence for this implying that additional
mechanisms are at work (Plath et al 2013)
One such mechanism could be intersexual selection and mate
choice experiments have indicated that migrants from sulphidic to
nonsulphidic habitats are at a significant disadvantage with fish from
nonsulphidic habitats avoiding potential mating partners from sul-
phidic ones (Plath et al 2013 Sommer-Trembo et al 2016) Such
assortative mating has been found to be critical during ecological spe-
ciation because it counteracts the homogenizing effects of gene flow
(van Doorn Edelaar amp Weissing 2009) yet how assortative mating
patterns arise often remains unclear In theory assortative mating can
arise through a variety of processes (Nosil 2012) ldquoMagic traitrdquo mecha-
nisms where mating preferences evolve as a by-product of divergent
selection on ecological traits that serve as cues have recently garnered
much attention (Servedio van Doorn Kopp Frame amp Nosil 2011)
Consistent with magic trait mechanisms assortative mating in P mexi-
cana appears to be linked to adaptive population differences in body
shape which serve as cues to distinguish potential mating partners
from different habitats (Greenway Drexler Arias-Rodriguez amp Tobler
2016) However neither sulphide spring fish (which are unlikely to
encounter migrants from nonsulphidic habitats due to H2S toxicity)
nor allopatric P mexicana from nonsulphidic waters (which have never
experienced migrants from sulphide spring populations) exhibit a
preference for mating partners from their own ecotype suggesting
that assortative mating in this system is a product of reinforcement
(ie direct selection for assortative mating Greenway et al 2016)
This is different from classic magic traits mechanisms because assorta-
tive mating is not a mere by-product of adaptation but adaptive traits
have instead been co-opted as mating signals in response to selection
on mating preferences (Maan amp Seehausen 2012) Moreover males of
some H2S-adapted populations have reduced aggressive behaviour
(Bierbach et al 2012 2018) In staged contests of size-matched males
from different ecotypes intruders had a significantly lower likelihood
to establish dominance than resident males independent of whether
or not they showed reduced aggressiveness overall Hence intrasexual
selection may represent another mechanism mediating reproductive
isolation between locally adapted ecotypes Overall H2S-adapted poe-
ciliids allow disentangling the relative contributions of different forms
of ecological and sexual selectionmdashand their interdependencymdashduring
ecological speciation
42 | Ensuing challenges quantifying postzygoticmechanisms of reproductive isolation
In contrast to prezygotic mechanisms little is known about postzy-
gotic mechanisms of reproductive isolation that potentially act to
reduce gene flow between adjacent populations in sulphidic and
nonsulphidic habitats Putative hybrids between sulphidic and non-
sulphidic ecotypes have occasionally been observed in nonsulphidic
habitats immediately adjacent to confluences with sulphide springs
(R Riesch M Plath and M Tobler personal observations) but it
remains unclear whether and through what mechanisms such hybrids
exhibit reduced fitness compared to parentals
Direct selection of mitochondrially encoded genes in the pres-
ence of H2S (Pfenninger et al 2014) should be accompanied by co-
evolution at nuclear loci that directly interact with mitochondrial
gene products in the formation of protein complexes involved in the
OXPHOS pathway (Sunnucks Morales Lamb Pavlova amp Greening
2017 Willett amp Burton 2004) Disruption of co-evolved gene com-
plexes in hybrids should therefore lead to intrinsic fitness reductions
through mito-nuclear incompatibilities (Burton Pereira amp Barreto
2013 Wolff Ladoukakis Enrıquez amp Dowling 2014) which have
been hypothesized to be a key driver in speciation (Chou amp Leu
2010 Gershoni Templeton amp Mishmar 2009) Empirical tests of
these ideas remain to be conducted in sulphide spring fishes just
like studies on potential ecological and sexual selection against
hybrids that arise from interpopulation mating events
5 | PERSPECTIVES LIFE IN SULPHIDICENVIRONMENTS BEYOND P MEXICANA
Research on sulphide spring fishes of the P mexicana species com-
plex in Mexico has provided insights into mechanisms underlying
adaptation and the links between adaptation and speciation Sul-
phide springs do not only occur in Mexico however but are found
TOBLER ET AL | 853
worldwide and have been colonized by 24 evolutionarily indepen-
dent fish lineages in North America the Neotropics and the Middle
East (Greenway et al 2014) The vast majority of lineages belong to
the order Cyprinodontiformes (95) and livebearing members of the
family Poeciliidae in particular (80) This potentially indicates that
internal gestation of young has facilitated the colonization of
extreme environments because it shields developing embryos from
physiochemical stressors (Blackburn 1999) Among the Poeciliidae
sulphide spring fishes include members of the genera Brachyrhaphis
Gambusia Poeciliopsis Priapichthys Pseudoxiphophorus Xiphophorus
and multiple subgenera of Poecilia (Figure 3 Greenway et al 2014)
spanning over 40 million years of evolution (based on Reznick Fur-
ness Meredith amp Springer 2017)
A handful of studies have started to address molecular and phe-
notypic evolution across a broader taxon sampling of sulphide spring
fishes finding evidence for convergent evolution in morphology
(Riesch et al 2016 Tobler amp Hastings 2011) life history traits
(Riesch Plath et al 2010 Riesch et al 2014) and oxygen transport
genes (Barts et al 2018) In addition ecological speciation in
response to adaptation to H2S-rich environments has also been doc-
umented in sulphide spring poeciliids other than P mexicana (Riesch
et al 2016) These studies have laid a fruitful foundation for the
investigation of mechanisms of adaptation to H2S-rich springs from
genes to phenotypic traits linking micro-evolutionary processes to
macro-evolutionary patterns
The availability of broad-scale evolutionary replication of popula-
tions that have invaded habitats with the same physiochemical stressors
allows for addressing some of the ensuing challenges discussed above
and investigating the factors that give rise to predictable evolutionary
outcomes Convergent evolution is a commonmdashbut not omnipresentmdash
theme in adaptive evolution and multiple factors can determine the
degree of convergence among replicated lineages exposed to the same
sources of selection (Kaeuffer et al 2012 Landry amp Bernatchez 2010
Oke Rolshausen LeBlond amp Hendry 2017 Rosenblum amp Harmon
2011) Thus leveraging replicated lineages that have colonized sulphide
springs to assess molecular mechanisms underlying convergent pheno-
typic changes analyse how environmental heterogeneity among sul-
phide spring habitats causes lineage-specific patterns of trait evolution
and test whether and by what mechanisms adaptation to extreme envi-
ronments is linked to speciation will provide insights into how ecologi-
cal genetic and functional factors contribute to evolutionary
convergence at different levels of organismal organization (Rosenblum
et al 2014) An expanded taxon sampling with population pairs that
span almost the full range of the speciation continuum from panmixia to
complete reproductive isolation (Riesch et al 2016) will also help
addressing questions about temporal aspects of H2S adaptation In par-
ticular comparing younger to older lineages will provide insights about
the order at which genetic and phenotypic modifications accrue during
the adaptation process
Animals have also invaded H2S-rich habitats in other aquatic
ecosystems including cold seeps and deep-sea hydrothermal vents
(Tobler et al 2016) Adaptation to H2S in the P mexicana species com-
plex has involved modification of physiological pathways associated
with H2S toxicity (OXPHOS pathway) and detoxification (sulphide
F IGURE 3 Phylogeny of the family Poeciliidae (adopted from Culumber amp Tobler 2017) Various lineages in the genera Poecilia (subgeneraAcanthophacelus Limia and Mollienesia) Priapichthys Brachyrhaphis Poeciliopsis Xiphophorus Pseudoxiphophorus and Gambusia have colonizedH2S-rich springs in different parts of the Americas (see Greenway et al 2014) Lineages highlighted in yellow represent endemic speciesrestricted to H2S-rich springs while red lineages represent species in which some populations have invaded sulphidic habitats Pictures depict arepresentative of each focal genussubgenus
854 | TOBLER ET AL
quinone oxidoreductase pathway) which have been highly conserved
during the diversification of life on Earth (Saraste 1999 Shahak amp
Hauska 2008) The high replicability of H2S as an environmental factor
that shaped animal evolution across vastly different ecological contexts
consequently offers the opportunity to investigate whether selection
mediated by the presence of H2S has repeatedly modified these con-
served physiological pathways across animal phyla that have diverged
during the Cambrian explosion spanning over 500 million years of evo-
lution Such comparisons would provide unprecedented insights into
the effects of a single physiochemical stressor on evolutionary change
irrespective of ecological or phylogenetic contexts
6 | CONCLUSIONS
H2S-rich environments and their inhabitants represent useful models
for research in organismal biology ecology and evolution illustrating
both opportunities (eg strong and explicit source of selection evolu-
tionary replication) and challenges (eg complexity of environments
and organismal responses) in asking basic question about the origins
of biodiversity Understanding how organisms thrive in apparently
inhospitable environments will provide us with insights into lifersquos
capacities and limitations to adapt to novel environments (Waterman
1999) This is particularly relevant considering that human activities
substantially contribute to the exacerbation of physiochemical stres-
sors at local regional and global scales impacting ecological and evo-
lutionary dynamics (Alberti 2015 Norberg Urban Vellend
Klausmeier amp Loeuille 2012) Thus a focus on ecological and evolu-
tionary consequences of natural stressors that push organisms to the
brink of their physiological capacities may be instrumental to predict
consequences of anthropogenic environmental change
ACKNOWLEDGEMENTS
We would like to thank Lenin Arias-Rodriguez the Universidad
Juarez Autonoma de Tabasco and the Centro de Investigacion E
Innovacion Para la Ense~nanza y El Aprendizaje for their continued
collaboration enabling our work on the P mexicana system Funding
for this review article has been provided by the National Science
Foundation (IOS-1557860 IOS-1557795) and the US Army
Research Office (W911NF-15-1-0175)
AUTHOR CONTRIBUTIONS
MT wrote the first draft of the paper all authors contributed text
and comments
ORCID
Michael Tobler httporcidorg0000-0002-0326-0890
Joanna L Kelley httporcidorg0000-0002-7731-605X
Reuroudiger Riesch httporcidorg0000-0002-0223-1254
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Ast G (2004) How did alternative splicing evolve Nature Reviews
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Bell E M (2012) Life at extremes Environments organisms and strategies
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Bierbach D Klein M Sassmannshausen V Schlupp I Riesch R
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Blackburn D G (1999) Viviparity and oviparity Evolution and reproduc-
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Brown A P Greenway R Morgan S Quackenbush C R Giordani
L Arias-Rodriguez L Kelley J L (2017) Genome-scale data
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Brown J H Marquet P A amp Taper M L (1993) Evolution of body
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nurev-ecolsys-110512-135758
Bush S J Chen L Tovar-Corona J M amp Urritia A O (2017) Alterna-
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Carroll S P Jorgensen P S Kinnison M T Bergstrom C T Denison
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Cartwright R A Schwartz R S Merry A L amp Howell M M (2017)
The importance of selection in the evolution of blindness in cavefish
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017-0876-4
Chan Y F Marks M E Jones F C Villarreal G Shapiro M D Brady
S D Kingsley D M (2010) Adaptive evolution of pelvic reduc-
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Chou J Y amp Leu J Y (2010) Speciation through cytonuclear incompat-
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BioEssays 32 401ndash411 httpsdoiorg101002bies200900162
Colosimo P F Hosemann K E Balabhadra S Villarreal G Dickson
M Grimwood J Kingsley D M (2005) Widespread parallel evo-
lution in sticklebacks by repeated fixation of ectodysplasin alleles
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Cooper C E amp Brown G C (2008) The inhibition of mitochondrial
cytochrome oxidase by the gases carbon monoxide nitric oxide
hydrogen cyanide and hydrogen sulfide Chemical mechanism and
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533ndash539 httpsdoiorg101007s10863-008-9166-6
Culumber Z W Hopper G W Barts N Passow C N Morgan S Brown
A Tobler M (2016) Habitat use of two extremophile highly ende-
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Freshwater Ecosystems 26 1155ndash1167 httpsdoiorg101002aqc
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Culumber Z W amp Tobler M (2017) Sex-specific evolution during the
diversification of live-bearing fishes Nature Ecology amp Evolution 1
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Dalziel A C Rogers S M amp Schulte P M (2009) Linking genotypes
to phenotypes and fitness How mechanistic biology can inform
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Darwin C (1859) On the origin of species based on natural selection or
the preservation of favoured races in the struggle of life 6th ed Lon-
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Dasmahapatra K K Walters J R Briscoe A D Davey J W Whibley
A Nadeau N J Jiggins C D (2012) Butterfly genome reveals
promiscuous exchange of mimicry adaptations among species Nature
487 94ndash98 httpsdoiorg101038nature11041
Doudna J A amp Charpentier E (2014) The new frontier of genome edit-
ing with CRISPR-Cas9 Science 346 1258096 httpsdoiorg10
1126science1258096
Dubilier N Giere O amp Grieshaber M (2005) Concomitant effects of sul-
fide and hypoxia on the aerobic metabolism of the marine oligochaete
Tubificoides benedii Journal of Experimental Zoology 269 287ndash298
Eales K L Hollinshead K E R amp Tennant D A (2016) Hypoxia and
metabolic adaptation of cancer cells Oncogenesis 5 e190 httpsd
oiorg101038oncsis201550
Eghbal M A Pennefather P S amp OrsquoBrien P J (2004) H2S cytotoxicity
mechanism involves reactive oxygen species formation and mito-
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1016jtox200405020
Feldman C R Brodie Jr E D Brodie III E D amp Pfrender M E (2012)
Constraint shapes convergence in tetrodotoxin-resistant sodium
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Flores K B Wolschin F amp Amdam G V (2013) The role of methyla-
tion of DNA in environmental adaptation Integrative and Comparative
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Fradin H Kiontke K Zegar C Gutwein M Lucas J Kovtun M
Piano F (2017) Genome architecture and evolution of a unichromo-
somal asexual nematode Current Biology 27(2928ndash2939) e2926
Gershoni M Templeton A amp Mishmar D (2009) Mitochondrial bioen-
ergetics as a major motive force of speciation BioEssays 31 642ndash
650 httpsdoiorg101002bies200800139
Ghalambor C K Walker J A amp Reznick D N (2003) Multi-trait selec-
tion adaptation and constraints on the evolution of burst swimming
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Gibert J amp Deharveng L (2002) Subterranean ecosystems A truncated
functional biodiversity BioScience 52 473ndash481 httpsdoiorg10
16410006-3568(2002)052[0473SEATFB]20CO2
Gjuvsland A B Vik J O Beard D A Hunter P J amp Omholt S W
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Grant P R Grant B R Markert J A Keller L F amp Petren K (2004)
Convergent evolution of Darwinrsquos finches caused by introgressive
hybridization and selection Evolution 58 1588ndash1599 httpsdoi
org101111j0014-38202004tb01738x
Greene H W (2005) Organisms in nature as a central focus for biology
Trends in Ecology and Evolution 20 23ndash27 httpsdoiorg101016j
tree200411005
Greenway R Arias-Rodriguez L Diaz P amp Tobler M (2014) Patterns
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Diversity 6 597ndash632 httpsdoiorg103390d6030597
Greenway R Drexler S Arias-Rodriguez L amp Tobler M (2016) Adap-
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to assortative mating preferences during ecological speciation Evolu-
tion 70 2809ndash2822 httpsdoiorg101111evo13087
Gunderson A R Armstrong E J amp Stillman J H (2016) Multiple stres-
sors in a changing world The need for an improved perspective on
physiological responses to the dynamic marine environment Annual
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nurev-marine-122414-033953
Hildebrandt T M amp Grieshaber M (2008) Three enzymatic activities
catalyze the oxidation of sulfide to thiosulfate in mammalian and
invertebrate mitochondria FEBS Journal 275 3352ndash3361 httpsd
oiorg101111j1742-4658200806482x
Houle D Govindaraju D R amp Omholt S (2010) Phenomics The next
challenge Nature Reviews Genetics 11 855ndash866 httpsdoiorg10
1038nrg2897
IUCN (2012) Red List of threatened species version 20132 www
iucnredlistorg
Johnston I A amp Bennett A F (1996) Animals and temperature Pheno-
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Jorgensen C Auer S K amp Reznick D N (2011) A model for optimal
offspring size in fish including live-bearing and parental effects Amer-
ican Naturalist 177 E119ndashE135 httpsdoiorg101086659622
Kaeuffer R Peichel C L Bolnick D I amp Hendry A P (2012) Parallel
and nonparallel aspects of ecological phenotypic and genetic diver-
gence across replicate population pairs of lake and stream stickle-
back Evolution 66 402ndash418 httpsdoiorg101111j1558-5646
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Kell D B amp Oliver S G (2004) Here is the evidence now what is the
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856 | TOBLER ET AL
Kelley J L Arias-Rodriguez L Patacsil Martin D Yee M C Busta-
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life in hydrogen sulfide rich environments Molecular Biology and Evo-
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Kelley J L Passow C Plath M Arias-Rodriguez L Yee M C amp
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1471-2164-13-652
Klerks P L Xie L amp Levinton J S (2011) Quantitative genetics
approaches to study evolutionary processes in ecotoxicology a per-
spective from research on the evolution of resistance Ecotoxicology
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Kump L R Pavlov A amp Arthur M A (2005) Massive release of hydrogen
sulfide to the surface ocean and atmosphere during intervals of oceanic
anoxia Geology 33 397ndash400 httpsdoiorg101130G212951
Keuroustner A Hoffmann M Fraser B A Kottler V A Sharma E Wei-
gel D amp Dreyer C (2016) The genome of the Trinidadian guppy
Poecilia reticulata and variation in the guanapo population PLoS ONE
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Landry L amp Bernatchez L (2010) Role of epibenthic resource opportu-
nities in the parallel evolution of lake whitefish species pairs (Core-
gonus sp) Journal of Evolutionary Biology 23 2602ndash2613 httpsdoi
org101111j1420-9101201002121x
Langerhans R B (2007) Evolutionary consequences of predation Avoid-
ance escape reproduction and diversification In A M T Elewa
(Ed) Predation in organisms Berlin Springer
Li L Rose P amp Moore P K (2011) Hydrogen sulfide and cell signaling
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httpsdoiorg101146annurev-pharmtox-010510-100505
Lin Q Fan S Zhang Y Xu M Zhang H Yang Y Gunter H M
(2016) The seahorse genome and the evolution of its specialized mor-
phology Nature 540 395ndash399 httpsdoiorg101038nature20595
Liu Y Ma S Wang X Chang J Gao J Shi R Zhao P (2014)
Highly efficient multiplex targeted mutagenesis and genomic struc-
ture variation in Bombyx mori cells using CRISPRCas9 Insect bio-
chemistry and molecular biology 49 35ndash42 httpsdoiorg101016j
ibmb201403010
Lopez-Maury L Marguerat S amp Beuroahler J (2008) Tuning gene expres-
sion to changing environments From rapid responses to evolutionary
adaptation Nature Reviews Genetics 9 583ndash593 httpsdoiorg10
1038nrg2398
Maan M E amp Seehausen O (2012) Magic traits versus magic prefer-
ences in speciation Evolutionary Ecology Research 14 779ndash785
Martyn U Weigel D amp Dreyer C (2006) In vitro culture of embryos
of the guppy Poecilia reticulata Developmental Dynamics 235 617ndash
622 httpsdoiorg101002(ISSN)1097-0177
Metzker M L (2010) Sequencing technologies The next generation
Nature Reviews Genetics 11 31ndash46 httpsdoiorg101038nrg2626
Moore M P Riesch R amp Martin R A (2016) The predictability and
magnitude of life-history divergence to ecological agents of selection
A meta-analysis in livebearing fishes Ecology Letters 19 435ndash442
httpsdoiorg101111ele12576
Mustafa A K Gadalla M M Sen N Kim S Mu W Gazi S K
Snyder S H (2009) H2S signals through protein S-sulfhydration
Science Signaling 2 ra72
Nesse R M amp Stearns S C (2008) The great opportunity Evolutionary
applications to medicine and public health Evolutionary Applications
1 28ndash48 httpsdoiorg101111j1752-4571200700006x
Nevo E (2011) Evolution under environmental stress at macro and
microscales Genome Biology and Evolution 3 1039ndash1052 httpsdoi
org101093gbeevr052
Norberg J Urban M C Vellend M Klausmeier C A amp Loeuille N (2012)
Eco-evolutionary responses of biodiversity to climate change Nature Cli-
mate Change 2 747ndash751 httpsdoiorg101038nclimate1588
Nosil P (2012) Ecological speciation Oxford UK Oxford University
Press httpsdoiorg101093acprofosobl9780199587100001
0001
Nosil P Vines T H amp Funk D J (2005) Perspective Reproductive
isolation caused by natural selection against immigrants from diver-
gent habitats Evolution 59 705ndash719
Oke K B Rolshausen G LeBlond C amp Hendry A P (2017) How par-
allel is parallel evolution A comparative analysis in fishes American
Naturalist 190 1ndash16 httpsdoiorg101086691989
Olson K R (2011) The therapeutic potential of hydrogen sulfide
Separating hype from hope American Journal of Physiology-Regulatory
Integrative and Comparative Physiology 301 R297ndashR312 httpsdoi
org101152ajpregu000452011
Olson K R Donald J A Dombkowski R A amp Perry S F (2012) Evo-
lutionary and comparative aspects of nitric oxide carbon monoxide
and hydrogen sulfide Respiratory Physiology amp Neurobiology 184
117ndash129 httpsdoiorg101016jresp201204004
Olson K R amp Straub K D (2015) The role of hydrogen sulfide in evo-
lution and the evolution of hydrogen sulfide in metabolism and sig-
naling Physiology 31 60ndash72
Palacios M Arias-Rodriguez L Plath M Eifert C Lerp H Lamboj A
Tobler M (2013) The rediscovery of a long described species
reveals additional complexity in speciation patterns of poeciliid fishes
in sulfide springs PLoS ONE 8 e71069 httpsdoiorg101371jour
nalpone0071069
Palacios M Voelker G Arias-Rodriguez L Mateos M amp Tobler M
(2016) Phylogenetic analyses of the subgenus Mollienesia (Poecilia
Poeciliidae Teleostei) reveal taxonomic inconsistencies cryptic biodi-
versity and spatio-temporal aspects of diversification in Middle
America Molecular Phylogenetics and Evolution 103 230ndash244
httpsdoiorg101016jympev201607025
Passow C Arias-Rodriguez L amp Tobler M (2017) Convergent evolu-
tion of reduced energy demands in extremophile fish PLoS ONE 12
e0186935 httpsdoiorg101371journalpone0186935
Passow C N Brown A Arias-Rodriguez L Yee M-C Sockell A
Schartl M Tobler M (2017) Complexities of gene expression
patterns in natural populations of an extremophile fish (Poecilia mexi-
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101111mec14198
Passow C Greenway R Arias-Rodriguez L Jeyasingh P D amp Tobler
M (2015) Reduction of energetic demands through modification of
body size and metabolic rates in locally adapted extremophile fishes
Physiological and Biochemical Zoology 88 371ndash383 httpsdoiorg
101086681053
Passow C N Henpita C Shaw J H Quakenbush C Warren W C
Schartl M Tobler M (2017) The roles of plasticity and evolu-
tionary change in shaping gene expression variation in natural popu-
lations of extremophile fish Molecular Ecology 26 6384ndash6399
Pavey S A Bernatchez L Aubin-Horth N amp Landry C R (2012)
What is needed for next-generation ecological and evolutionary
genomics Trends in Ecology amp Evolution 27 673ndash678 httpsdoi
org101016jtree201207014
Peers C Bauer C Boyle J P Scragg J L amp Dallas M L (2012)
Modulation of ion channels by hydrogen sulfide Antioxidants amp Redox
Signaling 17 95ndash105 httpsdoiorg101089ars20114359
Pfennig D W amp Pfennig K S (2012) Evolutionrsquos wedge competition and
the origins of diversity Berkeley CA University of California Press
httpsdoiorg101525california97805202741810010001
Pfenninger M Lerp H Tobler M Passow C Kelley J L Funke E
Plath M (2014) Parallel evolution of cox genes in H2S-tolerant
fish as key adaptation to a toxic environment Nature Communica-
tions 5 3873
Pfenninger M Patel S Arias-Rodriguez L Feldmeyer B Riesch R amp
Plath M (2015) Unique evolutionary trajectories in repeated adapta-
tion to hydrogen sulphide-toxic habitats of a neotropical fish (Poecilia
TOBLER ET AL | 857
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1111mec13397
Pietri R Roman-Morales E amp Lopez-Garriga J (2011) Hydrogen sul-
fide and heme proteins Knowledge and mysteries Antioxidants amp
Redox Signaling 15 393ndash404 httpsdoiorg101089ars20103698
Plath M (2008) Male mating behavior and costs of sexual harassment
for females in cavernicolous and extremophile populations of Atlantic
mollies (Poecilia mexicana) Behaviour 145 73ndash98 httpsdoiorg10
1163156853908782687241
Plath M Hermann C Schreurooder R Riesch R Tobler M Garcia de
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maintain small-scale genetic differentiation despite perturbation by a
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Plath M Pfenninger M Lerp H Riesch R Eschenbrenner C Slattery
P Tobler M (2013) Genetic differentiation and selection against
migrants in evolutionarily replicated extreme environments Evolution
67 2647ndash2661 httpsdoiorg101111evo12133
Plath M Tobler M Riesch R Garcia de Leon F J Giere O amp
Schlupp I (2007) Survival in an extreme habitat The role of beha-
viour and energy limitation Naturwissenschaften 94 991ndash996
httpsdoiorg101007s00114-007-0279-2
Powell E (1989) Oxygen sulfide and diffusion Why thiobiotic meiofauna
must be sulfide-insensitive first-order respirers Journal of Marine
Research 47 887ndash932 httpsdoiorg101357002224089785076082
Ramirez-Soriano A Ramos-Onsins S E Rozas J Calafell F amp Navarro A
(2008) Statistical power analysis of neutrality tests under demographic
expansions contractions and bottlenecks with recombination Genetics
179 555ndash567 httpsdoiorg101534genetics107083006
Reiffenstein R Hulbert W amp Roth S (1992) Toxicology of hydrogen
sulfide Annual Reviews of Pharmacology and Toxicology 1992 109ndash
134 httpsdoiorg101146annurevpa32040192000545
Reznick D N Furness A I Meredith R W amp Springer M S (2017)
The origin and biogeographic diversification of fishes in the family
Poeciliidae PLoS ONE 12 e0172546 httpsdoiorg101371journa
lpone0172546
Reznick D amp Yang A P (1993) The influence of fluctuating resources
on life history Patterns of allocation and plasticity in female guppies
Ecology 74 2011ndash2019 httpsdoiorg1023071940844
Riesch R Duwe V Herrmann N Padur L Ramm A Scharnweber K
Plath M (2009) Variation along the shy-bold continuum in extre-
mophile fishes (Poecilia mexicana Poecilia sulphuraria) Behavioral Ecol-
ogy and Sociobiology 63 1515ndash1526 httpsdoiorg101007
s00265-009-0780-z
Riesch R Oranth A Dzienko J Karau N Schiessl A Stadler S
Plath M (2010) Extreme habitats are not refuges Poeciliids suffer
from increased aerial predation risk in sulphidic southern Mexican
habitats Biological Journal of the Linnean Society 101 417ndash426
httpsdoiorg101111j1095-8312201001522x
Riesch R Plath M Garcia de Leon F J amp Schlupp I (2010) Conver-
gent life-history shifts Toxic environments result in big babies in two
clades of poeciliids Naturwissenschaften 97 133ndash141 httpsdoi
org101007s00114-009-0613-y
Riesch R Plath M Schlupp I Tobler M amp Langerhans R B (2014)
Colonization of toxic springs drives predictable life-history shift in
livebearing fishes (Poeciliidae) Ecology Letters 17 65ndash71 httpsdoi
org101111ele12209
Riesch R Tobler M Lerp H Jourdan J Doumas L T Nosil P Plath
M (2016) Extremophile Poeciliidae Multivariate insights into the com-
plexity of speciation along replicated ecological gradients BMC Evolu-
tionary Biology 16 136 httpsdoiorg101186s12862-016-0705-1
Riesch R Tobler M amp Plath M (2015) Extremophile fishes Ecology
evolution and physiology of teleosts in extreme environments p 290
Heidelberg Germany Springer httpsdoiorg101007978-3-319-
13362-1
Ritchie M E Holzinger E R Li R Pendergrass S A amp Kim S (2015)
Methods of integrating data to uncover genotype-phenotype interac-
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nrg3868
Roach K Tobler M amp Winemiller K O (2011) Hydrogen sulfide bac-
teria and fish A unique subterranean food chain Ecology 92 2056ndash
2062 httpsdoiorg10189011-02761
Rohner N Jarosz D F Kowalko J E Yoshizawa M Jeffery W R
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logical evolution HSP90 as a capacitor for loss of eyes in cavefish
Science 342 1372ndash1375 httpsdoiorg101126science1240276
Rosales Lagarde L (2012) Investigation of karst brackish-sulfidic springs
and their role in the hydrogeology subsurface water-rock interactions
and speleogenesis at northern Sierra de Chiapas New Mexico Institute
of Mining and Technology Mexico
Rosenblum E B amp Harmon L J (2011) ldquoSame but differentrdquo Repli-
cated ecological speciation at White Sands Evolution 65 946ndash960
httpsdoiorg101111j1558-5646201001190x
Rosenblum E B Parent C E amp Brandt E E (2014) The molecular
basis of convergent evolution Annual Review of Ecology Evolution and
Systematics 45 203ndash226 httpsdoiorg101146annurev-ecolsys-
120213-091851
Saltz J B Hessel F C amp Kelly M W (2017) Trait correlations in the
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Saraste M (1999) Oxidative phosphorylation at the fin de siecle Science
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Schartl M (2014) Beyond the zebrafish Diverse fish species for model-
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httpsdoiorg101242dmm012245
Schartl M Walter R B Shen Y Garcia T Catchen J Amores A
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1038ng2604
Schluter D (2009) Evidence for ecological speciation and its alternative
Science 323 737ndash741 httpsdoiorg101126science1160006
Schulz-Mirbach T Eifert C Riesch R Farnworth M S Zimmer C
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httpsdoiorg101111jzo12366
SEDESOL (1994) NORMA Oficial Mexicana NOM-059-ECOL-1994 que
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Servedio M R van Doorn G S Kopp M Frame A M amp Nosil P
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amp Evolution 26 389ndash397 httpsdoiorg101016jtree201104005
Shahak Y amp Hauska G (2008) Sulfide oxidation from cyanobacteria to
humans Sulfide-quinone oxidoreductase (SQR) In R Hell C Dahl D
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Shalem O Sanjana N E amp Zhang F (2015) High-throughput func-
tional genomics using CRISPR-Cas9 Nature Reviews Genetics 16
299ndash311 httpsdoiorg101038nrg3899
Silva G J J Bye A el Azzouzi H amp Wisloslashff U (2017) MicroRNAs as
important regulators of exercise adaptation Progress in Cardiovascular
Diseases 60 130ndash151 httpsdoiorg101016jpcad201706003
Simola D F Wissler L Donahue G Waterhouse R M Helmkampf
M Roux J Viljakainen L (2013) Social insect genomes exhibit
dramatic evolution in gene composition and regulation while preserv-
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1235ndash1247 httpsdoiorg101101gr155408113
858 | TOBLER ET AL
Sommer-Trembo C Bierbach D Arias Rodriguez L Verel Y Jourdan
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Steindachner F (1863) Beitreuroage zur Kentniss der Sciaenoiden Brasiliens
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Naturwissenschaftlichen Classe der Kaiserlichen Akademie der Wis-
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Stinchcombe J R amp Hoekstra H E (2008) Combining population geno-
mics and quantitative genetics Finding genes underlying ecologically
important traits Heredity 100 158ndash170 httpsdoiorg101038sj
hdy6800937
Stipanuk M H (2004) Sulfur amino acid metabolism Pathways for pro-
duction and removal of homocysteine and cysteine Annual Review of
Nutrition 24 539ndash577 httpsdoiorg101146annurevnutr24
012003132418
Stipanuk M H amp Ueki I (2011) Dealing with methioninehomocysteine
sulfur Cysteine metabolism to taurine and inorganic sulfur Journal of
Inherited Metabolic Disease 34 17ndash32 httpsdoiorg101007
s10545-009-9006-9
Stockinger B Di Meglio P Gialitakis M amp Duarte J H (2014) The
aryl hydrocarbon receptor Multitasking in the immune system
Annual Review of Immunology 32 403ndash432 httpsdoiorg101146
annurev-immunol-032713-120245
Storz J F amp Wheat C W (2010) Integrating evolutionary and
functional approaches to infer adaptation at specific loci Evolution
64 2489ndash2509 httpsdoiorg101111j1558-5646201001044x
Stuart Y E Veen T Weber J N Hanson D Ravinet M Lohman B
K Bolnick D I (2017) Contrasting effects of environment and
genetics generate a continuum of parallel evolution Nature Ecology amp
Evolution 1 0158 httpsdoiorg101038s41559-017-0158
Sunnucks P Morales H E Lamb A M Pavlova A amp Greening C
(2017) Integrative approaches for studying mitochondrial and nuclear
genome co-evolution in oxidative phosphorylation Frontiers in Genet-
ics 8 25
Tobler M amp Hastings L (2011) Convergent patterns of body shape dif-
ferentiation in four different clades of poeciliid fishes inhabiting sul-
fide springs Evolutionary Biology 38 412ndash421 httpsdoiorg10
1007s11692-011-9129-4
Tobler M Palacios M Chapman L J Mitrofanov I Bierbach D
Plath M Mateos M (2011) Evolution in extreme environments
Replicated phenotypic differentiation in livebearing fish inhabiting
sulfidic springs Evolution 65 2213ndash2228 httpsdoiorg101111j
1558-5646201101298x
Tobler M Passow C N Greenway R Kelley J L amp Shaw J H
(2016) The evolutionary ecology of animals inhabiting hydrogen sul-
fide-rich environments Annual Review of Ecology Evolution and Sys-
tematics 47 239ndash262 httpsdoiorg101146annurev-ecolsys-
121415-032418
Tobler M amp Plath M (2009a) Threatened fishes of the world Gambu-
sia eurystoma Miller 1975 (Poeciliidae) Environmental Biology of
Fishes 85 251 httpsdoiorg101007s10641-009-9481-8
Tobler M amp Plath M (2009b) Threatened fishes of the world Poecilia
sulphuraria (Alvarez 1948) (Poeciliidae) Environmental Biology of
Fishes 85 333ndash334 httpsdoiorg101007s10641-009-9506-3
Tobler M Plath M Riesch R Schlupp I Grasse A Munimanda G
Moodley Y (2014) Selection from parasites favors immunogenic diver-
sity but not divergence among locally adapted host populations Journal
of Evolutionary Biology 27 960ndash974 httpsdoiorg101111jeb12370
Tobler M Riesch R amp Plath M (2015) Extremophile fishes An inte-
grative synthesis In R Riesch M Tobler amp M Plath (Eds) Extremo-
phile fishes Ecology evolution and physiology of teleosts in extreme
environments (pp 279ndash296) Heidelberg Germany Springer
Tobler M Riesch R Tobler C M amp Plath M (2009) Compensatory
behaviour in response to sulfide-induced hypoxia affects time
budgets feeding efficiency and predation risk Evolutionary Ecology
Research 11 935ndash948
Tobler M Scharnweber K Greenway R Passow C Arias-Rodriguez
L amp Garcia-De-Leon F J (2015) Convergent changes in the
trophic ecology if extremophile fish along replicated environmental
gradients Freshwater Biology 60 768ndash780 httpsdoiorg10
1111fwb12530
Todgham A E amp Stillman J H (2013) Physiological responses to shifts
in multiple environmental stressors Relevance in a changing worlds
Integrative and Comparative Biology 53 539ndash544 httpsdoiorg10
1093icbict086
van Doorn G S Edelaar P amp Weissing F J (2009) On the origin of
species by natural selection and sexual selection Science 326 1704ndash
1707 httpsdoiorg101126science1181661
Wall D H amp Virginia R A (1999) Controls on soil biodiversity Insights
from extreme environments Applied Soil Ecology 13 137ndash150
httpsdoiorg101016S0929-1393(99)00029-3
Wallace J L amp Wang R (2015) Hydrogen-sulfide based therapeutics
Exploiting a unique but ubiquitous gasotransmitter Nature Reviews
Drug Discovery 14 329ndash345 httpsdoiorg101038nrd4433
Wang R (2012) Physiological implications of hydrogen sulfide A whiff
exploration that blossomed Physiological Reviews 92 791ndash896
httpsdoiorg101152physrev000172011
Wang Z Gerstein M amp Snyder M (2009) RNA-seq A revolutionary
tool for transcriptomics Nature Reviews Genetics 10 57ndash63
httpsdoiorg101038nrg2484
Waterman T H (1999) The evolutionary challenges of extreme environ-
ments (part 1) Journal of Experimental Zoology 285 326ndash359
httpsdoiorg101002(SICI)1097-010X(19991215)2854amplt326
AID-JEZ3ampgt30CO2-T
Werren J H Richards S Desjardins C A Niehuis O Gadau J Col-
bourne J K amp Group N G W (2010) Functional and evolutionary
insights from the genomes of three parasitoid Nasonia species
Science 327 343ndash348 httpsdoiorg101126science1178028
Whitehead A (2012) Comparative genomics in ecological physiology
Toward a more nuanced understanding of acclimation and adapta-
tion Journal of Experimental Biology 215 884ndash891 httpsdoiorg
101242jeb058735
Whitehead A amp Crawford D L (2006) Neutral and adaptive variation
in gene expression Proceedings of the National Academy of Sciences
USA 103 5425ndash5430 httpsdoiorg101073pnas0507648103
Willett C S amp Burton R S (2004) Evolution of interacting proteins in
the mitochondrial electron transport system in a marine copepod
Molecular Biology and Evolution 21 443ndash453 httpsdoiorg10
1093molbevmsh031
Willi Y Van Buskirk J amp Hoffmann A (2006) Limits to the adaptive
potential of small populations Annual Review of Ecology Evolution
and Systematics 37 433ndash458 httpsdoiorg101146annurevecol
sys37091305110145
Wolff J N Ladoukakis E D Enrıquez J A amp Dowling D K (2014)
Mitonuclear interactions Evolutionary consequences over multiple
biological scales Philosophical Transactions of the Royal Society B 369
20130443 httpsdoiorg101098rstb20130443
How to cite this article Tobler M Kelley JL Plath M Riesch
R Extreme environments and the origins of biodiversity
Adaptation and speciation in sulphide spring fishes Mol Ecol
201827843ndash859 httpsdoiorg101111mec14497
TOBLER ET AL | 859
different physiological traits (associated with resistance mitiga-
tion and regulation Table 1) in F2 crosses will further allow test-
ing what molecular mechanisms are necessary for tolerance
whether there are alternative strategies with equivalent fitness
benefits and whether there are complementary modifications
with additive effects on tolerance
2 Considering the number of genes and physiological pathways
likely involved in H2S tolerance assessing the functional conse-
quences of genetic and gene expression variation is a daunting
task At the same time such functional tests can significantly
contribute to closing the genotype-to-phenotype gap because
physiochemical stress responses can be assessed along the con-
tinuum of organismal organization (Gjuvsland Vik Beard Hunter
amp Omholt 2013) Standardized assays can be used to quantify
the biochemical makeup of organelles cells and tissues (eg pro-
tein abundance and enzyme activity) physiological performance
in vitro and in vivo (eg rates of H2S detoxification ATP produc-
tion and reactive oxygen production) and fitness-relevant proxies
of whole organism performance (eg H2S tolerance Figure 2)
This highlights the importance of methods development for the
quantification of complex phenotypic traits or even whole phe-
nomes that can keep pace with rapid data generation through
high-throughput sequencing (Houle Govindaraju amp Omholt
2010 Ritchie Holzinger Li Pendergrass amp Kim 2015) In addi-
tion genotypendashphenotype links established through such correla-
tive approaches will ideally be verified through targeted gene
editing While gene editing with CRISPRndashCas9 is now routinely
employed in model organisms (Doudna amp Charpentier 2014 Sha-
lem Sanjana amp Zhang 2015) applying this technology to non-
models still remains challenging especially in livebearing species
like poeciliid fishes where in vitro culture of embryos is not rou-
tinely employed (Martyn Weigel amp Dreyer 2006) Nonetheless
the effects of specific mutations can potentially be verified
through the establishment and manipulation of cells in culture
(Liu et al 2014) or by generating transgenics (Lin et al 2016)
3 Organismal responses to physiochemical stressors occur at multi-
ple hierarchical levels that can be plastically modulated in
response to endogenous and exogenous cues (Whitehead 2012)
Understanding adaptation to physiochemical stressors conse-
quently requires disentangling the effects of genetic variation
developmental plasticity and short-term acclimation in gene
expression patterns on the emerging physiological processes
Common garden experiments on sulphide spring fishes have
shown that many genes differentially expressed between popula-
tions from sulphidic and nonsulphidic habitats in nature remain
differentially expressed in laboratory-reared fish that have never
been exposed to H2S indicating evolved differences in constitu-
tive gene expression (Passow Henpita et al 2017) At the same
time expression patterns in the laboratory also exhibit evidence
for plasticity in response to short-term H2S exposure (Passow
Henpita et al 2017) Some of these plastic responses are shared
between populations from sulphidic and nonsulphidic habitats
(likely representing shared ancestral plasticity) while others have
been lost or gained during sulphide spring colonization A core
challenge for future research is to overcome logistical constraints
that have so far prevented long-term H2S exposures of fish in
the laboratory Exposing individuals throughout development will
be required to test how developmental plasticity and short-term
acclimation shape responses to H2S in individuals from adapted
and nonadapted populations Finally the next big scientific
advance in this context will also require the identification of reg-
ulatory mechanisms that shape expression differences during
adaptation including sequence changes in transcription factors
and their binding sites patterns of DNA methylation and chro-
matin accessibility as well as the production of microRNAs and
competing endogenous RNAs that can modulate transcript abun-
dance (Flores Wolschin amp Amdam 2013 Lopez-Maury Mar-
guerat amp Beuroahler 2008 Silva Bye el Azzouzi amp Wisloslashff 2017)
In addition the role of alternative splicing and other post-tran-
scriptional and post-translational modifications in mediating adap-
tation remains understudied by evolutionary biologists (Ast
2004 Bush Chen Tovar-Corona amp Urritia 2017) This is particu-
larly relevant because H2S has been hypothesized to play a role
in the post-translational modification of proteins with sulphur-
containing amino acids through sulfhydration (Mustafa et al
2009)
Bridging the genotypendashphenotype gap remains one of the major
challenges in biology (Gjuvsland et al 2013 Ritchie et al 2015) and
will provide opportunities to understand how organisms function in
the face of environmental stress and how evolution shapes these
functions during adaptation to different environmental conditions
Establishing the molecular mechanisms of adaptation across repli-
cated lineages exposed to the same sources of selection will also
provide insights into how evolutionary history demography and
genetics shape convergent evolutionary outcomes providing a better
understanding at what hierarchical levels of organismal organization
evolutionary change may actually be predictable (Rosenblum et al
2014)
3 | COMPLEX ORGANISMS IN A COMPLEXWORLD DISENTANGLING THE TANGLEDBANK
Mechanisms of adaptation can be studied through bottom-up
(screening for genetic modifications that allow for inferences about
relevant phenotypic traits) and top-down approaches (assessing fit-
ness-relevant phenotypic differences among populations without
knowledge about the genetic basis see Schluter 2009) Over the
years genomic and transcriptomic studies on sulphide spring popula-
tions of the P mexicana species complex have been complemented
with analyses of phenotypic variation Just as genetic analyses have
indicated that adaptation to H2S is mediated by the modification
and modulation of many genes associated with multiple physiological
pathways phenotypic studies uncovered a variety of differences
TOBLER ET AL | 849
between populations from sulphidic and nonsulphidic environments
Many of these phenotypic differences have evolved in convergence
across replicated sulphide spring lineages in different river drainages
Examples include modifications of morphological (body shape and
organ size Tobler et al 2011 Tobler Scharnweber et al 2015
Schulz-Mirbach et al 2016) physiological (tolerance and metabolic
rates Tobler et al 2011 Passow Arias-Rodriguez amp Tobler 2017)
behavioural (aggression and personality Riesch et al 2009 Bierbach
et al 2012 Sommer-Trembo et al 2016 Bierbach Arias Rodriguez
amp Plath 2018) and life history traits (fecundity and offspring size
Riesch Plath Garcia de Leon amp Schlupp 2010 Riesch et al 2014)
Hence proximate populations in different habitat types have
diverged in complex phenotypes that span many different suites of
traits and a key question is how divergence in multivariate pheno-
types arises In theory H2S could be a key source of selection driv-
ing multivariate phenotypic divergence either because it directly
exerts selection on a multitude of traits that are independently modi-
fied to reach new fitness peaks or because selection on a few fit-
ness-relevant traits causes simultaneous changes in correlated
characteristics Neither explanation seems sufficient because many
Abiotic environmentOxygen concentrations
Ionic compositionTemperature
Structure and flow
Biotic environmentTrophic resources
DemographyPredationParasitism
H2S
Genetic variation
Gene expression
Biochemistry
Physiologicalfunction
Organismal performance
Primary fitness components
Survivalgrowth
reproduction
Organismal function Environment
F IGURE 2 Understanding how organisms function in the context of their environment is a core question in evolutionary biology yet thecomplexities associated with organismal function and environmental variation provide significant challenges High-throughput sequencing hasallowed for the identification of candidate genes subject to selection and differential expression during adaptation to H2S The next step is tobridge the genotype-to-phenotype gap by quantifying the integrated responses to H2S across the continuum of organismal organization includingbiochemical phenotypes physiological functions whole organism performance and ultimately fitness In addition understanding adaptation toH2S-rich environments will also require acknowledging H2Srsquos relation to other abiotic and biotic environmental variables which can exert selectionand modulate aspects of organismal function This will allow for the establishment of cause-and-effect relationships between specific sources ofselection and evolutionary change in specific traits and contribute to our understanding of organismal function in natural systems
850 | TOBLER ET AL
of the divergent traits between populations from sulphidic and non-
sulphidic environments have no clearmdashor at best tenuousmdashlinks to
H2S toxicity and detoxification and there is only limited evidence
for within-population correlations between different traits groups
(Riesch et al 2016)
Evolutionary analyses frequently focus on seemingly simple
sources of selection juxtaposing populations that are assigned to dis-
crete habitat types to understand the evolution of specific traits
(Kaeuffer Peichel Bolnick amp Hendry 2012) But as Darwin (1859)
already observed in his ldquotangled bankrdquo allegory selection in natural
systems is rarely univariate but multifarious selective regimes con-
tribute to trait divergence even among populations that occur along
apparently simple environmental gradients Unquantified environmen-
tal variation that is subsumed in the categorization of habitat types
can profoundly affect trait variation in natural systems (Stuart et al
2017) highlighting the importance of considering and testing alterna-
tive hypotheses about the cause-and-effect relationship between
specific sources of selection and evolution of specific traits Thus
accurate natural history observations that consider organisms as holis-
tic entities consisting of interacting traits in the context of the abiotic
and biotic factors comprising their environment are critical to inform
the design and interpretation of empirical research in natural systems
(Greene 2005) Indeed adjacent sulphidic and nonsulphidic habitat
types do not merely differ in the presence or absence of H2S but also
in a variety of other abiotic and biotic factors (Figure 2) Unlike nonsul-
phidic habitats sulphide springs in southern Mexico are characterized
by extreme hypoxia elevated salinity and lower pH (Rosales Lagarde
2012 Tobler et al 2011) In addition the presence of physiochemical
stressors affects the biotic communities in these sulphide springs Spe-
cies richness of aquatic invertebrates and vertebrates is drastically
reduced as a consequence of H2S toxicity (Greenway Arias-Rodriguez
Diaz amp Tobler 2014) and primary production appears to be driven in
part by chemoautotrophic bacteria that use H2S as an energy source
rather than just by autochthonous and allochthonous photosynthetic
organisms (Roach Tobler amp Winemiller 2011) These changes in the
biotic environment affect the relative importance of intra-vs inter-
specific competition trophic resource use and exposure to predators
and parasites (Riesch Oranth et al 2010 Tobler et al 2014 Tobler
Scharnweber et al 2015)
31 | Alternative drivers of trait evolution
Acknowledging the complexity of selective regimes in sulphide
springs allows for a more nuanced understanding of trait evolution
and for the development of alternative hypotheses about the ulti-
mate mechanisms that cause divergence in specific genetic and phe-
notypic traits In some instances sources of selection other than H2S
can plausibly explain trait divergence between populations from sul-
phidic and nonsulphidic habitats For example hypoxia likely drives
increases in head size and gill surface area (Tobler et al 2011)
changes in the ionic composition of the water can explain differential
expression of genes associated with osmoregulation (Kelley et al
2016) and shifts in trophic resource use are associated with
modifications of the gastrointestinal system (Tobler Scharnweber
et al 2015) In other cases disentangling alternative hypotheses is
not trivial because selection mediated by H2S and other environmen-
tal factors are predicted to affect trait modifications in similar ways
Shifts towards anaerobic metabolism in sulphide spring fish could be
the consequence of inhibition of aerobic ATP production by H2S or
by constraints in oxygen availability Similarly reproductive life his-
tory shifts towards the production of larger but fewer offspring could
be driven by H2S (decreasing offspringrsquos surfacevolume ratio to mini-
mize the diffusion of H2S into the body Powell 1989) by habitat dif-
ferences in the quality and quantity of predation that can affect size-
specific rates of extrinsic mortality (Jorgensen Auer amp Reznick
2011) or by energetic constraints (see below) While comparisons
across different selective regimes can shed light into the ultimate dri-
vers of trait evolution (Moore Riesch amp Martin 2016) isolating the
effects of different sources of selection may not be possible because
they could act in concert and facilitate increases of organismal perfor-
mance in multiple contexts (Ghalambor Walker amp Reznick 2003)
When multifarious selection shapes multivariate phenotypic evo-
lution it is important to also consider the role of trade-offs both in
terms of trait expression (increased investment in one trait may
impose constraints on the expression of other traits) and in terms of
trait function (optimizing the trait for one function may impair the
traitrsquos utility for other functions) For example selection on large off-
spring size in sulphide spring fishes has inadvertent consequences
for fecundity (Riesch et al 2014) and selection for increased gill
surface areas due to hypoxia simultaneously increases exposure to
environmental H2S (Tobler et al 2011) Thus the outcome of phe-
notypic evolution is ultimately a result of balancing competing needs
in a manner that optimizes trait expression and organismal function
in multiple contexts to maximize fitness (Arnold 1983) and the pres-
ence of trade-offs can have cascading indirect effects with selection
on one or a few traits leading to the modification of a multitude of
apparently unrelated characteristics
In this context bioenergetics may represent a nexus in the evo-
lution of complex traits observed in sulphide spring fish and other
extreme environments Living in and adapting to sulphide springs
imposes constraints on organismal energy availability (Plath et al
2007) with according trade-offs for energy allocation Energy limita-
tion in sulphide springs does not arise from a scarcity of food
resources (chemoautotrophic bacteria eaten by the fish are abundant
in sulphide springs Tobler Scharnweber et al 2015) but from
behavioural and physiological adaptation A primary survival strategy
for fish in the toxic and hypoxic springs is to engage in aquatic sur-
face respiration where individuals breathe directly at the water sur-
face to maximize oxygen uptake and minimize H2S exposure This
behaviour mediates short-term survival (Plath et al 2007) but it
also constrains energy acquisition for benthic foragers like P mexi-
cana (Tobler Riesch Tobler amp Plath 2009) and increases susceptibil-
ity to bird predation (Riesch Oranth et al 2010) Costly investments
into H2S detoxification and tissue repair as well as a shift from aero-
bic to less efficient anaerobic metabolism further constrain energy
allocation to other functions (Passow Greenway Arias-Rodriguez
TOBLER ET AL | 851
Jeyasingh amp Tobler 2015) Theory predicts that energetic con-
straints ndash whether mediated by limitations in the rate of resource
acquisition or in the rate of energy allocation to reproduction (as
opposed to maintenance or growth) ndash should exert selection for a
reduction in organismal energy budgets which allows for the maxi-
mization of residual energy allocation for the production of offspring
(Brown Marquet amp Taper 1993) Several lines of evidence support
the hypothesis that energy limitation plays a critical role in pheno-
typic evolution of sulphide spring fish Compared to individuals in
nonsulphidic environments fish in sulphide springs have significantly
lower routine energy demands which is mediated both by reduc-
tions in body size and mass-specific metabolism (Passow Arias-
Rodriguez et al 2017) Reductions in other traits such as energeti-
cally costly behaviours (Bierbach et al 2012 Plath 2008) and the
size of energy-demanding organs (Schulz-Mirbach et al 2016) may
also contribute to the reduced energy demands and shifts in repro-
ductive life history traits (Riesch et al 2014) are consistent with
selection from energy limitation (Reznick amp Yang 1993)
32 | Ensuing challenges Disentanglingenvironmental and organismal complexities
Future research needs to empirically address how multifarious selec-
tive regimes shape the evolution of organisms with complex pheno-
types We have made great advances in our understanding of how
specific traits evolve and mediate adaptation (eg morphology Chan
et al 2010 coloration Dasmahapatra et al 2012 sensory organs
Cartwright Schwartz Merry amp Howell 2017) and we have a thor-
ough understanding of how single environmental factors can shape
organismal responses (eg climatic conditions Johnston amp Bennett
1996 predation Langerhans 2007 competition Pfennig amp Pfennig
2012) In contrast our understanding of phenotypic evolution in
response to multifarious selection is comparatively limited especially
because interactive effects of different environmental factors can lead
to organismal and evolutionary responses that are unpredictable based
on experiments that manipulate single environmental variables in iso-
lation (Gunderson Armstrong amp Stillman 2016 Todgham amp Stillman
2013) Future research thus needs to acknowledge the complexities of
environments and organismal design and address them explicitly in
empirical investigations to gain a robust understanding of biological
diversification (Culumber amp Tobler 2017 Stuart et al 2017)
4 | LINKING ADAPTATION TOSPECIATION
So far we primarily explored the causes of adaptive evolution but
what are its consequences Theory predicts that adaptation to local
environmental conditions can lead to speciation because reproduc-
tive isolation emerges incidentally as a by-product (ecological specia-
tion Nosil 2012) Population genetic analyses of Poecilia spp in
sulphide springs and adjacent populations in nonsulphidic environ-
ments have indicated strong genetic differentiation across small
spatial scales (in some instances lt100 m) and in the absence of
physical barriers that would prevent fish movement (Plath et al
2013) In addition population genetic structuring has been shown to
be stable even in the face of major environmental disturbance
BOX 1 Speciation in sulphide springs and conservation
biology
Adaptation to H2S-rich environments in poeciliids coincides
with ecological speciation giving rise to phenotypically dis-
tinct and reproductively isolated lineages In some
instances this has led to the evolution of highly distinct
micro-endemic species including Poecilia sulphuraria and
P thermalis Limia sulphurophila and Gambusia eurystoma
(Figure 3) Except for P sulphuraria which is found in two
spring complexes of the Rıo Pichucalco drainage of Mexico
(Tobler amp Plath 2009b) the distributions of these sulphide
spring endemics are restricted to single localities that
include only small areas of suitable habitat For example
the largest sulphide spring complex (the Ba~nos del Azufre
in southern Mexico which harbours P sulphuraria and G
eurystoma) includes ~18000 m2 of suitable habitat (equiva-
lent to lt35 football fields) while one of the smallest (La
Esperanza springs harbouring P thermalis) merely covers
~600 m2 (just over a tenth of a football field Brown et al
2017) Accordingly some sulphide spring endemics are
considered critically endangered (IUCN 2012) and pro-
tected by law (SEDESOL 1994)
Many threatened species with restricted ranges exhibit
low genetic diversity and high incidences of inbreeding
(Bouzat 2010 Willi Van Buskirk amp Hoffmann 2006) In
contrast at least P sulphuraria and P thermalismdashthe only
species investigated to datemdashactually exhibit relatively high
effective population sizes (Ne = 1200ndash3800 depending on
the population) and no evidence for inbreeding (FIS lt 0)
(Brown et al 2017) This is likely a consequence of the
long-term stability of sulphide springs and the high pro-
ductivity and low levels of interspecific competition also
support unusually high fish densities and thus population
census sizes (Brown et al 2017 Culumber et al 2016)
Nonetheless sulphide spring endemics are threatened by
habitat destruction which is mediated primarily by the
modification of springs for recreational purposes defor-
estation and conversion of land for agriculture and most
recently high-intensity palm oil plantations (Tobler amp Plath
2009ab) Despite the official protection of sulphide spring
endemics there have been no tangible conservation mea-
sures that foster the sustainable development of land
around sulphide springs with the well-being of the endemic
fishes in mind
852 | TOBLER ET AL
(catastrophic flood event Plath et al 2010) Population genetic dif-
ferentiation between adjacent sulphide-adapted and nonadapted
populations is indicative of ongoing ecological speciation (Box 1) but
different population pairs from sulphidic and nonsulphidic waters
have made variable progress along the speciation continuum towards
complete reproductive isolation (Plath et al 2013) This prompts
questions about the mechanisms involved in reducing gene flow
along the environmental gradient connecting the two habitat types
41 | Prezygotic mechanisms of reproductiveisolation
The simplest form of reproductive isolation between locally adapted
populations arises from selection against migrants between habitat
types (Nosil Vines amp Funk 2005) Translocation experiments in nat-
ural habitats have indicated strong survival selection against
migrants especially those translocated from nonsulphidic to sulphidic
habitats (Plath et al 2013) Fish translocated from nonsulphidic
environments face high mortality within short periods of time
(lt24 hr) which is likely caused by H2S toxicity and hypoxia in sul-
phide springs Hence low gene flow from populations inhabiting
nonsulphidic waters into populations inhabiting sulphidic habitats is
simply explained by a lack of adaptation to the extreme environmen-
tal conditions At least in some drainages there is also appreciable
mortality for sulphide spring fish translocated to nonsulphidic habi-
tats although mechanisms causing inviability are unclear and the
strength of selection appears to be lower (Plath et al 2013) If sur-
vival selection against immigrants was the sole source of reproduc-
tive isolation this should result in unidirectional gene flow from
H2S-adapted to nonadapted populations but population genetic
analyses provided no evidence for this implying that additional
mechanisms are at work (Plath et al 2013)
One such mechanism could be intersexual selection and mate
choice experiments have indicated that migrants from sulphidic to
nonsulphidic habitats are at a significant disadvantage with fish from
nonsulphidic habitats avoiding potential mating partners from sul-
phidic ones (Plath et al 2013 Sommer-Trembo et al 2016) Such
assortative mating has been found to be critical during ecological spe-
ciation because it counteracts the homogenizing effects of gene flow
(van Doorn Edelaar amp Weissing 2009) yet how assortative mating
patterns arise often remains unclear In theory assortative mating can
arise through a variety of processes (Nosil 2012) ldquoMagic traitrdquo mecha-
nisms where mating preferences evolve as a by-product of divergent
selection on ecological traits that serve as cues have recently garnered
much attention (Servedio van Doorn Kopp Frame amp Nosil 2011)
Consistent with magic trait mechanisms assortative mating in P mexi-
cana appears to be linked to adaptive population differences in body
shape which serve as cues to distinguish potential mating partners
from different habitats (Greenway Drexler Arias-Rodriguez amp Tobler
2016) However neither sulphide spring fish (which are unlikely to
encounter migrants from nonsulphidic habitats due to H2S toxicity)
nor allopatric P mexicana from nonsulphidic waters (which have never
experienced migrants from sulphide spring populations) exhibit a
preference for mating partners from their own ecotype suggesting
that assortative mating in this system is a product of reinforcement
(ie direct selection for assortative mating Greenway et al 2016)
This is different from classic magic traits mechanisms because assorta-
tive mating is not a mere by-product of adaptation but adaptive traits
have instead been co-opted as mating signals in response to selection
on mating preferences (Maan amp Seehausen 2012) Moreover males of
some H2S-adapted populations have reduced aggressive behaviour
(Bierbach et al 2012 2018) In staged contests of size-matched males
from different ecotypes intruders had a significantly lower likelihood
to establish dominance than resident males independent of whether
or not they showed reduced aggressiveness overall Hence intrasexual
selection may represent another mechanism mediating reproductive
isolation between locally adapted ecotypes Overall H2S-adapted poe-
ciliids allow disentangling the relative contributions of different forms
of ecological and sexual selectionmdashand their interdependencymdashduring
ecological speciation
42 | Ensuing challenges quantifying postzygoticmechanisms of reproductive isolation
In contrast to prezygotic mechanisms little is known about postzy-
gotic mechanisms of reproductive isolation that potentially act to
reduce gene flow between adjacent populations in sulphidic and
nonsulphidic habitats Putative hybrids between sulphidic and non-
sulphidic ecotypes have occasionally been observed in nonsulphidic
habitats immediately adjacent to confluences with sulphide springs
(R Riesch M Plath and M Tobler personal observations) but it
remains unclear whether and through what mechanisms such hybrids
exhibit reduced fitness compared to parentals
Direct selection of mitochondrially encoded genes in the pres-
ence of H2S (Pfenninger et al 2014) should be accompanied by co-
evolution at nuclear loci that directly interact with mitochondrial
gene products in the formation of protein complexes involved in the
OXPHOS pathway (Sunnucks Morales Lamb Pavlova amp Greening
2017 Willett amp Burton 2004) Disruption of co-evolved gene com-
plexes in hybrids should therefore lead to intrinsic fitness reductions
through mito-nuclear incompatibilities (Burton Pereira amp Barreto
2013 Wolff Ladoukakis Enrıquez amp Dowling 2014) which have
been hypothesized to be a key driver in speciation (Chou amp Leu
2010 Gershoni Templeton amp Mishmar 2009) Empirical tests of
these ideas remain to be conducted in sulphide spring fishes just
like studies on potential ecological and sexual selection against
hybrids that arise from interpopulation mating events
5 | PERSPECTIVES LIFE IN SULPHIDICENVIRONMENTS BEYOND P MEXICANA
Research on sulphide spring fishes of the P mexicana species com-
plex in Mexico has provided insights into mechanisms underlying
adaptation and the links between adaptation and speciation Sul-
phide springs do not only occur in Mexico however but are found
TOBLER ET AL | 853
worldwide and have been colonized by 24 evolutionarily indepen-
dent fish lineages in North America the Neotropics and the Middle
East (Greenway et al 2014) The vast majority of lineages belong to
the order Cyprinodontiformes (95) and livebearing members of the
family Poeciliidae in particular (80) This potentially indicates that
internal gestation of young has facilitated the colonization of
extreme environments because it shields developing embryos from
physiochemical stressors (Blackburn 1999) Among the Poeciliidae
sulphide spring fishes include members of the genera Brachyrhaphis
Gambusia Poeciliopsis Priapichthys Pseudoxiphophorus Xiphophorus
and multiple subgenera of Poecilia (Figure 3 Greenway et al 2014)
spanning over 40 million years of evolution (based on Reznick Fur-
ness Meredith amp Springer 2017)
A handful of studies have started to address molecular and phe-
notypic evolution across a broader taxon sampling of sulphide spring
fishes finding evidence for convergent evolution in morphology
(Riesch et al 2016 Tobler amp Hastings 2011) life history traits
(Riesch Plath et al 2010 Riesch et al 2014) and oxygen transport
genes (Barts et al 2018) In addition ecological speciation in
response to adaptation to H2S-rich environments has also been doc-
umented in sulphide spring poeciliids other than P mexicana (Riesch
et al 2016) These studies have laid a fruitful foundation for the
investigation of mechanisms of adaptation to H2S-rich springs from
genes to phenotypic traits linking micro-evolutionary processes to
macro-evolutionary patterns
The availability of broad-scale evolutionary replication of popula-
tions that have invaded habitats with the same physiochemical stressors
allows for addressing some of the ensuing challenges discussed above
and investigating the factors that give rise to predictable evolutionary
outcomes Convergent evolution is a commonmdashbut not omnipresentmdash
theme in adaptive evolution and multiple factors can determine the
degree of convergence among replicated lineages exposed to the same
sources of selection (Kaeuffer et al 2012 Landry amp Bernatchez 2010
Oke Rolshausen LeBlond amp Hendry 2017 Rosenblum amp Harmon
2011) Thus leveraging replicated lineages that have colonized sulphide
springs to assess molecular mechanisms underlying convergent pheno-
typic changes analyse how environmental heterogeneity among sul-
phide spring habitats causes lineage-specific patterns of trait evolution
and test whether and by what mechanisms adaptation to extreme envi-
ronments is linked to speciation will provide insights into how ecologi-
cal genetic and functional factors contribute to evolutionary
convergence at different levels of organismal organization (Rosenblum
et al 2014) An expanded taxon sampling with population pairs that
span almost the full range of the speciation continuum from panmixia to
complete reproductive isolation (Riesch et al 2016) will also help
addressing questions about temporal aspects of H2S adaptation In par-
ticular comparing younger to older lineages will provide insights about
the order at which genetic and phenotypic modifications accrue during
the adaptation process
Animals have also invaded H2S-rich habitats in other aquatic
ecosystems including cold seeps and deep-sea hydrothermal vents
(Tobler et al 2016) Adaptation to H2S in the P mexicana species com-
plex has involved modification of physiological pathways associated
with H2S toxicity (OXPHOS pathway) and detoxification (sulphide
F IGURE 3 Phylogeny of the family Poeciliidae (adopted from Culumber amp Tobler 2017) Various lineages in the genera Poecilia (subgeneraAcanthophacelus Limia and Mollienesia) Priapichthys Brachyrhaphis Poeciliopsis Xiphophorus Pseudoxiphophorus and Gambusia have colonizedH2S-rich springs in different parts of the Americas (see Greenway et al 2014) Lineages highlighted in yellow represent endemic speciesrestricted to H2S-rich springs while red lineages represent species in which some populations have invaded sulphidic habitats Pictures depict arepresentative of each focal genussubgenus
854 | TOBLER ET AL
quinone oxidoreductase pathway) which have been highly conserved
during the diversification of life on Earth (Saraste 1999 Shahak amp
Hauska 2008) The high replicability of H2S as an environmental factor
that shaped animal evolution across vastly different ecological contexts
consequently offers the opportunity to investigate whether selection
mediated by the presence of H2S has repeatedly modified these con-
served physiological pathways across animal phyla that have diverged
during the Cambrian explosion spanning over 500 million years of evo-
lution Such comparisons would provide unprecedented insights into
the effects of a single physiochemical stressor on evolutionary change
irrespective of ecological or phylogenetic contexts
6 | CONCLUSIONS
H2S-rich environments and their inhabitants represent useful models
for research in organismal biology ecology and evolution illustrating
both opportunities (eg strong and explicit source of selection evolu-
tionary replication) and challenges (eg complexity of environments
and organismal responses) in asking basic question about the origins
of biodiversity Understanding how organisms thrive in apparently
inhospitable environments will provide us with insights into lifersquos
capacities and limitations to adapt to novel environments (Waterman
1999) This is particularly relevant considering that human activities
substantially contribute to the exacerbation of physiochemical stres-
sors at local regional and global scales impacting ecological and evo-
lutionary dynamics (Alberti 2015 Norberg Urban Vellend
Klausmeier amp Loeuille 2012) Thus a focus on ecological and evolu-
tionary consequences of natural stressors that push organisms to the
brink of their physiological capacities may be instrumental to predict
consequences of anthropogenic environmental change
ACKNOWLEDGEMENTS
We would like to thank Lenin Arias-Rodriguez the Universidad
Juarez Autonoma de Tabasco and the Centro de Investigacion E
Innovacion Para la Ense~nanza y El Aprendizaje for their continued
collaboration enabling our work on the P mexicana system Funding
for this review article has been provided by the National Science
Foundation (IOS-1557860 IOS-1557795) and the US Army
Research Office (W911NF-15-1-0175)
AUTHOR CONTRIBUTIONS
MT wrote the first draft of the paper all authors contributed text
and comments
ORCID
Michael Tobler httporcidorg0000-0002-0326-0890
Joanna L Kelley httporcidorg0000-0002-7731-605X
Reuroudiger Riesch httporcidorg0000-0002-0223-1254
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Bell E M (2012) Life at extremes Environments organisms and strategies
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Blackburn D G (1999) Viviparity and oviparity Evolution and reproduc-
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Brown A P Greenway R Morgan S Quackenbush C R Giordani
L Arias-Rodriguez L Kelley J L (2017) Genome-scale data
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Brown J H Marquet P A amp Taper M L (1993) Evolution of body
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nurev-ecolsys-110512-135758
Bush S J Chen L Tovar-Corona J M amp Urritia A O (2017) Alterna-
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Carroll S P Jorgensen P S Kinnison M T Bergstrom C T Denison
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Cartwright R A Schwartz R S Merry A L amp Howell M M (2017)
The importance of selection in the evolution of blindness in cavefish
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017-0876-4
Chan Y F Marks M E Jones F C Villarreal G Shapiro M D Brady
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Chou J Y amp Leu J Y (2010) Speciation through cytonuclear incompat-
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Colosimo P F Hosemann K E Balabhadra S Villarreal G Dickson
M Grimwood J Kingsley D M (2005) Widespread parallel evo-
lution in sticklebacks by repeated fixation of ectodysplasin alleles
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Cooper C E amp Brown G C (2008) The inhibition of mitochondrial
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hydrogen cyanide and hydrogen sulfide Chemical mechanism and
physiological significance Journal of Bioenergy and Biomembranes 40
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Culumber Z W Hopper G W Barts N Passow C N Morgan S Brown
A Tobler M (2016) Habitat use of two extremophile highly ende-
mic and critically endangered fish species (Gambusia eurystoma and Poe-
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Freshwater Ecosystems 26 1155ndash1167 httpsdoiorg101002aqc
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Culumber Z W amp Tobler M (2017) Sex-specific evolution during the
diversification of live-bearing fishes Nature Ecology amp Evolution 1
1185ndash1191
Dalziel A C Rogers S M amp Schulte P M (2009) Linking genotypes
to phenotypes and fitness How mechanistic biology can inform
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Darwin C (1859) On the origin of species based on natural selection or
the preservation of favoured races in the struggle of life 6th ed Lon-
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Dasmahapatra K K Walters J R Briscoe A D Davey J W Whibley
A Nadeau N J Jiggins C D (2012) Butterfly genome reveals
promiscuous exchange of mimicry adaptations among species Nature
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Doudna J A amp Charpentier E (2014) The new frontier of genome edit-
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1126science1258096
Dubilier N Giere O amp Grieshaber M (2005) Concomitant effects of sul-
fide and hypoxia on the aerobic metabolism of the marine oligochaete
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Eales K L Hollinshead K E R amp Tennant D A (2016) Hypoxia and
metabolic adaptation of cancer cells Oncogenesis 5 e190 httpsd
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Eghbal M A Pennefather P S amp OrsquoBrien P J (2004) H2S cytotoxicity
mechanism involves reactive oxygen species formation and mito-
chondrial depolarisation Toxicology 203 69ndash76 httpsdoiorg10
1016jtox200405020
Feldman C R Brodie Jr E D Brodie III E D amp Pfrender M E (2012)
Constraint shapes convergence in tetrodotoxin-resistant sodium
channels of snakes Proceedings of the National Academy of Sciences
USA 109 4556ndash4561 httpsdoiorg101073pnas1113468109
Flores K B Wolschin F amp Amdam G V (2013) The role of methyla-
tion of DNA in environmental adaptation Integrative and Comparative
Biology 53 359ndash372 httpsdoiorg101093icbict019
Fradin H Kiontke K Zegar C Gutwein M Lucas J Kovtun M
Piano F (2017) Genome architecture and evolution of a unichromo-
somal asexual nematode Current Biology 27(2928ndash2939) e2926
Gershoni M Templeton A amp Mishmar D (2009) Mitochondrial bioen-
ergetics as a major motive force of speciation BioEssays 31 642ndash
650 httpsdoiorg101002bies200800139
Ghalambor C K Walker J A amp Reznick D N (2003) Multi-trait selec-
tion adaptation and constraints on the evolution of burst swimming
performance Integrative and Comparative Biology 43 431ndash438
httpsdoiorg101093icb433431
Gibert J amp Deharveng L (2002) Subterranean ecosystems A truncated
functional biodiversity BioScience 52 473ndash481 httpsdoiorg10
16410006-3568(2002)052[0473SEATFB]20CO2
Gjuvsland A B Vik J O Beard D A Hunter P J amp Omholt S W
(2013) Bridging the genotype-phenotype gap What does it take
Journal of Physiology 591 2055ndash2066 httpsdoiorg101113jphys
iol2012248864
Grant P R Grant B R Markert J A Keller L F amp Petren K (2004)
Convergent evolution of Darwinrsquos finches caused by introgressive
hybridization and selection Evolution 58 1588ndash1599 httpsdoi
org101111j0014-38202004tb01738x
Greene H W (2005) Organisms in nature as a central focus for biology
Trends in Ecology and Evolution 20 23ndash27 httpsdoiorg101016j
tree200411005
Greenway R Arias-Rodriguez L Diaz P amp Tobler M (2014) Patterns
of macroinvertebrate and fish diversity in freshwater sulphide springs
Diversity 6 597ndash632 httpsdoiorg103390d6030597
Greenway R Drexler S Arias-Rodriguez L amp Tobler M (2016) Adap-
tive but not condition-dependent body shape differences contribute
to assortative mating preferences during ecological speciation Evolu-
tion 70 2809ndash2822 httpsdoiorg101111evo13087
Gunderson A R Armstrong E J amp Stillman J H (2016) Multiple stres-
sors in a changing world The need for an improved perspective on
physiological responses to the dynamic marine environment Annual
Review of Marine Science 8 357ndash378 httpsdoiorg101146an
nurev-marine-122414-033953
Hildebrandt T M amp Grieshaber M (2008) Three enzymatic activities
catalyze the oxidation of sulfide to thiosulfate in mammalian and
invertebrate mitochondria FEBS Journal 275 3352ndash3361 httpsd
oiorg101111j1742-4658200806482x
Houle D Govindaraju D R amp Omholt S (2010) Phenomics The next
challenge Nature Reviews Genetics 11 855ndash866 httpsdoiorg10
1038nrg2897
IUCN (2012) Red List of threatened species version 20132 www
iucnredlistorg
Johnston I A amp Bennett A F (1996) Animals and temperature Pheno-
typic and evolutionary adaptation Cambridge Cambridge University
Press httpsdoiorg101017CBO9780511721854
Jorgensen C Auer S K amp Reznick D N (2011) A model for optimal
offspring size in fish including live-bearing and parental effects Amer-
ican Naturalist 177 E119ndashE135 httpsdoiorg101086659622
Kaeuffer R Peichel C L Bolnick D I amp Hendry A P (2012) Parallel
and nonparallel aspects of ecological phenotypic and genetic diver-
gence across replicate population pairs of lake and stream stickle-
back Evolution 66 402ndash418 httpsdoiorg101111j1558-5646
201101440x
Kell D B amp Oliver S G (2004) Here is the evidence now what is the
hypothesis The complementary roles of inductive and hypothesis-
driven science in the post-genomic era BioEssays 26 99ndash105
httpsdoiorg101002(ISSN)1521-1878
856 | TOBLER ET AL
Kelley J L Arias-Rodriguez L Patacsil Martin D Yee M C Busta-
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life in hydrogen sulfide rich environments Molecular Biology and Evo-
lution 33 1419ndash1434 httpsdoiorg101093molbevmsw020
Kelley J L Passow C Plath M Arias-Rodriguez L Yee M C amp
Tobler M (2012) Genomic resources for a model in adaptation and
speciation research The characterization of the Poecilia mexicana
transcriptome BMC Genomics 13 652 httpsdoiorg101186
1471-2164-13-652
Klerks P L Xie L amp Levinton J S (2011) Quantitative genetics
approaches to study evolutionary processes in ecotoxicology a per-
spective from research on the evolution of resistance Ecotoxicology
20 513ndash523 httpsdoiorg101007s10646-011-0640-2
Kump L R Pavlov A amp Arthur M A (2005) Massive release of hydrogen
sulfide to the surface ocean and atmosphere during intervals of oceanic
anoxia Geology 33 397ndash400 httpsdoiorg101130G212951
Keuroustner A Hoffmann M Fraser B A Kottler V A Sharma E Wei-
gel D amp Dreyer C (2016) The genome of the Trinidadian guppy
Poecilia reticulata and variation in the guanapo population PLoS ONE
11 e0169087 httpsdoiorg101371journalpone0169087
Landry L amp Bernatchez L (2010) Role of epibenthic resource opportu-
nities in the parallel evolution of lake whitefish species pairs (Core-
gonus sp) Journal of Evolutionary Biology 23 2602ndash2613 httpsdoi
org101111j1420-9101201002121x
Langerhans R B (2007) Evolutionary consequences of predation Avoid-
ance escape reproduction and diversification In A M T Elewa
(Ed) Predation in organisms Berlin Springer
Li L Rose P amp Moore P K (2011) Hydrogen sulfide and cell signaling
Annual Review of Pharmacology and Toxicology 51 169ndash187
httpsdoiorg101146annurev-pharmtox-010510-100505
Lin Q Fan S Zhang Y Xu M Zhang H Yang Y Gunter H M
(2016) The seahorse genome and the evolution of its specialized mor-
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Liu Y Ma S Wang X Chang J Gao J Shi R Zhao P (2014)
Highly efficient multiplex targeted mutagenesis and genomic struc-
ture variation in Bombyx mori cells using CRISPRCas9 Insect bio-
chemistry and molecular biology 49 35ndash42 httpsdoiorg101016j
ibmb201403010
Lopez-Maury L Marguerat S amp Beuroahler J (2008) Tuning gene expres-
sion to changing environments From rapid responses to evolutionary
adaptation Nature Reviews Genetics 9 583ndash593 httpsdoiorg10
1038nrg2398
Maan M E amp Seehausen O (2012) Magic traits versus magic prefer-
ences in speciation Evolutionary Ecology Research 14 779ndash785
Martyn U Weigel D amp Dreyer C (2006) In vitro culture of embryos
of the guppy Poecilia reticulata Developmental Dynamics 235 617ndash
622 httpsdoiorg101002(ISSN)1097-0177
Metzker M L (2010) Sequencing technologies The next generation
Nature Reviews Genetics 11 31ndash46 httpsdoiorg101038nrg2626
Moore M P Riesch R amp Martin R A (2016) The predictability and
magnitude of life-history divergence to ecological agents of selection
A meta-analysis in livebearing fishes Ecology Letters 19 435ndash442
httpsdoiorg101111ele12576
Mustafa A K Gadalla M M Sen N Kim S Mu W Gazi S K
Snyder S H (2009) H2S signals through protein S-sulfhydration
Science Signaling 2 ra72
Nesse R M amp Stearns S C (2008) The great opportunity Evolutionary
applications to medicine and public health Evolutionary Applications
1 28ndash48 httpsdoiorg101111j1752-4571200700006x
Nevo E (2011) Evolution under environmental stress at macro and
microscales Genome Biology and Evolution 3 1039ndash1052 httpsdoi
org101093gbeevr052
Norberg J Urban M C Vellend M Klausmeier C A amp Loeuille N (2012)
Eco-evolutionary responses of biodiversity to climate change Nature Cli-
mate Change 2 747ndash751 httpsdoiorg101038nclimate1588
Nosil P (2012) Ecological speciation Oxford UK Oxford University
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0001
Nosil P Vines T H amp Funk D J (2005) Perspective Reproductive
isolation caused by natural selection against immigrants from diver-
gent habitats Evolution 59 705ndash719
Oke K B Rolshausen G LeBlond C amp Hendry A P (2017) How par-
allel is parallel evolution A comparative analysis in fishes American
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Olson K R (2011) The therapeutic potential of hydrogen sulfide
Separating hype from hope American Journal of Physiology-Regulatory
Integrative and Comparative Physiology 301 R297ndashR312 httpsdoi
org101152ajpregu000452011
Olson K R Donald J A Dombkowski R A amp Perry S F (2012) Evo-
lutionary and comparative aspects of nitric oxide carbon monoxide
and hydrogen sulfide Respiratory Physiology amp Neurobiology 184
117ndash129 httpsdoiorg101016jresp201204004
Olson K R amp Straub K D (2015) The role of hydrogen sulfide in evo-
lution and the evolution of hydrogen sulfide in metabolism and sig-
naling Physiology 31 60ndash72
Palacios M Arias-Rodriguez L Plath M Eifert C Lerp H Lamboj A
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reveals additional complexity in speciation patterns of poeciliid fishes
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nalpone0071069
Palacios M Voelker G Arias-Rodriguez L Mateos M amp Tobler M
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versity and spatio-temporal aspects of diversification in Middle
America Molecular Phylogenetics and Evolution 103 230ndash244
httpsdoiorg101016jympev201607025
Passow C Arias-Rodriguez L amp Tobler M (2017) Convergent evolu-
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e0186935 httpsdoiorg101371journalpone0186935
Passow C N Brown A Arias-Rodriguez L Yee M-C Sockell A
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101111mec14198
Passow C Greenway R Arias-Rodriguez L Jeyasingh P D amp Tobler
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body size and metabolic rates in locally adapted extremophile fishes
Physiological and Biochemical Zoology 88 371ndash383 httpsdoiorg
101086681053
Passow C N Henpita C Shaw J H Quakenbush C Warren W C
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lations of extremophile fish Molecular Ecology 26 6384ndash6399
Pavey S A Bernatchez L Aubin-Horth N amp Landry C R (2012)
What is needed for next-generation ecological and evolutionary
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org101016jtree201207014
Peers C Bauer C Boyle J P Scragg J L amp Dallas M L (2012)
Modulation of ion channels by hydrogen sulfide Antioxidants amp Redox
Signaling 17 95ndash105 httpsdoiorg101089ars20114359
Pfennig D W amp Pfennig K S (2012) Evolutionrsquos wedge competition and
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httpsdoiorg101525california97805202741810010001
Pfenninger M Lerp H Tobler M Passow C Kelley J L Funke E
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Pfenninger M Patel S Arias-Rodriguez L Feldmeyer B Riesch R amp
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TOBLER ET AL | 857
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1111mec13397
Pietri R Roman-Morales E amp Lopez-Garriga J (2011) Hydrogen sul-
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Plath M (2008) Male mating behavior and costs of sexual harassment
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1163156853908782687241
Plath M Hermann C Schreurooder R Riesch R Tobler M Garcia de
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Plath M Pfenninger M Lerp H Riesch R Eschenbrenner C Slattery
P Tobler M (2013) Genetic differentiation and selection against
migrants in evolutionarily replicated extreme environments Evolution
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Plath M Tobler M Riesch R Garcia de Leon F J Giere O amp
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Powell E (1989) Oxygen sulfide and diffusion Why thiobiotic meiofauna
must be sulfide-insensitive first-order respirers Journal of Marine
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Ramirez-Soriano A Ramos-Onsins S E Rozas J Calafell F amp Navarro A
(2008) Statistical power analysis of neutrality tests under demographic
expansions contractions and bottlenecks with recombination Genetics
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Reiffenstein R Hulbert W amp Roth S (1992) Toxicology of hydrogen
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Reznick D N Furness A I Meredith R W amp Springer M S (2017)
The origin and biogeographic diversification of fishes in the family
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lpone0172546
Reznick D amp Yang A P (1993) The influence of fluctuating resources
on life history Patterns of allocation and plasticity in female guppies
Ecology 74 2011ndash2019 httpsdoiorg1023071940844
Riesch R Duwe V Herrmann N Padur L Ramm A Scharnweber K
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mophile fishes (Poecilia mexicana Poecilia sulphuraria) Behavioral Ecol-
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s00265-009-0780-z
Riesch R Oranth A Dzienko J Karau N Schiessl A Stadler S
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from increased aerial predation risk in sulphidic southern Mexican
habitats Biological Journal of the Linnean Society 101 417ndash426
httpsdoiorg101111j1095-8312201001522x
Riesch R Plath M Garcia de Leon F J amp Schlupp I (2010) Conver-
gent life-history shifts Toxic environments result in big babies in two
clades of poeciliids Naturwissenschaften 97 133ndash141 httpsdoi
org101007s00114-009-0613-y
Riesch R Plath M Schlupp I Tobler M amp Langerhans R B (2014)
Colonization of toxic springs drives predictable life-history shift in
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Riesch R Tobler M Lerp H Jourdan J Doumas L T Nosil P Plath
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plexity of speciation along replicated ecological gradients BMC Evolu-
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Riesch R Tobler M amp Plath M (2015) Extremophile fishes Ecology
evolution and physiology of teleosts in extreme environments p 290
Heidelberg Germany Springer httpsdoiorg101007978-3-319-
13362-1
Ritchie M E Holzinger E R Li R Pendergrass S A amp Kim S (2015)
Methods of integrating data to uncover genotype-phenotype interac-
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nrg3868
Roach K Tobler M amp Winemiller K O (2011) Hydrogen sulfide bac-
teria and fish A unique subterranean food chain Ecology 92 2056ndash
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Rohner N Jarosz D F Kowalko J E Yoshizawa M Jeffery W R
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and their role in the hydrogeology subsurface water-rock interactions
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of Mining and Technology Mexico
Rosenblum E B amp Harmon L J (2011) ldquoSame but differentrdquo Repli-
cated ecological speciation at White Sands Evolution 65 946ndash960
httpsdoiorg101111j1558-5646201001190x
Rosenblum E B Parent C E amp Brandt E E (2014) The molecular
basis of convergent evolution Annual Review of Ecology Evolution and
Systematics 45 203ndash226 httpsdoiorg101146annurev-ecolsys-
120213-091851
Saltz J B Hessel F C amp Kelly M W (2017) Trait correlations in the
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Saraste M (1999) Oxidative phosphorylation at the fin de siecle Science
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Schartl M (2014) Beyond the zebrafish Diverse fish species for model-
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Schartl M Walter R B Shen Y Garcia T Catchen J Amores A
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1038ng2604
Schluter D (2009) Evidence for ecological speciation and its alternative
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Schulz-Mirbach T Eifert C Riesch R Farnworth M S Zimmer C
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Servedio M R van Doorn G S Kopp M Frame A M amp Nosil P
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Shalem O Sanjana N E amp Zhang F (2015) High-throughput func-
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Silva G J J Bye A el Azzouzi H amp Wisloslashff U (2017) MicroRNAs as
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Simola D F Wissler L Donahue G Waterhouse R M Helmkampf
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858 | TOBLER ET AL
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Stinchcombe J R amp Hoekstra H E (2008) Combining population geno-
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Stipanuk M H (2004) Sulfur amino acid metabolism Pathways for pro-
duction and removal of homocysteine and cysteine Annual Review of
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Stipanuk M H amp Ueki I (2011) Dealing with methioninehomocysteine
sulfur Cysteine metabolism to taurine and inorganic sulfur Journal of
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s10545-009-9006-9
Stockinger B Di Meglio P Gialitakis M amp Duarte J H (2014) The
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Storz J F amp Wheat C W (2010) Integrating evolutionary and
functional approaches to infer adaptation at specific loci Evolution
64 2489ndash2509 httpsdoiorg101111j1558-5646201001044x
Stuart Y E Veen T Weber J N Hanson D Ravinet M Lohman B
K Bolnick D I (2017) Contrasting effects of environment and
genetics generate a continuum of parallel evolution Nature Ecology amp
Evolution 1 0158 httpsdoiorg101038s41559-017-0158
Sunnucks P Morales H E Lamb A M Pavlova A amp Greening C
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genome co-evolution in oxidative phosphorylation Frontiers in Genet-
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Tobler M amp Hastings L (2011) Convergent patterns of body shape dif-
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fide springs Evolutionary Biology 38 412ndash421 httpsdoiorg10
1007s11692-011-9129-4
Tobler M Palacios M Chapman L J Mitrofanov I Bierbach D
Plath M Mateos M (2011) Evolution in extreme environments
Replicated phenotypic differentiation in livebearing fish inhabiting
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1558-5646201101298x
Tobler M Passow C N Greenway R Kelley J L amp Shaw J H
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fide-rich environments Annual Review of Ecology Evolution and Sys-
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Tobler M amp Plath M (2009a) Threatened fishes of the world Gambu-
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Fishes 85 251 httpsdoiorg101007s10641-009-9481-8
Tobler M amp Plath M (2009b) Threatened fishes of the world Poecilia
sulphuraria (Alvarez 1948) (Poeciliidae) Environmental Biology of
Fishes 85 333ndash334 httpsdoiorg101007s10641-009-9506-3
Tobler M Plath M Riesch R Schlupp I Grasse A Munimanda G
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sity but not divergence among locally adapted host populations Journal
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budgets feeding efficiency and predation risk Evolutionary Ecology
Research 11 935ndash948
Tobler M Scharnweber K Greenway R Passow C Arias-Rodriguez
L amp Garcia-De-Leon F J (2015) Convergent changes in the
trophic ecology if extremophile fish along replicated environmental
gradients Freshwater Biology 60 768ndash780 httpsdoiorg10
1111fwb12530
Todgham A E amp Stillman J H (2013) Physiological responses to shifts
in multiple environmental stressors Relevance in a changing worlds
Integrative and Comparative Biology 53 539ndash544 httpsdoiorg10
1093icbict086
van Doorn G S Edelaar P amp Weissing F J (2009) On the origin of
species by natural selection and sexual selection Science 326 1704ndash
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Wall D H amp Virginia R A (1999) Controls on soil biodiversity Insights
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httpsdoiorg101016S0929-1393(99)00029-3
Wallace J L amp Wang R (2015) Hydrogen-sulfide based therapeutics
Exploiting a unique but ubiquitous gasotransmitter Nature Reviews
Drug Discovery 14 329ndash345 httpsdoiorg101038nrd4433
Wang R (2012) Physiological implications of hydrogen sulfide A whiff
exploration that blossomed Physiological Reviews 92 791ndash896
httpsdoiorg101152physrev000172011
Wang Z Gerstein M amp Snyder M (2009) RNA-seq A revolutionary
tool for transcriptomics Nature Reviews Genetics 10 57ndash63
httpsdoiorg101038nrg2484
Waterman T H (1999) The evolutionary challenges of extreme environ-
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AID-JEZ3ampgt30CO2-T
Werren J H Richards S Desjardins C A Niehuis O Gadau J Col-
bourne J K amp Group N G W (2010) Functional and evolutionary
insights from the genomes of three parasitoid Nasonia species
Science 327 343ndash348 httpsdoiorg101126science1178028
Whitehead A (2012) Comparative genomics in ecological physiology
Toward a more nuanced understanding of acclimation and adapta-
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Whitehead A amp Crawford D L (2006) Neutral and adaptive variation
in gene expression Proceedings of the National Academy of Sciences
USA 103 5425ndash5430 httpsdoiorg101073pnas0507648103
Willett C S amp Burton R S (2004) Evolution of interacting proteins in
the mitochondrial electron transport system in a marine copepod
Molecular Biology and Evolution 21 443ndash453 httpsdoiorg10
1093molbevmsh031
Willi Y Van Buskirk J amp Hoffmann A (2006) Limits to the adaptive
potential of small populations Annual Review of Ecology Evolution
and Systematics 37 433ndash458 httpsdoiorg101146annurevecol
sys37091305110145
Wolff J N Ladoukakis E D Enrıquez J A amp Dowling D K (2014)
Mitonuclear interactions Evolutionary consequences over multiple
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How to cite this article Tobler M Kelley JL Plath M Riesch
R Extreme environments and the origins of biodiversity
Adaptation and speciation in sulphide spring fishes Mol Ecol
201827843ndash859 httpsdoiorg101111mec14497
TOBLER ET AL | 859
between populations from sulphidic and nonsulphidic environments
Many of these phenotypic differences have evolved in convergence
across replicated sulphide spring lineages in different river drainages
Examples include modifications of morphological (body shape and
organ size Tobler et al 2011 Tobler Scharnweber et al 2015
Schulz-Mirbach et al 2016) physiological (tolerance and metabolic
rates Tobler et al 2011 Passow Arias-Rodriguez amp Tobler 2017)
behavioural (aggression and personality Riesch et al 2009 Bierbach
et al 2012 Sommer-Trembo et al 2016 Bierbach Arias Rodriguez
amp Plath 2018) and life history traits (fecundity and offspring size
Riesch Plath Garcia de Leon amp Schlupp 2010 Riesch et al 2014)
Hence proximate populations in different habitat types have
diverged in complex phenotypes that span many different suites of
traits and a key question is how divergence in multivariate pheno-
types arises In theory H2S could be a key source of selection driv-
ing multivariate phenotypic divergence either because it directly
exerts selection on a multitude of traits that are independently modi-
fied to reach new fitness peaks or because selection on a few fit-
ness-relevant traits causes simultaneous changes in correlated
characteristics Neither explanation seems sufficient because many
Abiotic environmentOxygen concentrations
Ionic compositionTemperature
Structure and flow
Biotic environmentTrophic resources
DemographyPredationParasitism
H2S
Genetic variation
Gene expression
Biochemistry
Physiologicalfunction
Organismal performance
Primary fitness components
Survivalgrowth
reproduction
Organismal function Environment
F IGURE 2 Understanding how organisms function in the context of their environment is a core question in evolutionary biology yet thecomplexities associated with organismal function and environmental variation provide significant challenges High-throughput sequencing hasallowed for the identification of candidate genes subject to selection and differential expression during adaptation to H2S The next step is tobridge the genotype-to-phenotype gap by quantifying the integrated responses to H2S across the continuum of organismal organization includingbiochemical phenotypes physiological functions whole organism performance and ultimately fitness In addition understanding adaptation toH2S-rich environments will also require acknowledging H2Srsquos relation to other abiotic and biotic environmental variables which can exert selectionand modulate aspects of organismal function This will allow for the establishment of cause-and-effect relationships between specific sources ofselection and evolutionary change in specific traits and contribute to our understanding of organismal function in natural systems
850 | TOBLER ET AL
of the divergent traits between populations from sulphidic and non-
sulphidic environments have no clearmdashor at best tenuousmdashlinks to
H2S toxicity and detoxification and there is only limited evidence
for within-population correlations between different traits groups
(Riesch et al 2016)
Evolutionary analyses frequently focus on seemingly simple
sources of selection juxtaposing populations that are assigned to dis-
crete habitat types to understand the evolution of specific traits
(Kaeuffer Peichel Bolnick amp Hendry 2012) But as Darwin (1859)
already observed in his ldquotangled bankrdquo allegory selection in natural
systems is rarely univariate but multifarious selective regimes con-
tribute to trait divergence even among populations that occur along
apparently simple environmental gradients Unquantified environmen-
tal variation that is subsumed in the categorization of habitat types
can profoundly affect trait variation in natural systems (Stuart et al
2017) highlighting the importance of considering and testing alterna-
tive hypotheses about the cause-and-effect relationship between
specific sources of selection and evolution of specific traits Thus
accurate natural history observations that consider organisms as holis-
tic entities consisting of interacting traits in the context of the abiotic
and biotic factors comprising their environment are critical to inform
the design and interpretation of empirical research in natural systems
(Greene 2005) Indeed adjacent sulphidic and nonsulphidic habitat
types do not merely differ in the presence or absence of H2S but also
in a variety of other abiotic and biotic factors (Figure 2) Unlike nonsul-
phidic habitats sulphide springs in southern Mexico are characterized
by extreme hypoxia elevated salinity and lower pH (Rosales Lagarde
2012 Tobler et al 2011) In addition the presence of physiochemical
stressors affects the biotic communities in these sulphide springs Spe-
cies richness of aquatic invertebrates and vertebrates is drastically
reduced as a consequence of H2S toxicity (Greenway Arias-Rodriguez
Diaz amp Tobler 2014) and primary production appears to be driven in
part by chemoautotrophic bacteria that use H2S as an energy source
rather than just by autochthonous and allochthonous photosynthetic
organisms (Roach Tobler amp Winemiller 2011) These changes in the
biotic environment affect the relative importance of intra-vs inter-
specific competition trophic resource use and exposure to predators
and parasites (Riesch Oranth et al 2010 Tobler et al 2014 Tobler
Scharnweber et al 2015)
31 | Alternative drivers of trait evolution
Acknowledging the complexity of selective regimes in sulphide
springs allows for a more nuanced understanding of trait evolution
and for the development of alternative hypotheses about the ulti-
mate mechanisms that cause divergence in specific genetic and phe-
notypic traits In some instances sources of selection other than H2S
can plausibly explain trait divergence between populations from sul-
phidic and nonsulphidic habitats For example hypoxia likely drives
increases in head size and gill surface area (Tobler et al 2011)
changes in the ionic composition of the water can explain differential
expression of genes associated with osmoregulation (Kelley et al
2016) and shifts in trophic resource use are associated with
modifications of the gastrointestinal system (Tobler Scharnweber
et al 2015) In other cases disentangling alternative hypotheses is
not trivial because selection mediated by H2S and other environmen-
tal factors are predicted to affect trait modifications in similar ways
Shifts towards anaerobic metabolism in sulphide spring fish could be
the consequence of inhibition of aerobic ATP production by H2S or
by constraints in oxygen availability Similarly reproductive life his-
tory shifts towards the production of larger but fewer offspring could
be driven by H2S (decreasing offspringrsquos surfacevolume ratio to mini-
mize the diffusion of H2S into the body Powell 1989) by habitat dif-
ferences in the quality and quantity of predation that can affect size-
specific rates of extrinsic mortality (Jorgensen Auer amp Reznick
2011) or by energetic constraints (see below) While comparisons
across different selective regimes can shed light into the ultimate dri-
vers of trait evolution (Moore Riesch amp Martin 2016) isolating the
effects of different sources of selection may not be possible because
they could act in concert and facilitate increases of organismal perfor-
mance in multiple contexts (Ghalambor Walker amp Reznick 2003)
When multifarious selection shapes multivariate phenotypic evo-
lution it is important to also consider the role of trade-offs both in
terms of trait expression (increased investment in one trait may
impose constraints on the expression of other traits) and in terms of
trait function (optimizing the trait for one function may impair the
traitrsquos utility for other functions) For example selection on large off-
spring size in sulphide spring fishes has inadvertent consequences
for fecundity (Riesch et al 2014) and selection for increased gill
surface areas due to hypoxia simultaneously increases exposure to
environmental H2S (Tobler et al 2011) Thus the outcome of phe-
notypic evolution is ultimately a result of balancing competing needs
in a manner that optimizes trait expression and organismal function
in multiple contexts to maximize fitness (Arnold 1983) and the pres-
ence of trade-offs can have cascading indirect effects with selection
on one or a few traits leading to the modification of a multitude of
apparently unrelated characteristics
In this context bioenergetics may represent a nexus in the evo-
lution of complex traits observed in sulphide spring fish and other
extreme environments Living in and adapting to sulphide springs
imposes constraints on organismal energy availability (Plath et al
2007) with according trade-offs for energy allocation Energy limita-
tion in sulphide springs does not arise from a scarcity of food
resources (chemoautotrophic bacteria eaten by the fish are abundant
in sulphide springs Tobler Scharnweber et al 2015) but from
behavioural and physiological adaptation A primary survival strategy
for fish in the toxic and hypoxic springs is to engage in aquatic sur-
face respiration where individuals breathe directly at the water sur-
face to maximize oxygen uptake and minimize H2S exposure This
behaviour mediates short-term survival (Plath et al 2007) but it
also constrains energy acquisition for benthic foragers like P mexi-
cana (Tobler Riesch Tobler amp Plath 2009) and increases susceptibil-
ity to bird predation (Riesch Oranth et al 2010) Costly investments
into H2S detoxification and tissue repair as well as a shift from aero-
bic to less efficient anaerobic metabolism further constrain energy
allocation to other functions (Passow Greenway Arias-Rodriguez
TOBLER ET AL | 851
Jeyasingh amp Tobler 2015) Theory predicts that energetic con-
straints ndash whether mediated by limitations in the rate of resource
acquisition or in the rate of energy allocation to reproduction (as
opposed to maintenance or growth) ndash should exert selection for a
reduction in organismal energy budgets which allows for the maxi-
mization of residual energy allocation for the production of offspring
(Brown Marquet amp Taper 1993) Several lines of evidence support
the hypothesis that energy limitation plays a critical role in pheno-
typic evolution of sulphide spring fish Compared to individuals in
nonsulphidic environments fish in sulphide springs have significantly
lower routine energy demands which is mediated both by reduc-
tions in body size and mass-specific metabolism (Passow Arias-
Rodriguez et al 2017) Reductions in other traits such as energeti-
cally costly behaviours (Bierbach et al 2012 Plath 2008) and the
size of energy-demanding organs (Schulz-Mirbach et al 2016) may
also contribute to the reduced energy demands and shifts in repro-
ductive life history traits (Riesch et al 2014) are consistent with
selection from energy limitation (Reznick amp Yang 1993)
32 | Ensuing challenges Disentanglingenvironmental and organismal complexities
Future research needs to empirically address how multifarious selec-
tive regimes shape the evolution of organisms with complex pheno-
types We have made great advances in our understanding of how
specific traits evolve and mediate adaptation (eg morphology Chan
et al 2010 coloration Dasmahapatra et al 2012 sensory organs
Cartwright Schwartz Merry amp Howell 2017) and we have a thor-
ough understanding of how single environmental factors can shape
organismal responses (eg climatic conditions Johnston amp Bennett
1996 predation Langerhans 2007 competition Pfennig amp Pfennig
2012) In contrast our understanding of phenotypic evolution in
response to multifarious selection is comparatively limited especially
because interactive effects of different environmental factors can lead
to organismal and evolutionary responses that are unpredictable based
on experiments that manipulate single environmental variables in iso-
lation (Gunderson Armstrong amp Stillman 2016 Todgham amp Stillman
2013) Future research thus needs to acknowledge the complexities of
environments and organismal design and address them explicitly in
empirical investigations to gain a robust understanding of biological
diversification (Culumber amp Tobler 2017 Stuart et al 2017)
4 | LINKING ADAPTATION TOSPECIATION
So far we primarily explored the causes of adaptive evolution but
what are its consequences Theory predicts that adaptation to local
environmental conditions can lead to speciation because reproduc-
tive isolation emerges incidentally as a by-product (ecological specia-
tion Nosil 2012) Population genetic analyses of Poecilia spp in
sulphide springs and adjacent populations in nonsulphidic environ-
ments have indicated strong genetic differentiation across small
spatial scales (in some instances lt100 m) and in the absence of
physical barriers that would prevent fish movement (Plath et al
2013) In addition population genetic structuring has been shown to
be stable even in the face of major environmental disturbance
BOX 1 Speciation in sulphide springs and conservation
biology
Adaptation to H2S-rich environments in poeciliids coincides
with ecological speciation giving rise to phenotypically dis-
tinct and reproductively isolated lineages In some
instances this has led to the evolution of highly distinct
micro-endemic species including Poecilia sulphuraria and
P thermalis Limia sulphurophila and Gambusia eurystoma
(Figure 3) Except for P sulphuraria which is found in two
spring complexes of the Rıo Pichucalco drainage of Mexico
(Tobler amp Plath 2009b) the distributions of these sulphide
spring endemics are restricted to single localities that
include only small areas of suitable habitat For example
the largest sulphide spring complex (the Ba~nos del Azufre
in southern Mexico which harbours P sulphuraria and G
eurystoma) includes ~18000 m2 of suitable habitat (equiva-
lent to lt35 football fields) while one of the smallest (La
Esperanza springs harbouring P thermalis) merely covers
~600 m2 (just over a tenth of a football field Brown et al
2017) Accordingly some sulphide spring endemics are
considered critically endangered (IUCN 2012) and pro-
tected by law (SEDESOL 1994)
Many threatened species with restricted ranges exhibit
low genetic diversity and high incidences of inbreeding
(Bouzat 2010 Willi Van Buskirk amp Hoffmann 2006) In
contrast at least P sulphuraria and P thermalismdashthe only
species investigated to datemdashactually exhibit relatively high
effective population sizes (Ne = 1200ndash3800 depending on
the population) and no evidence for inbreeding (FIS lt 0)
(Brown et al 2017) This is likely a consequence of the
long-term stability of sulphide springs and the high pro-
ductivity and low levels of interspecific competition also
support unusually high fish densities and thus population
census sizes (Brown et al 2017 Culumber et al 2016)
Nonetheless sulphide spring endemics are threatened by
habitat destruction which is mediated primarily by the
modification of springs for recreational purposes defor-
estation and conversion of land for agriculture and most
recently high-intensity palm oil plantations (Tobler amp Plath
2009ab) Despite the official protection of sulphide spring
endemics there have been no tangible conservation mea-
sures that foster the sustainable development of land
around sulphide springs with the well-being of the endemic
fishes in mind
852 | TOBLER ET AL
(catastrophic flood event Plath et al 2010) Population genetic dif-
ferentiation between adjacent sulphide-adapted and nonadapted
populations is indicative of ongoing ecological speciation (Box 1) but
different population pairs from sulphidic and nonsulphidic waters
have made variable progress along the speciation continuum towards
complete reproductive isolation (Plath et al 2013) This prompts
questions about the mechanisms involved in reducing gene flow
along the environmental gradient connecting the two habitat types
41 | Prezygotic mechanisms of reproductiveisolation
The simplest form of reproductive isolation between locally adapted
populations arises from selection against migrants between habitat
types (Nosil Vines amp Funk 2005) Translocation experiments in nat-
ural habitats have indicated strong survival selection against
migrants especially those translocated from nonsulphidic to sulphidic
habitats (Plath et al 2013) Fish translocated from nonsulphidic
environments face high mortality within short periods of time
(lt24 hr) which is likely caused by H2S toxicity and hypoxia in sul-
phide springs Hence low gene flow from populations inhabiting
nonsulphidic waters into populations inhabiting sulphidic habitats is
simply explained by a lack of adaptation to the extreme environmen-
tal conditions At least in some drainages there is also appreciable
mortality for sulphide spring fish translocated to nonsulphidic habi-
tats although mechanisms causing inviability are unclear and the
strength of selection appears to be lower (Plath et al 2013) If sur-
vival selection against immigrants was the sole source of reproduc-
tive isolation this should result in unidirectional gene flow from
H2S-adapted to nonadapted populations but population genetic
analyses provided no evidence for this implying that additional
mechanisms are at work (Plath et al 2013)
One such mechanism could be intersexual selection and mate
choice experiments have indicated that migrants from sulphidic to
nonsulphidic habitats are at a significant disadvantage with fish from
nonsulphidic habitats avoiding potential mating partners from sul-
phidic ones (Plath et al 2013 Sommer-Trembo et al 2016) Such
assortative mating has been found to be critical during ecological spe-
ciation because it counteracts the homogenizing effects of gene flow
(van Doorn Edelaar amp Weissing 2009) yet how assortative mating
patterns arise often remains unclear In theory assortative mating can
arise through a variety of processes (Nosil 2012) ldquoMagic traitrdquo mecha-
nisms where mating preferences evolve as a by-product of divergent
selection on ecological traits that serve as cues have recently garnered
much attention (Servedio van Doorn Kopp Frame amp Nosil 2011)
Consistent with magic trait mechanisms assortative mating in P mexi-
cana appears to be linked to adaptive population differences in body
shape which serve as cues to distinguish potential mating partners
from different habitats (Greenway Drexler Arias-Rodriguez amp Tobler
2016) However neither sulphide spring fish (which are unlikely to
encounter migrants from nonsulphidic habitats due to H2S toxicity)
nor allopatric P mexicana from nonsulphidic waters (which have never
experienced migrants from sulphide spring populations) exhibit a
preference for mating partners from their own ecotype suggesting
that assortative mating in this system is a product of reinforcement
(ie direct selection for assortative mating Greenway et al 2016)
This is different from classic magic traits mechanisms because assorta-
tive mating is not a mere by-product of adaptation but adaptive traits
have instead been co-opted as mating signals in response to selection
on mating preferences (Maan amp Seehausen 2012) Moreover males of
some H2S-adapted populations have reduced aggressive behaviour
(Bierbach et al 2012 2018) In staged contests of size-matched males
from different ecotypes intruders had a significantly lower likelihood
to establish dominance than resident males independent of whether
or not they showed reduced aggressiveness overall Hence intrasexual
selection may represent another mechanism mediating reproductive
isolation between locally adapted ecotypes Overall H2S-adapted poe-
ciliids allow disentangling the relative contributions of different forms
of ecological and sexual selectionmdashand their interdependencymdashduring
ecological speciation
42 | Ensuing challenges quantifying postzygoticmechanisms of reproductive isolation
In contrast to prezygotic mechanisms little is known about postzy-
gotic mechanisms of reproductive isolation that potentially act to
reduce gene flow between adjacent populations in sulphidic and
nonsulphidic habitats Putative hybrids between sulphidic and non-
sulphidic ecotypes have occasionally been observed in nonsulphidic
habitats immediately adjacent to confluences with sulphide springs
(R Riesch M Plath and M Tobler personal observations) but it
remains unclear whether and through what mechanisms such hybrids
exhibit reduced fitness compared to parentals
Direct selection of mitochondrially encoded genes in the pres-
ence of H2S (Pfenninger et al 2014) should be accompanied by co-
evolution at nuclear loci that directly interact with mitochondrial
gene products in the formation of protein complexes involved in the
OXPHOS pathway (Sunnucks Morales Lamb Pavlova amp Greening
2017 Willett amp Burton 2004) Disruption of co-evolved gene com-
plexes in hybrids should therefore lead to intrinsic fitness reductions
through mito-nuclear incompatibilities (Burton Pereira amp Barreto
2013 Wolff Ladoukakis Enrıquez amp Dowling 2014) which have
been hypothesized to be a key driver in speciation (Chou amp Leu
2010 Gershoni Templeton amp Mishmar 2009) Empirical tests of
these ideas remain to be conducted in sulphide spring fishes just
like studies on potential ecological and sexual selection against
hybrids that arise from interpopulation mating events
5 | PERSPECTIVES LIFE IN SULPHIDICENVIRONMENTS BEYOND P MEXICANA
Research on sulphide spring fishes of the P mexicana species com-
plex in Mexico has provided insights into mechanisms underlying
adaptation and the links between adaptation and speciation Sul-
phide springs do not only occur in Mexico however but are found
TOBLER ET AL | 853
worldwide and have been colonized by 24 evolutionarily indepen-
dent fish lineages in North America the Neotropics and the Middle
East (Greenway et al 2014) The vast majority of lineages belong to
the order Cyprinodontiformes (95) and livebearing members of the
family Poeciliidae in particular (80) This potentially indicates that
internal gestation of young has facilitated the colonization of
extreme environments because it shields developing embryos from
physiochemical stressors (Blackburn 1999) Among the Poeciliidae
sulphide spring fishes include members of the genera Brachyrhaphis
Gambusia Poeciliopsis Priapichthys Pseudoxiphophorus Xiphophorus
and multiple subgenera of Poecilia (Figure 3 Greenway et al 2014)
spanning over 40 million years of evolution (based on Reznick Fur-
ness Meredith amp Springer 2017)
A handful of studies have started to address molecular and phe-
notypic evolution across a broader taxon sampling of sulphide spring
fishes finding evidence for convergent evolution in morphology
(Riesch et al 2016 Tobler amp Hastings 2011) life history traits
(Riesch Plath et al 2010 Riesch et al 2014) and oxygen transport
genes (Barts et al 2018) In addition ecological speciation in
response to adaptation to H2S-rich environments has also been doc-
umented in sulphide spring poeciliids other than P mexicana (Riesch
et al 2016) These studies have laid a fruitful foundation for the
investigation of mechanisms of adaptation to H2S-rich springs from
genes to phenotypic traits linking micro-evolutionary processes to
macro-evolutionary patterns
The availability of broad-scale evolutionary replication of popula-
tions that have invaded habitats with the same physiochemical stressors
allows for addressing some of the ensuing challenges discussed above
and investigating the factors that give rise to predictable evolutionary
outcomes Convergent evolution is a commonmdashbut not omnipresentmdash
theme in adaptive evolution and multiple factors can determine the
degree of convergence among replicated lineages exposed to the same
sources of selection (Kaeuffer et al 2012 Landry amp Bernatchez 2010
Oke Rolshausen LeBlond amp Hendry 2017 Rosenblum amp Harmon
2011) Thus leveraging replicated lineages that have colonized sulphide
springs to assess molecular mechanisms underlying convergent pheno-
typic changes analyse how environmental heterogeneity among sul-
phide spring habitats causes lineage-specific patterns of trait evolution
and test whether and by what mechanisms adaptation to extreme envi-
ronments is linked to speciation will provide insights into how ecologi-
cal genetic and functional factors contribute to evolutionary
convergence at different levels of organismal organization (Rosenblum
et al 2014) An expanded taxon sampling with population pairs that
span almost the full range of the speciation continuum from panmixia to
complete reproductive isolation (Riesch et al 2016) will also help
addressing questions about temporal aspects of H2S adaptation In par-
ticular comparing younger to older lineages will provide insights about
the order at which genetic and phenotypic modifications accrue during
the adaptation process
Animals have also invaded H2S-rich habitats in other aquatic
ecosystems including cold seeps and deep-sea hydrothermal vents
(Tobler et al 2016) Adaptation to H2S in the P mexicana species com-
plex has involved modification of physiological pathways associated
with H2S toxicity (OXPHOS pathway) and detoxification (sulphide
F IGURE 3 Phylogeny of the family Poeciliidae (adopted from Culumber amp Tobler 2017) Various lineages in the genera Poecilia (subgeneraAcanthophacelus Limia and Mollienesia) Priapichthys Brachyrhaphis Poeciliopsis Xiphophorus Pseudoxiphophorus and Gambusia have colonizedH2S-rich springs in different parts of the Americas (see Greenway et al 2014) Lineages highlighted in yellow represent endemic speciesrestricted to H2S-rich springs while red lineages represent species in which some populations have invaded sulphidic habitats Pictures depict arepresentative of each focal genussubgenus
854 | TOBLER ET AL
quinone oxidoreductase pathway) which have been highly conserved
during the diversification of life on Earth (Saraste 1999 Shahak amp
Hauska 2008) The high replicability of H2S as an environmental factor
that shaped animal evolution across vastly different ecological contexts
consequently offers the opportunity to investigate whether selection
mediated by the presence of H2S has repeatedly modified these con-
served physiological pathways across animal phyla that have diverged
during the Cambrian explosion spanning over 500 million years of evo-
lution Such comparisons would provide unprecedented insights into
the effects of a single physiochemical stressor on evolutionary change
irrespective of ecological or phylogenetic contexts
6 | CONCLUSIONS
H2S-rich environments and their inhabitants represent useful models
for research in organismal biology ecology and evolution illustrating
both opportunities (eg strong and explicit source of selection evolu-
tionary replication) and challenges (eg complexity of environments
and organismal responses) in asking basic question about the origins
of biodiversity Understanding how organisms thrive in apparently
inhospitable environments will provide us with insights into lifersquos
capacities and limitations to adapt to novel environments (Waterman
1999) This is particularly relevant considering that human activities
substantially contribute to the exacerbation of physiochemical stres-
sors at local regional and global scales impacting ecological and evo-
lutionary dynamics (Alberti 2015 Norberg Urban Vellend
Klausmeier amp Loeuille 2012) Thus a focus on ecological and evolu-
tionary consequences of natural stressors that push organisms to the
brink of their physiological capacities may be instrumental to predict
consequences of anthropogenic environmental change
ACKNOWLEDGEMENTS
We would like to thank Lenin Arias-Rodriguez the Universidad
Juarez Autonoma de Tabasco and the Centro de Investigacion E
Innovacion Para la Ense~nanza y El Aprendizaje for their continued
collaboration enabling our work on the P mexicana system Funding
for this review article has been provided by the National Science
Foundation (IOS-1557860 IOS-1557795) and the US Army
Research Office (W911NF-15-1-0175)
AUTHOR CONTRIBUTIONS
MT wrote the first draft of the paper all authors contributed text
and comments
ORCID
Michael Tobler httporcidorg0000-0002-0326-0890
Joanna L Kelley httporcidorg0000-0002-7731-605X
Reuroudiger Riesch httporcidorg0000-0002-0223-1254
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Blackburn D G (1999) Viviparity and oviparity Evolution and reproduc-
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Brown A P Greenway R Morgan S Quackenbush C R Giordani
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Brown J H Marquet P A amp Taper M L (1993) Evolution of body
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Carroll S P Jorgensen P S Kinnison M T Bergstrom C T Denison
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Cartwright R A Schwartz R S Merry A L amp Howell M M (2017)
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Chan Y F Marks M E Jones F C Villarreal G Shapiro M D Brady
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Colosimo P F Hosemann K E Balabhadra S Villarreal G Dickson
M Grimwood J Kingsley D M (2005) Widespread parallel evo-
lution in sticklebacks by repeated fixation of ectodysplasin alleles
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Cooper C E amp Brown G C (2008) The inhibition of mitochondrial
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hydrogen cyanide and hydrogen sulfide Chemical mechanism and
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Culumber Z W Hopper G W Barts N Passow C N Morgan S Brown
A Tobler M (2016) Habitat use of two extremophile highly ende-
mic and critically endangered fish species (Gambusia eurystoma and Poe-
cilia sulphurophila Poeciliidae) Aquatic Conservation-Marine and
Freshwater Ecosystems 26 1155ndash1167 httpsdoiorg101002aqc
2640
Culumber Z W amp Tobler M (2017) Sex-specific evolution during the
diversification of live-bearing fishes Nature Ecology amp Evolution 1
1185ndash1191
Dalziel A C Rogers S M amp Schulte P M (2009) Linking genotypes
to phenotypes and fitness How mechanistic biology can inform
molecular ecology Molecular Ecology 18 4997ndash5017 httpsdoi
org101111j1365-294X200904427x
Darwin C (1859) On the origin of species based on natural selection or
the preservation of favoured races in the struggle of life 6th ed Lon-
don John Murray
Dasmahapatra K K Walters J R Briscoe A D Davey J W Whibley
A Nadeau N J Jiggins C D (2012) Butterfly genome reveals
promiscuous exchange of mimicry adaptations among species Nature
487 94ndash98 httpsdoiorg101038nature11041
Doudna J A amp Charpentier E (2014) The new frontier of genome edit-
ing with CRISPR-Cas9 Science 346 1258096 httpsdoiorg10
1126science1258096
Dubilier N Giere O amp Grieshaber M (2005) Concomitant effects of sul-
fide and hypoxia on the aerobic metabolism of the marine oligochaete
Tubificoides benedii Journal of Experimental Zoology 269 287ndash298
Eales K L Hollinshead K E R amp Tennant D A (2016) Hypoxia and
metabolic adaptation of cancer cells Oncogenesis 5 e190 httpsd
oiorg101038oncsis201550
Eghbal M A Pennefather P S amp OrsquoBrien P J (2004) H2S cytotoxicity
mechanism involves reactive oxygen species formation and mito-
chondrial depolarisation Toxicology 203 69ndash76 httpsdoiorg10
1016jtox200405020
Feldman C R Brodie Jr E D Brodie III E D amp Pfrender M E (2012)
Constraint shapes convergence in tetrodotoxin-resistant sodium
channels of snakes Proceedings of the National Academy of Sciences
USA 109 4556ndash4561 httpsdoiorg101073pnas1113468109
Flores K B Wolschin F amp Amdam G V (2013) The role of methyla-
tion of DNA in environmental adaptation Integrative and Comparative
Biology 53 359ndash372 httpsdoiorg101093icbict019
Fradin H Kiontke K Zegar C Gutwein M Lucas J Kovtun M
Piano F (2017) Genome architecture and evolution of a unichromo-
somal asexual nematode Current Biology 27(2928ndash2939) e2926
Gershoni M Templeton A amp Mishmar D (2009) Mitochondrial bioen-
ergetics as a major motive force of speciation BioEssays 31 642ndash
650 httpsdoiorg101002bies200800139
Ghalambor C K Walker J A amp Reznick D N (2003) Multi-trait selec-
tion adaptation and constraints on the evolution of burst swimming
performance Integrative and Comparative Biology 43 431ndash438
httpsdoiorg101093icb433431
Gibert J amp Deharveng L (2002) Subterranean ecosystems A truncated
functional biodiversity BioScience 52 473ndash481 httpsdoiorg10
16410006-3568(2002)052[0473SEATFB]20CO2
Gjuvsland A B Vik J O Beard D A Hunter P J amp Omholt S W
(2013) Bridging the genotype-phenotype gap What does it take
Journal of Physiology 591 2055ndash2066 httpsdoiorg101113jphys
iol2012248864
Grant P R Grant B R Markert J A Keller L F amp Petren K (2004)
Convergent evolution of Darwinrsquos finches caused by introgressive
hybridization and selection Evolution 58 1588ndash1599 httpsdoi
org101111j0014-38202004tb01738x
Greene H W (2005) Organisms in nature as a central focus for biology
Trends in Ecology and Evolution 20 23ndash27 httpsdoiorg101016j
tree200411005
Greenway R Arias-Rodriguez L Diaz P amp Tobler M (2014) Patterns
of macroinvertebrate and fish diversity in freshwater sulphide springs
Diversity 6 597ndash632 httpsdoiorg103390d6030597
Greenway R Drexler S Arias-Rodriguez L amp Tobler M (2016) Adap-
tive but not condition-dependent body shape differences contribute
to assortative mating preferences during ecological speciation Evolu-
tion 70 2809ndash2822 httpsdoiorg101111evo13087
Gunderson A R Armstrong E J amp Stillman J H (2016) Multiple stres-
sors in a changing world The need for an improved perspective on
physiological responses to the dynamic marine environment Annual
Review of Marine Science 8 357ndash378 httpsdoiorg101146an
nurev-marine-122414-033953
Hildebrandt T M amp Grieshaber M (2008) Three enzymatic activities
catalyze the oxidation of sulfide to thiosulfate in mammalian and
invertebrate mitochondria FEBS Journal 275 3352ndash3361 httpsd
oiorg101111j1742-4658200806482x
Houle D Govindaraju D R amp Omholt S (2010) Phenomics The next
challenge Nature Reviews Genetics 11 855ndash866 httpsdoiorg10
1038nrg2897
IUCN (2012) Red List of threatened species version 20132 www
iucnredlistorg
Johnston I A amp Bennett A F (1996) Animals and temperature Pheno-
typic and evolutionary adaptation Cambridge Cambridge University
Press httpsdoiorg101017CBO9780511721854
Jorgensen C Auer S K amp Reznick D N (2011) A model for optimal
offspring size in fish including live-bearing and parental effects Amer-
ican Naturalist 177 E119ndashE135 httpsdoiorg101086659622
Kaeuffer R Peichel C L Bolnick D I amp Hendry A P (2012) Parallel
and nonparallel aspects of ecological phenotypic and genetic diver-
gence across replicate population pairs of lake and stream stickle-
back Evolution 66 402ndash418 httpsdoiorg101111j1558-5646
201101440x
Kell D B amp Oliver S G (2004) Here is the evidence now what is the
hypothesis The complementary roles of inductive and hypothesis-
driven science in the post-genomic era BioEssays 26 99ndash105
httpsdoiorg101002(ISSN)1521-1878
856 | TOBLER ET AL
Kelley J L Arias-Rodriguez L Patacsil Martin D Yee M C Busta-
mante C amp Tobler M (2016) Mechanisms underlying adaptation to
life in hydrogen sulfide rich environments Molecular Biology and Evo-
lution 33 1419ndash1434 httpsdoiorg101093molbevmsw020
Kelley J L Passow C Plath M Arias-Rodriguez L Yee M C amp
Tobler M (2012) Genomic resources for a model in adaptation and
speciation research The characterization of the Poecilia mexicana
transcriptome BMC Genomics 13 652 httpsdoiorg101186
1471-2164-13-652
Klerks P L Xie L amp Levinton J S (2011) Quantitative genetics
approaches to study evolutionary processes in ecotoxicology a per-
spective from research on the evolution of resistance Ecotoxicology
20 513ndash523 httpsdoiorg101007s10646-011-0640-2
Kump L R Pavlov A amp Arthur M A (2005) Massive release of hydrogen
sulfide to the surface ocean and atmosphere during intervals of oceanic
anoxia Geology 33 397ndash400 httpsdoiorg101130G212951
Keuroustner A Hoffmann M Fraser B A Kottler V A Sharma E Wei-
gel D amp Dreyer C (2016) The genome of the Trinidadian guppy
Poecilia reticulata and variation in the guanapo population PLoS ONE
11 e0169087 httpsdoiorg101371journalpone0169087
Landry L amp Bernatchez L (2010) Role of epibenthic resource opportu-
nities in the parallel evolution of lake whitefish species pairs (Core-
gonus sp) Journal of Evolutionary Biology 23 2602ndash2613 httpsdoi
org101111j1420-9101201002121x
Langerhans R B (2007) Evolutionary consequences of predation Avoid-
ance escape reproduction and diversification In A M T Elewa
(Ed) Predation in organisms Berlin Springer
Li L Rose P amp Moore P K (2011) Hydrogen sulfide and cell signaling
Annual Review of Pharmacology and Toxicology 51 169ndash187
httpsdoiorg101146annurev-pharmtox-010510-100505
Lin Q Fan S Zhang Y Xu M Zhang H Yang Y Gunter H M
(2016) The seahorse genome and the evolution of its specialized mor-
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Liu Y Ma S Wang X Chang J Gao J Shi R Zhao P (2014)
Highly efficient multiplex targeted mutagenesis and genomic struc-
ture variation in Bombyx mori cells using CRISPRCas9 Insect bio-
chemistry and molecular biology 49 35ndash42 httpsdoiorg101016j
ibmb201403010
Lopez-Maury L Marguerat S amp Beuroahler J (2008) Tuning gene expres-
sion to changing environments From rapid responses to evolutionary
adaptation Nature Reviews Genetics 9 583ndash593 httpsdoiorg10
1038nrg2398
Maan M E amp Seehausen O (2012) Magic traits versus magic prefer-
ences in speciation Evolutionary Ecology Research 14 779ndash785
Martyn U Weigel D amp Dreyer C (2006) In vitro culture of embryos
of the guppy Poecilia reticulata Developmental Dynamics 235 617ndash
622 httpsdoiorg101002(ISSN)1097-0177
Metzker M L (2010) Sequencing technologies The next generation
Nature Reviews Genetics 11 31ndash46 httpsdoiorg101038nrg2626
Moore M P Riesch R amp Martin R A (2016) The predictability and
magnitude of life-history divergence to ecological agents of selection
A meta-analysis in livebearing fishes Ecology Letters 19 435ndash442
httpsdoiorg101111ele12576
Mustafa A K Gadalla M M Sen N Kim S Mu W Gazi S K
Snyder S H (2009) H2S signals through protein S-sulfhydration
Science Signaling 2 ra72
Nesse R M amp Stearns S C (2008) The great opportunity Evolutionary
applications to medicine and public health Evolutionary Applications
1 28ndash48 httpsdoiorg101111j1752-4571200700006x
Nevo E (2011) Evolution under environmental stress at macro and
microscales Genome Biology and Evolution 3 1039ndash1052 httpsdoi
org101093gbeevr052
Norberg J Urban M C Vellend M Klausmeier C A amp Loeuille N (2012)
Eco-evolutionary responses of biodiversity to climate change Nature Cli-
mate Change 2 747ndash751 httpsdoiorg101038nclimate1588
Nosil P (2012) Ecological speciation Oxford UK Oxford University
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0001
Nosil P Vines T H amp Funk D J (2005) Perspective Reproductive
isolation caused by natural selection against immigrants from diver-
gent habitats Evolution 59 705ndash719
Oke K B Rolshausen G LeBlond C amp Hendry A P (2017) How par-
allel is parallel evolution A comparative analysis in fishes American
Naturalist 190 1ndash16 httpsdoiorg101086691989
Olson K R (2011) The therapeutic potential of hydrogen sulfide
Separating hype from hope American Journal of Physiology-Regulatory
Integrative and Comparative Physiology 301 R297ndashR312 httpsdoi
org101152ajpregu000452011
Olson K R Donald J A Dombkowski R A amp Perry S F (2012) Evo-
lutionary and comparative aspects of nitric oxide carbon monoxide
and hydrogen sulfide Respiratory Physiology amp Neurobiology 184
117ndash129 httpsdoiorg101016jresp201204004
Olson K R amp Straub K D (2015) The role of hydrogen sulfide in evo-
lution and the evolution of hydrogen sulfide in metabolism and sig-
naling Physiology 31 60ndash72
Palacios M Arias-Rodriguez L Plath M Eifert C Lerp H Lamboj A
Tobler M (2013) The rediscovery of a long described species
reveals additional complexity in speciation patterns of poeciliid fishes
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nalpone0071069
Palacios M Voelker G Arias-Rodriguez L Mateos M amp Tobler M
(2016) Phylogenetic analyses of the subgenus Mollienesia (Poecilia
Poeciliidae Teleostei) reveal taxonomic inconsistencies cryptic biodi-
versity and spatio-temporal aspects of diversification in Middle
America Molecular Phylogenetics and Evolution 103 230ndash244
httpsdoiorg101016jympev201607025
Passow C Arias-Rodriguez L amp Tobler M (2017) Convergent evolu-
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e0186935 httpsdoiorg101371journalpone0186935
Passow C N Brown A Arias-Rodriguez L Yee M-C Sockell A
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patterns in natural populations of an extremophile fish (Poecilia mexi-
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101111mec14198
Passow C Greenway R Arias-Rodriguez L Jeyasingh P D amp Tobler
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body size and metabolic rates in locally adapted extremophile fishes
Physiological and Biochemical Zoology 88 371ndash383 httpsdoiorg
101086681053
Passow C N Henpita C Shaw J H Quakenbush C Warren W C
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lations of extremophile fish Molecular Ecology 26 6384ndash6399
Pavey S A Bernatchez L Aubin-Horth N amp Landry C R (2012)
What is needed for next-generation ecological and evolutionary
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org101016jtree201207014
Peers C Bauer C Boyle J P Scragg J L amp Dallas M L (2012)
Modulation of ion channels by hydrogen sulfide Antioxidants amp Redox
Signaling 17 95ndash105 httpsdoiorg101089ars20114359
Pfennig D W amp Pfennig K S (2012) Evolutionrsquos wedge competition and
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httpsdoiorg101525california97805202741810010001
Pfenninger M Lerp H Tobler M Passow C Kelley J L Funke E
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Pfenninger M Patel S Arias-Rodriguez L Feldmeyer B Riesch R amp
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TOBLER ET AL | 857
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1111mec13397
Pietri R Roman-Morales E amp Lopez-Garriga J (2011) Hydrogen sul-
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Redox Signaling 15 393ndash404 httpsdoiorg101089ars20103698
Plath M (2008) Male mating behavior and costs of sexual harassment
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Plath M Hermann C Schreurooder R Riesch R Tobler M Garcia de
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Plath M Pfenninger M Lerp H Riesch R Eschenbrenner C Slattery
P Tobler M (2013) Genetic differentiation and selection against
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Plath M Tobler M Riesch R Garcia de Leon F J Giere O amp
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Powell E (1989) Oxygen sulfide and diffusion Why thiobiotic meiofauna
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Ramirez-Soriano A Ramos-Onsins S E Rozas J Calafell F amp Navarro A
(2008) Statistical power analysis of neutrality tests under demographic
expansions contractions and bottlenecks with recombination Genetics
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Reiffenstein R Hulbert W amp Roth S (1992) Toxicology of hydrogen
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Reznick D N Furness A I Meredith R W amp Springer M S (2017)
The origin and biogeographic diversification of fishes in the family
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lpone0172546
Reznick D amp Yang A P (1993) The influence of fluctuating resources
on life history Patterns of allocation and plasticity in female guppies
Ecology 74 2011ndash2019 httpsdoiorg1023071940844
Riesch R Duwe V Herrmann N Padur L Ramm A Scharnweber K
Plath M (2009) Variation along the shy-bold continuum in extre-
mophile fishes (Poecilia mexicana Poecilia sulphuraria) Behavioral Ecol-
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s00265-009-0780-z
Riesch R Oranth A Dzienko J Karau N Schiessl A Stadler S
Plath M (2010) Extreme habitats are not refuges Poeciliids suffer
from increased aerial predation risk in sulphidic southern Mexican
habitats Biological Journal of the Linnean Society 101 417ndash426
httpsdoiorg101111j1095-8312201001522x
Riesch R Plath M Garcia de Leon F J amp Schlupp I (2010) Conver-
gent life-history shifts Toxic environments result in big babies in two
clades of poeciliids Naturwissenschaften 97 133ndash141 httpsdoi
org101007s00114-009-0613-y
Riesch R Plath M Schlupp I Tobler M amp Langerhans R B (2014)
Colonization of toxic springs drives predictable life-history shift in
livebearing fishes (Poeciliidae) Ecology Letters 17 65ndash71 httpsdoi
org101111ele12209
Riesch R Tobler M Lerp H Jourdan J Doumas L T Nosil P Plath
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plexity of speciation along replicated ecological gradients BMC Evolu-
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Riesch R Tobler M amp Plath M (2015) Extremophile fishes Ecology
evolution and physiology of teleosts in extreme environments p 290
Heidelberg Germany Springer httpsdoiorg101007978-3-319-
13362-1
Ritchie M E Holzinger E R Li R Pendergrass S A amp Kim S (2015)
Methods of integrating data to uncover genotype-phenotype interac-
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nrg3868
Roach K Tobler M amp Winemiller K O (2011) Hydrogen sulfide bac-
teria and fish A unique subterranean food chain Ecology 92 2056ndash
2062 httpsdoiorg10189011-02761
Rohner N Jarosz D F Kowalko J E Yoshizawa M Jeffery W R
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Science 342 1372ndash1375 httpsdoiorg101126science1240276
Rosales Lagarde L (2012) Investigation of karst brackish-sulfidic springs
and their role in the hydrogeology subsurface water-rock interactions
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of Mining and Technology Mexico
Rosenblum E B amp Harmon L J (2011) ldquoSame but differentrdquo Repli-
cated ecological speciation at White Sands Evolution 65 946ndash960
httpsdoiorg101111j1558-5646201001190x
Rosenblum E B Parent C E amp Brandt E E (2014) The molecular
basis of convergent evolution Annual Review of Ecology Evolution and
Systematics 45 203ndash226 httpsdoiorg101146annurev-ecolsys-
120213-091851
Saltz J B Hessel F C amp Kelly M W (2017) Trait correlations in the
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org101016jtree201612008
Saraste M (1999) Oxidative phosphorylation at the fin de siecle Science
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Schartl M (2014) Beyond the zebrafish Diverse fish species for model-
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Schartl M Walter R B Shen Y Garcia T Catchen J Amores A
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ulatus provides insights into evolutionary adaptation and several
complex traits Nature Genetics 45 567ndash572 httpsdoiorg10
1038ng2604
Schluter D (2009) Evidence for ecological speciation and its alternative
Science 323 737ndash741 httpsdoiorg101126science1160006
Schulz-Mirbach T Eifert C Riesch R Farnworth M S Zimmer C
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su proteccion Diario Oficial 16 May 1994 488 2ndash60
Servedio M R van Doorn G S Kopp M Frame A M amp Nosil P
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amp Evolution 26 389ndash397 httpsdoiorg101016jtree201104005
Shahak Y amp Hauska G (2008) Sulfide oxidation from cyanobacteria to
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Shalem O Sanjana N E amp Zhang F (2015) High-throughput func-
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Silva G J J Bye A el Azzouzi H amp Wisloslashff U (2017) MicroRNAs as
important regulators of exercise adaptation Progress in Cardiovascular
Diseases 60 130ndash151 httpsdoiorg101016jpcad201706003
Simola D F Wissler L Donahue G Waterhouse R M Helmkampf
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dramatic evolution in gene composition and regulation while preserv-
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1235ndash1247 httpsdoiorg101101gr155408113
858 | TOBLER ET AL
Sommer-Trembo C Bierbach D Arias Rodriguez L Verel Y Jourdan
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Naturwissenschaftlichen Classe der Kaiserlichen Akademie der Wis-
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Stinchcombe J R amp Hoekstra H E (2008) Combining population geno-
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hdy6800937
Stipanuk M H (2004) Sulfur amino acid metabolism Pathways for pro-
duction and removal of homocysteine and cysteine Annual Review of
Nutrition 24 539ndash577 httpsdoiorg101146annurevnutr24
012003132418
Stipanuk M H amp Ueki I (2011) Dealing with methioninehomocysteine
sulfur Cysteine metabolism to taurine and inorganic sulfur Journal of
Inherited Metabolic Disease 34 17ndash32 httpsdoiorg101007
s10545-009-9006-9
Stockinger B Di Meglio P Gialitakis M amp Duarte J H (2014) The
aryl hydrocarbon receptor Multitasking in the immune system
Annual Review of Immunology 32 403ndash432 httpsdoiorg101146
annurev-immunol-032713-120245
Storz J F amp Wheat C W (2010) Integrating evolutionary and
functional approaches to infer adaptation at specific loci Evolution
64 2489ndash2509 httpsdoiorg101111j1558-5646201001044x
Stuart Y E Veen T Weber J N Hanson D Ravinet M Lohman B
K Bolnick D I (2017) Contrasting effects of environment and
genetics generate a continuum of parallel evolution Nature Ecology amp
Evolution 1 0158 httpsdoiorg101038s41559-017-0158
Sunnucks P Morales H E Lamb A M Pavlova A amp Greening C
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genome co-evolution in oxidative phosphorylation Frontiers in Genet-
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Tobler M amp Hastings L (2011) Convergent patterns of body shape dif-
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fide springs Evolutionary Biology 38 412ndash421 httpsdoiorg10
1007s11692-011-9129-4
Tobler M Palacios M Chapman L J Mitrofanov I Bierbach D
Plath M Mateos M (2011) Evolution in extreme environments
Replicated phenotypic differentiation in livebearing fish inhabiting
sulfidic springs Evolution 65 2213ndash2228 httpsdoiorg101111j
1558-5646201101298x
Tobler M Passow C N Greenway R Kelley J L amp Shaw J H
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fide-rich environments Annual Review of Ecology Evolution and Sys-
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121415-032418
Tobler M amp Plath M (2009a) Threatened fishes of the world Gambu-
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Fishes 85 251 httpsdoiorg101007s10641-009-9481-8
Tobler M amp Plath M (2009b) Threatened fishes of the world Poecilia
sulphuraria (Alvarez 1948) (Poeciliidae) Environmental Biology of
Fishes 85 333ndash334 httpsdoiorg101007s10641-009-9506-3
Tobler M Plath M Riesch R Schlupp I Grasse A Munimanda G
Moodley Y (2014) Selection from parasites favors immunogenic diver-
sity but not divergence among locally adapted host populations Journal
of Evolutionary Biology 27 960ndash974 httpsdoiorg101111jeb12370
Tobler M Riesch R amp Plath M (2015) Extremophile fishes An inte-
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environments (pp 279ndash296) Heidelberg Germany Springer
Tobler M Riesch R Tobler C M amp Plath M (2009) Compensatory
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budgets feeding efficiency and predation risk Evolutionary Ecology
Research 11 935ndash948
Tobler M Scharnweber K Greenway R Passow C Arias-Rodriguez
L amp Garcia-De-Leon F J (2015) Convergent changes in the
trophic ecology if extremophile fish along replicated environmental
gradients Freshwater Biology 60 768ndash780 httpsdoiorg10
1111fwb12530
Todgham A E amp Stillman J H (2013) Physiological responses to shifts
in multiple environmental stressors Relevance in a changing worlds
Integrative and Comparative Biology 53 539ndash544 httpsdoiorg10
1093icbict086
van Doorn G S Edelaar P amp Weissing F J (2009) On the origin of
species by natural selection and sexual selection Science 326 1704ndash
1707 httpsdoiorg101126science1181661
Wall D H amp Virginia R A (1999) Controls on soil biodiversity Insights
from extreme environments Applied Soil Ecology 13 137ndash150
httpsdoiorg101016S0929-1393(99)00029-3
Wallace J L amp Wang R (2015) Hydrogen-sulfide based therapeutics
Exploiting a unique but ubiquitous gasotransmitter Nature Reviews
Drug Discovery 14 329ndash345 httpsdoiorg101038nrd4433
Wang R (2012) Physiological implications of hydrogen sulfide A whiff
exploration that blossomed Physiological Reviews 92 791ndash896
httpsdoiorg101152physrev000172011
Wang Z Gerstein M amp Snyder M (2009) RNA-seq A revolutionary
tool for transcriptomics Nature Reviews Genetics 10 57ndash63
httpsdoiorg101038nrg2484
Waterman T H (1999) The evolutionary challenges of extreme environ-
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httpsdoiorg101002(SICI)1097-010X(19991215)2854amplt326
AID-JEZ3ampgt30CO2-T
Werren J H Richards S Desjardins C A Niehuis O Gadau J Col-
bourne J K amp Group N G W (2010) Functional and evolutionary
insights from the genomes of three parasitoid Nasonia species
Science 327 343ndash348 httpsdoiorg101126science1178028
Whitehead A (2012) Comparative genomics in ecological physiology
Toward a more nuanced understanding of acclimation and adapta-
tion Journal of Experimental Biology 215 884ndash891 httpsdoiorg
101242jeb058735
Whitehead A amp Crawford D L (2006) Neutral and adaptive variation
in gene expression Proceedings of the National Academy of Sciences
USA 103 5425ndash5430 httpsdoiorg101073pnas0507648103
Willett C S amp Burton R S (2004) Evolution of interacting proteins in
the mitochondrial electron transport system in a marine copepod
Molecular Biology and Evolution 21 443ndash453 httpsdoiorg10
1093molbevmsh031
Willi Y Van Buskirk J amp Hoffmann A (2006) Limits to the adaptive
potential of small populations Annual Review of Ecology Evolution
and Systematics 37 433ndash458 httpsdoiorg101146annurevecol
sys37091305110145
Wolff J N Ladoukakis E D Enrıquez J A amp Dowling D K (2014)
Mitonuclear interactions Evolutionary consequences over multiple
biological scales Philosophical Transactions of the Royal Society B 369
20130443 httpsdoiorg101098rstb20130443
How to cite this article Tobler M Kelley JL Plath M Riesch
R Extreme environments and the origins of biodiversity
Adaptation and speciation in sulphide spring fishes Mol Ecol
201827843ndash859 httpsdoiorg101111mec14497
TOBLER ET AL | 859
of the divergent traits between populations from sulphidic and non-
sulphidic environments have no clearmdashor at best tenuousmdashlinks to
H2S toxicity and detoxification and there is only limited evidence
for within-population correlations between different traits groups
(Riesch et al 2016)
Evolutionary analyses frequently focus on seemingly simple
sources of selection juxtaposing populations that are assigned to dis-
crete habitat types to understand the evolution of specific traits
(Kaeuffer Peichel Bolnick amp Hendry 2012) But as Darwin (1859)
already observed in his ldquotangled bankrdquo allegory selection in natural
systems is rarely univariate but multifarious selective regimes con-
tribute to trait divergence even among populations that occur along
apparently simple environmental gradients Unquantified environmen-
tal variation that is subsumed in the categorization of habitat types
can profoundly affect trait variation in natural systems (Stuart et al
2017) highlighting the importance of considering and testing alterna-
tive hypotheses about the cause-and-effect relationship between
specific sources of selection and evolution of specific traits Thus
accurate natural history observations that consider organisms as holis-
tic entities consisting of interacting traits in the context of the abiotic
and biotic factors comprising their environment are critical to inform
the design and interpretation of empirical research in natural systems
(Greene 2005) Indeed adjacent sulphidic and nonsulphidic habitat
types do not merely differ in the presence or absence of H2S but also
in a variety of other abiotic and biotic factors (Figure 2) Unlike nonsul-
phidic habitats sulphide springs in southern Mexico are characterized
by extreme hypoxia elevated salinity and lower pH (Rosales Lagarde
2012 Tobler et al 2011) In addition the presence of physiochemical
stressors affects the biotic communities in these sulphide springs Spe-
cies richness of aquatic invertebrates and vertebrates is drastically
reduced as a consequence of H2S toxicity (Greenway Arias-Rodriguez
Diaz amp Tobler 2014) and primary production appears to be driven in
part by chemoautotrophic bacteria that use H2S as an energy source
rather than just by autochthonous and allochthonous photosynthetic
organisms (Roach Tobler amp Winemiller 2011) These changes in the
biotic environment affect the relative importance of intra-vs inter-
specific competition trophic resource use and exposure to predators
and parasites (Riesch Oranth et al 2010 Tobler et al 2014 Tobler
Scharnweber et al 2015)
31 | Alternative drivers of trait evolution
Acknowledging the complexity of selective regimes in sulphide
springs allows for a more nuanced understanding of trait evolution
and for the development of alternative hypotheses about the ulti-
mate mechanisms that cause divergence in specific genetic and phe-
notypic traits In some instances sources of selection other than H2S
can plausibly explain trait divergence between populations from sul-
phidic and nonsulphidic habitats For example hypoxia likely drives
increases in head size and gill surface area (Tobler et al 2011)
changes in the ionic composition of the water can explain differential
expression of genes associated with osmoregulation (Kelley et al
2016) and shifts in trophic resource use are associated with
modifications of the gastrointestinal system (Tobler Scharnweber
et al 2015) In other cases disentangling alternative hypotheses is
not trivial because selection mediated by H2S and other environmen-
tal factors are predicted to affect trait modifications in similar ways
Shifts towards anaerobic metabolism in sulphide spring fish could be
the consequence of inhibition of aerobic ATP production by H2S or
by constraints in oxygen availability Similarly reproductive life his-
tory shifts towards the production of larger but fewer offspring could
be driven by H2S (decreasing offspringrsquos surfacevolume ratio to mini-
mize the diffusion of H2S into the body Powell 1989) by habitat dif-
ferences in the quality and quantity of predation that can affect size-
specific rates of extrinsic mortality (Jorgensen Auer amp Reznick
2011) or by energetic constraints (see below) While comparisons
across different selective regimes can shed light into the ultimate dri-
vers of trait evolution (Moore Riesch amp Martin 2016) isolating the
effects of different sources of selection may not be possible because
they could act in concert and facilitate increases of organismal perfor-
mance in multiple contexts (Ghalambor Walker amp Reznick 2003)
When multifarious selection shapes multivariate phenotypic evo-
lution it is important to also consider the role of trade-offs both in
terms of trait expression (increased investment in one trait may
impose constraints on the expression of other traits) and in terms of
trait function (optimizing the trait for one function may impair the
traitrsquos utility for other functions) For example selection on large off-
spring size in sulphide spring fishes has inadvertent consequences
for fecundity (Riesch et al 2014) and selection for increased gill
surface areas due to hypoxia simultaneously increases exposure to
environmental H2S (Tobler et al 2011) Thus the outcome of phe-
notypic evolution is ultimately a result of balancing competing needs
in a manner that optimizes trait expression and organismal function
in multiple contexts to maximize fitness (Arnold 1983) and the pres-
ence of trade-offs can have cascading indirect effects with selection
on one or a few traits leading to the modification of a multitude of
apparently unrelated characteristics
In this context bioenergetics may represent a nexus in the evo-
lution of complex traits observed in sulphide spring fish and other
extreme environments Living in and adapting to sulphide springs
imposes constraints on organismal energy availability (Plath et al
2007) with according trade-offs for energy allocation Energy limita-
tion in sulphide springs does not arise from a scarcity of food
resources (chemoautotrophic bacteria eaten by the fish are abundant
in sulphide springs Tobler Scharnweber et al 2015) but from
behavioural and physiological adaptation A primary survival strategy
for fish in the toxic and hypoxic springs is to engage in aquatic sur-
face respiration where individuals breathe directly at the water sur-
face to maximize oxygen uptake and minimize H2S exposure This
behaviour mediates short-term survival (Plath et al 2007) but it
also constrains energy acquisition for benthic foragers like P mexi-
cana (Tobler Riesch Tobler amp Plath 2009) and increases susceptibil-
ity to bird predation (Riesch Oranth et al 2010) Costly investments
into H2S detoxification and tissue repair as well as a shift from aero-
bic to less efficient anaerobic metabolism further constrain energy
allocation to other functions (Passow Greenway Arias-Rodriguez
TOBLER ET AL | 851
Jeyasingh amp Tobler 2015) Theory predicts that energetic con-
straints ndash whether mediated by limitations in the rate of resource
acquisition or in the rate of energy allocation to reproduction (as
opposed to maintenance or growth) ndash should exert selection for a
reduction in organismal energy budgets which allows for the maxi-
mization of residual energy allocation for the production of offspring
(Brown Marquet amp Taper 1993) Several lines of evidence support
the hypothesis that energy limitation plays a critical role in pheno-
typic evolution of sulphide spring fish Compared to individuals in
nonsulphidic environments fish in sulphide springs have significantly
lower routine energy demands which is mediated both by reduc-
tions in body size and mass-specific metabolism (Passow Arias-
Rodriguez et al 2017) Reductions in other traits such as energeti-
cally costly behaviours (Bierbach et al 2012 Plath 2008) and the
size of energy-demanding organs (Schulz-Mirbach et al 2016) may
also contribute to the reduced energy demands and shifts in repro-
ductive life history traits (Riesch et al 2014) are consistent with
selection from energy limitation (Reznick amp Yang 1993)
32 | Ensuing challenges Disentanglingenvironmental and organismal complexities
Future research needs to empirically address how multifarious selec-
tive regimes shape the evolution of organisms with complex pheno-
types We have made great advances in our understanding of how
specific traits evolve and mediate adaptation (eg morphology Chan
et al 2010 coloration Dasmahapatra et al 2012 sensory organs
Cartwright Schwartz Merry amp Howell 2017) and we have a thor-
ough understanding of how single environmental factors can shape
organismal responses (eg climatic conditions Johnston amp Bennett
1996 predation Langerhans 2007 competition Pfennig amp Pfennig
2012) In contrast our understanding of phenotypic evolution in
response to multifarious selection is comparatively limited especially
because interactive effects of different environmental factors can lead
to organismal and evolutionary responses that are unpredictable based
on experiments that manipulate single environmental variables in iso-
lation (Gunderson Armstrong amp Stillman 2016 Todgham amp Stillman
2013) Future research thus needs to acknowledge the complexities of
environments and organismal design and address them explicitly in
empirical investigations to gain a robust understanding of biological
diversification (Culumber amp Tobler 2017 Stuart et al 2017)
4 | LINKING ADAPTATION TOSPECIATION
So far we primarily explored the causes of adaptive evolution but
what are its consequences Theory predicts that adaptation to local
environmental conditions can lead to speciation because reproduc-
tive isolation emerges incidentally as a by-product (ecological specia-
tion Nosil 2012) Population genetic analyses of Poecilia spp in
sulphide springs and adjacent populations in nonsulphidic environ-
ments have indicated strong genetic differentiation across small
spatial scales (in some instances lt100 m) and in the absence of
physical barriers that would prevent fish movement (Plath et al
2013) In addition population genetic structuring has been shown to
be stable even in the face of major environmental disturbance
BOX 1 Speciation in sulphide springs and conservation
biology
Adaptation to H2S-rich environments in poeciliids coincides
with ecological speciation giving rise to phenotypically dis-
tinct and reproductively isolated lineages In some
instances this has led to the evolution of highly distinct
micro-endemic species including Poecilia sulphuraria and
P thermalis Limia sulphurophila and Gambusia eurystoma
(Figure 3) Except for P sulphuraria which is found in two
spring complexes of the Rıo Pichucalco drainage of Mexico
(Tobler amp Plath 2009b) the distributions of these sulphide
spring endemics are restricted to single localities that
include only small areas of suitable habitat For example
the largest sulphide spring complex (the Ba~nos del Azufre
in southern Mexico which harbours P sulphuraria and G
eurystoma) includes ~18000 m2 of suitable habitat (equiva-
lent to lt35 football fields) while one of the smallest (La
Esperanza springs harbouring P thermalis) merely covers
~600 m2 (just over a tenth of a football field Brown et al
2017) Accordingly some sulphide spring endemics are
considered critically endangered (IUCN 2012) and pro-
tected by law (SEDESOL 1994)
Many threatened species with restricted ranges exhibit
low genetic diversity and high incidences of inbreeding
(Bouzat 2010 Willi Van Buskirk amp Hoffmann 2006) In
contrast at least P sulphuraria and P thermalismdashthe only
species investigated to datemdashactually exhibit relatively high
effective population sizes (Ne = 1200ndash3800 depending on
the population) and no evidence for inbreeding (FIS lt 0)
(Brown et al 2017) This is likely a consequence of the
long-term stability of sulphide springs and the high pro-
ductivity and low levels of interspecific competition also
support unusually high fish densities and thus population
census sizes (Brown et al 2017 Culumber et al 2016)
Nonetheless sulphide spring endemics are threatened by
habitat destruction which is mediated primarily by the
modification of springs for recreational purposes defor-
estation and conversion of land for agriculture and most
recently high-intensity palm oil plantations (Tobler amp Plath
2009ab) Despite the official protection of sulphide spring
endemics there have been no tangible conservation mea-
sures that foster the sustainable development of land
around sulphide springs with the well-being of the endemic
fishes in mind
852 | TOBLER ET AL
(catastrophic flood event Plath et al 2010) Population genetic dif-
ferentiation between adjacent sulphide-adapted and nonadapted
populations is indicative of ongoing ecological speciation (Box 1) but
different population pairs from sulphidic and nonsulphidic waters
have made variable progress along the speciation continuum towards
complete reproductive isolation (Plath et al 2013) This prompts
questions about the mechanisms involved in reducing gene flow
along the environmental gradient connecting the two habitat types
41 | Prezygotic mechanisms of reproductiveisolation
The simplest form of reproductive isolation between locally adapted
populations arises from selection against migrants between habitat
types (Nosil Vines amp Funk 2005) Translocation experiments in nat-
ural habitats have indicated strong survival selection against
migrants especially those translocated from nonsulphidic to sulphidic
habitats (Plath et al 2013) Fish translocated from nonsulphidic
environments face high mortality within short periods of time
(lt24 hr) which is likely caused by H2S toxicity and hypoxia in sul-
phide springs Hence low gene flow from populations inhabiting
nonsulphidic waters into populations inhabiting sulphidic habitats is
simply explained by a lack of adaptation to the extreme environmen-
tal conditions At least in some drainages there is also appreciable
mortality for sulphide spring fish translocated to nonsulphidic habi-
tats although mechanisms causing inviability are unclear and the
strength of selection appears to be lower (Plath et al 2013) If sur-
vival selection against immigrants was the sole source of reproduc-
tive isolation this should result in unidirectional gene flow from
H2S-adapted to nonadapted populations but population genetic
analyses provided no evidence for this implying that additional
mechanisms are at work (Plath et al 2013)
One such mechanism could be intersexual selection and mate
choice experiments have indicated that migrants from sulphidic to
nonsulphidic habitats are at a significant disadvantage with fish from
nonsulphidic habitats avoiding potential mating partners from sul-
phidic ones (Plath et al 2013 Sommer-Trembo et al 2016) Such
assortative mating has been found to be critical during ecological spe-
ciation because it counteracts the homogenizing effects of gene flow
(van Doorn Edelaar amp Weissing 2009) yet how assortative mating
patterns arise often remains unclear In theory assortative mating can
arise through a variety of processes (Nosil 2012) ldquoMagic traitrdquo mecha-
nisms where mating preferences evolve as a by-product of divergent
selection on ecological traits that serve as cues have recently garnered
much attention (Servedio van Doorn Kopp Frame amp Nosil 2011)
Consistent with magic trait mechanisms assortative mating in P mexi-
cana appears to be linked to adaptive population differences in body
shape which serve as cues to distinguish potential mating partners
from different habitats (Greenway Drexler Arias-Rodriguez amp Tobler
2016) However neither sulphide spring fish (which are unlikely to
encounter migrants from nonsulphidic habitats due to H2S toxicity)
nor allopatric P mexicana from nonsulphidic waters (which have never
experienced migrants from sulphide spring populations) exhibit a
preference for mating partners from their own ecotype suggesting
that assortative mating in this system is a product of reinforcement
(ie direct selection for assortative mating Greenway et al 2016)
This is different from classic magic traits mechanisms because assorta-
tive mating is not a mere by-product of adaptation but adaptive traits
have instead been co-opted as mating signals in response to selection
on mating preferences (Maan amp Seehausen 2012) Moreover males of
some H2S-adapted populations have reduced aggressive behaviour
(Bierbach et al 2012 2018) In staged contests of size-matched males
from different ecotypes intruders had a significantly lower likelihood
to establish dominance than resident males independent of whether
or not they showed reduced aggressiveness overall Hence intrasexual
selection may represent another mechanism mediating reproductive
isolation between locally adapted ecotypes Overall H2S-adapted poe-
ciliids allow disentangling the relative contributions of different forms
of ecological and sexual selectionmdashand their interdependencymdashduring
ecological speciation
42 | Ensuing challenges quantifying postzygoticmechanisms of reproductive isolation
In contrast to prezygotic mechanisms little is known about postzy-
gotic mechanisms of reproductive isolation that potentially act to
reduce gene flow between adjacent populations in sulphidic and
nonsulphidic habitats Putative hybrids between sulphidic and non-
sulphidic ecotypes have occasionally been observed in nonsulphidic
habitats immediately adjacent to confluences with sulphide springs
(R Riesch M Plath and M Tobler personal observations) but it
remains unclear whether and through what mechanisms such hybrids
exhibit reduced fitness compared to parentals
Direct selection of mitochondrially encoded genes in the pres-
ence of H2S (Pfenninger et al 2014) should be accompanied by co-
evolution at nuclear loci that directly interact with mitochondrial
gene products in the formation of protein complexes involved in the
OXPHOS pathway (Sunnucks Morales Lamb Pavlova amp Greening
2017 Willett amp Burton 2004) Disruption of co-evolved gene com-
plexes in hybrids should therefore lead to intrinsic fitness reductions
through mito-nuclear incompatibilities (Burton Pereira amp Barreto
2013 Wolff Ladoukakis Enrıquez amp Dowling 2014) which have
been hypothesized to be a key driver in speciation (Chou amp Leu
2010 Gershoni Templeton amp Mishmar 2009) Empirical tests of
these ideas remain to be conducted in sulphide spring fishes just
like studies on potential ecological and sexual selection against
hybrids that arise from interpopulation mating events
5 | PERSPECTIVES LIFE IN SULPHIDICENVIRONMENTS BEYOND P MEXICANA
Research on sulphide spring fishes of the P mexicana species com-
plex in Mexico has provided insights into mechanisms underlying
adaptation and the links between adaptation and speciation Sul-
phide springs do not only occur in Mexico however but are found
TOBLER ET AL | 853
worldwide and have been colonized by 24 evolutionarily indepen-
dent fish lineages in North America the Neotropics and the Middle
East (Greenway et al 2014) The vast majority of lineages belong to
the order Cyprinodontiformes (95) and livebearing members of the
family Poeciliidae in particular (80) This potentially indicates that
internal gestation of young has facilitated the colonization of
extreme environments because it shields developing embryos from
physiochemical stressors (Blackburn 1999) Among the Poeciliidae
sulphide spring fishes include members of the genera Brachyrhaphis
Gambusia Poeciliopsis Priapichthys Pseudoxiphophorus Xiphophorus
and multiple subgenera of Poecilia (Figure 3 Greenway et al 2014)
spanning over 40 million years of evolution (based on Reznick Fur-
ness Meredith amp Springer 2017)
A handful of studies have started to address molecular and phe-
notypic evolution across a broader taxon sampling of sulphide spring
fishes finding evidence for convergent evolution in morphology
(Riesch et al 2016 Tobler amp Hastings 2011) life history traits
(Riesch Plath et al 2010 Riesch et al 2014) and oxygen transport
genes (Barts et al 2018) In addition ecological speciation in
response to adaptation to H2S-rich environments has also been doc-
umented in sulphide spring poeciliids other than P mexicana (Riesch
et al 2016) These studies have laid a fruitful foundation for the
investigation of mechanisms of adaptation to H2S-rich springs from
genes to phenotypic traits linking micro-evolutionary processes to
macro-evolutionary patterns
The availability of broad-scale evolutionary replication of popula-
tions that have invaded habitats with the same physiochemical stressors
allows for addressing some of the ensuing challenges discussed above
and investigating the factors that give rise to predictable evolutionary
outcomes Convergent evolution is a commonmdashbut not omnipresentmdash
theme in adaptive evolution and multiple factors can determine the
degree of convergence among replicated lineages exposed to the same
sources of selection (Kaeuffer et al 2012 Landry amp Bernatchez 2010
Oke Rolshausen LeBlond amp Hendry 2017 Rosenblum amp Harmon
2011) Thus leveraging replicated lineages that have colonized sulphide
springs to assess molecular mechanisms underlying convergent pheno-
typic changes analyse how environmental heterogeneity among sul-
phide spring habitats causes lineage-specific patterns of trait evolution
and test whether and by what mechanisms adaptation to extreme envi-
ronments is linked to speciation will provide insights into how ecologi-
cal genetic and functional factors contribute to evolutionary
convergence at different levels of organismal organization (Rosenblum
et al 2014) An expanded taxon sampling with population pairs that
span almost the full range of the speciation continuum from panmixia to
complete reproductive isolation (Riesch et al 2016) will also help
addressing questions about temporal aspects of H2S adaptation In par-
ticular comparing younger to older lineages will provide insights about
the order at which genetic and phenotypic modifications accrue during
the adaptation process
Animals have also invaded H2S-rich habitats in other aquatic
ecosystems including cold seeps and deep-sea hydrothermal vents
(Tobler et al 2016) Adaptation to H2S in the P mexicana species com-
plex has involved modification of physiological pathways associated
with H2S toxicity (OXPHOS pathway) and detoxification (sulphide
F IGURE 3 Phylogeny of the family Poeciliidae (adopted from Culumber amp Tobler 2017) Various lineages in the genera Poecilia (subgeneraAcanthophacelus Limia and Mollienesia) Priapichthys Brachyrhaphis Poeciliopsis Xiphophorus Pseudoxiphophorus and Gambusia have colonizedH2S-rich springs in different parts of the Americas (see Greenway et al 2014) Lineages highlighted in yellow represent endemic speciesrestricted to H2S-rich springs while red lineages represent species in which some populations have invaded sulphidic habitats Pictures depict arepresentative of each focal genussubgenus
854 | TOBLER ET AL
quinone oxidoreductase pathway) which have been highly conserved
during the diversification of life on Earth (Saraste 1999 Shahak amp
Hauska 2008) The high replicability of H2S as an environmental factor
that shaped animal evolution across vastly different ecological contexts
consequently offers the opportunity to investigate whether selection
mediated by the presence of H2S has repeatedly modified these con-
served physiological pathways across animal phyla that have diverged
during the Cambrian explosion spanning over 500 million years of evo-
lution Such comparisons would provide unprecedented insights into
the effects of a single physiochemical stressor on evolutionary change
irrespective of ecological or phylogenetic contexts
6 | CONCLUSIONS
H2S-rich environments and their inhabitants represent useful models
for research in organismal biology ecology and evolution illustrating
both opportunities (eg strong and explicit source of selection evolu-
tionary replication) and challenges (eg complexity of environments
and organismal responses) in asking basic question about the origins
of biodiversity Understanding how organisms thrive in apparently
inhospitable environments will provide us with insights into lifersquos
capacities and limitations to adapt to novel environments (Waterman
1999) This is particularly relevant considering that human activities
substantially contribute to the exacerbation of physiochemical stres-
sors at local regional and global scales impacting ecological and evo-
lutionary dynamics (Alberti 2015 Norberg Urban Vellend
Klausmeier amp Loeuille 2012) Thus a focus on ecological and evolu-
tionary consequences of natural stressors that push organisms to the
brink of their physiological capacities may be instrumental to predict
consequences of anthropogenic environmental change
ACKNOWLEDGEMENTS
We would like to thank Lenin Arias-Rodriguez the Universidad
Juarez Autonoma de Tabasco and the Centro de Investigacion E
Innovacion Para la Ense~nanza y El Aprendizaje for their continued
collaboration enabling our work on the P mexicana system Funding
for this review article has been provided by the National Science
Foundation (IOS-1557860 IOS-1557795) and the US Army
Research Office (W911NF-15-1-0175)
AUTHOR CONTRIBUTIONS
MT wrote the first draft of the paper all authors contributed text
and comments
ORCID
Michael Tobler httporcidorg0000-0002-0326-0890
Joanna L Kelley httporcidorg0000-0002-7731-605X
Reuroudiger Riesch httporcidorg0000-0002-0223-1254
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Brown A P Greenway R Morgan S Quackenbush C R Giordani
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Carroll S P Jorgensen P S Kinnison M T Bergstrom C T Denison
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Chan Y F Marks M E Jones F C Villarreal G Shapiro M D Brady
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M Grimwood J Kingsley D M (2005) Widespread parallel evo-
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Cooper C E amp Brown G C (2008) The inhibition of mitochondrial
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Culumber Z W Hopper G W Barts N Passow C N Morgan S Brown
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Freshwater Ecosystems 26 1155ndash1167 httpsdoiorg101002aqc
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Culumber Z W amp Tobler M (2017) Sex-specific evolution during the
diversification of live-bearing fishes Nature Ecology amp Evolution 1
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Dalziel A C Rogers S M amp Schulte P M (2009) Linking genotypes
to phenotypes and fitness How mechanistic biology can inform
molecular ecology Molecular Ecology 18 4997ndash5017 httpsdoi
org101111j1365-294X200904427x
Darwin C (1859) On the origin of species based on natural selection or
the preservation of favoured races in the struggle of life 6th ed Lon-
don John Murray
Dasmahapatra K K Walters J R Briscoe A D Davey J W Whibley
A Nadeau N J Jiggins C D (2012) Butterfly genome reveals
promiscuous exchange of mimicry adaptations among species Nature
487 94ndash98 httpsdoiorg101038nature11041
Doudna J A amp Charpentier E (2014) The new frontier of genome edit-
ing with CRISPR-Cas9 Science 346 1258096 httpsdoiorg10
1126science1258096
Dubilier N Giere O amp Grieshaber M (2005) Concomitant effects of sul-
fide and hypoxia on the aerobic metabolism of the marine oligochaete
Tubificoides benedii Journal of Experimental Zoology 269 287ndash298
Eales K L Hollinshead K E R amp Tennant D A (2016) Hypoxia and
metabolic adaptation of cancer cells Oncogenesis 5 e190 httpsd
oiorg101038oncsis201550
Eghbal M A Pennefather P S amp OrsquoBrien P J (2004) H2S cytotoxicity
mechanism involves reactive oxygen species formation and mito-
chondrial depolarisation Toxicology 203 69ndash76 httpsdoiorg10
1016jtox200405020
Feldman C R Brodie Jr E D Brodie III E D amp Pfrender M E (2012)
Constraint shapes convergence in tetrodotoxin-resistant sodium
channels of snakes Proceedings of the National Academy of Sciences
USA 109 4556ndash4561 httpsdoiorg101073pnas1113468109
Flores K B Wolschin F amp Amdam G V (2013) The role of methyla-
tion of DNA in environmental adaptation Integrative and Comparative
Biology 53 359ndash372 httpsdoiorg101093icbict019
Fradin H Kiontke K Zegar C Gutwein M Lucas J Kovtun M
Piano F (2017) Genome architecture and evolution of a unichromo-
somal asexual nematode Current Biology 27(2928ndash2939) e2926
Gershoni M Templeton A amp Mishmar D (2009) Mitochondrial bioen-
ergetics as a major motive force of speciation BioEssays 31 642ndash
650 httpsdoiorg101002bies200800139
Ghalambor C K Walker J A amp Reznick D N (2003) Multi-trait selec-
tion adaptation and constraints on the evolution of burst swimming
performance Integrative and Comparative Biology 43 431ndash438
httpsdoiorg101093icb433431
Gibert J amp Deharveng L (2002) Subterranean ecosystems A truncated
functional biodiversity BioScience 52 473ndash481 httpsdoiorg10
16410006-3568(2002)052[0473SEATFB]20CO2
Gjuvsland A B Vik J O Beard D A Hunter P J amp Omholt S W
(2013) Bridging the genotype-phenotype gap What does it take
Journal of Physiology 591 2055ndash2066 httpsdoiorg101113jphys
iol2012248864
Grant P R Grant B R Markert J A Keller L F amp Petren K (2004)
Convergent evolution of Darwinrsquos finches caused by introgressive
hybridization and selection Evolution 58 1588ndash1599 httpsdoi
org101111j0014-38202004tb01738x
Greene H W (2005) Organisms in nature as a central focus for biology
Trends in Ecology and Evolution 20 23ndash27 httpsdoiorg101016j
tree200411005
Greenway R Arias-Rodriguez L Diaz P amp Tobler M (2014) Patterns
of macroinvertebrate and fish diversity in freshwater sulphide springs
Diversity 6 597ndash632 httpsdoiorg103390d6030597
Greenway R Drexler S Arias-Rodriguez L amp Tobler M (2016) Adap-
tive but not condition-dependent body shape differences contribute
to assortative mating preferences during ecological speciation Evolu-
tion 70 2809ndash2822 httpsdoiorg101111evo13087
Gunderson A R Armstrong E J amp Stillman J H (2016) Multiple stres-
sors in a changing world The need for an improved perspective on
physiological responses to the dynamic marine environment Annual
Review of Marine Science 8 357ndash378 httpsdoiorg101146an
nurev-marine-122414-033953
Hildebrandt T M amp Grieshaber M (2008) Three enzymatic activities
catalyze the oxidation of sulfide to thiosulfate in mammalian and
invertebrate mitochondria FEBS Journal 275 3352ndash3361 httpsd
oiorg101111j1742-4658200806482x
Houle D Govindaraju D R amp Omholt S (2010) Phenomics The next
challenge Nature Reviews Genetics 11 855ndash866 httpsdoiorg10
1038nrg2897
IUCN (2012) Red List of threatened species version 20132 www
iucnredlistorg
Johnston I A amp Bennett A F (1996) Animals and temperature Pheno-
typic and evolutionary adaptation Cambridge Cambridge University
Press httpsdoiorg101017CBO9780511721854
Jorgensen C Auer S K amp Reznick D N (2011) A model for optimal
offspring size in fish including live-bearing and parental effects Amer-
ican Naturalist 177 E119ndashE135 httpsdoiorg101086659622
Kaeuffer R Peichel C L Bolnick D I amp Hendry A P (2012) Parallel
and nonparallel aspects of ecological phenotypic and genetic diver-
gence across replicate population pairs of lake and stream stickle-
back Evolution 66 402ndash418 httpsdoiorg101111j1558-5646
201101440x
Kell D B amp Oliver S G (2004) Here is the evidence now what is the
hypothesis The complementary roles of inductive and hypothesis-
driven science in the post-genomic era BioEssays 26 99ndash105
httpsdoiorg101002(ISSN)1521-1878
856 | TOBLER ET AL
Kelley J L Arias-Rodriguez L Patacsil Martin D Yee M C Busta-
mante C amp Tobler M (2016) Mechanisms underlying adaptation to
life in hydrogen sulfide rich environments Molecular Biology and Evo-
lution 33 1419ndash1434 httpsdoiorg101093molbevmsw020
Kelley J L Passow C Plath M Arias-Rodriguez L Yee M C amp
Tobler M (2012) Genomic resources for a model in adaptation and
speciation research The characterization of the Poecilia mexicana
transcriptome BMC Genomics 13 652 httpsdoiorg101186
1471-2164-13-652
Klerks P L Xie L amp Levinton J S (2011) Quantitative genetics
approaches to study evolutionary processes in ecotoxicology a per-
spective from research on the evolution of resistance Ecotoxicology
20 513ndash523 httpsdoiorg101007s10646-011-0640-2
Kump L R Pavlov A amp Arthur M A (2005) Massive release of hydrogen
sulfide to the surface ocean and atmosphere during intervals of oceanic
anoxia Geology 33 397ndash400 httpsdoiorg101130G212951
Keuroustner A Hoffmann M Fraser B A Kottler V A Sharma E Wei-
gel D amp Dreyer C (2016) The genome of the Trinidadian guppy
Poecilia reticulata and variation in the guanapo population PLoS ONE
11 e0169087 httpsdoiorg101371journalpone0169087
Landry L amp Bernatchez L (2010) Role of epibenthic resource opportu-
nities in the parallel evolution of lake whitefish species pairs (Core-
gonus sp) Journal of Evolutionary Biology 23 2602ndash2613 httpsdoi
org101111j1420-9101201002121x
Langerhans R B (2007) Evolutionary consequences of predation Avoid-
ance escape reproduction and diversification In A M T Elewa
(Ed) Predation in organisms Berlin Springer
Li L Rose P amp Moore P K (2011) Hydrogen sulfide and cell signaling
Annual Review of Pharmacology and Toxicology 51 169ndash187
httpsdoiorg101146annurev-pharmtox-010510-100505
Lin Q Fan S Zhang Y Xu M Zhang H Yang Y Gunter H M
(2016) The seahorse genome and the evolution of its specialized mor-
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Liu Y Ma S Wang X Chang J Gao J Shi R Zhao P (2014)
Highly efficient multiplex targeted mutagenesis and genomic struc-
ture variation in Bombyx mori cells using CRISPRCas9 Insect bio-
chemistry and molecular biology 49 35ndash42 httpsdoiorg101016j
ibmb201403010
Lopez-Maury L Marguerat S amp Beuroahler J (2008) Tuning gene expres-
sion to changing environments From rapid responses to evolutionary
adaptation Nature Reviews Genetics 9 583ndash593 httpsdoiorg10
1038nrg2398
Maan M E amp Seehausen O (2012) Magic traits versus magic prefer-
ences in speciation Evolutionary Ecology Research 14 779ndash785
Martyn U Weigel D amp Dreyer C (2006) In vitro culture of embryos
of the guppy Poecilia reticulata Developmental Dynamics 235 617ndash
622 httpsdoiorg101002(ISSN)1097-0177
Metzker M L (2010) Sequencing technologies The next generation
Nature Reviews Genetics 11 31ndash46 httpsdoiorg101038nrg2626
Moore M P Riesch R amp Martin R A (2016) The predictability and
magnitude of life-history divergence to ecological agents of selection
A meta-analysis in livebearing fishes Ecology Letters 19 435ndash442
httpsdoiorg101111ele12576
Mustafa A K Gadalla M M Sen N Kim S Mu W Gazi S K
Snyder S H (2009) H2S signals through protein S-sulfhydration
Science Signaling 2 ra72
Nesse R M amp Stearns S C (2008) The great opportunity Evolutionary
applications to medicine and public health Evolutionary Applications
1 28ndash48 httpsdoiorg101111j1752-4571200700006x
Nevo E (2011) Evolution under environmental stress at macro and
microscales Genome Biology and Evolution 3 1039ndash1052 httpsdoi
org101093gbeevr052
Norberg J Urban M C Vellend M Klausmeier C A amp Loeuille N (2012)
Eco-evolutionary responses of biodiversity to climate change Nature Cli-
mate Change 2 747ndash751 httpsdoiorg101038nclimate1588
Nosil P (2012) Ecological speciation Oxford UK Oxford University
Press httpsdoiorg101093acprofosobl9780199587100001
0001
Nosil P Vines T H amp Funk D J (2005) Perspective Reproductive
isolation caused by natural selection against immigrants from diver-
gent habitats Evolution 59 705ndash719
Oke K B Rolshausen G LeBlond C amp Hendry A P (2017) How par-
allel is parallel evolution A comparative analysis in fishes American
Naturalist 190 1ndash16 httpsdoiorg101086691989
Olson K R (2011) The therapeutic potential of hydrogen sulfide
Separating hype from hope American Journal of Physiology-Regulatory
Integrative and Comparative Physiology 301 R297ndashR312 httpsdoi
org101152ajpregu000452011
Olson K R Donald J A Dombkowski R A amp Perry S F (2012) Evo-
lutionary and comparative aspects of nitric oxide carbon monoxide
and hydrogen sulfide Respiratory Physiology amp Neurobiology 184
117ndash129 httpsdoiorg101016jresp201204004
Olson K R amp Straub K D (2015) The role of hydrogen sulfide in evo-
lution and the evolution of hydrogen sulfide in metabolism and sig-
naling Physiology 31 60ndash72
Palacios M Arias-Rodriguez L Plath M Eifert C Lerp H Lamboj A
Tobler M (2013) The rediscovery of a long described species
reveals additional complexity in speciation patterns of poeciliid fishes
in sulfide springs PLoS ONE 8 e71069 httpsdoiorg101371jour
nalpone0071069
Palacios M Voelker G Arias-Rodriguez L Mateos M amp Tobler M
(2016) Phylogenetic analyses of the subgenus Mollienesia (Poecilia
Poeciliidae Teleostei) reveal taxonomic inconsistencies cryptic biodi-
versity and spatio-temporal aspects of diversification in Middle
America Molecular Phylogenetics and Evolution 103 230ndash244
httpsdoiorg101016jympev201607025
Passow C Arias-Rodriguez L amp Tobler M (2017) Convergent evolu-
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e0186935 httpsdoiorg101371journalpone0186935
Passow C N Brown A Arias-Rodriguez L Yee M-C Sockell A
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patterns in natural populations of an extremophile fish (Poecilia mexi-
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101111mec14198
Passow C Greenway R Arias-Rodriguez L Jeyasingh P D amp Tobler
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body size and metabolic rates in locally adapted extremophile fishes
Physiological and Biochemical Zoology 88 371ndash383 httpsdoiorg
101086681053
Passow C N Henpita C Shaw J H Quakenbush C Warren W C
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lations of extremophile fish Molecular Ecology 26 6384ndash6399
Pavey S A Bernatchez L Aubin-Horth N amp Landry C R (2012)
What is needed for next-generation ecological and evolutionary
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org101016jtree201207014
Peers C Bauer C Boyle J P Scragg J L amp Dallas M L (2012)
Modulation of ion channels by hydrogen sulfide Antioxidants amp Redox
Signaling 17 95ndash105 httpsdoiorg101089ars20114359
Pfennig D W amp Pfennig K S (2012) Evolutionrsquos wedge competition and
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httpsdoiorg101525california97805202741810010001
Pfenninger M Lerp H Tobler M Passow C Kelley J L Funke E
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Pfenninger M Patel S Arias-Rodriguez L Feldmeyer B Riesch R amp
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TOBLER ET AL | 857
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1111mec13397
Pietri R Roman-Morales E amp Lopez-Garriga J (2011) Hydrogen sul-
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Redox Signaling 15 393ndash404 httpsdoiorg101089ars20103698
Plath M (2008) Male mating behavior and costs of sexual harassment
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1163156853908782687241
Plath M Hermann C Schreurooder R Riesch R Tobler M Garcia de
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Plath M Pfenninger M Lerp H Riesch R Eschenbrenner C Slattery
P Tobler M (2013) Genetic differentiation and selection against
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Plath M Tobler M Riesch R Garcia de Leon F J Giere O amp
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Powell E (1989) Oxygen sulfide and diffusion Why thiobiotic meiofauna
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Ramirez-Soriano A Ramos-Onsins S E Rozas J Calafell F amp Navarro A
(2008) Statistical power analysis of neutrality tests under demographic
expansions contractions and bottlenecks with recombination Genetics
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Reiffenstein R Hulbert W amp Roth S (1992) Toxicology of hydrogen
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Reznick D N Furness A I Meredith R W amp Springer M S (2017)
The origin and biogeographic diversification of fishes in the family
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lpone0172546
Reznick D amp Yang A P (1993) The influence of fluctuating resources
on life history Patterns of allocation and plasticity in female guppies
Ecology 74 2011ndash2019 httpsdoiorg1023071940844
Riesch R Duwe V Herrmann N Padur L Ramm A Scharnweber K
Plath M (2009) Variation along the shy-bold continuum in extre-
mophile fishes (Poecilia mexicana Poecilia sulphuraria) Behavioral Ecol-
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s00265-009-0780-z
Riesch R Oranth A Dzienko J Karau N Schiessl A Stadler S
Plath M (2010) Extreme habitats are not refuges Poeciliids suffer
from increased aerial predation risk in sulphidic southern Mexican
habitats Biological Journal of the Linnean Society 101 417ndash426
httpsdoiorg101111j1095-8312201001522x
Riesch R Plath M Garcia de Leon F J amp Schlupp I (2010) Conver-
gent life-history shifts Toxic environments result in big babies in two
clades of poeciliids Naturwissenschaften 97 133ndash141 httpsdoi
org101007s00114-009-0613-y
Riesch R Plath M Schlupp I Tobler M amp Langerhans R B (2014)
Colonization of toxic springs drives predictable life-history shift in
livebearing fishes (Poeciliidae) Ecology Letters 17 65ndash71 httpsdoi
org101111ele12209
Riesch R Tobler M Lerp H Jourdan J Doumas L T Nosil P Plath
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plexity of speciation along replicated ecological gradients BMC Evolu-
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Riesch R Tobler M amp Plath M (2015) Extremophile fishes Ecology
evolution and physiology of teleosts in extreme environments p 290
Heidelberg Germany Springer httpsdoiorg101007978-3-319-
13362-1
Ritchie M E Holzinger E R Li R Pendergrass S A amp Kim S (2015)
Methods of integrating data to uncover genotype-phenotype interac-
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nrg3868
Roach K Tobler M amp Winemiller K O (2011) Hydrogen sulfide bac-
teria and fish A unique subterranean food chain Ecology 92 2056ndash
2062 httpsdoiorg10189011-02761
Rohner N Jarosz D F Kowalko J E Yoshizawa M Jeffery W R
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Science 342 1372ndash1375 httpsdoiorg101126science1240276
Rosales Lagarde L (2012) Investigation of karst brackish-sulfidic springs
and their role in the hydrogeology subsurface water-rock interactions
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of Mining and Technology Mexico
Rosenblum E B amp Harmon L J (2011) ldquoSame but differentrdquo Repli-
cated ecological speciation at White Sands Evolution 65 946ndash960
httpsdoiorg101111j1558-5646201001190x
Rosenblum E B Parent C E amp Brandt E E (2014) The molecular
basis of convergent evolution Annual Review of Ecology Evolution and
Systematics 45 203ndash226 httpsdoiorg101146annurev-ecolsys-
120213-091851
Saltz J B Hessel F C amp Kelly M W (2017) Trait correlations in the
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Saraste M (1999) Oxidative phosphorylation at the fin de siecle Science
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Schartl M (2014) Beyond the zebrafish Diverse fish species for model-
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Schartl M Walter R B Shen Y Garcia T Catchen J Amores A
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complex traits Nature Genetics 45 567ndash572 httpsdoiorg10
1038ng2604
Schluter D (2009) Evidence for ecological speciation and its alternative
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Schulz-Mirbach T Eifert C Riesch R Farnworth M S Zimmer C
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su proteccion Diario Oficial 16 May 1994 488 2ndash60
Servedio M R van Doorn G S Kopp M Frame A M amp Nosil P
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amp Evolution 26 389ndash397 httpsdoiorg101016jtree201104005
Shahak Y amp Hauska G (2008) Sulfide oxidation from cyanobacteria to
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Shalem O Sanjana N E amp Zhang F (2015) High-throughput func-
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Silva G J J Bye A el Azzouzi H amp Wisloslashff U (2017) MicroRNAs as
important regulators of exercise adaptation Progress in Cardiovascular
Diseases 60 130ndash151 httpsdoiorg101016jpcad201706003
Simola D F Wissler L Donahue G Waterhouse R M Helmkampf
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dramatic evolution in gene composition and regulation while preserv-
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1235ndash1247 httpsdoiorg101101gr155408113
858 | TOBLER ET AL
Sommer-Trembo C Bierbach D Arias Rodriguez L Verel Y Jourdan
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Naturwissenschaftlichen Classe der Kaiserlichen Akademie der Wis-
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Stinchcombe J R amp Hoekstra H E (2008) Combining population geno-
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hdy6800937
Stipanuk M H (2004) Sulfur amino acid metabolism Pathways for pro-
duction and removal of homocysteine and cysteine Annual Review of
Nutrition 24 539ndash577 httpsdoiorg101146annurevnutr24
012003132418
Stipanuk M H amp Ueki I (2011) Dealing with methioninehomocysteine
sulfur Cysteine metabolism to taurine and inorganic sulfur Journal of
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s10545-009-9006-9
Stockinger B Di Meglio P Gialitakis M amp Duarte J H (2014) The
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Storz J F amp Wheat C W (2010) Integrating evolutionary and
functional approaches to infer adaptation at specific loci Evolution
64 2489ndash2509 httpsdoiorg101111j1558-5646201001044x
Stuart Y E Veen T Weber J N Hanson D Ravinet M Lohman B
K Bolnick D I (2017) Contrasting effects of environment and
genetics generate a continuum of parallel evolution Nature Ecology amp
Evolution 1 0158 httpsdoiorg101038s41559-017-0158
Sunnucks P Morales H E Lamb A M Pavlova A amp Greening C
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genome co-evolution in oxidative phosphorylation Frontiers in Genet-
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Tobler M amp Hastings L (2011) Convergent patterns of body shape dif-
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fide springs Evolutionary Biology 38 412ndash421 httpsdoiorg10
1007s11692-011-9129-4
Tobler M Palacios M Chapman L J Mitrofanov I Bierbach D
Plath M Mateos M (2011) Evolution in extreme environments
Replicated phenotypic differentiation in livebearing fish inhabiting
sulfidic springs Evolution 65 2213ndash2228 httpsdoiorg101111j
1558-5646201101298x
Tobler M Passow C N Greenway R Kelley J L amp Shaw J H
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fide-rich environments Annual Review of Ecology Evolution and Sys-
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Tobler M amp Plath M (2009a) Threatened fishes of the world Gambu-
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Fishes 85 251 httpsdoiorg101007s10641-009-9481-8
Tobler M amp Plath M (2009b) Threatened fishes of the world Poecilia
sulphuraria (Alvarez 1948) (Poeciliidae) Environmental Biology of
Fishes 85 333ndash334 httpsdoiorg101007s10641-009-9506-3
Tobler M Plath M Riesch R Schlupp I Grasse A Munimanda G
Moodley Y (2014) Selection from parasites favors immunogenic diver-
sity but not divergence among locally adapted host populations Journal
of Evolutionary Biology 27 960ndash974 httpsdoiorg101111jeb12370
Tobler M Riesch R amp Plath M (2015) Extremophile fishes An inte-
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phile fishes Ecology evolution and physiology of teleosts in extreme
environments (pp 279ndash296) Heidelberg Germany Springer
Tobler M Riesch R Tobler C M amp Plath M (2009) Compensatory
behaviour in response to sulfide-induced hypoxia affects time
budgets feeding efficiency and predation risk Evolutionary Ecology
Research 11 935ndash948
Tobler M Scharnweber K Greenway R Passow C Arias-Rodriguez
L amp Garcia-De-Leon F J (2015) Convergent changes in the
trophic ecology if extremophile fish along replicated environmental
gradients Freshwater Biology 60 768ndash780 httpsdoiorg10
1111fwb12530
Todgham A E amp Stillman J H (2013) Physiological responses to shifts
in multiple environmental stressors Relevance in a changing worlds
Integrative and Comparative Biology 53 539ndash544 httpsdoiorg10
1093icbict086
van Doorn G S Edelaar P amp Weissing F J (2009) On the origin of
species by natural selection and sexual selection Science 326 1704ndash
1707 httpsdoiorg101126science1181661
Wall D H amp Virginia R A (1999) Controls on soil biodiversity Insights
from extreme environments Applied Soil Ecology 13 137ndash150
httpsdoiorg101016S0929-1393(99)00029-3
Wallace J L amp Wang R (2015) Hydrogen-sulfide based therapeutics
Exploiting a unique but ubiquitous gasotransmitter Nature Reviews
Drug Discovery 14 329ndash345 httpsdoiorg101038nrd4433
Wang R (2012) Physiological implications of hydrogen sulfide A whiff
exploration that blossomed Physiological Reviews 92 791ndash896
httpsdoiorg101152physrev000172011
Wang Z Gerstein M amp Snyder M (2009) RNA-seq A revolutionary
tool for transcriptomics Nature Reviews Genetics 10 57ndash63
httpsdoiorg101038nrg2484
Waterman T H (1999) The evolutionary challenges of extreme environ-
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httpsdoiorg101002(SICI)1097-010X(19991215)2854amplt326
AID-JEZ3ampgt30CO2-T
Werren J H Richards S Desjardins C A Niehuis O Gadau J Col-
bourne J K amp Group N G W (2010) Functional and evolutionary
insights from the genomes of three parasitoid Nasonia species
Science 327 343ndash348 httpsdoiorg101126science1178028
Whitehead A (2012) Comparative genomics in ecological physiology
Toward a more nuanced understanding of acclimation and adapta-
tion Journal of Experimental Biology 215 884ndash891 httpsdoiorg
101242jeb058735
Whitehead A amp Crawford D L (2006) Neutral and adaptive variation
in gene expression Proceedings of the National Academy of Sciences
USA 103 5425ndash5430 httpsdoiorg101073pnas0507648103
Willett C S amp Burton R S (2004) Evolution of interacting proteins in
the mitochondrial electron transport system in a marine copepod
Molecular Biology and Evolution 21 443ndash453 httpsdoiorg10
1093molbevmsh031
Willi Y Van Buskirk J amp Hoffmann A (2006) Limits to the adaptive
potential of small populations Annual Review of Ecology Evolution
and Systematics 37 433ndash458 httpsdoiorg101146annurevecol
sys37091305110145
Wolff J N Ladoukakis E D Enrıquez J A amp Dowling D K (2014)
Mitonuclear interactions Evolutionary consequences over multiple
biological scales Philosophical Transactions of the Royal Society B 369
20130443 httpsdoiorg101098rstb20130443
How to cite this article Tobler M Kelley JL Plath M Riesch
R Extreme environments and the origins of biodiversity
Adaptation and speciation in sulphide spring fishes Mol Ecol
201827843ndash859 httpsdoiorg101111mec14497
TOBLER ET AL | 859
Jeyasingh amp Tobler 2015) Theory predicts that energetic con-
straints ndash whether mediated by limitations in the rate of resource
acquisition or in the rate of energy allocation to reproduction (as
opposed to maintenance or growth) ndash should exert selection for a
reduction in organismal energy budgets which allows for the maxi-
mization of residual energy allocation for the production of offspring
(Brown Marquet amp Taper 1993) Several lines of evidence support
the hypothesis that energy limitation plays a critical role in pheno-
typic evolution of sulphide spring fish Compared to individuals in
nonsulphidic environments fish in sulphide springs have significantly
lower routine energy demands which is mediated both by reduc-
tions in body size and mass-specific metabolism (Passow Arias-
Rodriguez et al 2017) Reductions in other traits such as energeti-
cally costly behaviours (Bierbach et al 2012 Plath 2008) and the
size of energy-demanding organs (Schulz-Mirbach et al 2016) may
also contribute to the reduced energy demands and shifts in repro-
ductive life history traits (Riesch et al 2014) are consistent with
selection from energy limitation (Reznick amp Yang 1993)
32 | Ensuing challenges Disentanglingenvironmental and organismal complexities
Future research needs to empirically address how multifarious selec-
tive regimes shape the evolution of organisms with complex pheno-
types We have made great advances in our understanding of how
specific traits evolve and mediate adaptation (eg morphology Chan
et al 2010 coloration Dasmahapatra et al 2012 sensory organs
Cartwright Schwartz Merry amp Howell 2017) and we have a thor-
ough understanding of how single environmental factors can shape
organismal responses (eg climatic conditions Johnston amp Bennett
1996 predation Langerhans 2007 competition Pfennig amp Pfennig
2012) In contrast our understanding of phenotypic evolution in
response to multifarious selection is comparatively limited especially
because interactive effects of different environmental factors can lead
to organismal and evolutionary responses that are unpredictable based
on experiments that manipulate single environmental variables in iso-
lation (Gunderson Armstrong amp Stillman 2016 Todgham amp Stillman
2013) Future research thus needs to acknowledge the complexities of
environments and organismal design and address them explicitly in
empirical investigations to gain a robust understanding of biological
diversification (Culumber amp Tobler 2017 Stuart et al 2017)
4 | LINKING ADAPTATION TOSPECIATION
So far we primarily explored the causes of adaptive evolution but
what are its consequences Theory predicts that adaptation to local
environmental conditions can lead to speciation because reproduc-
tive isolation emerges incidentally as a by-product (ecological specia-
tion Nosil 2012) Population genetic analyses of Poecilia spp in
sulphide springs and adjacent populations in nonsulphidic environ-
ments have indicated strong genetic differentiation across small
spatial scales (in some instances lt100 m) and in the absence of
physical barriers that would prevent fish movement (Plath et al
2013) In addition population genetic structuring has been shown to
be stable even in the face of major environmental disturbance
BOX 1 Speciation in sulphide springs and conservation
biology
Adaptation to H2S-rich environments in poeciliids coincides
with ecological speciation giving rise to phenotypically dis-
tinct and reproductively isolated lineages In some
instances this has led to the evolution of highly distinct
micro-endemic species including Poecilia sulphuraria and
P thermalis Limia sulphurophila and Gambusia eurystoma
(Figure 3) Except for P sulphuraria which is found in two
spring complexes of the Rıo Pichucalco drainage of Mexico
(Tobler amp Plath 2009b) the distributions of these sulphide
spring endemics are restricted to single localities that
include only small areas of suitable habitat For example
the largest sulphide spring complex (the Ba~nos del Azufre
in southern Mexico which harbours P sulphuraria and G
eurystoma) includes ~18000 m2 of suitable habitat (equiva-
lent to lt35 football fields) while one of the smallest (La
Esperanza springs harbouring P thermalis) merely covers
~600 m2 (just over a tenth of a football field Brown et al
2017) Accordingly some sulphide spring endemics are
considered critically endangered (IUCN 2012) and pro-
tected by law (SEDESOL 1994)
Many threatened species with restricted ranges exhibit
low genetic diversity and high incidences of inbreeding
(Bouzat 2010 Willi Van Buskirk amp Hoffmann 2006) In
contrast at least P sulphuraria and P thermalismdashthe only
species investigated to datemdashactually exhibit relatively high
effective population sizes (Ne = 1200ndash3800 depending on
the population) and no evidence for inbreeding (FIS lt 0)
(Brown et al 2017) This is likely a consequence of the
long-term stability of sulphide springs and the high pro-
ductivity and low levels of interspecific competition also
support unusually high fish densities and thus population
census sizes (Brown et al 2017 Culumber et al 2016)
Nonetheless sulphide spring endemics are threatened by
habitat destruction which is mediated primarily by the
modification of springs for recreational purposes defor-
estation and conversion of land for agriculture and most
recently high-intensity palm oil plantations (Tobler amp Plath
2009ab) Despite the official protection of sulphide spring
endemics there have been no tangible conservation mea-
sures that foster the sustainable development of land
around sulphide springs with the well-being of the endemic
fishes in mind
852 | TOBLER ET AL
(catastrophic flood event Plath et al 2010) Population genetic dif-
ferentiation between adjacent sulphide-adapted and nonadapted
populations is indicative of ongoing ecological speciation (Box 1) but
different population pairs from sulphidic and nonsulphidic waters
have made variable progress along the speciation continuum towards
complete reproductive isolation (Plath et al 2013) This prompts
questions about the mechanisms involved in reducing gene flow
along the environmental gradient connecting the two habitat types
41 | Prezygotic mechanisms of reproductiveisolation
The simplest form of reproductive isolation between locally adapted
populations arises from selection against migrants between habitat
types (Nosil Vines amp Funk 2005) Translocation experiments in nat-
ural habitats have indicated strong survival selection against
migrants especially those translocated from nonsulphidic to sulphidic
habitats (Plath et al 2013) Fish translocated from nonsulphidic
environments face high mortality within short periods of time
(lt24 hr) which is likely caused by H2S toxicity and hypoxia in sul-
phide springs Hence low gene flow from populations inhabiting
nonsulphidic waters into populations inhabiting sulphidic habitats is
simply explained by a lack of adaptation to the extreme environmen-
tal conditions At least in some drainages there is also appreciable
mortality for sulphide spring fish translocated to nonsulphidic habi-
tats although mechanisms causing inviability are unclear and the
strength of selection appears to be lower (Plath et al 2013) If sur-
vival selection against immigrants was the sole source of reproduc-
tive isolation this should result in unidirectional gene flow from
H2S-adapted to nonadapted populations but population genetic
analyses provided no evidence for this implying that additional
mechanisms are at work (Plath et al 2013)
One such mechanism could be intersexual selection and mate
choice experiments have indicated that migrants from sulphidic to
nonsulphidic habitats are at a significant disadvantage with fish from
nonsulphidic habitats avoiding potential mating partners from sul-
phidic ones (Plath et al 2013 Sommer-Trembo et al 2016) Such
assortative mating has been found to be critical during ecological spe-
ciation because it counteracts the homogenizing effects of gene flow
(van Doorn Edelaar amp Weissing 2009) yet how assortative mating
patterns arise often remains unclear In theory assortative mating can
arise through a variety of processes (Nosil 2012) ldquoMagic traitrdquo mecha-
nisms where mating preferences evolve as a by-product of divergent
selection on ecological traits that serve as cues have recently garnered
much attention (Servedio van Doorn Kopp Frame amp Nosil 2011)
Consistent with magic trait mechanisms assortative mating in P mexi-
cana appears to be linked to adaptive population differences in body
shape which serve as cues to distinguish potential mating partners
from different habitats (Greenway Drexler Arias-Rodriguez amp Tobler
2016) However neither sulphide spring fish (which are unlikely to
encounter migrants from nonsulphidic habitats due to H2S toxicity)
nor allopatric P mexicana from nonsulphidic waters (which have never
experienced migrants from sulphide spring populations) exhibit a
preference for mating partners from their own ecotype suggesting
that assortative mating in this system is a product of reinforcement
(ie direct selection for assortative mating Greenway et al 2016)
This is different from classic magic traits mechanisms because assorta-
tive mating is not a mere by-product of adaptation but adaptive traits
have instead been co-opted as mating signals in response to selection
on mating preferences (Maan amp Seehausen 2012) Moreover males of
some H2S-adapted populations have reduced aggressive behaviour
(Bierbach et al 2012 2018) In staged contests of size-matched males
from different ecotypes intruders had a significantly lower likelihood
to establish dominance than resident males independent of whether
or not they showed reduced aggressiveness overall Hence intrasexual
selection may represent another mechanism mediating reproductive
isolation between locally adapted ecotypes Overall H2S-adapted poe-
ciliids allow disentangling the relative contributions of different forms
of ecological and sexual selectionmdashand their interdependencymdashduring
ecological speciation
42 | Ensuing challenges quantifying postzygoticmechanisms of reproductive isolation
In contrast to prezygotic mechanisms little is known about postzy-
gotic mechanisms of reproductive isolation that potentially act to
reduce gene flow between adjacent populations in sulphidic and
nonsulphidic habitats Putative hybrids between sulphidic and non-
sulphidic ecotypes have occasionally been observed in nonsulphidic
habitats immediately adjacent to confluences with sulphide springs
(R Riesch M Plath and M Tobler personal observations) but it
remains unclear whether and through what mechanisms such hybrids
exhibit reduced fitness compared to parentals
Direct selection of mitochondrially encoded genes in the pres-
ence of H2S (Pfenninger et al 2014) should be accompanied by co-
evolution at nuclear loci that directly interact with mitochondrial
gene products in the formation of protein complexes involved in the
OXPHOS pathway (Sunnucks Morales Lamb Pavlova amp Greening
2017 Willett amp Burton 2004) Disruption of co-evolved gene com-
plexes in hybrids should therefore lead to intrinsic fitness reductions
through mito-nuclear incompatibilities (Burton Pereira amp Barreto
2013 Wolff Ladoukakis Enrıquez amp Dowling 2014) which have
been hypothesized to be a key driver in speciation (Chou amp Leu
2010 Gershoni Templeton amp Mishmar 2009) Empirical tests of
these ideas remain to be conducted in sulphide spring fishes just
like studies on potential ecological and sexual selection against
hybrids that arise from interpopulation mating events
5 | PERSPECTIVES LIFE IN SULPHIDICENVIRONMENTS BEYOND P MEXICANA
Research on sulphide spring fishes of the P mexicana species com-
plex in Mexico has provided insights into mechanisms underlying
adaptation and the links between adaptation and speciation Sul-
phide springs do not only occur in Mexico however but are found
TOBLER ET AL | 853
worldwide and have been colonized by 24 evolutionarily indepen-
dent fish lineages in North America the Neotropics and the Middle
East (Greenway et al 2014) The vast majority of lineages belong to
the order Cyprinodontiformes (95) and livebearing members of the
family Poeciliidae in particular (80) This potentially indicates that
internal gestation of young has facilitated the colonization of
extreme environments because it shields developing embryos from
physiochemical stressors (Blackburn 1999) Among the Poeciliidae
sulphide spring fishes include members of the genera Brachyrhaphis
Gambusia Poeciliopsis Priapichthys Pseudoxiphophorus Xiphophorus
and multiple subgenera of Poecilia (Figure 3 Greenway et al 2014)
spanning over 40 million years of evolution (based on Reznick Fur-
ness Meredith amp Springer 2017)
A handful of studies have started to address molecular and phe-
notypic evolution across a broader taxon sampling of sulphide spring
fishes finding evidence for convergent evolution in morphology
(Riesch et al 2016 Tobler amp Hastings 2011) life history traits
(Riesch Plath et al 2010 Riesch et al 2014) and oxygen transport
genes (Barts et al 2018) In addition ecological speciation in
response to adaptation to H2S-rich environments has also been doc-
umented in sulphide spring poeciliids other than P mexicana (Riesch
et al 2016) These studies have laid a fruitful foundation for the
investigation of mechanisms of adaptation to H2S-rich springs from
genes to phenotypic traits linking micro-evolutionary processes to
macro-evolutionary patterns
The availability of broad-scale evolutionary replication of popula-
tions that have invaded habitats with the same physiochemical stressors
allows for addressing some of the ensuing challenges discussed above
and investigating the factors that give rise to predictable evolutionary
outcomes Convergent evolution is a commonmdashbut not omnipresentmdash
theme in adaptive evolution and multiple factors can determine the
degree of convergence among replicated lineages exposed to the same
sources of selection (Kaeuffer et al 2012 Landry amp Bernatchez 2010
Oke Rolshausen LeBlond amp Hendry 2017 Rosenblum amp Harmon
2011) Thus leveraging replicated lineages that have colonized sulphide
springs to assess molecular mechanisms underlying convergent pheno-
typic changes analyse how environmental heterogeneity among sul-
phide spring habitats causes lineage-specific patterns of trait evolution
and test whether and by what mechanisms adaptation to extreme envi-
ronments is linked to speciation will provide insights into how ecologi-
cal genetic and functional factors contribute to evolutionary
convergence at different levels of organismal organization (Rosenblum
et al 2014) An expanded taxon sampling with population pairs that
span almost the full range of the speciation continuum from panmixia to
complete reproductive isolation (Riesch et al 2016) will also help
addressing questions about temporal aspects of H2S adaptation In par-
ticular comparing younger to older lineages will provide insights about
the order at which genetic and phenotypic modifications accrue during
the adaptation process
Animals have also invaded H2S-rich habitats in other aquatic
ecosystems including cold seeps and deep-sea hydrothermal vents
(Tobler et al 2016) Adaptation to H2S in the P mexicana species com-
plex has involved modification of physiological pathways associated
with H2S toxicity (OXPHOS pathway) and detoxification (sulphide
F IGURE 3 Phylogeny of the family Poeciliidae (adopted from Culumber amp Tobler 2017) Various lineages in the genera Poecilia (subgeneraAcanthophacelus Limia and Mollienesia) Priapichthys Brachyrhaphis Poeciliopsis Xiphophorus Pseudoxiphophorus and Gambusia have colonizedH2S-rich springs in different parts of the Americas (see Greenway et al 2014) Lineages highlighted in yellow represent endemic speciesrestricted to H2S-rich springs while red lineages represent species in which some populations have invaded sulphidic habitats Pictures depict arepresentative of each focal genussubgenus
854 | TOBLER ET AL
quinone oxidoreductase pathway) which have been highly conserved
during the diversification of life on Earth (Saraste 1999 Shahak amp
Hauska 2008) The high replicability of H2S as an environmental factor
that shaped animal evolution across vastly different ecological contexts
consequently offers the opportunity to investigate whether selection
mediated by the presence of H2S has repeatedly modified these con-
served physiological pathways across animal phyla that have diverged
during the Cambrian explosion spanning over 500 million years of evo-
lution Such comparisons would provide unprecedented insights into
the effects of a single physiochemical stressor on evolutionary change
irrespective of ecological or phylogenetic contexts
6 | CONCLUSIONS
H2S-rich environments and their inhabitants represent useful models
for research in organismal biology ecology and evolution illustrating
both opportunities (eg strong and explicit source of selection evolu-
tionary replication) and challenges (eg complexity of environments
and organismal responses) in asking basic question about the origins
of biodiversity Understanding how organisms thrive in apparently
inhospitable environments will provide us with insights into lifersquos
capacities and limitations to adapt to novel environments (Waterman
1999) This is particularly relevant considering that human activities
substantially contribute to the exacerbation of physiochemical stres-
sors at local regional and global scales impacting ecological and evo-
lutionary dynamics (Alberti 2015 Norberg Urban Vellend
Klausmeier amp Loeuille 2012) Thus a focus on ecological and evolu-
tionary consequences of natural stressors that push organisms to the
brink of their physiological capacities may be instrumental to predict
consequences of anthropogenic environmental change
ACKNOWLEDGEMENTS
We would like to thank Lenin Arias-Rodriguez the Universidad
Juarez Autonoma de Tabasco and the Centro de Investigacion E
Innovacion Para la Ense~nanza y El Aprendizaje for their continued
collaboration enabling our work on the P mexicana system Funding
for this review article has been provided by the National Science
Foundation (IOS-1557860 IOS-1557795) and the US Army
Research Office (W911NF-15-1-0175)
AUTHOR CONTRIBUTIONS
MT wrote the first draft of the paper all authors contributed text
and comments
ORCID
Michael Tobler httporcidorg0000-0002-0326-0890
Joanna L Kelley httporcidorg0000-0002-7731-605X
Reuroudiger Riesch httporcidorg0000-0002-0223-1254
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Cooper C E amp Brown G C (2008) The inhibition of mitochondrial
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Culumber Z W Hopper G W Barts N Passow C N Morgan S Brown
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Culumber Z W amp Tobler M (2017) Sex-specific evolution during the
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Dalziel A C Rogers S M amp Schulte P M (2009) Linking genotypes
to phenotypes and fitness How mechanistic biology can inform
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Darwin C (1859) On the origin of species based on natural selection or
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A Nadeau N J Jiggins C D (2012) Butterfly genome reveals
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Doudna J A amp Charpentier E (2014) The new frontier of genome edit-
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Eales K L Hollinshead K E R amp Tennant D A (2016) Hypoxia and
metabolic adaptation of cancer cells Oncogenesis 5 e190 httpsd
oiorg101038oncsis201550
Eghbal M A Pennefather P S amp OrsquoBrien P J (2004) H2S cytotoxicity
mechanism involves reactive oxygen species formation and mito-
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1016jtox200405020
Feldman C R Brodie Jr E D Brodie III E D amp Pfrender M E (2012)
Constraint shapes convergence in tetrodotoxin-resistant sodium
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Fradin H Kiontke K Zegar C Gutwein M Lucas J Kovtun M
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Gershoni M Templeton A amp Mishmar D (2009) Mitochondrial bioen-
ergetics as a major motive force of speciation BioEssays 31 642ndash
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Ghalambor C K Walker J A amp Reznick D N (2003) Multi-trait selec-
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Gibert J amp Deharveng L (2002) Subterranean ecosystems A truncated
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Grant P R Grant B R Markert J A Keller L F amp Petren K (2004)
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Greene H W (2005) Organisms in nature as a central focus for biology
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to assortative mating preferences during ecological speciation Evolu-
tion 70 2809ndash2822 httpsdoiorg101111evo13087
Gunderson A R Armstrong E J amp Stillman J H (2016) Multiple stres-
sors in a changing world The need for an improved perspective on
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nurev-marine-122414-033953
Hildebrandt T M amp Grieshaber M (2008) Three enzymatic activities
catalyze the oxidation of sulfide to thiosulfate in mammalian and
invertebrate mitochondria FEBS Journal 275 3352ndash3361 httpsd
oiorg101111j1742-4658200806482x
Houle D Govindaraju D R amp Omholt S (2010) Phenomics The next
challenge Nature Reviews Genetics 11 855ndash866 httpsdoiorg10
1038nrg2897
IUCN (2012) Red List of threatened species version 20132 www
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Press httpsdoiorg101017CBO9780511721854
Jorgensen C Auer S K amp Reznick D N (2011) A model for optimal
offspring size in fish including live-bearing and parental effects Amer-
ican Naturalist 177 E119ndashE135 httpsdoiorg101086659622
Kaeuffer R Peichel C L Bolnick D I amp Hendry A P (2012) Parallel
and nonparallel aspects of ecological phenotypic and genetic diver-
gence across replicate population pairs of lake and stream stickle-
back Evolution 66 402ndash418 httpsdoiorg101111j1558-5646
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driven science in the post-genomic era BioEssays 26 99ndash105
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856 | TOBLER ET AL
Kelley J L Arias-Rodriguez L Patacsil Martin D Yee M C Busta-
mante C amp Tobler M (2016) Mechanisms underlying adaptation to
life in hydrogen sulfide rich environments Molecular Biology and Evo-
lution 33 1419ndash1434 httpsdoiorg101093molbevmsw020
Kelley J L Passow C Plath M Arias-Rodriguez L Yee M C amp
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speciation research The characterization of the Poecilia mexicana
transcriptome BMC Genomics 13 652 httpsdoiorg101186
1471-2164-13-652
Klerks P L Xie L amp Levinton J S (2011) Quantitative genetics
approaches to study evolutionary processes in ecotoxicology a per-
spective from research on the evolution of resistance Ecotoxicology
20 513ndash523 httpsdoiorg101007s10646-011-0640-2
Kump L R Pavlov A amp Arthur M A (2005) Massive release of hydrogen
sulfide to the surface ocean and atmosphere during intervals of oceanic
anoxia Geology 33 397ndash400 httpsdoiorg101130G212951
Keuroustner A Hoffmann M Fraser B A Kottler V A Sharma E Wei-
gel D amp Dreyer C (2016) The genome of the Trinidadian guppy
Poecilia reticulata and variation in the guanapo population PLoS ONE
11 e0169087 httpsdoiorg101371journalpone0169087
Landry L amp Bernatchez L (2010) Role of epibenthic resource opportu-
nities in the parallel evolution of lake whitefish species pairs (Core-
gonus sp) Journal of Evolutionary Biology 23 2602ndash2613 httpsdoi
org101111j1420-9101201002121x
Langerhans R B (2007) Evolutionary consequences of predation Avoid-
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(Ed) Predation in organisms Berlin Springer
Li L Rose P amp Moore P K (2011) Hydrogen sulfide and cell signaling
Annual Review of Pharmacology and Toxicology 51 169ndash187
httpsdoiorg101146annurev-pharmtox-010510-100505
Lin Q Fan S Zhang Y Xu M Zhang H Yang Y Gunter H M
(2016) The seahorse genome and the evolution of its specialized mor-
phology Nature 540 395ndash399 httpsdoiorg101038nature20595
Liu Y Ma S Wang X Chang J Gao J Shi R Zhao P (2014)
Highly efficient multiplex targeted mutagenesis and genomic struc-
ture variation in Bombyx mori cells using CRISPRCas9 Insect bio-
chemistry and molecular biology 49 35ndash42 httpsdoiorg101016j
ibmb201403010
Lopez-Maury L Marguerat S amp Beuroahler J (2008) Tuning gene expres-
sion to changing environments From rapid responses to evolutionary
adaptation Nature Reviews Genetics 9 583ndash593 httpsdoiorg10
1038nrg2398
Maan M E amp Seehausen O (2012) Magic traits versus magic prefer-
ences in speciation Evolutionary Ecology Research 14 779ndash785
Martyn U Weigel D amp Dreyer C (2006) In vitro culture of embryos
of the guppy Poecilia reticulata Developmental Dynamics 235 617ndash
622 httpsdoiorg101002(ISSN)1097-0177
Metzker M L (2010) Sequencing technologies The next generation
Nature Reviews Genetics 11 31ndash46 httpsdoiorg101038nrg2626
Moore M P Riesch R amp Martin R A (2016) The predictability and
magnitude of life-history divergence to ecological agents of selection
A meta-analysis in livebearing fishes Ecology Letters 19 435ndash442
httpsdoiorg101111ele12576
Mustafa A K Gadalla M M Sen N Kim S Mu W Gazi S K
Snyder S H (2009) H2S signals through protein S-sulfhydration
Science Signaling 2 ra72
Nesse R M amp Stearns S C (2008) The great opportunity Evolutionary
applications to medicine and public health Evolutionary Applications
1 28ndash48 httpsdoiorg101111j1752-4571200700006x
Nevo E (2011) Evolution under environmental stress at macro and
microscales Genome Biology and Evolution 3 1039ndash1052 httpsdoi
org101093gbeevr052
Norberg J Urban M C Vellend M Klausmeier C A amp Loeuille N (2012)
Eco-evolutionary responses of biodiversity to climate change Nature Cli-
mate Change 2 747ndash751 httpsdoiorg101038nclimate1588
Nosil P (2012) Ecological speciation Oxford UK Oxford University
Press httpsdoiorg101093acprofosobl9780199587100001
0001
Nosil P Vines T H amp Funk D J (2005) Perspective Reproductive
isolation caused by natural selection against immigrants from diver-
gent habitats Evolution 59 705ndash719
Oke K B Rolshausen G LeBlond C amp Hendry A P (2017) How par-
allel is parallel evolution A comparative analysis in fishes American
Naturalist 190 1ndash16 httpsdoiorg101086691989
Olson K R (2011) The therapeutic potential of hydrogen sulfide
Separating hype from hope American Journal of Physiology-Regulatory
Integrative and Comparative Physiology 301 R297ndashR312 httpsdoi
org101152ajpregu000452011
Olson K R Donald J A Dombkowski R A amp Perry S F (2012) Evo-
lutionary and comparative aspects of nitric oxide carbon monoxide
and hydrogen sulfide Respiratory Physiology amp Neurobiology 184
117ndash129 httpsdoiorg101016jresp201204004
Olson K R amp Straub K D (2015) The role of hydrogen sulfide in evo-
lution and the evolution of hydrogen sulfide in metabolism and sig-
naling Physiology 31 60ndash72
Palacios M Arias-Rodriguez L Plath M Eifert C Lerp H Lamboj A
Tobler M (2013) The rediscovery of a long described species
reveals additional complexity in speciation patterns of poeciliid fishes
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nalpone0071069
Palacios M Voelker G Arias-Rodriguez L Mateos M amp Tobler M
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versity and spatio-temporal aspects of diversification in Middle
America Molecular Phylogenetics and Evolution 103 230ndash244
httpsdoiorg101016jympev201607025
Passow C Arias-Rodriguez L amp Tobler M (2017) Convergent evolu-
tion of reduced energy demands in extremophile fish PLoS ONE 12
e0186935 httpsdoiorg101371journalpone0186935
Passow C N Brown A Arias-Rodriguez L Yee M-C Sockell A
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patterns in natural populations of an extremophile fish (Poecilia mexi-
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101111mec14198
Passow C Greenway R Arias-Rodriguez L Jeyasingh P D amp Tobler
M (2015) Reduction of energetic demands through modification of
body size and metabolic rates in locally adapted extremophile fishes
Physiological and Biochemical Zoology 88 371ndash383 httpsdoiorg
101086681053
Passow C N Henpita C Shaw J H Quakenbush C Warren W C
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lations of extremophile fish Molecular Ecology 26 6384ndash6399
Pavey S A Bernatchez L Aubin-Horth N amp Landry C R (2012)
What is needed for next-generation ecological and evolutionary
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Peers C Bauer C Boyle J P Scragg J L amp Dallas M L (2012)
Modulation of ion channels by hydrogen sulfide Antioxidants amp Redox
Signaling 17 95ndash105 httpsdoiorg101089ars20114359
Pfennig D W amp Pfennig K S (2012) Evolutionrsquos wedge competition and
the origins of diversity Berkeley CA University of California Press
httpsdoiorg101525california97805202741810010001
Pfenninger M Lerp H Tobler M Passow C Kelley J L Funke E
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fish as key adaptation to a toxic environment Nature Communica-
tions 5 3873
Pfenninger M Patel S Arias-Rodriguez L Feldmeyer B Riesch R amp
Plath M (2015) Unique evolutionary trajectories in repeated adapta-
tion to hydrogen sulphide-toxic habitats of a neotropical fish (Poecilia
TOBLER ET AL | 857
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1111mec13397
Pietri R Roman-Morales E amp Lopez-Garriga J (2011) Hydrogen sul-
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Redox Signaling 15 393ndash404 httpsdoiorg101089ars20103698
Plath M (2008) Male mating behavior and costs of sexual harassment
for females in cavernicolous and extremophile populations of Atlantic
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1163156853908782687241
Plath M Hermann C Schreurooder R Riesch R Tobler M Garcia de
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maintain small-scale genetic differentiation despite perturbation by a
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Plath M Pfenninger M Lerp H Riesch R Eschenbrenner C Slattery
P Tobler M (2013) Genetic differentiation and selection against
migrants in evolutionarily replicated extreme environments Evolution
67 2647ndash2661 httpsdoiorg101111evo12133
Plath M Tobler M Riesch R Garcia de Leon F J Giere O amp
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httpsdoiorg101007s00114-007-0279-2
Powell E (1989) Oxygen sulfide and diffusion Why thiobiotic meiofauna
must be sulfide-insensitive first-order respirers Journal of Marine
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Ramirez-Soriano A Ramos-Onsins S E Rozas J Calafell F amp Navarro A
(2008) Statistical power analysis of neutrality tests under demographic
expansions contractions and bottlenecks with recombination Genetics
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Reiffenstein R Hulbert W amp Roth S (1992) Toxicology of hydrogen
sulfide Annual Reviews of Pharmacology and Toxicology 1992 109ndash
134 httpsdoiorg101146annurevpa32040192000545
Reznick D N Furness A I Meredith R W amp Springer M S (2017)
The origin and biogeographic diversification of fishes in the family
Poeciliidae PLoS ONE 12 e0172546 httpsdoiorg101371journa
lpone0172546
Reznick D amp Yang A P (1993) The influence of fluctuating resources
on life history Patterns of allocation and plasticity in female guppies
Ecology 74 2011ndash2019 httpsdoiorg1023071940844
Riesch R Duwe V Herrmann N Padur L Ramm A Scharnweber K
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s00265-009-0780-z
Riesch R Oranth A Dzienko J Karau N Schiessl A Stadler S
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from increased aerial predation risk in sulphidic southern Mexican
habitats Biological Journal of the Linnean Society 101 417ndash426
httpsdoiorg101111j1095-8312201001522x
Riesch R Plath M Garcia de Leon F J amp Schlupp I (2010) Conver-
gent life-history shifts Toxic environments result in big babies in two
clades of poeciliids Naturwissenschaften 97 133ndash141 httpsdoi
org101007s00114-009-0613-y
Riesch R Plath M Schlupp I Tobler M amp Langerhans R B (2014)
Colonization of toxic springs drives predictable life-history shift in
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org101111ele12209
Riesch R Tobler M Lerp H Jourdan J Doumas L T Nosil P Plath
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plexity of speciation along replicated ecological gradients BMC Evolu-
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Riesch R Tobler M amp Plath M (2015) Extremophile fishes Ecology
evolution and physiology of teleosts in extreme environments p 290
Heidelberg Germany Springer httpsdoiorg101007978-3-319-
13362-1
Ritchie M E Holzinger E R Li R Pendergrass S A amp Kim S (2015)
Methods of integrating data to uncover genotype-phenotype interac-
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nrg3868
Roach K Tobler M amp Winemiller K O (2011) Hydrogen sulfide bac-
teria and fish A unique subterranean food chain Ecology 92 2056ndash
2062 httpsdoiorg10189011-02761
Rohner N Jarosz D F Kowalko J E Yoshizawa M Jeffery W R
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Science 342 1372ndash1375 httpsdoiorg101126science1240276
Rosales Lagarde L (2012) Investigation of karst brackish-sulfidic springs
and their role in the hydrogeology subsurface water-rock interactions
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of Mining and Technology Mexico
Rosenblum E B amp Harmon L J (2011) ldquoSame but differentrdquo Repli-
cated ecological speciation at White Sands Evolution 65 946ndash960
httpsdoiorg101111j1558-5646201001190x
Rosenblum E B Parent C E amp Brandt E E (2014) The molecular
basis of convergent evolution Annual Review of Ecology Evolution and
Systematics 45 203ndash226 httpsdoiorg101146annurev-ecolsys-
120213-091851
Saltz J B Hessel F C amp Kelly M W (2017) Trait correlations in the
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Saraste M (1999) Oxidative phosphorylation at the fin de siecle Science
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Schartl M (2014) Beyond the zebrafish Diverse fish species for model-
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Schartl M Walter R B Shen Y Garcia T Catchen J Amores A
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1038ng2604
Schluter D (2009) Evidence for ecological speciation and its alternative
Science 323 737ndash741 httpsdoiorg101126science1160006
Schulz-Mirbach T Eifert C Riesch R Farnworth M S Zimmer C
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Servedio M R van Doorn G S Kopp M Frame A M amp Nosil P
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amp Evolution 26 389ndash397 httpsdoiorg101016jtree201104005
Shahak Y amp Hauska G (2008) Sulfide oxidation from cyanobacteria to
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Shalem O Sanjana N E amp Zhang F (2015) High-throughput func-
tional genomics using CRISPR-Cas9 Nature Reviews Genetics 16
299ndash311 httpsdoiorg101038nrg3899
Silva G J J Bye A el Azzouzi H amp Wisloslashff U (2017) MicroRNAs as
important regulators of exercise adaptation Progress in Cardiovascular
Diseases 60 130ndash151 httpsdoiorg101016jpcad201706003
Simola D F Wissler L Donahue G Waterhouse R M Helmkampf
M Roux J Viljakainen L (2013) Social insect genomes exhibit
dramatic evolution in gene composition and regulation while preserv-
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1235ndash1247 httpsdoiorg101101gr155408113
858 | TOBLER ET AL
Sommer-Trembo C Bierbach D Arias Rodriguez L Verel Y Jourdan
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Steindachner F (1863) Beitreuroage zur Kentniss der Sciaenoiden Brasiliens
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Naturwissenschaftlichen Classe der Kaiserlichen Akademie der Wis-
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Stinchcombe J R amp Hoekstra H E (2008) Combining population geno-
mics and quantitative genetics Finding genes underlying ecologically
important traits Heredity 100 158ndash170 httpsdoiorg101038sj
hdy6800937
Stipanuk M H (2004) Sulfur amino acid metabolism Pathways for pro-
duction and removal of homocysteine and cysteine Annual Review of
Nutrition 24 539ndash577 httpsdoiorg101146annurevnutr24
012003132418
Stipanuk M H amp Ueki I (2011) Dealing with methioninehomocysteine
sulfur Cysteine metabolism to taurine and inorganic sulfur Journal of
Inherited Metabolic Disease 34 17ndash32 httpsdoiorg101007
s10545-009-9006-9
Stockinger B Di Meglio P Gialitakis M amp Duarte J H (2014) The
aryl hydrocarbon receptor Multitasking in the immune system
Annual Review of Immunology 32 403ndash432 httpsdoiorg101146
annurev-immunol-032713-120245
Storz J F amp Wheat C W (2010) Integrating evolutionary and
functional approaches to infer adaptation at specific loci Evolution
64 2489ndash2509 httpsdoiorg101111j1558-5646201001044x
Stuart Y E Veen T Weber J N Hanson D Ravinet M Lohman B
K Bolnick D I (2017) Contrasting effects of environment and
genetics generate a continuum of parallel evolution Nature Ecology amp
Evolution 1 0158 httpsdoiorg101038s41559-017-0158
Sunnucks P Morales H E Lamb A M Pavlova A amp Greening C
(2017) Integrative approaches for studying mitochondrial and nuclear
genome co-evolution in oxidative phosphorylation Frontiers in Genet-
ics 8 25
Tobler M amp Hastings L (2011) Convergent patterns of body shape dif-
ferentiation in four different clades of poeciliid fishes inhabiting sul-
fide springs Evolutionary Biology 38 412ndash421 httpsdoiorg10
1007s11692-011-9129-4
Tobler M Palacios M Chapman L J Mitrofanov I Bierbach D
Plath M Mateos M (2011) Evolution in extreme environments
Replicated phenotypic differentiation in livebearing fish inhabiting
sulfidic springs Evolution 65 2213ndash2228 httpsdoiorg101111j
1558-5646201101298x
Tobler M Passow C N Greenway R Kelley J L amp Shaw J H
(2016) The evolutionary ecology of animals inhabiting hydrogen sul-
fide-rich environments Annual Review of Ecology Evolution and Sys-
tematics 47 239ndash262 httpsdoiorg101146annurev-ecolsys-
121415-032418
Tobler M amp Plath M (2009a) Threatened fishes of the world Gambu-
sia eurystoma Miller 1975 (Poeciliidae) Environmental Biology of
Fishes 85 251 httpsdoiorg101007s10641-009-9481-8
Tobler M amp Plath M (2009b) Threatened fishes of the world Poecilia
sulphuraria (Alvarez 1948) (Poeciliidae) Environmental Biology of
Fishes 85 333ndash334 httpsdoiorg101007s10641-009-9506-3
Tobler M Plath M Riesch R Schlupp I Grasse A Munimanda G
Moodley Y (2014) Selection from parasites favors immunogenic diver-
sity but not divergence among locally adapted host populations Journal
of Evolutionary Biology 27 960ndash974 httpsdoiorg101111jeb12370
Tobler M Riesch R amp Plath M (2015) Extremophile fishes An inte-
grative synthesis In R Riesch M Tobler amp M Plath (Eds) Extremo-
phile fishes Ecology evolution and physiology of teleosts in extreme
environments (pp 279ndash296) Heidelberg Germany Springer
Tobler M Riesch R Tobler C M amp Plath M (2009) Compensatory
behaviour in response to sulfide-induced hypoxia affects time
budgets feeding efficiency and predation risk Evolutionary Ecology
Research 11 935ndash948
Tobler M Scharnweber K Greenway R Passow C Arias-Rodriguez
L amp Garcia-De-Leon F J (2015) Convergent changes in the
trophic ecology if extremophile fish along replicated environmental
gradients Freshwater Biology 60 768ndash780 httpsdoiorg10
1111fwb12530
Todgham A E amp Stillman J H (2013) Physiological responses to shifts
in multiple environmental stressors Relevance in a changing worlds
Integrative and Comparative Biology 53 539ndash544 httpsdoiorg10
1093icbict086
van Doorn G S Edelaar P amp Weissing F J (2009) On the origin of
species by natural selection and sexual selection Science 326 1704ndash
1707 httpsdoiorg101126science1181661
Wall D H amp Virginia R A (1999) Controls on soil biodiversity Insights
from extreme environments Applied Soil Ecology 13 137ndash150
httpsdoiorg101016S0929-1393(99)00029-3
Wallace J L amp Wang R (2015) Hydrogen-sulfide based therapeutics
Exploiting a unique but ubiquitous gasotransmitter Nature Reviews
Drug Discovery 14 329ndash345 httpsdoiorg101038nrd4433
Wang R (2012) Physiological implications of hydrogen sulfide A whiff
exploration that blossomed Physiological Reviews 92 791ndash896
httpsdoiorg101152physrev000172011
Wang Z Gerstein M amp Snyder M (2009) RNA-seq A revolutionary
tool for transcriptomics Nature Reviews Genetics 10 57ndash63
httpsdoiorg101038nrg2484
Waterman T H (1999) The evolutionary challenges of extreme environ-
ments (part 1) Journal of Experimental Zoology 285 326ndash359
httpsdoiorg101002(SICI)1097-010X(19991215)2854amplt326
AID-JEZ3ampgt30CO2-T
Werren J H Richards S Desjardins C A Niehuis O Gadau J Col-
bourne J K amp Group N G W (2010) Functional and evolutionary
insights from the genomes of three parasitoid Nasonia species
Science 327 343ndash348 httpsdoiorg101126science1178028
Whitehead A (2012) Comparative genomics in ecological physiology
Toward a more nuanced understanding of acclimation and adapta-
tion Journal of Experimental Biology 215 884ndash891 httpsdoiorg
101242jeb058735
Whitehead A amp Crawford D L (2006) Neutral and adaptive variation
in gene expression Proceedings of the National Academy of Sciences
USA 103 5425ndash5430 httpsdoiorg101073pnas0507648103
Willett C S amp Burton R S (2004) Evolution of interacting proteins in
the mitochondrial electron transport system in a marine copepod
Molecular Biology and Evolution 21 443ndash453 httpsdoiorg10
1093molbevmsh031
Willi Y Van Buskirk J amp Hoffmann A (2006) Limits to the adaptive
potential of small populations Annual Review of Ecology Evolution
and Systematics 37 433ndash458 httpsdoiorg101146annurevecol
sys37091305110145
Wolff J N Ladoukakis E D Enrıquez J A amp Dowling D K (2014)
Mitonuclear interactions Evolutionary consequences over multiple
biological scales Philosophical Transactions of the Royal Society B 369
20130443 httpsdoiorg101098rstb20130443
How to cite this article Tobler M Kelley JL Plath M Riesch
R Extreme environments and the origins of biodiversity
Adaptation and speciation in sulphide spring fishes Mol Ecol
201827843ndash859 httpsdoiorg101111mec14497
TOBLER ET AL | 859
(catastrophic flood event Plath et al 2010) Population genetic dif-
ferentiation between adjacent sulphide-adapted and nonadapted
populations is indicative of ongoing ecological speciation (Box 1) but
different population pairs from sulphidic and nonsulphidic waters
have made variable progress along the speciation continuum towards
complete reproductive isolation (Plath et al 2013) This prompts
questions about the mechanisms involved in reducing gene flow
along the environmental gradient connecting the two habitat types
41 | Prezygotic mechanisms of reproductiveisolation
The simplest form of reproductive isolation between locally adapted
populations arises from selection against migrants between habitat
types (Nosil Vines amp Funk 2005) Translocation experiments in nat-
ural habitats have indicated strong survival selection against
migrants especially those translocated from nonsulphidic to sulphidic
habitats (Plath et al 2013) Fish translocated from nonsulphidic
environments face high mortality within short periods of time
(lt24 hr) which is likely caused by H2S toxicity and hypoxia in sul-
phide springs Hence low gene flow from populations inhabiting
nonsulphidic waters into populations inhabiting sulphidic habitats is
simply explained by a lack of adaptation to the extreme environmen-
tal conditions At least in some drainages there is also appreciable
mortality for sulphide spring fish translocated to nonsulphidic habi-
tats although mechanisms causing inviability are unclear and the
strength of selection appears to be lower (Plath et al 2013) If sur-
vival selection against immigrants was the sole source of reproduc-
tive isolation this should result in unidirectional gene flow from
H2S-adapted to nonadapted populations but population genetic
analyses provided no evidence for this implying that additional
mechanisms are at work (Plath et al 2013)
One such mechanism could be intersexual selection and mate
choice experiments have indicated that migrants from sulphidic to
nonsulphidic habitats are at a significant disadvantage with fish from
nonsulphidic habitats avoiding potential mating partners from sul-
phidic ones (Plath et al 2013 Sommer-Trembo et al 2016) Such
assortative mating has been found to be critical during ecological spe-
ciation because it counteracts the homogenizing effects of gene flow
(van Doorn Edelaar amp Weissing 2009) yet how assortative mating
patterns arise often remains unclear In theory assortative mating can
arise through a variety of processes (Nosil 2012) ldquoMagic traitrdquo mecha-
nisms where mating preferences evolve as a by-product of divergent
selection on ecological traits that serve as cues have recently garnered
much attention (Servedio van Doorn Kopp Frame amp Nosil 2011)
Consistent with magic trait mechanisms assortative mating in P mexi-
cana appears to be linked to adaptive population differences in body
shape which serve as cues to distinguish potential mating partners
from different habitats (Greenway Drexler Arias-Rodriguez amp Tobler
2016) However neither sulphide spring fish (which are unlikely to
encounter migrants from nonsulphidic habitats due to H2S toxicity)
nor allopatric P mexicana from nonsulphidic waters (which have never
experienced migrants from sulphide spring populations) exhibit a
preference for mating partners from their own ecotype suggesting
that assortative mating in this system is a product of reinforcement
(ie direct selection for assortative mating Greenway et al 2016)
This is different from classic magic traits mechanisms because assorta-
tive mating is not a mere by-product of adaptation but adaptive traits
have instead been co-opted as mating signals in response to selection
on mating preferences (Maan amp Seehausen 2012) Moreover males of
some H2S-adapted populations have reduced aggressive behaviour
(Bierbach et al 2012 2018) In staged contests of size-matched males
from different ecotypes intruders had a significantly lower likelihood
to establish dominance than resident males independent of whether
or not they showed reduced aggressiveness overall Hence intrasexual
selection may represent another mechanism mediating reproductive
isolation between locally adapted ecotypes Overall H2S-adapted poe-
ciliids allow disentangling the relative contributions of different forms
of ecological and sexual selectionmdashand their interdependencymdashduring
ecological speciation
42 | Ensuing challenges quantifying postzygoticmechanisms of reproductive isolation
In contrast to prezygotic mechanisms little is known about postzy-
gotic mechanisms of reproductive isolation that potentially act to
reduce gene flow between adjacent populations in sulphidic and
nonsulphidic habitats Putative hybrids between sulphidic and non-
sulphidic ecotypes have occasionally been observed in nonsulphidic
habitats immediately adjacent to confluences with sulphide springs
(R Riesch M Plath and M Tobler personal observations) but it
remains unclear whether and through what mechanisms such hybrids
exhibit reduced fitness compared to parentals
Direct selection of mitochondrially encoded genes in the pres-
ence of H2S (Pfenninger et al 2014) should be accompanied by co-
evolution at nuclear loci that directly interact with mitochondrial
gene products in the formation of protein complexes involved in the
OXPHOS pathway (Sunnucks Morales Lamb Pavlova amp Greening
2017 Willett amp Burton 2004) Disruption of co-evolved gene com-
plexes in hybrids should therefore lead to intrinsic fitness reductions
through mito-nuclear incompatibilities (Burton Pereira amp Barreto
2013 Wolff Ladoukakis Enrıquez amp Dowling 2014) which have
been hypothesized to be a key driver in speciation (Chou amp Leu
2010 Gershoni Templeton amp Mishmar 2009) Empirical tests of
these ideas remain to be conducted in sulphide spring fishes just
like studies on potential ecological and sexual selection against
hybrids that arise from interpopulation mating events
5 | PERSPECTIVES LIFE IN SULPHIDICENVIRONMENTS BEYOND P MEXICANA
Research on sulphide spring fishes of the P mexicana species com-
plex in Mexico has provided insights into mechanisms underlying
adaptation and the links between adaptation and speciation Sul-
phide springs do not only occur in Mexico however but are found
TOBLER ET AL | 853
worldwide and have been colonized by 24 evolutionarily indepen-
dent fish lineages in North America the Neotropics and the Middle
East (Greenway et al 2014) The vast majority of lineages belong to
the order Cyprinodontiformes (95) and livebearing members of the
family Poeciliidae in particular (80) This potentially indicates that
internal gestation of young has facilitated the colonization of
extreme environments because it shields developing embryos from
physiochemical stressors (Blackburn 1999) Among the Poeciliidae
sulphide spring fishes include members of the genera Brachyrhaphis
Gambusia Poeciliopsis Priapichthys Pseudoxiphophorus Xiphophorus
and multiple subgenera of Poecilia (Figure 3 Greenway et al 2014)
spanning over 40 million years of evolution (based on Reznick Fur-
ness Meredith amp Springer 2017)
A handful of studies have started to address molecular and phe-
notypic evolution across a broader taxon sampling of sulphide spring
fishes finding evidence for convergent evolution in morphology
(Riesch et al 2016 Tobler amp Hastings 2011) life history traits
(Riesch Plath et al 2010 Riesch et al 2014) and oxygen transport
genes (Barts et al 2018) In addition ecological speciation in
response to adaptation to H2S-rich environments has also been doc-
umented in sulphide spring poeciliids other than P mexicana (Riesch
et al 2016) These studies have laid a fruitful foundation for the
investigation of mechanisms of adaptation to H2S-rich springs from
genes to phenotypic traits linking micro-evolutionary processes to
macro-evolutionary patterns
The availability of broad-scale evolutionary replication of popula-
tions that have invaded habitats with the same physiochemical stressors
allows for addressing some of the ensuing challenges discussed above
and investigating the factors that give rise to predictable evolutionary
outcomes Convergent evolution is a commonmdashbut not omnipresentmdash
theme in adaptive evolution and multiple factors can determine the
degree of convergence among replicated lineages exposed to the same
sources of selection (Kaeuffer et al 2012 Landry amp Bernatchez 2010
Oke Rolshausen LeBlond amp Hendry 2017 Rosenblum amp Harmon
2011) Thus leveraging replicated lineages that have colonized sulphide
springs to assess molecular mechanisms underlying convergent pheno-
typic changes analyse how environmental heterogeneity among sul-
phide spring habitats causes lineage-specific patterns of trait evolution
and test whether and by what mechanisms adaptation to extreme envi-
ronments is linked to speciation will provide insights into how ecologi-
cal genetic and functional factors contribute to evolutionary
convergence at different levels of organismal organization (Rosenblum
et al 2014) An expanded taxon sampling with population pairs that
span almost the full range of the speciation continuum from panmixia to
complete reproductive isolation (Riesch et al 2016) will also help
addressing questions about temporal aspects of H2S adaptation In par-
ticular comparing younger to older lineages will provide insights about
the order at which genetic and phenotypic modifications accrue during
the adaptation process
Animals have also invaded H2S-rich habitats in other aquatic
ecosystems including cold seeps and deep-sea hydrothermal vents
(Tobler et al 2016) Adaptation to H2S in the P mexicana species com-
plex has involved modification of physiological pathways associated
with H2S toxicity (OXPHOS pathway) and detoxification (sulphide
F IGURE 3 Phylogeny of the family Poeciliidae (adopted from Culumber amp Tobler 2017) Various lineages in the genera Poecilia (subgeneraAcanthophacelus Limia and Mollienesia) Priapichthys Brachyrhaphis Poeciliopsis Xiphophorus Pseudoxiphophorus and Gambusia have colonizedH2S-rich springs in different parts of the Americas (see Greenway et al 2014) Lineages highlighted in yellow represent endemic speciesrestricted to H2S-rich springs while red lineages represent species in which some populations have invaded sulphidic habitats Pictures depict arepresentative of each focal genussubgenus
854 | TOBLER ET AL
quinone oxidoreductase pathway) which have been highly conserved
during the diversification of life on Earth (Saraste 1999 Shahak amp
Hauska 2008) The high replicability of H2S as an environmental factor
that shaped animal evolution across vastly different ecological contexts
consequently offers the opportunity to investigate whether selection
mediated by the presence of H2S has repeatedly modified these con-
served physiological pathways across animal phyla that have diverged
during the Cambrian explosion spanning over 500 million years of evo-
lution Such comparisons would provide unprecedented insights into
the effects of a single physiochemical stressor on evolutionary change
irrespective of ecological or phylogenetic contexts
6 | CONCLUSIONS
H2S-rich environments and their inhabitants represent useful models
for research in organismal biology ecology and evolution illustrating
both opportunities (eg strong and explicit source of selection evolu-
tionary replication) and challenges (eg complexity of environments
and organismal responses) in asking basic question about the origins
of biodiversity Understanding how organisms thrive in apparently
inhospitable environments will provide us with insights into lifersquos
capacities and limitations to adapt to novel environments (Waterman
1999) This is particularly relevant considering that human activities
substantially contribute to the exacerbation of physiochemical stres-
sors at local regional and global scales impacting ecological and evo-
lutionary dynamics (Alberti 2015 Norberg Urban Vellend
Klausmeier amp Loeuille 2012) Thus a focus on ecological and evolu-
tionary consequences of natural stressors that push organisms to the
brink of their physiological capacities may be instrumental to predict
consequences of anthropogenic environmental change
ACKNOWLEDGEMENTS
We would like to thank Lenin Arias-Rodriguez the Universidad
Juarez Autonoma de Tabasco and the Centro de Investigacion E
Innovacion Para la Ense~nanza y El Aprendizaje for their continued
collaboration enabling our work on the P mexicana system Funding
for this review article has been provided by the National Science
Foundation (IOS-1557860 IOS-1557795) and the US Army
Research Office (W911NF-15-1-0175)
AUTHOR CONTRIBUTIONS
MT wrote the first draft of the paper all authors contributed text
and comments
ORCID
Michael Tobler httporcidorg0000-0002-0326-0890
Joanna L Kelley httporcidorg0000-0002-7731-605X
Reuroudiger Riesch httporcidorg0000-0002-0223-1254
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Brown J H Marquet P A amp Taper M L (1993) Evolution of body
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Carroll S P Jorgensen P S Kinnison M T Bergstrom C T Denison
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Chou J Y amp Leu J Y (2010) Speciation through cytonuclear incompat-
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BioEssays 32 401ndash411 httpsdoiorg101002bies200900162
Colosimo P F Hosemann K E Balabhadra S Villarreal G Dickson
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Cooper C E amp Brown G C (2008) The inhibition of mitochondrial
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hydrogen cyanide and hydrogen sulfide Chemical mechanism and
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Culumber Z W Hopper G W Barts N Passow C N Morgan S Brown
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Culumber Z W amp Tobler M (2017) Sex-specific evolution during the
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Dalziel A C Rogers S M amp Schulte P M (2009) Linking genotypes
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Doudna J A amp Charpentier E (2014) The new frontier of genome edit-
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Eales K L Hollinshead K E R amp Tennant D A (2016) Hypoxia and
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Eghbal M A Pennefather P S amp OrsquoBrien P J (2004) H2S cytotoxicity
mechanism involves reactive oxygen species formation and mito-
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Feldman C R Brodie Jr E D Brodie III E D amp Pfrender M E (2012)
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Fradin H Kiontke K Zegar C Gutwein M Lucas J Kovtun M
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Gershoni M Templeton A amp Mishmar D (2009) Mitochondrial bioen-
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Ghalambor C K Walker J A amp Reznick D N (2003) Multi-trait selec-
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Gibert J amp Deharveng L (2002) Subterranean ecosystems A truncated
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Grant P R Grant B R Markert J A Keller L F amp Petren K (2004)
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Greene H W (2005) Organisms in nature as a central focus for biology
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Gunderson A R Armstrong E J amp Stillman J H (2016) Multiple stres-
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Hildebrandt T M amp Grieshaber M (2008) Three enzymatic activities
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856 | TOBLER ET AL
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Metzker M L (2010) Sequencing technologies The next generation
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Nosil P (2012) Ecological speciation Oxford UK Oxford University
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Nosil P Vines T H amp Funk D J (2005) Perspective Reproductive
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Oke K B Rolshausen G LeBlond C amp Hendry A P (2017) How par-
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Olson K R Donald J A Dombkowski R A amp Perry S F (2012) Evo-
lutionary and comparative aspects of nitric oxide carbon monoxide
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Olson K R amp Straub K D (2015) The role of hydrogen sulfide in evo-
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Palacios M Arias-Rodriguez L Plath M Eifert C Lerp H Lamboj A
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nalpone0071069
Palacios M Voelker G Arias-Rodriguez L Mateos M amp Tobler M
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Passow C Arias-Rodriguez L amp Tobler M (2017) Convergent evolu-
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Passow C N Brown A Arias-Rodriguez L Yee M-C Sockell A
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Pfenninger M Patel S Arias-Rodriguez L Feldmeyer B Riesch R amp
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TOBLER ET AL | 857
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Reznick D amp Yang A P (1993) The influence of fluctuating resources
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mophile fishes (Poecilia mexicana Poecilia sulphuraria) Behavioral Ecol-
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from increased aerial predation risk in sulphidic southern Mexican
habitats Biological Journal of the Linnean Society 101 417ndash426
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Riesch R Plath M Garcia de Leon F J amp Schlupp I (2010) Conver-
gent life-history shifts Toxic environments result in big babies in two
clades of poeciliids Naturwissenschaften 97 133ndash141 httpsdoi
org101007s00114-009-0613-y
Riesch R Plath M Schlupp I Tobler M amp Langerhans R B (2014)
Colonization of toxic springs drives predictable life-history shift in
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org101111ele12209
Riesch R Tobler M Lerp H Jourdan J Doumas L T Nosil P Plath
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plexity of speciation along replicated ecological gradients BMC Evolu-
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Riesch R Tobler M amp Plath M (2015) Extremophile fishes Ecology
evolution and physiology of teleosts in extreme environments p 290
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13362-1
Ritchie M E Holzinger E R Li R Pendergrass S A amp Kim S (2015)
Methods of integrating data to uncover genotype-phenotype interac-
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nrg3868
Roach K Tobler M amp Winemiller K O (2011) Hydrogen sulfide bac-
teria and fish A unique subterranean food chain Ecology 92 2056ndash
2062 httpsdoiorg10189011-02761
Rohner N Jarosz D F Kowalko J E Yoshizawa M Jeffery W R
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Rosenblum E B amp Harmon L J (2011) ldquoSame but differentrdquo Repli-
cated ecological speciation at White Sands Evolution 65 946ndash960
httpsdoiorg101111j1558-5646201001190x
Rosenblum E B Parent C E amp Brandt E E (2014) The molecular
basis of convergent evolution Annual Review of Ecology Evolution and
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1038ng2604
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Schulz-Mirbach T Eifert C Riesch R Farnworth M S Zimmer C
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Servedio M R van Doorn G S Kopp M Frame A M amp Nosil P
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858 | TOBLER ET AL
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Stipanuk M H (2004) Sulfur amino acid metabolism Pathways for pro-
duction and removal of homocysteine and cysteine Annual Review of
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Stipanuk M H amp Ueki I (2011) Dealing with methioninehomocysteine
sulfur Cysteine metabolism to taurine and inorganic sulfur Journal of
Inherited Metabolic Disease 34 17ndash32 httpsdoiorg101007
s10545-009-9006-9
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Storz J F amp Wheat C W (2010) Integrating evolutionary and
functional approaches to infer adaptation at specific loci Evolution
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Stuart Y E Veen T Weber J N Hanson D Ravinet M Lohman B
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genetics generate a continuum of parallel evolution Nature Ecology amp
Evolution 1 0158 httpsdoiorg101038s41559-017-0158
Sunnucks P Morales H E Lamb A M Pavlova A amp Greening C
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genome co-evolution in oxidative phosphorylation Frontiers in Genet-
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Tobler M amp Hastings L (2011) Convergent patterns of body shape dif-
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1007s11692-011-9129-4
Tobler M Palacios M Chapman L J Mitrofanov I Bierbach D
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Replicated phenotypic differentiation in livebearing fish inhabiting
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Tobler M Passow C N Greenway R Kelley J L amp Shaw J H
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Tobler M amp Plath M (2009a) Threatened fishes of the world Gambu-
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Tobler M amp Plath M (2009b) Threatened fishes of the world Poecilia
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Tobler M Riesch R amp Plath M (2015) Extremophile fishes An inte-
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Tobler M Scharnweber K Greenway R Passow C Arias-Rodriguez
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1093icbict086
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Waterman T H (1999) The evolutionary challenges of extreme environ-
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bourne J K amp Group N G W (2010) Functional and evolutionary
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Toward a more nuanced understanding of acclimation and adapta-
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101242jeb058735
Whitehead A amp Crawford D L (2006) Neutral and adaptive variation
in gene expression Proceedings of the National Academy of Sciences
USA 103 5425ndash5430 httpsdoiorg101073pnas0507648103
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the mitochondrial electron transport system in a marine copepod
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1093molbevmsh031
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potential of small populations Annual Review of Ecology Evolution
and Systematics 37 433ndash458 httpsdoiorg101146annurevecol
sys37091305110145
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How to cite this article Tobler M Kelley JL Plath M Riesch
R Extreme environments and the origins of biodiversity
Adaptation and speciation in sulphide spring fishes Mol Ecol
201827843ndash859 httpsdoiorg101111mec14497
TOBLER ET AL | 859
worldwide and have been colonized by 24 evolutionarily indepen-
dent fish lineages in North America the Neotropics and the Middle
East (Greenway et al 2014) The vast majority of lineages belong to
the order Cyprinodontiformes (95) and livebearing members of the
family Poeciliidae in particular (80) This potentially indicates that
internal gestation of young has facilitated the colonization of
extreme environments because it shields developing embryos from
physiochemical stressors (Blackburn 1999) Among the Poeciliidae
sulphide spring fishes include members of the genera Brachyrhaphis
Gambusia Poeciliopsis Priapichthys Pseudoxiphophorus Xiphophorus
and multiple subgenera of Poecilia (Figure 3 Greenway et al 2014)
spanning over 40 million years of evolution (based on Reznick Fur-
ness Meredith amp Springer 2017)
A handful of studies have started to address molecular and phe-
notypic evolution across a broader taxon sampling of sulphide spring
fishes finding evidence for convergent evolution in morphology
(Riesch et al 2016 Tobler amp Hastings 2011) life history traits
(Riesch Plath et al 2010 Riesch et al 2014) and oxygen transport
genes (Barts et al 2018) In addition ecological speciation in
response to adaptation to H2S-rich environments has also been doc-
umented in sulphide spring poeciliids other than P mexicana (Riesch
et al 2016) These studies have laid a fruitful foundation for the
investigation of mechanisms of adaptation to H2S-rich springs from
genes to phenotypic traits linking micro-evolutionary processes to
macro-evolutionary patterns
The availability of broad-scale evolutionary replication of popula-
tions that have invaded habitats with the same physiochemical stressors
allows for addressing some of the ensuing challenges discussed above
and investigating the factors that give rise to predictable evolutionary
outcomes Convergent evolution is a commonmdashbut not omnipresentmdash
theme in adaptive evolution and multiple factors can determine the
degree of convergence among replicated lineages exposed to the same
sources of selection (Kaeuffer et al 2012 Landry amp Bernatchez 2010
Oke Rolshausen LeBlond amp Hendry 2017 Rosenblum amp Harmon
2011) Thus leveraging replicated lineages that have colonized sulphide
springs to assess molecular mechanisms underlying convergent pheno-
typic changes analyse how environmental heterogeneity among sul-
phide spring habitats causes lineage-specific patterns of trait evolution
and test whether and by what mechanisms adaptation to extreme envi-
ronments is linked to speciation will provide insights into how ecologi-
cal genetic and functional factors contribute to evolutionary
convergence at different levels of organismal organization (Rosenblum
et al 2014) An expanded taxon sampling with population pairs that
span almost the full range of the speciation continuum from panmixia to
complete reproductive isolation (Riesch et al 2016) will also help
addressing questions about temporal aspects of H2S adaptation In par-
ticular comparing younger to older lineages will provide insights about
the order at which genetic and phenotypic modifications accrue during
the adaptation process
Animals have also invaded H2S-rich habitats in other aquatic
ecosystems including cold seeps and deep-sea hydrothermal vents
(Tobler et al 2016) Adaptation to H2S in the P mexicana species com-
plex has involved modification of physiological pathways associated
with H2S toxicity (OXPHOS pathway) and detoxification (sulphide
F IGURE 3 Phylogeny of the family Poeciliidae (adopted from Culumber amp Tobler 2017) Various lineages in the genera Poecilia (subgeneraAcanthophacelus Limia and Mollienesia) Priapichthys Brachyrhaphis Poeciliopsis Xiphophorus Pseudoxiphophorus and Gambusia have colonizedH2S-rich springs in different parts of the Americas (see Greenway et al 2014) Lineages highlighted in yellow represent endemic speciesrestricted to H2S-rich springs while red lineages represent species in which some populations have invaded sulphidic habitats Pictures depict arepresentative of each focal genussubgenus
854 | TOBLER ET AL
quinone oxidoreductase pathway) which have been highly conserved
during the diversification of life on Earth (Saraste 1999 Shahak amp
Hauska 2008) The high replicability of H2S as an environmental factor
that shaped animal evolution across vastly different ecological contexts
consequently offers the opportunity to investigate whether selection
mediated by the presence of H2S has repeatedly modified these con-
served physiological pathways across animal phyla that have diverged
during the Cambrian explosion spanning over 500 million years of evo-
lution Such comparisons would provide unprecedented insights into
the effects of a single physiochemical stressor on evolutionary change
irrespective of ecological or phylogenetic contexts
6 | CONCLUSIONS
H2S-rich environments and their inhabitants represent useful models
for research in organismal biology ecology and evolution illustrating
both opportunities (eg strong and explicit source of selection evolu-
tionary replication) and challenges (eg complexity of environments
and organismal responses) in asking basic question about the origins
of biodiversity Understanding how organisms thrive in apparently
inhospitable environments will provide us with insights into lifersquos
capacities and limitations to adapt to novel environments (Waterman
1999) This is particularly relevant considering that human activities
substantially contribute to the exacerbation of physiochemical stres-
sors at local regional and global scales impacting ecological and evo-
lutionary dynamics (Alberti 2015 Norberg Urban Vellend
Klausmeier amp Loeuille 2012) Thus a focus on ecological and evolu-
tionary consequences of natural stressors that push organisms to the
brink of their physiological capacities may be instrumental to predict
consequences of anthropogenic environmental change
ACKNOWLEDGEMENTS
We would like to thank Lenin Arias-Rodriguez the Universidad
Juarez Autonoma de Tabasco and the Centro de Investigacion E
Innovacion Para la Ense~nanza y El Aprendizaje for their continued
collaboration enabling our work on the P mexicana system Funding
for this review article has been provided by the National Science
Foundation (IOS-1557860 IOS-1557795) and the US Army
Research Office (W911NF-15-1-0175)
AUTHOR CONTRIBUTIONS
MT wrote the first draft of the paper all authors contributed text
and comments
ORCID
Michael Tobler httporcidorg0000-0002-0326-0890
Joanna L Kelley httporcidorg0000-0002-7731-605X
Reuroudiger Riesch httporcidorg0000-0002-0223-1254
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Colosimo P F Hosemann K E Balabhadra S Villarreal G Dickson
M Grimwood J Kingsley D M (2005) Widespread parallel evo-
lution in sticklebacks by repeated fixation of ectodysplasin alleles
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Cooper C E amp Brown G C (2008) The inhibition of mitochondrial
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hydrogen cyanide and hydrogen sulfide Chemical mechanism and
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Culumber Z W amp Tobler M (2017) Sex-specific evolution during the
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Dalziel A C Rogers S M amp Schulte P M (2009) Linking genotypes
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A Nadeau N J Jiggins C D (2012) Butterfly genome reveals
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Eales K L Hollinshead K E R amp Tennant D A (2016) Hypoxia and
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Eghbal M A Pennefather P S amp OrsquoBrien P J (2004) H2S cytotoxicity
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Feldman C R Brodie Jr E D Brodie III E D amp Pfrender M E (2012)
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Fradin H Kiontke K Zegar C Gutwein M Lucas J Kovtun M
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ergetics as a major motive force of speciation BioEssays 31 642ndash
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Ghalambor C K Walker J A amp Reznick D N (2003) Multi-trait selec-
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Hildebrandt T M amp Grieshaber M (2008) Three enzymatic activities
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oiorg101111j1742-4658200806482x
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1038nrg2897
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offspring size in fish including live-bearing and parental effects Amer-
ican Naturalist 177 E119ndashE135 httpsdoiorg101086659622
Kaeuffer R Peichel C L Bolnick D I amp Hendry A P (2012) Parallel
and nonparallel aspects of ecological phenotypic and genetic diver-
gence across replicate population pairs of lake and stream stickle-
back Evolution 66 402ndash418 httpsdoiorg101111j1558-5646
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Kell D B amp Oliver S G (2004) Here is the evidence now what is the
hypothesis The complementary roles of inductive and hypothesis-
driven science in the post-genomic era BioEssays 26 99ndash105
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856 | TOBLER ET AL
Kelley J L Arias-Rodriguez L Patacsil Martin D Yee M C Busta-
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Kelley J L Passow C Plath M Arias-Rodriguez L Yee M C amp
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spective from research on the evolution of resistance Ecotoxicology
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Keuroustner A Hoffmann M Fraser B A Kottler V A Sharma E Wei-
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Physiological and Biochemical Zoology 88 371ndash383 httpsdoiorg
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TOBLER ET AL | 857
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Ritchie M E Holzinger E R Li R Pendergrass S A amp Kim S (2015)
Methods of integrating data to uncover genotype-phenotype interac-
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Rohner N Jarosz D F Kowalko J E Yoshizawa M Jeffery W R
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basis of convergent evolution Annual Review of Ecology Evolution and
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Saltz J B Hessel F C amp Kelly M W (2017) Trait correlations in the
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858 | TOBLER ET AL
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Stipanuk M H (2004) Sulfur amino acid metabolism Pathways for pro-
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sulfur Cysteine metabolism to taurine and inorganic sulfur Journal of
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Storz J F amp Wheat C W (2010) Integrating evolutionary and
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Stuart Y E Veen T Weber J N Hanson D Ravinet M Lohman B
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Evolution 1 0158 httpsdoiorg101038s41559-017-0158
Sunnucks P Morales H E Lamb A M Pavlova A amp Greening C
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genome co-evolution in oxidative phosphorylation Frontiers in Genet-
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Tobler M amp Hastings L (2011) Convergent patterns of body shape dif-
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1007s11692-011-9129-4
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Replicated phenotypic differentiation in livebearing fish inhabiting
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Tobler M Passow C N Greenway R Kelley J L amp Shaw J H
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fide-rich environments Annual Review of Ecology Evolution and Sys-
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Tobler M Scharnweber K Greenway R Passow C Arias-Rodriguez
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1093icbict086
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Wall D H amp Virginia R A (1999) Controls on soil biodiversity Insights
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Wallace J L amp Wang R (2015) Hydrogen-sulfide based therapeutics
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Wang R (2012) Physiological implications of hydrogen sulfide A whiff
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Wang Z Gerstein M amp Snyder M (2009) RNA-seq A revolutionary
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Waterman T H (1999) The evolutionary challenges of extreme environ-
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Toward a more nuanced understanding of acclimation and adapta-
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Whitehead A amp Crawford D L (2006) Neutral and adaptive variation
in gene expression Proceedings of the National Academy of Sciences
USA 103 5425ndash5430 httpsdoiorg101073pnas0507648103
Willett C S amp Burton R S (2004) Evolution of interacting proteins in
the mitochondrial electron transport system in a marine copepod
Molecular Biology and Evolution 21 443ndash453 httpsdoiorg10
1093molbevmsh031
Willi Y Van Buskirk J amp Hoffmann A (2006) Limits to the adaptive
potential of small populations Annual Review of Ecology Evolution
and Systematics 37 433ndash458 httpsdoiorg101146annurevecol
sys37091305110145
Wolff J N Ladoukakis E D Enrıquez J A amp Dowling D K (2014)
Mitonuclear interactions Evolutionary consequences over multiple
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How to cite this article Tobler M Kelley JL Plath M Riesch
R Extreme environments and the origins of biodiversity
Adaptation and speciation in sulphide spring fishes Mol Ecol
201827843ndash859 httpsdoiorg101111mec14497
TOBLER ET AL | 859
quinone oxidoreductase pathway) which have been highly conserved
during the diversification of life on Earth (Saraste 1999 Shahak amp
Hauska 2008) The high replicability of H2S as an environmental factor
that shaped animal evolution across vastly different ecological contexts
consequently offers the opportunity to investigate whether selection
mediated by the presence of H2S has repeatedly modified these con-
served physiological pathways across animal phyla that have diverged
during the Cambrian explosion spanning over 500 million years of evo-
lution Such comparisons would provide unprecedented insights into
the effects of a single physiochemical stressor on evolutionary change
irrespective of ecological or phylogenetic contexts
6 | CONCLUSIONS
H2S-rich environments and their inhabitants represent useful models
for research in organismal biology ecology and evolution illustrating
both opportunities (eg strong and explicit source of selection evolu-
tionary replication) and challenges (eg complexity of environments
and organismal responses) in asking basic question about the origins
of biodiversity Understanding how organisms thrive in apparently
inhospitable environments will provide us with insights into lifersquos
capacities and limitations to adapt to novel environments (Waterman
1999) This is particularly relevant considering that human activities
substantially contribute to the exacerbation of physiochemical stres-
sors at local regional and global scales impacting ecological and evo-
lutionary dynamics (Alberti 2015 Norberg Urban Vellend
Klausmeier amp Loeuille 2012) Thus a focus on ecological and evolu-
tionary consequences of natural stressors that push organisms to the
brink of their physiological capacities may be instrumental to predict
consequences of anthropogenic environmental change
ACKNOWLEDGEMENTS
We would like to thank Lenin Arias-Rodriguez the Universidad
Juarez Autonoma de Tabasco and the Centro de Investigacion E
Innovacion Para la Ense~nanza y El Aprendizaje for their continued
collaboration enabling our work on the P mexicana system Funding
for this review article has been provided by the National Science
Foundation (IOS-1557860 IOS-1557795) and the US Army
Research Office (W911NF-15-1-0175)
AUTHOR CONTRIBUTIONS
MT wrote the first draft of the paper all authors contributed text
and comments
ORCID
Michael Tobler httporcidorg0000-0002-0326-0890
Joanna L Kelley httporcidorg0000-0002-7731-605X
Reuroudiger Riesch httporcidorg0000-0002-0223-1254
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Barts N Greenway R Passow C N Arias Rodriguez L Kelley J L amp
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transport genes in livebearing fishes (Poeciliidae) from hydrogen sul-
fide rich springs Genome httpsdoiorg101139gen-2017-0051
[Epub ahead of print]
Beauchamp R O Bus J S Popp C S Boreiko C J amp Andjelkovich
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CRC Critical Reviews in Toxicology 13 25ndash97 httpsdoiorg10
310910408448409029321
Bell E M (2012) Life at extremes Environments organisms and strategies
for survival Oxfordshire CABI httpsdoiorg101079
97818459381470000
Bierbach D Arias Rodriguez L amp Plath M (2018) Intrasexual competi-
tion enhances reproductive isolation between locally adapted popula-
tions Current Zoology httpsdoiorg101093czzox071 [Epub
ahead of print]
Bierbach D Klein M Sassmannshausen V Schlupp I Riesch R
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sive behavior Another reproductive isolation barrier in extremophile
poeciliid fishes International Journal of Evolutionary Biology 2012
148745
Blackburn D G (1999) Viviparity and oviparity Evolution and reproduc-
tive strategies In T E Knobil amp J D Neill (Eds) Encyclopedia of
reproduction Vol 4 (pp 994ndash1003) New York NY Academic Press
Bouzat J L (2010) Conservation genetics of population bottlenecks
The role of chance selection and history Conservation Genetics 11
463ndash478
Brown A P Greenway R Morgan S Quackenbush C R Giordani
L Arias-Rodriguez L Kelley J L (2017) Genome-scale data
reveals that endemic Poecilia populations from small sulfide spring
display no evidence of inbreeding Molecular Ecology 26 4920ndash
4934
Brown J H Marquet P A amp Taper M L (1993) Evolution of body
size Consequences of an energetic definition of fitness American
Naturalist 142 573ndash584 httpsdoiorg101086285558
Burton R S Pereira R J amp Barreto F S (2013) Cytonuclear genomic
interactions and hybrid breakdown Annual Review of Ecology
TOBLER ET AL | 855
Evolution and Systematics 44 281ndash302 httpsdoiorg101146an
nurev-ecolsys-110512-135758
Bush S J Chen L Tovar-Corona J M amp Urritia A O (2017) Alterna-
tive splicing and the evolution of phenotypic novelty Philosophical
Transactions of the Royal Society B 372 20150474 httpsdoiorg
101098rstb20150474
Carroll S P Jorgensen P S Kinnison M T Bergstrom C T Denison
R F Gluckman P Tabashnik B E (2014) Applying evolutionary
biology to address global challenges Science 346 313 httpsdoi
org101364FIO2014JW3A13
Cartwright R A Schwartz R S Merry A L amp Howell M M (2017)
The importance of selection in the evolution of blindness in cavefish
Bmc Evolutionary Biology 17 45 httpsdoiorg101186s12862-
017-0876-4
Chan Y F Marks M E Jones F C Villarreal G Shapiro M D Brady
S D Kingsley D M (2010) Adaptive evolution of pelvic reduc-
tion in sticklebacks by recurrent deletion of Pitx1 enhancer Science
327 302ndash305 httpsdoiorg101126science1182213
Chou J Y amp Leu J Y (2010) Speciation through cytonuclear incompat-
ibility Insights from yeast and implications for higher eukaryotes
BioEssays 32 401ndash411 httpsdoiorg101002bies200900162
Colosimo P F Hosemann K E Balabhadra S Villarreal G Dickson
M Grimwood J Kingsley D M (2005) Widespread parallel evo-
lution in sticklebacks by repeated fixation of ectodysplasin alleles
Science 307 1928ndash1933 httpsdoiorg101126science1107239
Cooper C E amp Brown G C (2008) The inhibition of mitochondrial
cytochrome oxidase by the gases carbon monoxide nitric oxide
hydrogen cyanide and hydrogen sulfide Chemical mechanism and
physiological significance Journal of Bioenergy and Biomembranes 40
533ndash539 httpsdoiorg101007s10863-008-9166-6
Culumber Z W Hopper G W Barts N Passow C N Morgan S Brown
A Tobler M (2016) Habitat use of two extremophile highly ende-
mic and critically endangered fish species (Gambusia eurystoma and Poe-
cilia sulphurophila Poeciliidae) Aquatic Conservation-Marine and
Freshwater Ecosystems 26 1155ndash1167 httpsdoiorg101002aqc
2640
Culumber Z W amp Tobler M (2017) Sex-specific evolution during the
diversification of live-bearing fishes Nature Ecology amp Evolution 1
1185ndash1191
Dalziel A C Rogers S M amp Schulte P M (2009) Linking genotypes
to phenotypes and fitness How mechanistic biology can inform
molecular ecology Molecular Ecology 18 4997ndash5017 httpsdoi
org101111j1365-294X200904427x
Darwin C (1859) On the origin of species based on natural selection or
the preservation of favoured races in the struggle of life 6th ed Lon-
don John Murray
Dasmahapatra K K Walters J R Briscoe A D Davey J W Whibley
A Nadeau N J Jiggins C D (2012) Butterfly genome reveals
promiscuous exchange of mimicry adaptations among species Nature
487 94ndash98 httpsdoiorg101038nature11041
Doudna J A amp Charpentier E (2014) The new frontier of genome edit-
ing with CRISPR-Cas9 Science 346 1258096 httpsdoiorg10
1126science1258096
Dubilier N Giere O amp Grieshaber M (2005) Concomitant effects of sul-
fide and hypoxia on the aerobic metabolism of the marine oligochaete
Tubificoides benedii Journal of Experimental Zoology 269 287ndash298
Eales K L Hollinshead K E R amp Tennant D A (2016) Hypoxia and
metabolic adaptation of cancer cells Oncogenesis 5 e190 httpsd
oiorg101038oncsis201550
Eghbal M A Pennefather P S amp OrsquoBrien P J (2004) H2S cytotoxicity
mechanism involves reactive oxygen species formation and mito-
chondrial depolarisation Toxicology 203 69ndash76 httpsdoiorg10
1016jtox200405020
Feldman C R Brodie Jr E D Brodie III E D amp Pfrender M E (2012)
Constraint shapes convergence in tetrodotoxin-resistant sodium
channels of snakes Proceedings of the National Academy of Sciences
USA 109 4556ndash4561 httpsdoiorg101073pnas1113468109
Flores K B Wolschin F amp Amdam G V (2013) The role of methyla-
tion of DNA in environmental adaptation Integrative and Comparative
Biology 53 359ndash372 httpsdoiorg101093icbict019
Fradin H Kiontke K Zegar C Gutwein M Lucas J Kovtun M
Piano F (2017) Genome architecture and evolution of a unichromo-
somal asexual nematode Current Biology 27(2928ndash2939) e2926
Gershoni M Templeton A amp Mishmar D (2009) Mitochondrial bioen-
ergetics as a major motive force of speciation BioEssays 31 642ndash
650 httpsdoiorg101002bies200800139
Ghalambor C K Walker J A amp Reznick D N (2003) Multi-trait selec-
tion adaptation and constraints on the evolution of burst swimming
performance Integrative and Comparative Biology 43 431ndash438
httpsdoiorg101093icb433431
Gibert J amp Deharveng L (2002) Subterranean ecosystems A truncated
functional biodiversity BioScience 52 473ndash481 httpsdoiorg10
16410006-3568(2002)052[0473SEATFB]20CO2
Gjuvsland A B Vik J O Beard D A Hunter P J amp Omholt S W
(2013) Bridging the genotype-phenotype gap What does it take
Journal of Physiology 591 2055ndash2066 httpsdoiorg101113jphys
iol2012248864
Grant P R Grant B R Markert J A Keller L F amp Petren K (2004)
Convergent evolution of Darwinrsquos finches caused by introgressive
hybridization and selection Evolution 58 1588ndash1599 httpsdoi
org101111j0014-38202004tb01738x
Greene H W (2005) Organisms in nature as a central focus for biology
Trends in Ecology and Evolution 20 23ndash27 httpsdoiorg101016j
tree200411005
Greenway R Arias-Rodriguez L Diaz P amp Tobler M (2014) Patterns
of macroinvertebrate and fish diversity in freshwater sulphide springs
Diversity 6 597ndash632 httpsdoiorg103390d6030597
Greenway R Drexler S Arias-Rodriguez L amp Tobler M (2016) Adap-
tive but not condition-dependent body shape differences contribute
to assortative mating preferences during ecological speciation Evolu-
tion 70 2809ndash2822 httpsdoiorg101111evo13087
Gunderson A R Armstrong E J amp Stillman J H (2016) Multiple stres-
sors in a changing world The need for an improved perspective on
physiological responses to the dynamic marine environment Annual
Review of Marine Science 8 357ndash378 httpsdoiorg101146an
nurev-marine-122414-033953
Hildebrandt T M amp Grieshaber M (2008) Three enzymatic activities
catalyze the oxidation of sulfide to thiosulfate in mammalian and
invertebrate mitochondria FEBS Journal 275 3352ndash3361 httpsd
oiorg101111j1742-4658200806482x
Houle D Govindaraju D R amp Omholt S (2010) Phenomics The next
challenge Nature Reviews Genetics 11 855ndash866 httpsdoiorg10
1038nrg2897
IUCN (2012) Red List of threatened species version 20132 www
iucnredlistorg
Johnston I A amp Bennett A F (1996) Animals and temperature Pheno-
typic and evolutionary adaptation Cambridge Cambridge University
Press httpsdoiorg101017CBO9780511721854
Jorgensen C Auer S K amp Reznick D N (2011) A model for optimal
offspring size in fish including live-bearing and parental effects Amer-
ican Naturalist 177 E119ndashE135 httpsdoiorg101086659622
Kaeuffer R Peichel C L Bolnick D I amp Hendry A P (2012) Parallel
and nonparallel aspects of ecological phenotypic and genetic diver-
gence across replicate population pairs of lake and stream stickle-
back Evolution 66 402ndash418 httpsdoiorg101111j1558-5646
201101440x
Kell D B amp Oliver S G (2004) Here is the evidence now what is the
hypothesis The complementary roles of inductive and hypothesis-
driven science in the post-genomic era BioEssays 26 99ndash105
httpsdoiorg101002(ISSN)1521-1878
856 | TOBLER ET AL
Kelley J L Arias-Rodriguez L Patacsil Martin D Yee M C Busta-
mante C amp Tobler M (2016) Mechanisms underlying adaptation to
life in hydrogen sulfide rich environments Molecular Biology and Evo-
lution 33 1419ndash1434 httpsdoiorg101093molbevmsw020
Kelley J L Passow C Plath M Arias-Rodriguez L Yee M C amp
Tobler M (2012) Genomic resources for a model in adaptation and
speciation research The characterization of the Poecilia mexicana
transcriptome BMC Genomics 13 652 httpsdoiorg101186
1471-2164-13-652
Klerks P L Xie L amp Levinton J S (2011) Quantitative genetics
approaches to study evolutionary processes in ecotoxicology a per-
spective from research on the evolution of resistance Ecotoxicology
20 513ndash523 httpsdoiorg101007s10646-011-0640-2
Kump L R Pavlov A amp Arthur M A (2005) Massive release of hydrogen
sulfide to the surface ocean and atmosphere during intervals of oceanic
anoxia Geology 33 397ndash400 httpsdoiorg101130G212951
Keuroustner A Hoffmann M Fraser B A Kottler V A Sharma E Wei-
gel D amp Dreyer C (2016) The genome of the Trinidadian guppy
Poecilia reticulata and variation in the guanapo population PLoS ONE
11 e0169087 httpsdoiorg101371journalpone0169087
Landry L amp Bernatchez L (2010) Role of epibenthic resource opportu-
nities in the parallel evolution of lake whitefish species pairs (Core-
gonus sp) Journal of Evolutionary Biology 23 2602ndash2613 httpsdoi
org101111j1420-9101201002121x
Langerhans R B (2007) Evolutionary consequences of predation Avoid-
ance escape reproduction and diversification In A M T Elewa
(Ed) Predation in organisms Berlin Springer
Li L Rose P amp Moore P K (2011) Hydrogen sulfide and cell signaling
Annual Review of Pharmacology and Toxicology 51 169ndash187
httpsdoiorg101146annurev-pharmtox-010510-100505
Lin Q Fan S Zhang Y Xu M Zhang H Yang Y Gunter H M
(2016) The seahorse genome and the evolution of its specialized mor-
phology Nature 540 395ndash399 httpsdoiorg101038nature20595
Liu Y Ma S Wang X Chang J Gao J Shi R Zhao P (2014)
Highly efficient multiplex targeted mutagenesis and genomic struc-
ture variation in Bombyx mori cells using CRISPRCas9 Insect bio-
chemistry and molecular biology 49 35ndash42 httpsdoiorg101016j
ibmb201403010
Lopez-Maury L Marguerat S amp Beuroahler J (2008) Tuning gene expres-
sion to changing environments From rapid responses to evolutionary
adaptation Nature Reviews Genetics 9 583ndash593 httpsdoiorg10
1038nrg2398
Maan M E amp Seehausen O (2012) Magic traits versus magic prefer-
ences in speciation Evolutionary Ecology Research 14 779ndash785
Martyn U Weigel D amp Dreyer C (2006) In vitro culture of embryos
of the guppy Poecilia reticulata Developmental Dynamics 235 617ndash
622 httpsdoiorg101002(ISSN)1097-0177
Metzker M L (2010) Sequencing technologies The next generation
Nature Reviews Genetics 11 31ndash46 httpsdoiorg101038nrg2626
Moore M P Riesch R amp Martin R A (2016) The predictability and
magnitude of life-history divergence to ecological agents of selection
A meta-analysis in livebearing fishes Ecology Letters 19 435ndash442
httpsdoiorg101111ele12576
Mustafa A K Gadalla M M Sen N Kim S Mu W Gazi S K
Snyder S H (2009) H2S signals through protein S-sulfhydration
Science Signaling 2 ra72
Nesse R M amp Stearns S C (2008) The great opportunity Evolutionary
applications to medicine and public health Evolutionary Applications
1 28ndash48 httpsdoiorg101111j1752-4571200700006x
Nevo E (2011) Evolution under environmental stress at macro and
microscales Genome Biology and Evolution 3 1039ndash1052 httpsdoi
org101093gbeevr052
Norberg J Urban M C Vellend M Klausmeier C A amp Loeuille N (2012)
Eco-evolutionary responses of biodiversity to climate change Nature Cli-
mate Change 2 747ndash751 httpsdoiorg101038nclimate1588
Nosil P (2012) Ecological speciation Oxford UK Oxford University
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0001
Nosil P Vines T H amp Funk D J (2005) Perspective Reproductive
isolation caused by natural selection against immigrants from diver-
gent habitats Evolution 59 705ndash719
Oke K B Rolshausen G LeBlond C amp Hendry A P (2017) How par-
allel is parallel evolution A comparative analysis in fishes American
Naturalist 190 1ndash16 httpsdoiorg101086691989
Olson K R (2011) The therapeutic potential of hydrogen sulfide
Separating hype from hope American Journal of Physiology-Regulatory
Integrative and Comparative Physiology 301 R297ndashR312 httpsdoi
org101152ajpregu000452011
Olson K R Donald J A Dombkowski R A amp Perry S F (2012) Evo-
lutionary and comparative aspects of nitric oxide carbon monoxide
and hydrogen sulfide Respiratory Physiology amp Neurobiology 184
117ndash129 httpsdoiorg101016jresp201204004
Olson K R amp Straub K D (2015) The role of hydrogen sulfide in evo-
lution and the evolution of hydrogen sulfide in metabolism and sig-
naling Physiology 31 60ndash72
Palacios M Arias-Rodriguez L Plath M Eifert C Lerp H Lamboj A
Tobler M (2013) The rediscovery of a long described species
reveals additional complexity in speciation patterns of poeciliid fishes
in sulfide springs PLoS ONE 8 e71069 httpsdoiorg101371jour
nalpone0071069
Palacios M Voelker G Arias-Rodriguez L Mateos M amp Tobler M
(2016) Phylogenetic analyses of the subgenus Mollienesia (Poecilia
Poeciliidae Teleostei) reveal taxonomic inconsistencies cryptic biodi-
versity and spatio-temporal aspects of diversification in Middle
America Molecular Phylogenetics and Evolution 103 230ndash244
httpsdoiorg101016jympev201607025
Passow C Arias-Rodriguez L amp Tobler M (2017) Convergent evolu-
tion of reduced energy demands in extremophile fish PLoS ONE 12
e0186935 httpsdoiorg101371journalpone0186935
Passow C N Brown A Arias-Rodriguez L Yee M-C Sockell A
Schartl M Tobler M (2017) Complexities of gene expression
patterns in natural populations of an extremophile fish (Poecilia mexi-
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101111mec14198
Passow C Greenway R Arias-Rodriguez L Jeyasingh P D amp Tobler
M (2015) Reduction of energetic demands through modification of
body size and metabolic rates in locally adapted extremophile fishes
Physiological and Biochemical Zoology 88 371ndash383 httpsdoiorg
101086681053
Passow C N Henpita C Shaw J H Quakenbush C Warren W C
Schartl M Tobler M (2017) The roles of plasticity and evolu-
tionary change in shaping gene expression variation in natural popu-
lations of extremophile fish Molecular Ecology 26 6384ndash6399
Pavey S A Bernatchez L Aubin-Horth N amp Landry C R (2012)
What is needed for next-generation ecological and evolutionary
genomics Trends in Ecology amp Evolution 27 673ndash678 httpsdoi
org101016jtree201207014
Peers C Bauer C Boyle J P Scragg J L amp Dallas M L (2012)
Modulation of ion channels by hydrogen sulfide Antioxidants amp Redox
Signaling 17 95ndash105 httpsdoiorg101089ars20114359
Pfennig D W amp Pfennig K S (2012) Evolutionrsquos wedge competition and
the origins of diversity Berkeley CA University of California Press
httpsdoiorg101525california97805202741810010001
Pfenninger M Lerp H Tobler M Passow C Kelley J L Funke E
Plath M (2014) Parallel evolution of cox genes in H2S-tolerant
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tions 5 3873
Pfenninger M Patel S Arias-Rodriguez L Feldmeyer B Riesch R amp
Plath M (2015) Unique evolutionary trajectories in repeated adapta-
tion to hydrogen sulphide-toxic habitats of a neotropical fish (Poecilia
TOBLER ET AL | 857
mexicana) Molecular Ecology 24 5446ndash5459 httpsdoiorg10
1111mec13397
Pietri R Roman-Morales E amp Lopez-Garriga J (2011) Hydrogen sul-
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Redox Signaling 15 393ndash404 httpsdoiorg101089ars20103698
Plath M (2008) Male mating behavior and costs of sexual harassment
for females in cavernicolous and extremophile populations of Atlantic
mollies (Poecilia mexicana) Behaviour 145 73ndash98 httpsdoiorg10
1163156853908782687241
Plath M Hermann C Schreurooder R Riesch R Tobler M Garcia de
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maintain small-scale genetic differentiation despite perturbation by a
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oiorg1011861471-2148-10-256
Plath M Pfenninger M Lerp H Riesch R Eschenbrenner C Slattery
P Tobler M (2013) Genetic differentiation and selection against
migrants in evolutionarily replicated extreme environments Evolution
67 2647ndash2661 httpsdoiorg101111evo12133
Plath M Tobler M Riesch R Garcia de Leon F J Giere O amp
Schlupp I (2007) Survival in an extreme habitat The role of beha-
viour and energy limitation Naturwissenschaften 94 991ndash996
httpsdoiorg101007s00114-007-0279-2
Powell E (1989) Oxygen sulfide and diffusion Why thiobiotic meiofauna
must be sulfide-insensitive first-order respirers Journal of Marine
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Ramirez-Soriano A Ramos-Onsins S E Rozas J Calafell F amp Navarro A
(2008) Statistical power analysis of neutrality tests under demographic
expansions contractions and bottlenecks with recombination Genetics
179 555ndash567 httpsdoiorg101534genetics107083006
Reiffenstein R Hulbert W amp Roth S (1992) Toxicology of hydrogen
sulfide Annual Reviews of Pharmacology and Toxicology 1992 109ndash
134 httpsdoiorg101146annurevpa32040192000545
Reznick D N Furness A I Meredith R W amp Springer M S (2017)
The origin and biogeographic diversification of fishes in the family
Poeciliidae PLoS ONE 12 e0172546 httpsdoiorg101371journa
lpone0172546
Reznick D amp Yang A P (1993) The influence of fluctuating resources
on life history Patterns of allocation and plasticity in female guppies
Ecology 74 2011ndash2019 httpsdoiorg1023071940844
Riesch R Duwe V Herrmann N Padur L Ramm A Scharnweber K
Plath M (2009) Variation along the shy-bold continuum in extre-
mophile fishes (Poecilia mexicana Poecilia sulphuraria) Behavioral Ecol-
ogy and Sociobiology 63 1515ndash1526 httpsdoiorg101007
s00265-009-0780-z
Riesch R Oranth A Dzienko J Karau N Schiessl A Stadler S
Plath M (2010) Extreme habitats are not refuges Poeciliids suffer
from increased aerial predation risk in sulphidic southern Mexican
habitats Biological Journal of the Linnean Society 101 417ndash426
httpsdoiorg101111j1095-8312201001522x
Riesch R Plath M Garcia de Leon F J amp Schlupp I (2010) Conver-
gent life-history shifts Toxic environments result in big babies in two
clades of poeciliids Naturwissenschaften 97 133ndash141 httpsdoi
org101007s00114-009-0613-y
Riesch R Plath M Schlupp I Tobler M amp Langerhans R B (2014)
Colonization of toxic springs drives predictable life-history shift in
livebearing fishes (Poeciliidae) Ecology Letters 17 65ndash71 httpsdoi
org101111ele12209
Riesch R Tobler M Lerp H Jourdan J Doumas L T Nosil P Plath
M (2016) Extremophile Poeciliidae Multivariate insights into the com-
plexity of speciation along replicated ecological gradients BMC Evolu-
tionary Biology 16 136 httpsdoiorg101186s12862-016-0705-1
Riesch R Tobler M amp Plath M (2015) Extremophile fishes Ecology
evolution and physiology of teleosts in extreme environments p 290
Heidelberg Germany Springer httpsdoiorg101007978-3-319-
13362-1
Ritchie M E Holzinger E R Li R Pendergrass S A amp Kim S (2015)
Methods of integrating data to uncover genotype-phenotype interac-
tions Nature Reviews Genetics 16 85ndash97 httpsdoiorg101038
nrg3868
Roach K Tobler M amp Winemiller K O (2011) Hydrogen sulfide bac-
teria and fish A unique subterranean food chain Ecology 92 2056ndash
2062 httpsdoiorg10189011-02761
Rohner N Jarosz D F Kowalko J E Yoshizawa M Jeffery W R
Borowsky R L Tabin C J (2013) Cryptic variation in morpho-
logical evolution HSP90 as a capacitor for loss of eyes in cavefish
Science 342 1372ndash1375 httpsdoiorg101126science1240276
Rosales Lagarde L (2012) Investigation of karst brackish-sulfidic springs
and their role in the hydrogeology subsurface water-rock interactions
and speleogenesis at northern Sierra de Chiapas New Mexico Institute
of Mining and Technology Mexico
Rosenblum E B amp Harmon L J (2011) ldquoSame but differentrdquo Repli-
cated ecological speciation at White Sands Evolution 65 946ndash960
httpsdoiorg101111j1558-5646201001190x
Rosenblum E B Parent C E amp Brandt E E (2014) The molecular
basis of convergent evolution Annual Review of Ecology Evolution and
Systematics 45 203ndash226 httpsdoiorg101146annurev-ecolsys-
120213-091851
Saltz J B Hessel F C amp Kelly M W (2017) Trait correlations in the
genomics era Trends in Ecology amp Evolution 32 279ndash290 httpsdoi
org101016jtree201612008
Saraste M (1999) Oxidative phosphorylation at the fin de siecle Science
283 1488ndash1493 httpsdoiorg101126science28354071488
Schartl M (2014) Beyond the zebrafish Diverse fish species for model-
ing human disease Disease Models and Mechanisms 7 181ndash192
httpsdoiorg101242dmm012245
Schartl M Walter R B Shen Y Garcia T Catchen J Amores A
Warren W C (2013) The genome of the platyfish Xiphophorus mac-
ulatus provides insights into evolutionary adaptation and several
complex traits Nature Genetics 45 567ndash572 httpsdoiorg10
1038ng2604
Schluter D (2009) Evidence for ecological speciation and its alternative
Science 323 737ndash741 httpsdoiorg101126science1160006
Schulz-Mirbach T Eifert C Riesch R Farnworth M S Zimmer C
Bierbach D Plath M (2016) Toxic hydrogen sulphide shapes
brain anatomy A comparative study of sulphide-adapted ecotypes in
the Poecilia mexicana complex Journal of Zoology 300 163ndash176
httpsdoiorg101111jzo12366
SEDESOL (1994) NORMA Oficial Mexicana NOM-059-ECOL-1994 que
determina las especies y subspecies de flora y fauna silvestres ter-
restres y acuaticas en peligro de extincion amenazadas raras y las
sujetas a proteccion especial y que establece especificaciones para
su proteccion Diario Oficial 16 May 1994 488 2ndash60
Servedio M R van Doorn G S Kopp M Frame A M amp Nosil P
(2011) Magic traits in speciation lsquomagicrsquo but not rare Trends in Ecology
amp Evolution 26 389ndash397 httpsdoiorg101016jtree201104005
Shahak Y amp Hauska G (2008) Sulfide oxidation from cyanobacteria to
humans Sulfide-quinone oxidoreductase (SQR) In R Hell C Dahl D
B Knaff amp Th Leustek (Eds) Advances in photosynthesis and respira-
tion (pp 319ndash335) Heidelberg Springer
Shalem O Sanjana N E amp Zhang F (2015) High-throughput func-
tional genomics using CRISPR-Cas9 Nature Reviews Genetics 16
299ndash311 httpsdoiorg101038nrg3899
Silva G J J Bye A el Azzouzi H amp Wisloslashff U (2017) MicroRNAs as
important regulators of exercise adaptation Progress in Cardiovascular
Diseases 60 130ndash151 httpsdoiorg101016jpcad201706003
Simola D F Wissler L Donahue G Waterhouse R M Helmkampf
M Roux J Viljakainen L (2013) Social insect genomes exhibit
dramatic evolution in gene composition and regulation while preserv-
ing regulatory features linked to sociality Genome Research 23
1235ndash1247 httpsdoiorg101101gr155408113
858 | TOBLER ET AL
Sommer-Trembo C Bierbach D Arias Rodriguez L Verel Y Jourdan
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Steindachner F (1863) Beitreuroage zur Kentniss der Sciaenoiden Brasiliens
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Naturwissenschaftlichen Classe der Kaiserlichen Akademie der Wis-
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Stinchcombe J R amp Hoekstra H E (2008) Combining population geno-
mics and quantitative genetics Finding genes underlying ecologically
important traits Heredity 100 158ndash170 httpsdoiorg101038sj
hdy6800937
Stipanuk M H (2004) Sulfur amino acid metabolism Pathways for pro-
duction and removal of homocysteine and cysteine Annual Review of
Nutrition 24 539ndash577 httpsdoiorg101146annurevnutr24
012003132418
Stipanuk M H amp Ueki I (2011) Dealing with methioninehomocysteine
sulfur Cysteine metabolism to taurine and inorganic sulfur Journal of
Inherited Metabolic Disease 34 17ndash32 httpsdoiorg101007
s10545-009-9006-9
Stockinger B Di Meglio P Gialitakis M amp Duarte J H (2014) The
aryl hydrocarbon receptor Multitasking in the immune system
Annual Review of Immunology 32 403ndash432 httpsdoiorg101146
annurev-immunol-032713-120245
Storz J F amp Wheat C W (2010) Integrating evolutionary and
functional approaches to infer adaptation at specific loci Evolution
64 2489ndash2509 httpsdoiorg101111j1558-5646201001044x
Stuart Y E Veen T Weber J N Hanson D Ravinet M Lohman B
K Bolnick D I (2017) Contrasting effects of environment and
genetics generate a continuum of parallel evolution Nature Ecology amp
Evolution 1 0158 httpsdoiorg101038s41559-017-0158
Sunnucks P Morales H E Lamb A M Pavlova A amp Greening C
(2017) Integrative approaches for studying mitochondrial and nuclear
genome co-evolution in oxidative phosphorylation Frontiers in Genet-
ics 8 25
Tobler M amp Hastings L (2011) Convergent patterns of body shape dif-
ferentiation in four different clades of poeciliid fishes inhabiting sul-
fide springs Evolutionary Biology 38 412ndash421 httpsdoiorg10
1007s11692-011-9129-4
Tobler M Palacios M Chapman L J Mitrofanov I Bierbach D
Plath M Mateos M (2011) Evolution in extreme environments
Replicated phenotypic differentiation in livebearing fish inhabiting
sulfidic springs Evolution 65 2213ndash2228 httpsdoiorg101111j
1558-5646201101298x
Tobler M Passow C N Greenway R Kelley J L amp Shaw J H
(2016) The evolutionary ecology of animals inhabiting hydrogen sul-
fide-rich environments Annual Review of Ecology Evolution and Sys-
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121415-032418
Tobler M amp Plath M (2009a) Threatened fishes of the world Gambu-
sia eurystoma Miller 1975 (Poeciliidae) Environmental Biology of
Fishes 85 251 httpsdoiorg101007s10641-009-9481-8
Tobler M amp Plath M (2009b) Threatened fishes of the world Poecilia
sulphuraria (Alvarez 1948) (Poeciliidae) Environmental Biology of
Fishes 85 333ndash334 httpsdoiorg101007s10641-009-9506-3
Tobler M Plath M Riesch R Schlupp I Grasse A Munimanda G
Moodley Y (2014) Selection from parasites favors immunogenic diver-
sity but not divergence among locally adapted host populations Journal
of Evolutionary Biology 27 960ndash974 httpsdoiorg101111jeb12370
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behaviour in response to sulfide-induced hypoxia affects time
budgets feeding efficiency and predation risk Evolutionary Ecology
Research 11 935ndash948
Tobler M Scharnweber K Greenway R Passow C Arias-Rodriguez
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trophic ecology if extremophile fish along replicated environmental
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1111fwb12530
Todgham A E amp Stillman J H (2013) Physiological responses to shifts
in multiple environmental stressors Relevance in a changing worlds
Integrative and Comparative Biology 53 539ndash544 httpsdoiorg10
1093icbict086
van Doorn G S Edelaar P amp Weissing F J (2009) On the origin of
species by natural selection and sexual selection Science 326 1704ndash
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from extreme environments Applied Soil Ecology 13 137ndash150
httpsdoiorg101016S0929-1393(99)00029-3
Wallace J L amp Wang R (2015) Hydrogen-sulfide based therapeutics
Exploiting a unique but ubiquitous gasotransmitter Nature Reviews
Drug Discovery 14 329ndash345 httpsdoiorg101038nrd4433
Wang R (2012) Physiological implications of hydrogen sulfide A whiff
exploration that blossomed Physiological Reviews 92 791ndash896
httpsdoiorg101152physrev000172011
Wang Z Gerstein M amp Snyder M (2009) RNA-seq A revolutionary
tool for transcriptomics Nature Reviews Genetics 10 57ndash63
httpsdoiorg101038nrg2484
Waterman T H (1999) The evolutionary challenges of extreme environ-
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AID-JEZ3ampgt30CO2-T
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insights from the genomes of three parasitoid Nasonia species
Science 327 343ndash348 httpsdoiorg101126science1178028
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Toward a more nuanced understanding of acclimation and adapta-
tion Journal of Experimental Biology 215 884ndash891 httpsdoiorg
101242jeb058735
Whitehead A amp Crawford D L (2006) Neutral and adaptive variation
in gene expression Proceedings of the National Academy of Sciences
USA 103 5425ndash5430 httpsdoiorg101073pnas0507648103
Willett C S amp Burton R S (2004) Evolution of interacting proteins in
the mitochondrial electron transport system in a marine copepod
Molecular Biology and Evolution 21 443ndash453 httpsdoiorg10
1093molbevmsh031
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potential of small populations Annual Review of Ecology Evolution
and Systematics 37 433ndash458 httpsdoiorg101146annurevecol
sys37091305110145
Wolff J N Ladoukakis E D Enrıquez J A amp Dowling D K (2014)
Mitonuclear interactions Evolutionary consequences over multiple
biological scales Philosophical Transactions of the Royal Society B 369
20130443 httpsdoiorg101098rstb20130443
How to cite this article Tobler M Kelley JL Plath M Riesch
R Extreme environments and the origins of biodiversity
Adaptation and speciation in sulphide spring fishes Mol Ecol
201827843ndash859 httpsdoiorg101111mec14497
TOBLER ET AL | 859
Evolution and Systematics 44 281ndash302 httpsdoiorg101146an
nurev-ecolsys-110512-135758
Bush S J Chen L Tovar-Corona J M amp Urritia A O (2017) Alterna-
tive splicing and the evolution of phenotypic novelty Philosophical
Transactions of the Royal Society B 372 20150474 httpsdoiorg
101098rstb20150474
Carroll S P Jorgensen P S Kinnison M T Bergstrom C T Denison
R F Gluckman P Tabashnik B E (2014) Applying evolutionary
biology to address global challenges Science 346 313 httpsdoi
org101364FIO2014JW3A13
Cartwright R A Schwartz R S Merry A L amp Howell M M (2017)
The importance of selection in the evolution of blindness in cavefish
Bmc Evolutionary Biology 17 45 httpsdoiorg101186s12862-
017-0876-4
Chan Y F Marks M E Jones F C Villarreal G Shapiro M D Brady
S D Kingsley D M (2010) Adaptive evolution of pelvic reduc-
tion in sticklebacks by recurrent deletion of Pitx1 enhancer Science
327 302ndash305 httpsdoiorg101126science1182213
Chou J Y amp Leu J Y (2010) Speciation through cytonuclear incompat-
ibility Insights from yeast and implications for higher eukaryotes
BioEssays 32 401ndash411 httpsdoiorg101002bies200900162
Colosimo P F Hosemann K E Balabhadra S Villarreal G Dickson
M Grimwood J Kingsley D M (2005) Widespread parallel evo-
lution in sticklebacks by repeated fixation of ectodysplasin alleles
Science 307 1928ndash1933 httpsdoiorg101126science1107239
Cooper C E amp Brown G C (2008) The inhibition of mitochondrial
cytochrome oxidase by the gases carbon monoxide nitric oxide
hydrogen cyanide and hydrogen sulfide Chemical mechanism and
physiological significance Journal of Bioenergy and Biomembranes 40
533ndash539 httpsdoiorg101007s10863-008-9166-6
Culumber Z W Hopper G W Barts N Passow C N Morgan S Brown
A Tobler M (2016) Habitat use of two extremophile highly ende-
mic and critically endangered fish species (Gambusia eurystoma and Poe-
cilia sulphurophila Poeciliidae) Aquatic Conservation-Marine and
Freshwater Ecosystems 26 1155ndash1167 httpsdoiorg101002aqc
2640
Culumber Z W amp Tobler M (2017) Sex-specific evolution during the
diversification of live-bearing fishes Nature Ecology amp Evolution 1
1185ndash1191
Dalziel A C Rogers S M amp Schulte P M (2009) Linking genotypes
to phenotypes and fitness How mechanistic biology can inform
molecular ecology Molecular Ecology 18 4997ndash5017 httpsdoi
org101111j1365-294X200904427x
Darwin C (1859) On the origin of species based on natural selection or
the preservation of favoured races in the struggle of life 6th ed Lon-
don John Murray
Dasmahapatra K K Walters J R Briscoe A D Davey J W Whibley
A Nadeau N J Jiggins C D (2012) Butterfly genome reveals
promiscuous exchange of mimicry adaptations among species Nature
487 94ndash98 httpsdoiorg101038nature11041
Doudna J A amp Charpentier E (2014) The new frontier of genome edit-
ing with CRISPR-Cas9 Science 346 1258096 httpsdoiorg10
1126science1258096
Dubilier N Giere O amp Grieshaber M (2005) Concomitant effects of sul-
fide and hypoxia on the aerobic metabolism of the marine oligochaete
Tubificoides benedii Journal of Experimental Zoology 269 287ndash298
Eales K L Hollinshead K E R amp Tennant D A (2016) Hypoxia and
metabolic adaptation of cancer cells Oncogenesis 5 e190 httpsd
oiorg101038oncsis201550
Eghbal M A Pennefather P S amp OrsquoBrien P J (2004) H2S cytotoxicity
mechanism involves reactive oxygen species formation and mito-
chondrial depolarisation Toxicology 203 69ndash76 httpsdoiorg10
1016jtox200405020
Feldman C R Brodie Jr E D Brodie III E D amp Pfrender M E (2012)
Constraint shapes convergence in tetrodotoxin-resistant sodium
channels of snakes Proceedings of the National Academy of Sciences
USA 109 4556ndash4561 httpsdoiorg101073pnas1113468109
Flores K B Wolschin F amp Amdam G V (2013) The role of methyla-
tion of DNA in environmental adaptation Integrative and Comparative
Biology 53 359ndash372 httpsdoiorg101093icbict019
Fradin H Kiontke K Zegar C Gutwein M Lucas J Kovtun M
Piano F (2017) Genome architecture and evolution of a unichromo-
somal asexual nematode Current Biology 27(2928ndash2939) e2926
Gershoni M Templeton A amp Mishmar D (2009) Mitochondrial bioen-
ergetics as a major motive force of speciation BioEssays 31 642ndash
650 httpsdoiorg101002bies200800139
Ghalambor C K Walker J A amp Reznick D N (2003) Multi-trait selec-
tion adaptation and constraints on the evolution of burst swimming
performance Integrative and Comparative Biology 43 431ndash438
httpsdoiorg101093icb433431
Gibert J amp Deharveng L (2002) Subterranean ecosystems A truncated
functional biodiversity BioScience 52 473ndash481 httpsdoiorg10
16410006-3568(2002)052[0473SEATFB]20CO2
Gjuvsland A B Vik J O Beard D A Hunter P J amp Omholt S W
(2013) Bridging the genotype-phenotype gap What does it take
Journal of Physiology 591 2055ndash2066 httpsdoiorg101113jphys
iol2012248864
Grant P R Grant B R Markert J A Keller L F amp Petren K (2004)
Convergent evolution of Darwinrsquos finches caused by introgressive
hybridization and selection Evolution 58 1588ndash1599 httpsdoi
org101111j0014-38202004tb01738x
Greene H W (2005) Organisms in nature as a central focus for biology
Trends in Ecology and Evolution 20 23ndash27 httpsdoiorg101016j
tree200411005
Greenway R Arias-Rodriguez L Diaz P amp Tobler M (2014) Patterns
of macroinvertebrate and fish diversity in freshwater sulphide springs
Diversity 6 597ndash632 httpsdoiorg103390d6030597
Greenway R Drexler S Arias-Rodriguez L amp Tobler M (2016) Adap-
tive but not condition-dependent body shape differences contribute
to assortative mating preferences during ecological speciation Evolu-
tion 70 2809ndash2822 httpsdoiorg101111evo13087
Gunderson A R Armstrong E J amp Stillman J H (2016) Multiple stres-
sors in a changing world The need for an improved perspective on
physiological responses to the dynamic marine environment Annual
Review of Marine Science 8 357ndash378 httpsdoiorg101146an
nurev-marine-122414-033953
Hildebrandt T M amp Grieshaber M (2008) Three enzymatic activities
catalyze the oxidation of sulfide to thiosulfate in mammalian and
invertebrate mitochondria FEBS Journal 275 3352ndash3361 httpsd
oiorg101111j1742-4658200806482x
Houle D Govindaraju D R amp Omholt S (2010) Phenomics The next
challenge Nature Reviews Genetics 11 855ndash866 httpsdoiorg10
1038nrg2897
IUCN (2012) Red List of threatened species version 20132 www
iucnredlistorg
Johnston I A amp Bennett A F (1996) Animals and temperature Pheno-
typic and evolutionary adaptation Cambridge Cambridge University
Press httpsdoiorg101017CBO9780511721854
Jorgensen C Auer S K amp Reznick D N (2011) A model for optimal
offspring size in fish including live-bearing and parental effects Amer-
ican Naturalist 177 E119ndashE135 httpsdoiorg101086659622
Kaeuffer R Peichel C L Bolnick D I amp Hendry A P (2012) Parallel
and nonparallel aspects of ecological phenotypic and genetic diver-
gence across replicate population pairs of lake and stream stickle-
back Evolution 66 402ndash418 httpsdoiorg101111j1558-5646
201101440x
Kell D B amp Oliver S G (2004) Here is the evidence now what is the
hypothesis The complementary roles of inductive and hypothesis-
driven science in the post-genomic era BioEssays 26 99ndash105
httpsdoiorg101002(ISSN)1521-1878
856 | TOBLER ET AL
Kelley J L Arias-Rodriguez L Patacsil Martin D Yee M C Busta-
mante C amp Tobler M (2016) Mechanisms underlying adaptation to
life in hydrogen sulfide rich environments Molecular Biology and Evo-
lution 33 1419ndash1434 httpsdoiorg101093molbevmsw020
Kelley J L Passow C Plath M Arias-Rodriguez L Yee M C amp
Tobler M (2012) Genomic resources for a model in adaptation and
speciation research The characterization of the Poecilia mexicana
transcriptome BMC Genomics 13 652 httpsdoiorg101186
1471-2164-13-652
Klerks P L Xie L amp Levinton J S (2011) Quantitative genetics
approaches to study evolutionary processes in ecotoxicology a per-
spective from research on the evolution of resistance Ecotoxicology
20 513ndash523 httpsdoiorg101007s10646-011-0640-2
Kump L R Pavlov A amp Arthur M A (2005) Massive release of hydrogen
sulfide to the surface ocean and atmosphere during intervals of oceanic
anoxia Geology 33 397ndash400 httpsdoiorg101130G212951
Keuroustner A Hoffmann M Fraser B A Kottler V A Sharma E Wei-
gel D amp Dreyer C (2016) The genome of the Trinidadian guppy
Poecilia reticulata and variation in the guanapo population PLoS ONE
11 e0169087 httpsdoiorg101371journalpone0169087
Landry L amp Bernatchez L (2010) Role of epibenthic resource opportu-
nities in the parallel evolution of lake whitefish species pairs (Core-
gonus sp) Journal of Evolutionary Biology 23 2602ndash2613 httpsdoi
org101111j1420-9101201002121x
Langerhans R B (2007) Evolutionary consequences of predation Avoid-
ance escape reproduction and diversification In A M T Elewa
(Ed) Predation in organisms Berlin Springer
Li L Rose P amp Moore P K (2011) Hydrogen sulfide and cell signaling
Annual Review of Pharmacology and Toxicology 51 169ndash187
httpsdoiorg101146annurev-pharmtox-010510-100505
Lin Q Fan S Zhang Y Xu M Zhang H Yang Y Gunter H M
(2016) The seahorse genome and the evolution of its specialized mor-
phology Nature 540 395ndash399 httpsdoiorg101038nature20595
Liu Y Ma S Wang X Chang J Gao J Shi R Zhao P (2014)
Highly efficient multiplex targeted mutagenesis and genomic struc-
ture variation in Bombyx mori cells using CRISPRCas9 Insect bio-
chemistry and molecular biology 49 35ndash42 httpsdoiorg101016j
ibmb201403010
Lopez-Maury L Marguerat S amp Beuroahler J (2008) Tuning gene expres-
sion to changing environments From rapid responses to evolutionary
adaptation Nature Reviews Genetics 9 583ndash593 httpsdoiorg10
1038nrg2398
Maan M E amp Seehausen O (2012) Magic traits versus magic prefer-
ences in speciation Evolutionary Ecology Research 14 779ndash785
Martyn U Weigel D amp Dreyer C (2006) In vitro culture of embryos
of the guppy Poecilia reticulata Developmental Dynamics 235 617ndash
622 httpsdoiorg101002(ISSN)1097-0177
Metzker M L (2010) Sequencing technologies The next generation
Nature Reviews Genetics 11 31ndash46 httpsdoiorg101038nrg2626
Moore M P Riesch R amp Martin R A (2016) The predictability and
magnitude of life-history divergence to ecological agents of selection
A meta-analysis in livebearing fishes Ecology Letters 19 435ndash442
httpsdoiorg101111ele12576
Mustafa A K Gadalla M M Sen N Kim S Mu W Gazi S K
Snyder S H (2009) H2S signals through protein S-sulfhydration
Science Signaling 2 ra72
Nesse R M amp Stearns S C (2008) The great opportunity Evolutionary
applications to medicine and public health Evolutionary Applications
1 28ndash48 httpsdoiorg101111j1752-4571200700006x
Nevo E (2011) Evolution under environmental stress at macro and
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org101093gbeevr052
Norberg J Urban M C Vellend M Klausmeier C A amp Loeuille N (2012)
Eco-evolutionary responses of biodiversity to climate change Nature Cli-
mate Change 2 747ndash751 httpsdoiorg101038nclimate1588
Nosil P (2012) Ecological speciation Oxford UK Oxford University
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0001
Nosil P Vines T H amp Funk D J (2005) Perspective Reproductive
isolation caused by natural selection against immigrants from diver-
gent habitats Evolution 59 705ndash719
Oke K B Rolshausen G LeBlond C amp Hendry A P (2017) How par-
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Olson K R (2011) The therapeutic potential of hydrogen sulfide
Separating hype from hope American Journal of Physiology-Regulatory
Integrative and Comparative Physiology 301 R297ndashR312 httpsdoi
org101152ajpregu000452011
Olson K R Donald J A Dombkowski R A amp Perry S F (2012) Evo-
lutionary and comparative aspects of nitric oxide carbon monoxide
and hydrogen sulfide Respiratory Physiology amp Neurobiology 184
117ndash129 httpsdoiorg101016jresp201204004
Olson K R amp Straub K D (2015) The role of hydrogen sulfide in evo-
lution and the evolution of hydrogen sulfide in metabolism and sig-
naling Physiology 31 60ndash72
Palacios M Arias-Rodriguez L Plath M Eifert C Lerp H Lamboj A
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reveals additional complexity in speciation patterns of poeciliid fishes
in sulfide springs PLoS ONE 8 e71069 httpsdoiorg101371jour
nalpone0071069
Palacios M Voelker G Arias-Rodriguez L Mateos M amp Tobler M
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versity and spatio-temporal aspects of diversification in Middle
America Molecular Phylogenetics and Evolution 103 230ndash244
httpsdoiorg101016jympev201607025
Passow C Arias-Rodriguez L amp Tobler M (2017) Convergent evolu-
tion of reduced energy demands in extremophile fish PLoS ONE 12
e0186935 httpsdoiorg101371journalpone0186935
Passow C N Brown A Arias-Rodriguez L Yee M-C Sockell A
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patterns in natural populations of an extremophile fish (Poecilia mexi-
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Passow C Greenway R Arias-Rodriguez L Jeyasingh P D amp Tobler
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body size and metabolic rates in locally adapted extremophile fishes
Physiological and Biochemical Zoology 88 371ndash383 httpsdoiorg
101086681053
Passow C N Henpita C Shaw J H Quakenbush C Warren W C
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Pavey S A Bernatchez L Aubin-Horth N amp Landry C R (2012)
What is needed for next-generation ecological and evolutionary
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org101016jtree201207014
Peers C Bauer C Boyle J P Scragg J L amp Dallas M L (2012)
Modulation of ion channels by hydrogen sulfide Antioxidants amp Redox
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Pfennig D W amp Pfennig K S (2012) Evolutionrsquos wedge competition and
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httpsdoiorg101525california97805202741810010001
Pfenninger M Lerp H Tobler M Passow C Kelley J L Funke E
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Pfenninger M Patel S Arias-Rodriguez L Feldmeyer B Riesch R amp
Plath M (2015) Unique evolutionary trajectories in repeated adapta-
tion to hydrogen sulphide-toxic habitats of a neotropical fish (Poecilia
TOBLER ET AL | 857
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1111mec13397
Pietri R Roman-Morales E amp Lopez-Garriga J (2011) Hydrogen sul-
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Plath M (2008) Male mating behavior and costs of sexual harassment
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1163156853908782687241
Plath M Hermann C Schreurooder R Riesch R Tobler M Garcia de
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Plath M Pfenninger M Lerp H Riesch R Eschenbrenner C Slattery
P Tobler M (2013) Genetic differentiation and selection against
migrants in evolutionarily replicated extreme environments Evolution
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Plath M Tobler M Riesch R Garcia de Leon F J Giere O amp
Schlupp I (2007) Survival in an extreme habitat The role of beha-
viour and energy limitation Naturwissenschaften 94 991ndash996
httpsdoiorg101007s00114-007-0279-2
Powell E (1989) Oxygen sulfide and diffusion Why thiobiotic meiofauna
must be sulfide-insensitive first-order respirers Journal of Marine
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Ramirez-Soriano A Ramos-Onsins S E Rozas J Calafell F amp Navarro A
(2008) Statistical power analysis of neutrality tests under demographic
expansions contractions and bottlenecks with recombination Genetics
179 555ndash567 httpsdoiorg101534genetics107083006
Reiffenstein R Hulbert W amp Roth S (1992) Toxicology of hydrogen
sulfide Annual Reviews of Pharmacology and Toxicology 1992 109ndash
134 httpsdoiorg101146annurevpa32040192000545
Reznick D N Furness A I Meredith R W amp Springer M S (2017)
The origin and biogeographic diversification of fishes in the family
Poeciliidae PLoS ONE 12 e0172546 httpsdoiorg101371journa
lpone0172546
Reznick D amp Yang A P (1993) The influence of fluctuating resources
on life history Patterns of allocation and plasticity in female guppies
Ecology 74 2011ndash2019 httpsdoiorg1023071940844
Riesch R Duwe V Herrmann N Padur L Ramm A Scharnweber K
Plath M (2009) Variation along the shy-bold continuum in extre-
mophile fishes (Poecilia mexicana Poecilia sulphuraria) Behavioral Ecol-
ogy and Sociobiology 63 1515ndash1526 httpsdoiorg101007
s00265-009-0780-z
Riesch R Oranth A Dzienko J Karau N Schiessl A Stadler S
Plath M (2010) Extreme habitats are not refuges Poeciliids suffer
from increased aerial predation risk in sulphidic southern Mexican
habitats Biological Journal of the Linnean Society 101 417ndash426
httpsdoiorg101111j1095-8312201001522x
Riesch R Plath M Garcia de Leon F J amp Schlupp I (2010) Conver-
gent life-history shifts Toxic environments result in big babies in two
clades of poeciliids Naturwissenschaften 97 133ndash141 httpsdoi
org101007s00114-009-0613-y
Riesch R Plath M Schlupp I Tobler M amp Langerhans R B (2014)
Colonization of toxic springs drives predictable life-history shift in
livebearing fishes (Poeciliidae) Ecology Letters 17 65ndash71 httpsdoi
org101111ele12209
Riesch R Tobler M Lerp H Jourdan J Doumas L T Nosil P Plath
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plexity of speciation along replicated ecological gradients BMC Evolu-
tionary Biology 16 136 httpsdoiorg101186s12862-016-0705-1
Riesch R Tobler M amp Plath M (2015) Extremophile fishes Ecology
evolution and physiology of teleosts in extreme environments p 290
Heidelberg Germany Springer httpsdoiorg101007978-3-319-
13362-1
Ritchie M E Holzinger E R Li R Pendergrass S A amp Kim S (2015)
Methods of integrating data to uncover genotype-phenotype interac-
tions Nature Reviews Genetics 16 85ndash97 httpsdoiorg101038
nrg3868
Roach K Tobler M amp Winemiller K O (2011) Hydrogen sulfide bac-
teria and fish A unique subterranean food chain Ecology 92 2056ndash
2062 httpsdoiorg10189011-02761
Rohner N Jarosz D F Kowalko J E Yoshizawa M Jeffery W R
Borowsky R L Tabin C J (2013) Cryptic variation in morpho-
logical evolution HSP90 as a capacitor for loss of eyes in cavefish
Science 342 1372ndash1375 httpsdoiorg101126science1240276
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and their role in the hydrogeology subsurface water-rock interactions
and speleogenesis at northern Sierra de Chiapas New Mexico Institute
of Mining and Technology Mexico
Rosenblum E B amp Harmon L J (2011) ldquoSame but differentrdquo Repli-
cated ecological speciation at White Sands Evolution 65 946ndash960
httpsdoiorg101111j1558-5646201001190x
Rosenblum E B Parent C E amp Brandt E E (2014) The molecular
basis of convergent evolution Annual Review of Ecology Evolution and
Systematics 45 203ndash226 httpsdoiorg101146annurev-ecolsys-
120213-091851
Saltz J B Hessel F C amp Kelly M W (2017) Trait correlations in the
genomics era Trends in Ecology amp Evolution 32 279ndash290 httpsdoi
org101016jtree201612008
Saraste M (1999) Oxidative phosphorylation at the fin de siecle Science
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Schartl M (2014) Beyond the zebrafish Diverse fish species for model-
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Schartl M Walter R B Shen Y Garcia T Catchen J Amores A
Warren W C (2013) The genome of the platyfish Xiphophorus mac-
ulatus provides insights into evolutionary adaptation and several
complex traits Nature Genetics 45 567ndash572 httpsdoiorg10
1038ng2604
Schluter D (2009) Evidence for ecological speciation and its alternative
Science 323 737ndash741 httpsdoiorg101126science1160006
Schulz-Mirbach T Eifert C Riesch R Farnworth M S Zimmer C
Bierbach D Plath M (2016) Toxic hydrogen sulphide shapes
brain anatomy A comparative study of sulphide-adapted ecotypes in
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determina las especies y subspecies de flora y fauna silvestres ter-
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sujetas a proteccion especial y que establece especificaciones para
su proteccion Diario Oficial 16 May 1994 488 2ndash60
Servedio M R van Doorn G S Kopp M Frame A M amp Nosil P
(2011) Magic traits in speciation lsquomagicrsquo but not rare Trends in Ecology
amp Evolution 26 389ndash397 httpsdoiorg101016jtree201104005
Shahak Y amp Hauska G (2008) Sulfide oxidation from cyanobacteria to
humans Sulfide-quinone oxidoreductase (SQR) In R Hell C Dahl D
B Knaff amp Th Leustek (Eds) Advances in photosynthesis and respira-
tion (pp 319ndash335) Heidelberg Springer
Shalem O Sanjana N E amp Zhang F (2015) High-throughput func-
tional genomics using CRISPR-Cas9 Nature Reviews Genetics 16
299ndash311 httpsdoiorg101038nrg3899
Silva G J J Bye A el Azzouzi H amp Wisloslashff U (2017) MicroRNAs as
important regulators of exercise adaptation Progress in Cardiovascular
Diseases 60 130ndash151 httpsdoiorg101016jpcad201706003
Simola D F Wissler L Donahue G Waterhouse R M Helmkampf
M Roux J Viljakainen L (2013) Social insect genomes exhibit
dramatic evolution in gene composition and regulation while preserv-
ing regulatory features linked to sociality Genome Research 23
1235ndash1247 httpsdoiorg101101gr155408113
858 | TOBLER ET AL
Sommer-Trembo C Bierbach D Arias Rodriguez L Verel Y Jourdan
J Zimmer C Plath M (2016) Does personality affect premating
isolation between locally-adapted populations BMC Evolutionary Biol-
ogy 16 138 httpsdoiorg101186s12862-016-0712-2
Steindachner F (1863) Beitreuroage zur Kentniss der Sciaenoiden Brasiliens
und der Cyprinodonten Mejicos Sitzungsberichte der Mathematisch-
Naturwissenschaftlichen Classe der Kaiserlichen Akademie der Wis-
senschaften 48 162ndash185
Stinchcombe J R amp Hoekstra H E (2008) Combining population geno-
mics and quantitative genetics Finding genes underlying ecologically
important traits Heredity 100 158ndash170 httpsdoiorg101038sj
hdy6800937
Stipanuk M H (2004) Sulfur amino acid metabolism Pathways for pro-
duction and removal of homocysteine and cysteine Annual Review of
Nutrition 24 539ndash577 httpsdoiorg101146annurevnutr24
012003132418
Stipanuk M H amp Ueki I (2011) Dealing with methioninehomocysteine
sulfur Cysteine metabolism to taurine and inorganic sulfur Journal of
Inherited Metabolic Disease 34 17ndash32 httpsdoiorg101007
s10545-009-9006-9
Stockinger B Di Meglio P Gialitakis M amp Duarte J H (2014) The
aryl hydrocarbon receptor Multitasking in the immune system
Annual Review of Immunology 32 403ndash432 httpsdoiorg101146
annurev-immunol-032713-120245
Storz J F amp Wheat C W (2010) Integrating evolutionary and
functional approaches to infer adaptation at specific loci Evolution
64 2489ndash2509 httpsdoiorg101111j1558-5646201001044x
Stuart Y E Veen T Weber J N Hanson D Ravinet M Lohman B
K Bolnick D I (2017) Contrasting effects of environment and
genetics generate a continuum of parallel evolution Nature Ecology amp
Evolution 1 0158 httpsdoiorg101038s41559-017-0158
Sunnucks P Morales H E Lamb A M Pavlova A amp Greening C
(2017) Integrative approaches for studying mitochondrial and nuclear
genome co-evolution in oxidative phosphorylation Frontiers in Genet-
ics 8 25
Tobler M amp Hastings L (2011) Convergent patterns of body shape dif-
ferentiation in four different clades of poeciliid fishes inhabiting sul-
fide springs Evolutionary Biology 38 412ndash421 httpsdoiorg10
1007s11692-011-9129-4
Tobler M Palacios M Chapman L J Mitrofanov I Bierbach D
Plath M Mateos M (2011) Evolution in extreme environments
Replicated phenotypic differentiation in livebearing fish inhabiting
sulfidic springs Evolution 65 2213ndash2228 httpsdoiorg101111j
1558-5646201101298x
Tobler M Passow C N Greenway R Kelley J L amp Shaw J H
(2016) The evolutionary ecology of animals inhabiting hydrogen sul-
fide-rich environments Annual Review of Ecology Evolution and Sys-
tematics 47 239ndash262 httpsdoiorg101146annurev-ecolsys-
121415-032418
Tobler M amp Plath M (2009a) Threatened fishes of the world Gambu-
sia eurystoma Miller 1975 (Poeciliidae) Environmental Biology of
Fishes 85 251 httpsdoiorg101007s10641-009-9481-8
Tobler M amp Plath M (2009b) Threatened fishes of the world Poecilia
sulphuraria (Alvarez 1948) (Poeciliidae) Environmental Biology of
Fishes 85 333ndash334 httpsdoiorg101007s10641-009-9506-3
Tobler M Plath M Riesch R Schlupp I Grasse A Munimanda G
Moodley Y (2014) Selection from parasites favors immunogenic diver-
sity but not divergence among locally adapted host populations Journal
of Evolutionary Biology 27 960ndash974 httpsdoiorg101111jeb12370
Tobler M Riesch R amp Plath M (2015) Extremophile fishes An inte-
grative synthesis In R Riesch M Tobler amp M Plath (Eds) Extremo-
phile fishes Ecology evolution and physiology of teleosts in extreme
environments (pp 279ndash296) Heidelberg Germany Springer
Tobler M Riesch R Tobler C M amp Plath M (2009) Compensatory
behaviour in response to sulfide-induced hypoxia affects time
budgets feeding efficiency and predation risk Evolutionary Ecology
Research 11 935ndash948
Tobler M Scharnweber K Greenway R Passow C Arias-Rodriguez
L amp Garcia-De-Leon F J (2015) Convergent changes in the
trophic ecology if extremophile fish along replicated environmental
gradients Freshwater Biology 60 768ndash780 httpsdoiorg10
1111fwb12530
Todgham A E amp Stillman J H (2013) Physiological responses to shifts
in multiple environmental stressors Relevance in a changing worlds
Integrative and Comparative Biology 53 539ndash544 httpsdoiorg10
1093icbict086
van Doorn G S Edelaar P amp Weissing F J (2009) On the origin of
species by natural selection and sexual selection Science 326 1704ndash
1707 httpsdoiorg101126science1181661
Wall D H amp Virginia R A (1999) Controls on soil biodiversity Insights
from extreme environments Applied Soil Ecology 13 137ndash150
httpsdoiorg101016S0929-1393(99)00029-3
Wallace J L amp Wang R (2015) Hydrogen-sulfide based therapeutics
Exploiting a unique but ubiquitous gasotransmitter Nature Reviews
Drug Discovery 14 329ndash345 httpsdoiorg101038nrd4433
Wang R (2012) Physiological implications of hydrogen sulfide A whiff
exploration that blossomed Physiological Reviews 92 791ndash896
httpsdoiorg101152physrev000172011
Wang Z Gerstein M amp Snyder M (2009) RNA-seq A revolutionary
tool for transcriptomics Nature Reviews Genetics 10 57ndash63
httpsdoiorg101038nrg2484
Waterman T H (1999) The evolutionary challenges of extreme environ-
ments (part 1) Journal of Experimental Zoology 285 326ndash359
httpsdoiorg101002(SICI)1097-010X(19991215)2854amplt326
AID-JEZ3ampgt30CO2-T
Werren J H Richards S Desjardins C A Niehuis O Gadau J Col-
bourne J K amp Group N G W (2010) Functional and evolutionary
insights from the genomes of three parasitoid Nasonia species
Science 327 343ndash348 httpsdoiorg101126science1178028
Whitehead A (2012) Comparative genomics in ecological physiology
Toward a more nuanced understanding of acclimation and adapta-
tion Journal of Experimental Biology 215 884ndash891 httpsdoiorg
101242jeb058735
Whitehead A amp Crawford D L (2006) Neutral and adaptive variation
in gene expression Proceedings of the National Academy of Sciences
USA 103 5425ndash5430 httpsdoiorg101073pnas0507648103
Willett C S amp Burton R S (2004) Evolution of interacting proteins in
the mitochondrial electron transport system in a marine copepod
Molecular Biology and Evolution 21 443ndash453 httpsdoiorg10
1093molbevmsh031
Willi Y Van Buskirk J amp Hoffmann A (2006) Limits to the adaptive
potential of small populations Annual Review of Ecology Evolution
and Systematics 37 433ndash458 httpsdoiorg101146annurevecol
sys37091305110145
Wolff J N Ladoukakis E D Enrıquez J A amp Dowling D K (2014)
Mitonuclear interactions Evolutionary consequences over multiple
biological scales Philosophical Transactions of the Royal Society B 369
20130443 httpsdoiorg101098rstb20130443
How to cite this article Tobler M Kelley JL Plath M Riesch
R Extreme environments and the origins of biodiversity
Adaptation and speciation in sulphide spring fishes Mol Ecol
201827843ndash859 httpsdoiorg101111mec14497
TOBLER ET AL | 859
Kelley J L Arias-Rodriguez L Patacsil Martin D Yee M C Busta-
mante C amp Tobler M (2016) Mechanisms underlying adaptation to
life in hydrogen sulfide rich environments Molecular Biology and Evo-
lution 33 1419ndash1434 httpsdoiorg101093molbevmsw020
Kelley J L Passow C Plath M Arias-Rodriguez L Yee M C amp
Tobler M (2012) Genomic resources for a model in adaptation and
speciation research The characterization of the Poecilia mexicana
transcriptome BMC Genomics 13 652 httpsdoiorg101186
1471-2164-13-652
Klerks P L Xie L amp Levinton J S (2011) Quantitative genetics
approaches to study evolutionary processes in ecotoxicology a per-
spective from research on the evolution of resistance Ecotoxicology
20 513ndash523 httpsdoiorg101007s10646-011-0640-2
Kump L R Pavlov A amp Arthur M A (2005) Massive release of hydrogen
sulfide to the surface ocean and atmosphere during intervals of oceanic
anoxia Geology 33 397ndash400 httpsdoiorg101130G212951
Keuroustner A Hoffmann M Fraser B A Kottler V A Sharma E Wei-
gel D amp Dreyer C (2016) The genome of the Trinidadian guppy
Poecilia reticulata and variation in the guanapo population PLoS ONE
11 e0169087 httpsdoiorg101371journalpone0169087
Landry L amp Bernatchez L (2010) Role of epibenthic resource opportu-
nities in the parallel evolution of lake whitefish species pairs (Core-
gonus sp) Journal of Evolutionary Biology 23 2602ndash2613 httpsdoi
org101111j1420-9101201002121x
Langerhans R B (2007) Evolutionary consequences of predation Avoid-
ance escape reproduction and diversification In A M T Elewa
(Ed) Predation in organisms Berlin Springer
Li L Rose P amp Moore P K (2011) Hydrogen sulfide and cell signaling
Annual Review of Pharmacology and Toxicology 51 169ndash187
httpsdoiorg101146annurev-pharmtox-010510-100505
Lin Q Fan S Zhang Y Xu M Zhang H Yang Y Gunter H M
(2016) The seahorse genome and the evolution of its specialized mor-
phology Nature 540 395ndash399 httpsdoiorg101038nature20595
Liu Y Ma S Wang X Chang J Gao J Shi R Zhao P (2014)
Highly efficient multiplex targeted mutagenesis and genomic struc-
ture variation in Bombyx mori cells using CRISPRCas9 Insect bio-
chemistry and molecular biology 49 35ndash42 httpsdoiorg101016j
ibmb201403010
Lopez-Maury L Marguerat S amp Beuroahler J (2008) Tuning gene expres-
sion to changing environments From rapid responses to evolutionary
adaptation Nature Reviews Genetics 9 583ndash593 httpsdoiorg10
1038nrg2398
Maan M E amp Seehausen O (2012) Magic traits versus magic prefer-
ences in speciation Evolutionary Ecology Research 14 779ndash785
Martyn U Weigel D amp Dreyer C (2006) In vitro culture of embryos
of the guppy Poecilia reticulata Developmental Dynamics 235 617ndash
622 httpsdoiorg101002(ISSN)1097-0177
Metzker M L (2010) Sequencing technologies The next generation
Nature Reviews Genetics 11 31ndash46 httpsdoiorg101038nrg2626
Moore M P Riesch R amp Martin R A (2016) The predictability and
magnitude of life-history divergence to ecological agents of selection
A meta-analysis in livebearing fishes Ecology Letters 19 435ndash442
httpsdoiorg101111ele12576
Mustafa A K Gadalla M M Sen N Kim S Mu W Gazi S K
Snyder S H (2009) H2S signals through protein S-sulfhydration
Science Signaling 2 ra72
Nesse R M amp Stearns S C (2008) The great opportunity Evolutionary
applications to medicine and public health Evolutionary Applications
1 28ndash48 httpsdoiorg101111j1752-4571200700006x
Nevo E (2011) Evolution under environmental stress at macro and
microscales Genome Biology and Evolution 3 1039ndash1052 httpsdoi
org101093gbeevr052
Norberg J Urban M C Vellend M Klausmeier C A amp Loeuille N (2012)
Eco-evolutionary responses of biodiversity to climate change Nature Cli-
mate Change 2 747ndash751 httpsdoiorg101038nclimate1588
Nosil P (2012) Ecological speciation Oxford UK Oxford University
Press httpsdoiorg101093acprofosobl9780199587100001
0001
Nosil P Vines T H amp Funk D J (2005) Perspective Reproductive
isolation caused by natural selection against immigrants from diver-
gent habitats Evolution 59 705ndash719
Oke K B Rolshausen G LeBlond C amp Hendry A P (2017) How par-
allel is parallel evolution A comparative analysis in fishes American
Naturalist 190 1ndash16 httpsdoiorg101086691989
Olson K R (2011) The therapeutic potential of hydrogen sulfide
Separating hype from hope American Journal of Physiology-Regulatory
Integrative and Comparative Physiology 301 R297ndashR312 httpsdoi
org101152ajpregu000452011
Olson K R Donald J A Dombkowski R A amp Perry S F (2012) Evo-
lutionary and comparative aspects of nitric oxide carbon monoxide
and hydrogen sulfide Respiratory Physiology amp Neurobiology 184
117ndash129 httpsdoiorg101016jresp201204004
Olson K R amp Straub K D (2015) The role of hydrogen sulfide in evo-
lution and the evolution of hydrogen sulfide in metabolism and sig-
naling Physiology 31 60ndash72
Palacios M Arias-Rodriguez L Plath M Eifert C Lerp H Lamboj A
Tobler M (2013) The rediscovery of a long described species
reveals additional complexity in speciation patterns of poeciliid fishes
in sulfide springs PLoS ONE 8 e71069 httpsdoiorg101371jour
nalpone0071069
Palacios M Voelker G Arias-Rodriguez L Mateos M amp Tobler M
(2016) Phylogenetic analyses of the subgenus Mollienesia (Poecilia
Poeciliidae Teleostei) reveal taxonomic inconsistencies cryptic biodi-
versity and spatio-temporal aspects of diversification in Middle
America Molecular Phylogenetics and Evolution 103 230ndash244
httpsdoiorg101016jympev201607025
Passow C Arias-Rodriguez L amp Tobler M (2017) Convergent evolu-
tion of reduced energy demands in extremophile fish PLoS ONE 12
e0186935 httpsdoiorg101371journalpone0186935
Passow C N Brown A Arias-Rodriguez L Yee M-C Sockell A
Schartl M Tobler M (2017) Complexities of gene expression
patterns in natural populations of an extremophile fish (Poecilia mexi-
cana Poeciliidae) Molecular Ecology 26 4211ndash4225 httpsdoiorg
101111mec14198
Passow C Greenway R Arias-Rodriguez L Jeyasingh P D amp Tobler
M (2015) Reduction of energetic demands through modification of
body size and metabolic rates in locally adapted extremophile fishes
Physiological and Biochemical Zoology 88 371ndash383 httpsdoiorg
101086681053
Passow C N Henpita C Shaw J H Quakenbush C Warren W C
Schartl M Tobler M (2017) The roles of plasticity and evolu-
tionary change in shaping gene expression variation in natural popu-
lations of extremophile fish Molecular Ecology 26 6384ndash6399
Pavey S A Bernatchez L Aubin-Horth N amp Landry C R (2012)
What is needed for next-generation ecological and evolutionary
genomics Trends in Ecology amp Evolution 27 673ndash678 httpsdoi
org101016jtree201207014
Peers C Bauer C Boyle J P Scragg J L amp Dallas M L (2012)
Modulation of ion channels by hydrogen sulfide Antioxidants amp Redox
Signaling 17 95ndash105 httpsdoiorg101089ars20114359
Pfennig D W amp Pfennig K S (2012) Evolutionrsquos wedge competition and
the origins of diversity Berkeley CA University of California Press
httpsdoiorg101525california97805202741810010001
Pfenninger M Lerp H Tobler M Passow C Kelley J L Funke E
Plath M (2014) Parallel evolution of cox genes in H2S-tolerant
fish as key adaptation to a toxic environment Nature Communica-
tions 5 3873
Pfenninger M Patel S Arias-Rodriguez L Feldmeyer B Riesch R amp
Plath M (2015) Unique evolutionary trajectories in repeated adapta-
tion to hydrogen sulphide-toxic habitats of a neotropical fish (Poecilia
TOBLER ET AL | 857
mexicana) Molecular Ecology 24 5446ndash5459 httpsdoiorg10
1111mec13397
Pietri R Roman-Morales E amp Lopez-Garriga J (2011) Hydrogen sul-
fide and heme proteins Knowledge and mysteries Antioxidants amp
Redox Signaling 15 393ndash404 httpsdoiorg101089ars20103698
Plath M (2008) Male mating behavior and costs of sexual harassment
for females in cavernicolous and extremophile populations of Atlantic
mollies (Poecilia mexicana) Behaviour 145 73ndash98 httpsdoiorg10
1163156853908782687241
Plath M Hermann C Schreurooder R Riesch R Tobler M Garcia de
Leon F J Tiedemann R (2010) Locally adapted fish populations
maintain small-scale genetic differentiation despite perturbation by a
catastrophic flood event BMC Evolutionary Biology 10 256 httpsd
oiorg1011861471-2148-10-256
Plath M Pfenninger M Lerp H Riesch R Eschenbrenner C Slattery
P Tobler M (2013) Genetic differentiation and selection against
migrants in evolutionarily replicated extreme environments Evolution
67 2647ndash2661 httpsdoiorg101111evo12133
Plath M Tobler M Riesch R Garcia de Leon F J Giere O amp
Schlupp I (2007) Survival in an extreme habitat The role of beha-
viour and energy limitation Naturwissenschaften 94 991ndash996
httpsdoiorg101007s00114-007-0279-2
Powell E (1989) Oxygen sulfide and diffusion Why thiobiotic meiofauna
must be sulfide-insensitive first-order respirers Journal of Marine
Research 47 887ndash932 httpsdoiorg101357002224089785076082
Ramirez-Soriano A Ramos-Onsins S E Rozas J Calafell F amp Navarro A
(2008) Statistical power analysis of neutrality tests under demographic
expansions contractions and bottlenecks with recombination Genetics
179 555ndash567 httpsdoiorg101534genetics107083006
Reiffenstein R Hulbert W amp Roth S (1992) Toxicology of hydrogen
sulfide Annual Reviews of Pharmacology and Toxicology 1992 109ndash
134 httpsdoiorg101146annurevpa32040192000545
Reznick D N Furness A I Meredith R W amp Springer M S (2017)
The origin and biogeographic diversification of fishes in the family
Poeciliidae PLoS ONE 12 e0172546 httpsdoiorg101371journa
lpone0172546
Reznick D amp Yang A P (1993) The influence of fluctuating resources
on life history Patterns of allocation and plasticity in female guppies
Ecology 74 2011ndash2019 httpsdoiorg1023071940844
Riesch R Duwe V Herrmann N Padur L Ramm A Scharnweber K
Plath M (2009) Variation along the shy-bold continuum in extre-
mophile fishes (Poecilia mexicana Poecilia sulphuraria) Behavioral Ecol-
ogy and Sociobiology 63 1515ndash1526 httpsdoiorg101007
s00265-009-0780-z
Riesch R Oranth A Dzienko J Karau N Schiessl A Stadler S
Plath M (2010) Extreme habitats are not refuges Poeciliids suffer
from increased aerial predation risk in sulphidic southern Mexican
habitats Biological Journal of the Linnean Society 101 417ndash426
httpsdoiorg101111j1095-8312201001522x
Riesch R Plath M Garcia de Leon F J amp Schlupp I (2010) Conver-
gent life-history shifts Toxic environments result in big babies in two
clades of poeciliids Naturwissenschaften 97 133ndash141 httpsdoi
org101007s00114-009-0613-y
Riesch R Plath M Schlupp I Tobler M amp Langerhans R B (2014)
Colonization of toxic springs drives predictable life-history shift in
livebearing fishes (Poeciliidae) Ecology Letters 17 65ndash71 httpsdoi
org101111ele12209
Riesch R Tobler M Lerp H Jourdan J Doumas L T Nosil P Plath
M (2016) Extremophile Poeciliidae Multivariate insights into the com-
plexity of speciation along replicated ecological gradients BMC Evolu-
tionary Biology 16 136 httpsdoiorg101186s12862-016-0705-1
Riesch R Tobler M amp Plath M (2015) Extremophile fishes Ecology
evolution and physiology of teleosts in extreme environments p 290
Heidelberg Germany Springer httpsdoiorg101007978-3-319-
13362-1
Ritchie M E Holzinger E R Li R Pendergrass S A amp Kim S (2015)
Methods of integrating data to uncover genotype-phenotype interac-
tions Nature Reviews Genetics 16 85ndash97 httpsdoiorg101038
nrg3868
Roach K Tobler M amp Winemiller K O (2011) Hydrogen sulfide bac-
teria and fish A unique subterranean food chain Ecology 92 2056ndash
2062 httpsdoiorg10189011-02761
Rohner N Jarosz D F Kowalko J E Yoshizawa M Jeffery W R
Borowsky R L Tabin C J (2013) Cryptic variation in morpho-
logical evolution HSP90 as a capacitor for loss of eyes in cavefish
Science 342 1372ndash1375 httpsdoiorg101126science1240276
Rosales Lagarde L (2012) Investigation of karst brackish-sulfidic springs
and their role in the hydrogeology subsurface water-rock interactions
and speleogenesis at northern Sierra de Chiapas New Mexico Institute
of Mining and Technology Mexico
Rosenblum E B amp Harmon L J (2011) ldquoSame but differentrdquo Repli-
cated ecological speciation at White Sands Evolution 65 946ndash960
httpsdoiorg101111j1558-5646201001190x
Rosenblum E B Parent C E amp Brandt E E (2014) The molecular
basis of convergent evolution Annual Review of Ecology Evolution and
Systematics 45 203ndash226 httpsdoiorg101146annurev-ecolsys-
120213-091851
Saltz J B Hessel F C amp Kelly M W (2017) Trait correlations in the
genomics era Trends in Ecology amp Evolution 32 279ndash290 httpsdoi
org101016jtree201612008
Saraste M (1999) Oxidative phosphorylation at the fin de siecle Science
283 1488ndash1493 httpsdoiorg101126science28354071488
Schartl M (2014) Beyond the zebrafish Diverse fish species for model-
ing human disease Disease Models and Mechanisms 7 181ndash192
httpsdoiorg101242dmm012245
Schartl M Walter R B Shen Y Garcia T Catchen J Amores A
Warren W C (2013) The genome of the platyfish Xiphophorus mac-
ulatus provides insights into evolutionary adaptation and several
complex traits Nature Genetics 45 567ndash572 httpsdoiorg10
1038ng2604
Schluter D (2009) Evidence for ecological speciation and its alternative
Science 323 737ndash741 httpsdoiorg101126science1160006
Schulz-Mirbach T Eifert C Riesch R Farnworth M S Zimmer C
Bierbach D Plath M (2016) Toxic hydrogen sulphide shapes
brain anatomy A comparative study of sulphide-adapted ecotypes in
the Poecilia mexicana complex Journal of Zoology 300 163ndash176
httpsdoiorg101111jzo12366
SEDESOL (1994) NORMA Oficial Mexicana NOM-059-ECOL-1994 que
determina las especies y subspecies de flora y fauna silvestres ter-
restres y acuaticas en peligro de extincion amenazadas raras y las
sujetas a proteccion especial y que establece especificaciones para
su proteccion Diario Oficial 16 May 1994 488 2ndash60
Servedio M R van Doorn G S Kopp M Frame A M amp Nosil P
(2011) Magic traits in speciation lsquomagicrsquo but not rare Trends in Ecology
amp Evolution 26 389ndash397 httpsdoiorg101016jtree201104005
Shahak Y amp Hauska G (2008) Sulfide oxidation from cyanobacteria to
humans Sulfide-quinone oxidoreductase (SQR) In R Hell C Dahl D
B Knaff amp Th Leustek (Eds) Advances in photosynthesis and respira-
tion (pp 319ndash335) Heidelberg Springer
Shalem O Sanjana N E amp Zhang F (2015) High-throughput func-
tional genomics using CRISPR-Cas9 Nature Reviews Genetics 16
299ndash311 httpsdoiorg101038nrg3899
Silva G J J Bye A el Azzouzi H amp Wisloslashff U (2017) MicroRNAs as
important regulators of exercise adaptation Progress in Cardiovascular
Diseases 60 130ndash151 httpsdoiorg101016jpcad201706003
Simola D F Wissler L Donahue G Waterhouse R M Helmkampf
M Roux J Viljakainen L (2013) Social insect genomes exhibit
dramatic evolution in gene composition and regulation while preserv-
ing regulatory features linked to sociality Genome Research 23
1235ndash1247 httpsdoiorg101101gr155408113
858 | TOBLER ET AL
Sommer-Trembo C Bierbach D Arias Rodriguez L Verel Y Jourdan
J Zimmer C Plath M (2016) Does personality affect premating
isolation between locally-adapted populations BMC Evolutionary Biol-
ogy 16 138 httpsdoiorg101186s12862-016-0712-2
Steindachner F (1863) Beitreuroage zur Kentniss der Sciaenoiden Brasiliens
und der Cyprinodonten Mejicos Sitzungsberichte der Mathematisch-
Naturwissenschaftlichen Classe der Kaiserlichen Akademie der Wis-
senschaften 48 162ndash185
Stinchcombe J R amp Hoekstra H E (2008) Combining population geno-
mics and quantitative genetics Finding genes underlying ecologically
important traits Heredity 100 158ndash170 httpsdoiorg101038sj
hdy6800937
Stipanuk M H (2004) Sulfur amino acid metabolism Pathways for pro-
duction and removal of homocysteine and cysteine Annual Review of
Nutrition 24 539ndash577 httpsdoiorg101146annurevnutr24
012003132418
Stipanuk M H amp Ueki I (2011) Dealing with methioninehomocysteine
sulfur Cysteine metabolism to taurine and inorganic sulfur Journal of
Inherited Metabolic Disease 34 17ndash32 httpsdoiorg101007
s10545-009-9006-9
Stockinger B Di Meglio P Gialitakis M amp Duarte J H (2014) The
aryl hydrocarbon receptor Multitasking in the immune system
Annual Review of Immunology 32 403ndash432 httpsdoiorg101146
annurev-immunol-032713-120245
Storz J F amp Wheat C W (2010) Integrating evolutionary and
functional approaches to infer adaptation at specific loci Evolution
64 2489ndash2509 httpsdoiorg101111j1558-5646201001044x
Stuart Y E Veen T Weber J N Hanson D Ravinet M Lohman B
K Bolnick D I (2017) Contrasting effects of environment and
genetics generate a continuum of parallel evolution Nature Ecology amp
Evolution 1 0158 httpsdoiorg101038s41559-017-0158
Sunnucks P Morales H E Lamb A M Pavlova A amp Greening C
(2017) Integrative approaches for studying mitochondrial and nuclear
genome co-evolution in oxidative phosphorylation Frontiers in Genet-
ics 8 25
Tobler M amp Hastings L (2011) Convergent patterns of body shape dif-
ferentiation in four different clades of poeciliid fishes inhabiting sul-
fide springs Evolutionary Biology 38 412ndash421 httpsdoiorg10
1007s11692-011-9129-4
Tobler M Palacios M Chapman L J Mitrofanov I Bierbach D
Plath M Mateos M (2011) Evolution in extreme environments
Replicated phenotypic differentiation in livebearing fish inhabiting
sulfidic springs Evolution 65 2213ndash2228 httpsdoiorg101111j
1558-5646201101298x
Tobler M Passow C N Greenway R Kelley J L amp Shaw J H
(2016) The evolutionary ecology of animals inhabiting hydrogen sul-
fide-rich environments Annual Review of Ecology Evolution and Sys-
tematics 47 239ndash262 httpsdoiorg101146annurev-ecolsys-
121415-032418
Tobler M amp Plath M (2009a) Threatened fishes of the world Gambu-
sia eurystoma Miller 1975 (Poeciliidae) Environmental Biology of
Fishes 85 251 httpsdoiorg101007s10641-009-9481-8
Tobler M amp Plath M (2009b) Threatened fishes of the world Poecilia
sulphuraria (Alvarez 1948) (Poeciliidae) Environmental Biology of
Fishes 85 333ndash334 httpsdoiorg101007s10641-009-9506-3
Tobler M Plath M Riesch R Schlupp I Grasse A Munimanda G
Moodley Y (2014) Selection from parasites favors immunogenic diver-
sity but not divergence among locally adapted host populations Journal
of Evolutionary Biology 27 960ndash974 httpsdoiorg101111jeb12370
Tobler M Riesch R amp Plath M (2015) Extremophile fishes An inte-
grative synthesis In R Riesch M Tobler amp M Plath (Eds) Extremo-
phile fishes Ecology evolution and physiology of teleosts in extreme
environments (pp 279ndash296) Heidelberg Germany Springer
Tobler M Riesch R Tobler C M amp Plath M (2009) Compensatory
behaviour in response to sulfide-induced hypoxia affects time
budgets feeding efficiency and predation risk Evolutionary Ecology
Research 11 935ndash948
Tobler M Scharnweber K Greenway R Passow C Arias-Rodriguez
L amp Garcia-De-Leon F J (2015) Convergent changes in the
trophic ecology if extremophile fish along replicated environmental
gradients Freshwater Biology 60 768ndash780 httpsdoiorg10
1111fwb12530
Todgham A E amp Stillman J H (2013) Physiological responses to shifts
in multiple environmental stressors Relevance in a changing worlds
Integrative and Comparative Biology 53 539ndash544 httpsdoiorg10
1093icbict086
van Doorn G S Edelaar P amp Weissing F J (2009) On the origin of
species by natural selection and sexual selection Science 326 1704ndash
1707 httpsdoiorg101126science1181661
Wall D H amp Virginia R A (1999) Controls on soil biodiversity Insights
from extreme environments Applied Soil Ecology 13 137ndash150
httpsdoiorg101016S0929-1393(99)00029-3
Wallace J L amp Wang R (2015) Hydrogen-sulfide based therapeutics
Exploiting a unique but ubiquitous gasotransmitter Nature Reviews
Drug Discovery 14 329ndash345 httpsdoiorg101038nrd4433
Wang R (2012) Physiological implications of hydrogen sulfide A whiff
exploration that blossomed Physiological Reviews 92 791ndash896
httpsdoiorg101152physrev000172011
Wang Z Gerstein M amp Snyder M (2009) RNA-seq A revolutionary
tool for transcriptomics Nature Reviews Genetics 10 57ndash63
httpsdoiorg101038nrg2484
Waterman T H (1999) The evolutionary challenges of extreme environ-
ments (part 1) Journal of Experimental Zoology 285 326ndash359
httpsdoiorg101002(SICI)1097-010X(19991215)2854amplt326
AID-JEZ3ampgt30CO2-T
Werren J H Richards S Desjardins C A Niehuis O Gadau J Col-
bourne J K amp Group N G W (2010) Functional and evolutionary
insights from the genomes of three parasitoid Nasonia species
Science 327 343ndash348 httpsdoiorg101126science1178028
Whitehead A (2012) Comparative genomics in ecological physiology
Toward a more nuanced understanding of acclimation and adapta-
tion Journal of Experimental Biology 215 884ndash891 httpsdoiorg
101242jeb058735
Whitehead A amp Crawford D L (2006) Neutral and adaptive variation
in gene expression Proceedings of the National Academy of Sciences
USA 103 5425ndash5430 httpsdoiorg101073pnas0507648103
Willett C S amp Burton R S (2004) Evolution of interacting proteins in
the mitochondrial electron transport system in a marine copepod
Molecular Biology and Evolution 21 443ndash453 httpsdoiorg10
1093molbevmsh031
Willi Y Van Buskirk J amp Hoffmann A (2006) Limits to the adaptive
potential of small populations Annual Review of Ecology Evolution
and Systematics 37 433ndash458 httpsdoiorg101146annurevecol
sys37091305110145
Wolff J N Ladoukakis E D Enrıquez J A amp Dowling D K (2014)
Mitonuclear interactions Evolutionary consequences over multiple
biological scales Philosophical Transactions of the Royal Society B 369
20130443 httpsdoiorg101098rstb20130443
How to cite this article Tobler M Kelley JL Plath M Riesch
R Extreme environments and the origins of biodiversity
Adaptation and speciation in sulphide spring fishes Mol Ecol
201827843ndash859 httpsdoiorg101111mec14497
TOBLER ET AL | 859
mexicana) Molecular Ecology 24 5446ndash5459 httpsdoiorg10
1111mec13397
Pietri R Roman-Morales E amp Lopez-Garriga J (2011) Hydrogen sul-
fide and heme proteins Knowledge and mysteries Antioxidants amp
Redox Signaling 15 393ndash404 httpsdoiorg101089ars20103698
Plath M (2008) Male mating behavior and costs of sexual harassment
for females in cavernicolous and extremophile populations of Atlantic
mollies (Poecilia mexicana) Behaviour 145 73ndash98 httpsdoiorg10
1163156853908782687241
Plath M Hermann C Schreurooder R Riesch R Tobler M Garcia de
Leon F J Tiedemann R (2010) Locally adapted fish populations
maintain small-scale genetic differentiation despite perturbation by a
catastrophic flood event BMC Evolutionary Biology 10 256 httpsd
oiorg1011861471-2148-10-256
Plath M Pfenninger M Lerp H Riesch R Eschenbrenner C Slattery
P Tobler M (2013) Genetic differentiation and selection against
migrants in evolutionarily replicated extreme environments Evolution
67 2647ndash2661 httpsdoiorg101111evo12133
Plath M Tobler M Riesch R Garcia de Leon F J Giere O amp
Schlupp I (2007) Survival in an extreme habitat The role of beha-
viour and energy limitation Naturwissenschaften 94 991ndash996
httpsdoiorg101007s00114-007-0279-2
Powell E (1989) Oxygen sulfide and diffusion Why thiobiotic meiofauna
must be sulfide-insensitive first-order respirers Journal of Marine
Research 47 887ndash932 httpsdoiorg101357002224089785076082
Ramirez-Soriano A Ramos-Onsins S E Rozas J Calafell F amp Navarro A
(2008) Statistical power analysis of neutrality tests under demographic
expansions contractions and bottlenecks with recombination Genetics
179 555ndash567 httpsdoiorg101534genetics107083006
Reiffenstein R Hulbert W amp Roth S (1992) Toxicology of hydrogen
sulfide Annual Reviews of Pharmacology and Toxicology 1992 109ndash
134 httpsdoiorg101146annurevpa32040192000545
Reznick D N Furness A I Meredith R W amp Springer M S (2017)
The origin and biogeographic diversification of fishes in the family
Poeciliidae PLoS ONE 12 e0172546 httpsdoiorg101371journa
lpone0172546
Reznick D amp Yang A P (1993) The influence of fluctuating resources
on life history Patterns of allocation and plasticity in female guppies
Ecology 74 2011ndash2019 httpsdoiorg1023071940844
Riesch R Duwe V Herrmann N Padur L Ramm A Scharnweber K
Plath M (2009) Variation along the shy-bold continuum in extre-
mophile fishes (Poecilia mexicana Poecilia sulphuraria) Behavioral Ecol-
ogy and Sociobiology 63 1515ndash1526 httpsdoiorg101007
s00265-009-0780-z
Riesch R Oranth A Dzienko J Karau N Schiessl A Stadler S
Plath M (2010) Extreme habitats are not refuges Poeciliids suffer
from increased aerial predation risk in sulphidic southern Mexican
habitats Biological Journal of the Linnean Society 101 417ndash426
httpsdoiorg101111j1095-8312201001522x
Riesch R Plath M Garcia de Leon F J amp Schlupp I (2010) Conver-
gent life-history shifts Toxic environments result in big babies in two
clades of poeciliids Naturwissenschaften 97 133ndash141 httpsdoi
org101007s00114-009-0613-y
Riesch R Plath M Schlupp I Tobler M amp Langerhans R B (2014)
Colonization of toxic springs drives predictable life-history shift in
livebearing fishes (Poeciliidae) Ecology Letters 17 65ndash71 httpsdoi
org101111ele12209
Riesch R Tobler M Lerp H Jourdan J Doumas L T Nosil P Plath
M (2016) Extremophile Poeciliidae Multivariate insights into the com-
plexity of speciation along replicated ecological gradients BMC Evolu-
tionary Biology 16 136 httpsdoiorg101186s12862-016-0705-1
Riesch R Tobler M amp Plath M (2015) Extremophile fishes Ecology
evolution and physiology of teleosts in extreme environments p 290
Heidelberg Germany Springer httpsdoiorg101007978-3-319-
13362-1
Ritchie M E Holzinger E R Li R Pendergrass S A amp Kim S (2015)
Methods of integrating data to uncover genotype-phenotype interac-
tions Nature Reviews Genetics 16 85ndash97 httpsdoiorg101038
nrg3868
Roach K Tobler M amp Winemiller K O (2011) Hydrogen sulfide bac-
teria and fish A unique subterranean food chain Ecology 92 2056ndash
2062 httpsdoiorg10189011-02761
Rohner N Jarosz D F Kowalko J E Yoshizawa M Jeffery W R
Borowsky R L Tabin C J (2013) Cryptic variation in morpho-
logical evolution HSP90 as a capacitor for loss of eyes in cavefish
Science 342 1372ndash1375 httpsdoiorg101126science1240276
Rosales Lagarde L (2012) Investigation of karst brackish-sulfidic springs
and their role in the hydrogeology subsurface water-rock interactions
and speleogenesis at northern Sierra de Chiapas New Mexico Institute
of Mining and Technology Mexico
Rosenblum E B amp Harmon L J (2011) ldquoSame but differentrdquo Repli-
cated ecological speciation at White Sands Evolution 65 946ndash960
httpsdoiorg101111j1558-5646201001190x
Rosenblum E B Parent C E amp Brandt E E (2014) The molecular
basis of convergent evolution Annual Review of Ecology Evolution and
Systematics 45 203ndash226 httpsdoiorg101146annurev-ecolsys-
120213-091851
Saltz J B Hessel F C amp Kelly M W (2017) Trait correlations in the
genomics era Trends in Ecology amp Evolution 32 279ndash290 httpsdoi
org101016jtree201612008
Saraste M (1999) Oxidative phosphorylation at the fin de siecle Science
283 1488ndash1493 httpsdoiorg101126science28354071488
Schartl M (2014) Beyond the zebrafish Diverse fish species for model-
ing human disease Disease Models and Mechanisms 7 181ndash192
httpsdoiorg101242dmm012245
Schartl M Walter R B Shen Y Garcia T Catchen J Amores A
Warren W C (2013) The genome of the platyfish Xiphophorus mac-
ulatus provides insights into evolutionary adaptation and several
complex traits Nature Genetics 45 567ndash572 httpsdoiorg10
1038ng2604
Schluter D (2009) Evidence for ecological speciation and its alternative
Science 323 737ndash741 httpsdoiorg101126science1160006
Schulz-Mirbach T Eifert C Riesch R Farnworth M S Zimmer C
Bierbach D Plath M (2016) Toxic hydrogen sulphide shapes
brain anatomy A comparative study of sulphide-adapted ecotypes in
the Poecilia mexicana complex Journal of Zoology 300 163ndash176
httpsdoiorg101111jzo12366
SEDESOL (1994) NORMA Oficial Mexicana NOM-059-ECOL-1994 que
determina las especies y subspecies de flora y fauna silvestres ter-
restres y acuaticas en peligro de extincion amenazadas raras y las
sujetas a proteccion especial y que establece especificaciones para
su proteccion Diario Oficial 16 May 1994 488 2ndash60
Servedio M R van Doorn G S Kopp M Frame A M amp Nosil P
(2011) Magic traits in speciation lsquomagicrsquo but not rare Trends in Ecology
amp Evolution 26 389ndash397 httpsdoiorg101016jtree201104005
Shahak Y amp Hauska G (2008) Sulfide oxidation from cyanobacteria to
humans Sulfide-quinone oxidoreductase (SQR) In R Hell C Dahl D
B Knaff amp Th Leustek (Eds) Advances in photosynthesis and respira-
tion (pp 319ndash335) Heidelberg Springer
Shalem O Sanjana N E amp Zhang F (2015) High-throughput func-
tional genomics using CRISPR-Cas9 Nature Reviews Genetics 16
299ndash311 httpsdoiorg101038nrg3899
Silva G J J Bye A el Azzouzi H amp Wisloslashff U (2017) MicroRNAs as
important regulators of exercise adaptation Progress in Cardiovascular
Diseases 60 130ndash151 httpsdoiorg101016jpcad201706003
Simola D F Wissler L Donahue G Waterhouse R M Helmkampf
M Roux J Viljakainen L (2013) Social insect genomes exhibit
dramatic evolution in gene composition and regulation while preserv-
ing regulatory features linked to sociality Genome Research 23
1235ndash1247 httpsdoiorg101101gr155408113
858 | TOBLER ET AL
Sommer-Trembo C Bierbach D Arias Rodriguez L Verel Y Jourdan
J Zimmer C Plath M (2016) Does personality affect premating
isolation between locally-adapted populations BMC Evolutionary Biol-
ogy 16 138 httpsdoiorg101186s12862-016-0712-2
Steindachner F (1863) Beitreuroage zur Kentniss der Sciaenoiden Brasiliens
und der Cyprinodonten Mejicos Sitzungsberichte der Mathematisch-
Naturwissenschaftlichen Classe der Kaiserlichen Akademie der Wis-
senschaften 48 162ndash185
Stinchcombe J R amp Hoekstra H E (2008) Combining population geno-
mics and quantitative genetics Finding genes underlying ecologically
important traits Heredity 100 158ndash170 httpsdoiorg101038sj
hdy6800937
Stipanuk M H (2004) Sulfur amino acid metabolism Pathways for pro-
duction and removal of homocysteine and cysteine Annual Review of
Nutrition 24 539ndash577 httpsdoiorg101146annurevnutr24
012003132418
Stipanuk M H amp Ueki I (2011) Dealing with methioninehomocysteine
sulfur Cysteine metabolism to taurine and inorganic sulfur Journal of
Inherited Metabolic Disease 34 17ndash32 httpsdoiorg101007
s10545-009-9006-9
Stockinger B Di Meglio P Gialitakis M amp Duarte J H (2014) The
aryl hydrocarbon receptor Multitasking in the immune system
Annual Review of Immunology 32 403ndash432 httpsdoiorg101146
annurev-immunol-032713-120245
Storz J F amp Wheat C W (2010) Integrating evolutionary and
functional approaches to infer adaptation at specific loci Evolution
64 2489ndash2509 httpsdoiorg101111j1558-5646201001044x
Stuart Y E Veen T Weber J N Hanson D Ravinet M Lohman B
K Bolnick D I (2017) Contrasting effects of environment and
genetics generate a continuum of parallel evolution Nature Ecology amp
Evolution 1 0158 httpsdoiorg101038s41559-017-0158
Sunnucks P Morales H E Lamb A M Pavlova A amp Greening C
(2017) Integrative approaches for studying mitochondrial and nuclear
genome co-evolution in oxidative phosphorylation Frontiers in Genet-
ics 8 25
Tobler M amp Hastings L (2011) Convergent patterns of body shape dif-
ferentiation in four different clades of poeciliid fishes inhabiting sul-
fide springs Evolutionary Biology 38 412ndash421 httpsdoiorg10
1007s11692-011-9129-4
Tobler M Palacios M Chapman L J Mitrofanov I Bierbach D
Plath M Mateos M (2011) Evolution in extreme environments
Replicated phenotypic differentiation in livebearing fish inhabiting
sulfidic springs Evolution 65 2213ndash2228 httpsdoiorg101111j
1558-5646201101298x
Tobler M Passow C N Greenway R Kelley J L amp Shaw J H
(2016) The evolutionary ecology of animals inhabiting hydrogen sul-
fide-rich environments Annual Review of Ecology Evolution and Sys-
tematics 47 239ndash262 httpsdoiorg101146annurev-ecolsys-
121415-032418
Tobler M amp Plath M (2009a) Threatened fishes of the world Gambu-
sia eurystoma Miller 1975 (Poeciliidae) Environmental Biology of
Fishes 85 251 httpsdoiorg101007s10641-009-9481-8
Tobler M amp Plath M (2009b) Threatened fishes of the world Poecilia
sulphuraria (Alvarez 1948) (Poeciliidae) Environmental Biology of
Fishes 85 333ndash334 httpsdoiorg101007s10641-009-9506-3
Tobler M Plath M Riesch R Schlupp I Grasse A Munimanda G
Moodley Y (2014) Selection from parasites favors immunogenic diver-
sity but not divergence among locally adapted host populations Journal
of Evolutionary Biology 27 960ndash974 httpsdoiorg101111jeb12370
Tobler M Riesch R amp Plath M (2015) Extremophile fishes An inte-
grative synthesis In R Riesch M Tobler amp M Plath (Eds) Extremo-
phile fishes Ecology evolution and physiology of teleosts in extreme
environments (pp 279ndash296) Heidelberg Germany Springer
Tobler M Riesch R Tobler C M amp Plath M (2009) Compensatory
behaviour in response to sulfide-induced hypoxia affects time
budgets feeding efficiency and predation risk Evolutionary Ecology
Research 11 935ndash948
Tobler M Scharnweber K Greenway R Passow C Arias-Rodriguez
L amp Garcia-De-Leon F J (2015) Convergent changes in the
trophic ecology if extremophile fish along replicated environmental
gradients Freshwater Biology 60 768ndash780 httpsdoiorg10
1111fwb12530
Todgham A E amp Stillman J H (2013) Physiological responses to shifts
in multiple environmental stressors Relevance in a changing worlds
Integrative and Comparative Biology 53 539ndash544 httpsdoiorg10
1093icbict086
van Doorn G S Edelaar P amp Weissing F J (2009) On the origin of
species by natural selection and sexual selection Science 326 1704ndash
1707 httpsdoiorg101126science1181661
Wall D H amp Virginia R A (1999) Controls on soil biodiversity Insights
from extreme environments Applied Soil Ecology 13 137ndash150
httpsdoiorg101016S0929-1393(99)00029-3
Wallace J L amp Wang R (2015) Hydrogen-sulfide based therapeutics
Exploiting a unique but ubiquitous gasotransmitter Nature Reviews
Drug Discovery 14 329ndash345 httpsdoiorg101038nrd4433
Wang R (2012) Physiological implications of hydrogen sulfide A whiff
exploration that blossomed Physiological Reviews 92 791ndash896
httpsdoiorg101152physrev000172011
Wang Z Gerstein M amp Snyder M (2009) RNA-seq A revolutionary
tool for transcriptomics Nature Reviews Genetics 10 57ndash63
httpsdoiorg101038nrg2484
Waterman T H (1999) The evolutionary challenges of extreme environ-
ments (part 1) Journal of Experimental Zoology 285 326ndash359
httpsdoiorg101002(SICI)1097-010X(19991215)2854amplt326
AID-JEZ3ampgt30CO2-T
Werren J H Richards S Desjardins C A Niehuis O Gadau J Col-
bourne J K amp Group N G W (2010) Functional and evolutionary
insights from the genomes of three parasitoid Nasonia species
Science 327 343ndash348 httpsdoiorg101126science1178028
Whitehead A (2012) Comparative genomics in ecological physiology
Toward a more nuanced understanding of acclimation and adapta-
tion Journal of Experimental Biology 215 884ndash891 httpsdoiorg
101242jeb058735
Whitehead A amp Crawford D L (2006) Neutral and adaptive variation
in gene expression Proceedings of the National Academy of Sciences
USA 103 5425ndash5430 httpsdoiorg101073pnas0507648103
Willett C S amp Burton R S (2004) Evolution of interacting proteins in
the mitochondrial electron transport system in a marine copepod
Molecular Biology and Evolution 21 443ndash453 httpsdoiorg10
1093molbevmsh031
Willi Y Van Buskirk J amp Hoffmann A (2006) Limits to the adaptive
potential of small populations Annual Review of Ecology Evolution
and Systematics 37 433ndash458 httpsdoiorg101146annurevecol
sys37091305110145
Wolff J N Ladoukakis E D Enrıquez J A amp Dowling D K (2014)
Mitonuclear interactions Evolutionary consequences over multiple
biological scales Philosophical Transactions of the Royal Society B 369
20130443 httpsdoiorg101098rstb20130443
How to cite this article Tobler M Kelley JL Plath M Riesch
R Extreme environments and the origins of biodiversity
Adaptation and speciation in sulphide spring fishes Mol Ecol
201827843ndash859 httpsdoiorg101111mec14497
TOBLER ET AL | 859
Sommer-Trembo C Bierbach D Arias Rodriguez L Verel Y Jourdan
J Zimmer C Plath M (2016) Does personality affect premating
isolation between locally-adapted populations BMC Evolutionary Biol-
ogy 16 138 httpsdoiorg101186s12862-016-0712-2
Steindachner F (1863) Beitreuroage zur Kentniss der Sciaenoiden Brasiliens
und der Cyprinodonten Mejicos Sitzungsberichte der Mathematisch-
Naturwissenschaftlichen Classe der Kaiserlichen Akademie der Wis-
senschaften 48 162ndash185
Stinchcombe J R amp Hoekstra H E (2008) Combining population geno-
mics and quantitative genetics Finding genes underlying ecologically
important traits Heredity 100 158ndash170 httpsdoiorg101038sj
hdy6800937
Stipanuk M H (2004) Sulfur amino acid metabolism Pathways for pro-
duction and removal of homocysteine and cysteine Annual Review of
Nutrition 24 539ndash577 httpsdoiorg101146annurevnutr24
012003132418
Stipanuk M H amp Ueki I (2011) Dealing with methioninehomocysteine
sulfur Cysteine metabolism to taurine and inorganic sulfur Journal of
Inherited Metabolic Disease 34 17ndash32 httpsdoiorg101007
s10545-009-9006-9
Stockinger B Di Meglio P Gialitakis M amp Duarte J H (2014) The
aryl hydrocarbon receptor Multitasking in the immune system
Annual Review of Immunology 32 403ndash432 httpsdoiorg101146
annurev-immunol-032713-120245
Storz J F amp Wheat C W (2010) Integrating evolutionary and
functional approaches to infer adaptation at specific loci Evolution
64 2489ndash2509 httpsdoiorg101111j1558-5646201001044x
Stuart Y E Veen T Weber J N Hanson D Ravinet M Lohman B
K Bolnick D I (2017) Contrasting effects of environment and
genetics generate a continuum of parallel evolution Nature Ecology amp
Evolution 1 0158 httpsdoiorg101038s41559-017-0158
Sunnucks P Morales H E Lamb A M Pavlova A amp Greening C
(2017) Integrative approaches for studying mitochondrial and nuclear
genome co-evolution in oxidative phosphorylation Frontiers in Genet-
ics 8 25
Tobler M amp Hastings L (2011) Convergent patterns of body shape dif-
ferentiation in four different clades of poeciliid fishes inhabiting sul-
fide springs Evolutionary Biology 38 412ndash421 httpsdoiorg10
1007s11692-011-9129-4
Tobler M Palacios M Chapman L J Mitrofanov I Bierbach D
Plath M Mateos M (2011) Evolution in extreme environments
Replicated phenotypic differentiation in livebearing fish inhabiting
sulfidic springs Evolution 65 2213ndash2228 httpsdoiorg101111j
1558-5646201101298x
Tobler M Passow C N Greenway R Kelley J L amp Shaw J H
(2016) The evolutionary ecology of animals inhabiting hydrogen sul-
fide-rich environments Annual Review of Ecology Evolution and Sys-
tematics 47 239ndash262 httpsdoiorg101146annurev-ecolsys-
121415-032418
Tobler M amp Plath M (2009a) Threatened fishes of the world Gambu-
sia eurystoma Miller 1975 (Poeciliidae) Environmental Biology of
Fishes 85 251 httpsdoiorg101007s10641-009-9481-8
Tobler M amp Plath M (2009b) Threatened fishes of the world Poecilia
sulphuraria (Alvarez 1948) (Poeciliidae) Environmental Biology of
Fishes 85 333ndash334 httpsdoiorg101007s10641-009-9506-3
Tobler M Plath M Riesch R Schlupp I Grasse A Munimanda G
Moodley Y (2014) Selection from parasites favors immunogenic diver-
sity but not divergence among locally adapted host populations Journal
of Evolutionary Biology 27 960ndash974 httpsdoiorg101111jeb12370
Tobler M Riesch R amp Plath M (2015) Extremophile fishes An inte-
grative synthesis In R Riesch M Tobler amp M Plath (Eds) Extremo-
phile fishes Ecology evolution and physiology of teleosts in extreme
environments (pp 279ndash296) Heidelberg Germany Springer
Tobler M Riesch R Tobler C M amp Plath M (2009) Compensatory
behaviour in response to sulfide-induced hypoxia affects time
budgets feeding efficiency and predation risk Evolutionary Ecology
Research 11 935ndash948
Tobler M Scharnweber K Greenway R Passow C Arias-Rodriguez
L amp Garcia-De-Leon F J (2015) Convergent changes in the
trophic ecology if extremophile fish along replicated environmental
gradients Freshwater Biology 60 768ndash780 httpsdoiorg10
1111fwb12530
Todgham A E amp Stillman J H (2013) Physiological responses to shifts
in multiple environmental stressors Relevance in a changing worlds
Integrative and Comparative Biology 53 539ndash544 httpsdoiorg10
1093icbict086
van Doorn G S Edelaar P amp Weissing F J (2009) On the origin of
species by natural selection and sexual selection Science 326 1704ndash
1707 httpsdoiorg101126science1181661
Wall D H amp Virginia R A (1999) Controls on soil biodiversity Insights
from extreme environments Applied Soil Ecology 13 137ndash150
httpsdoiorg101016S0929-1393(99)00029-3
Wallace J L amp Wang R (2015) Hydrogen-sulfide based therapeutics
Exploiting a unique but ubiquitous gasotransmitter Nature Reviews
Drug Discovery 14 329ndash345 httpsdoiorg101038nrd4433
Wang R (2012) Physiological implications of hydrogen sulfide A whiff
exploration that blossomed Physiological Reviews 92 791ndash896
httpsdoiorg101152physrev000172011
Wang Z Gerstein M amp Snyder M (2009) RNA-seq A revolutionary
tool for transcriptomics Nature Reviews Genetics 10 57ndash63
httpsdoiorg101038nrg2484
Waterman T H (1999) The evolutionary challenges of extreme environ-
ments (part 1) Journal of Experimental Zoology 285 326ndash359
httpsdoiorg101002(SICI)1097-010X(19991215)2854amplt326
AID-JEZ3ampgt30CO2-T
Werren J H Richards S Desjardins C A Niehuis O Gadau J Col-
bourne J K amp Group N G W (2010) Functional and evolutionary
insights from the genomes of three parasitoid Nasonia species
Science 327 343ndash348 httpsdoiorg101126science1178028
Whitehead A (2012) Comparative genomics in ecological physiology
Toward a more nuanced understanding of acclimation and adapta-
tion Journal of Experimental Biology 215 884ndash891 httpsdoiorg
101242jeb058735
Whitehead A amp Crawford D L (2006) Neutral and adaptive variation
in gene expression Proceedings of the National Academy of Sciences
USA 103 5425ndash5430 httpsdoiorg101073pnas0507648103
Willett C S amp Burton R S (2004) Evolution of interacting proteins in
the mitochondrial electron transport system in a marine copepod
Molecular Biology and Evolution 21 443ndash453 httpsdoiorg10
1093molbevmsh031
Willi Y Van Buskirk J amp Hoffmann A (2006) Limits to the adaptive
potential of small populations Annual Review of Ecology Evolution
and Systematics 37 433ndash458 httpsdoiorg101146annurevecol
sys37091305110145
Wolff J N Ladoukakis E D Enrıquez J A amp Dowling D K (2014)
Mitonuclear interactions Evolutionary consequences over multiple
biological scales Philosophical Transactions of the Royal Society B 369
20130443 httpsdoiorg101098rstb20130443
How to cite this article Tobler M Kelley JL Plath M Riesch
R Extreme environments and the origins of biodiversity
Adaptation and speciation in sulphide spring fishes Mol Ecol
201827843ndash859 httpsdoiorg101111mec14497
TOBLER ET AL | 859