Human disturbance causes the formation of a hybrid swarm … · 2018-03-13 · Human disturbance...
Transcript of Human disturbance causes the formation of a hybrid swarm … · 2018-03-13 · Human disturbance...
Human disturbance causes the formation of a hybridswarm between two naturally sympatric fish species
DANIEL J . HASSELMAN,* EMILY E. ARGO,* MEGHAN C. MCBRIDE,† PAUL BENTZEN,†
THOMAS F. SCHULTZ,‡ ANNA A. PEREZ-UMPHREY‡ and ERIC P. PALKOVACS*
*Department of Ecology and Evolutionary Biology, University of California, Santa Cruz, CA 95060, USA, †Marine Gene Probe
Laboratory, Biology Department, Dalhousie University, Halifax, NS B3H 4R2, Canada, ‡Marine Conservation Molecular
Facility, Duke University Marine Laboratory, Beaufort, NC 28516, USA
Abstract
Most evidence for hybrid swarm formation stemming from anthropogenic habitat dis-
turbance comes from the breakdown of reproductive isolation between incipient spe-
cies, or introgression between allopatric species following secondary contact. Human
impacts on hybridization between divergent species that naturally occur in sympatry
have received considerably less attention. Theory predicts that reinforcement should
act to preserve reproductive isolation under such circumstances, potentially making
reproductive barriers resistant to human habitat alteration. Using 15 microsatellites, we
examined hybridization between sympatric populations of alewife (Alosa pseudoharen-gus) and blueback herring (A. aestivalis) to test whether the frequency of hybridiza-
tion and pattern of introgression have been impacted by the construction of a dam that
isolated formerly anadromous populations of both species in a landlocked freshwater
reservoir. The frequency of hybridization and pattern of introgression differed mark-
edly between anadromous and landlocked populations. The rangewide frequency of
hybridization among anadromous populations was generally 0–8%, whereas all land-
locked individuals were hybrids. Although neutral introgression was observed among
anadromous hybrids, directional introgression leading to increased prevalence of ale-
wife genotypes was detected among landlocked hybrids. We demonstrate that habitat
alteration can lead to hybrid swarm formation between divergent species that naturally
occur sympatrically, and provide empirical evidence that reinforcement does not
always sustain reproductive isolation under such circumstances.
Keywords: Alosa, anadromous, hybrid swarm, hybridization, introgression, landlocked
Received 30 October 2013; revision received 13 January 2014; accepted 17 January 2014
Introduction
Much of the world’s biodiversity is of recent evolution-
ary origin and is sustained by divergent adaptation in
heterogeneous environments (Rundle & Nosil 2005;
Seehausen et al. 2008). However, rugged adaptive land-
scapes that promote and maintain diversification in nat-
ure can be smoothed by human activities, hindering
adaptive radiation (Hendry et al. 2006; De Le�on et al.
2011) and contributing to the loss of evolutionary
lineages through ‘reverse speciation’ (Seehausen 2006;
Taylor et al. 2006; Seehausen et al. 2008).
Most evidence for reverse speciation comes from
studies of introgressive hybridization between incipient
fish species that have recently (i.e. postglacially)
diverged in sympatry through disruptive selection in
heterogeneous environments (e.g. threespine stickleback
benthic–limnetic pairs (Taylor et al. 2006); Laurentian
Great Lakes ciscoes (Seehausen et al. 2008); European
whitefish (Vonlanthen et al. 2012); African cichlids
(Seehausen et al. 1997)). Although reproductive isolation
between incipient species may be incomplete, diver-
gence can be reinforced through prezygotic (e.g. assor-
tative mating) and postzygotic (e.g. reduced hybridCorrespondence: Daniel J. Hasselman, Fax: (831) 459 3833;
E-mail: [email protected]
© 2014 John Wiley & Sons Ltd
Molecular Ecology (2014) 23, 1137–1152 doi: 10.1111/mec.12674
fitness) isolating mechanisms when gene flow is low
(Gow et al. 2006). However, the loss of environmental
heterogeneity can relax disruptive selection and elimi-
nate reproductive isolating mechanisms, facilitating
introgressive hybridization and the collapse of incipient
species into a hybrid swarm (Seehausen et al. 2008).
Indeed, several studies have shown how habitat
homogenization through human activities (e.g. species
introductions, eutrophication) can weaken assortative
mating and facilitate reverse speciation for multiple
incipient species (Todd & Stedman 1989; Seehausen
et al. 1997; Behm et al. 2010).
Introgressive hybridization stemming from anthropo-
genic habitat disturbance also threatens the integrity of
species with deeper evolutionary histories (Wiegand
1935; Anderson 1948; Rhymer & Simberloff 1996). For
example, introgression between grey wolf (Canis lupus)
and coyote (C. latrans), species that diverged 1.4–
2.1 million years ago, occurs across a broad range
where forests have been converted to agricultural land
(Lehman et al. 1991). Similarly, despite a 2.6-million-
year evolutionary history of ecologically maintained
parapatry, hybridization between forest elephant
(Loxodonta cyclotis) and savannah elephant (L. africana)
threatens species integrity in regions of ongoing defor-
estation in Africa (Roca et al. 2005). However, the for-
mation of a hybrid swarm resulting from the
breakdown of reproductive isolation between divergent
species that naturally occur in sympatry is not well
established (Seehausen 2006). Indeed, under these
conditions, theory predicts that reinforcement should
sustain reproductive isolation, potentially making repro-
ductive barriers resistant to habitat disturbance.
The classic definition of reinforcement implies selec-
tion against hybrids through a variety of intrinsic (e.g.
hybrid inviability, sterility, behavioural dysfunctions) or
extrinsic (e.g. lowered fitness associated with a specific
set of ecological conditions) mechanisms (see Rundle &
Schluter 1998). However, this restrictive definition does
not take into account processes that reflect classic crite-
ria for reinforcement (see Servedio & Noor 2003). Here,
we refer to reinforcement in the ‘broad sense’ (sensu
Servedio & Noor 2003), where reinforcement constitutes
‘an increase in prezygotic isolation between hybridizing
[species] in response to any type of selection against
interspecific matings, regardless of whether hybrids
themselves [exhibit reduced fitness]’. Under this defini-
tion, reinforcement could manifest as reproductive iso-
lation via spatiotemporal differences in breeding for
divergent species in sympatry.
River herrings (alewife, Alosa pseudoharengus, and
blueback herring, A. aestivalis) provide an opportunity
to empirically examine hybridization between divergent
lineages that naturally occur in sympatry, and in
response to anthropogenic habitat disturbance. Alewife
and blueback herring are anadromous (migratory sea-
run) species native to the Atlantic coast of North Amer-
ica. Although they co-occur in rivers over much of their
historical range (alewife: Labrador to South Carolina;
blueback herring: Gulf of St. Lawrence to Florida),
reproductive isolation is maintained by differences in
spawning time (temperature dependent) and spawning
habitat (reviewed by Loesch 1987). In terms of timing,
alewife spawn at cooler temperatures (5–10 °C) than
blueback herring (10–15 °C) and typically begin spawn-
ing 3–4 weeks earlier. However, peak spawn timing
only differs by 2–3 weeks, resulting in considerable
temporal overlap in freshwater (Loesch 1987). In terms
of spawning habitat, alewife preferentially select lentic
(i.e. still-water) habitats for spawning, whereas blueback
herring prefer lotic (i.e. flowing-water) habitats (Loesch
1987). Prior molecular research revealed that these spe-
cies diverged up to 1 million years ago (Faria et al.
2006), but also detected a shared mitochondrial DNA
haplotype, suggesting either incomplete lineage sorting
(Chapman et al. 1994) or introgressive hybridization
(Faria et al. 2006).
A dam constructed on the Roanoke River in 1953
created novel conditions for river herring in Kerr Reser-
voir (North Carolina/Virginia, USA). Alewife have
repeatedly established landlocked populations in lakes
(Palkovacs et al. 2008), and landlocked blueback herring
populations persist in several reservoirs following intro-
duction (Prince & Barwick 1981; Guest & Drenner
1991). To our knowledge, Kerr Reservoir is the only
instance where these species have become landlocked
in sympatry. Further, the absence of fish passage has
prevented the immigration of anadromous alewife and
blueback herring for approximately 60 years. Thus, Kerr
Reservoir provides a unique opportunity to examine
the effects of anthropogenic habitat disturbance on the
frequency of hybridization and pattern of introgression
between divergent species that naturally occur in symp-
atry. Alewife and blueback herring populations have
declined dramatically in recent decades (Limburg &
Waldman 2009; Hall et al. 2012), making these species a
conservation concern (Atlantic States Marine Fisheries
Commission 2012; Palkovacs et al. 2013). Evidence for
human impacts on hybridization could hold important
implications in setting future conservation guidelines
(Allendorf et al. 2001) and for prioritizing river restora-
tion projects through dam removal.
Using 15 microsatellite loci, we (i) examine the extent
and spatial distribution of hybridization between anadro-
mous alewife and blueback herring across both species’
ranges, including Kerr Reservoir, (ii) determine whether
the frequency of hybridization and pattern of introgres-
sion differ between anadromous and landlocked
© 2014 John Wiley & Sons Ltd
1138 D. J . HASSELMAN ET AL.
scenarios and (iii) resolve whether anthropogenic habitat
disturbance can result in the formation of a hybrid
swarm between divergent species that naturally occur in
sympatry.
Materials and methods
Sample collections
Sampling was conducted across the ranges of both spe-
cies, and targeted a minimum of 50 individuals per col-
lection (i.e. river�year�1). Individuals were initially
assigned to species based on diagnostic peritoneal col-
oration (Jordan & Evermann 1896; Scott & Crossman
1973; Messieh 1977), but were ultimately identified as
alewife, blueback herring or hybrid using Bayesian clus-
tering analyses (see below). Collections were replicated
for a subset of rivers over successive years and pooled
following an analysis of molecular variance (AMOVA) that
indicated nonsignificant (P > 0.05) genetic variation
among years within rivers (Palkovacs et al. 2013). Sam-
ples were obtained in freshwater from adult specimens
on their spawning run, with the exception of those
collected from Veazie Dam and Souadabscook Falls
(Penobscot River) that comprised young-of-the-year
specimens. Sampling effort from 2005 to 2012 provided
tissue for 5358 alewife and 1529 blueback herring
from 56 to 30 anadromous populations, respectively, as
well as 76 and 43 presumed alewife and blueback
herring, respectively, from Kerr Reservoir (Table 1;
Fig. 1). Tissue was preserved in 95% ethanol until DNA
extraction.
Laboratory protocols
DNA extraction and genotyping. Laboratory procedures
were conducted at separate institutions (i.e. Dalhousie
University and Duke University Marine Laboratory) as
parts of independent studies of river herring genetic
variation in the northeastern USA and Canada (McBride
2013) and the USA (Labbe 2012; Palkovacs et al. 2013).
Details regarding DNA isolation and genotyping proto-
cols were reported previously (Palkovacs et al. 2013;
McBride 2013). We examined the variation at 21 poly-
morphic microsatellites: 15 loci developed for alewife
(Ap010, Ap033, Ap037, Ap038, Ap047, Ap058, Ap070,
Ap071) and blueback herring (Aa046, Aa070, Aa074,
Aa081, Aa082, Aa091, Aa093) (A’Hara et al. 2012) that
were common across studies and six additional loci
developed for alewife (Aps1, Aps2A; Bentzen & Paterson
2005), blueback herring (Aa039; A’Hara et al. 2012) and
American shad (A. sapidissima) [AsaD042, AsaC249
(Julian & Bartron 2007), Asa8 (Waters et al. 2000)] that
were examined by McBride (2013) alone. The data sets
of McBride (2013) and Palkovacs et al. (2013) were
combined after a correction factor for allele scores had
been applied for each locus common to both studies.
Briefly, small (30 ll) aliquots of pure genomic DNA
for alewife (n = 46) and blueback herring (n = 44) of
known genotype originally scored at Dalhousie Uni-
versity were genotyped at the Duke University Marine
Laboratory following the protocol of Palkovacs et al.
(2013). These data were then used to establish a cor-
rection factor to adjust the allele scores of the McBride
(2013) data set.
Data analyses
Overview. Following conformance of the data to model
assumptions, we simulated data for purebred alewife
and blueback herring and various hybrid classes (F1, F2
and backcrosses) using confirmed purebred individuals
from the empirical data set (identified using Bayesian
methods; see below). We then analysed the simulated
data using two Bayesian methods to determine the
appropriate threshold (Tq) for designating individuals
as either purebred or admixed. Next, we applied Tq to
the empirical data set to identify purebred and admixed
individuals, examined the extent of hybridization across
the ranges of alewife and blueback herring and deter-
mined whether the frequency of hybridization differed
between anadromous and landlocked scenarios. We
then used the genomic clines method (Gompert &
Buerkle 2009) to identify whether the pattern of intro-
gression differed among anadromous and landlocked
hybrids.
Data conformance to model assumptions. Genotyping arte-
facts were assessed using MICROCHECKER version 2.2.3
(Van Oosterhout et al. 2004). Departures from Hardy–
Weinberg equilibrium (HWE) and linkage disequilib-
rium (LD) were assessed within rivers (where n > 20)
for each species using GENEPOP version 4.0.6 (Rousset
2007) with default parameters for all tests. Sequential
Bonferroni adjustments were used to judge significance
levels for all simultaneous tests (Holm 1979; Rice 1989).
Selective neutrality of the microsatellite markers was
confirmed previously (Palkovacs et al. 2013).
Simulations and hybrid identification. Hybrid identifica-
tion depends on the genetic markers used and the
degree of differentiation between parental species
(Anderson & Thompson 2002; V€ah€a & Primmer 2006).
To assess the utility of each microsatellite locus to dis-
criminate alewife and blueback herring, we identified
purebred individuals using Bayesian clustering methods
(see below) and estimated the Kullback–Leibler diver-
gence (Kullback & Leibler 1951) following Anderson &
© 2014 John Wiley & Sons Ltd
HYBRIDIZATION AND INTROGRESSION IN ALOSINES 1139
Table 1 Sampling locations and sample sizes (N) for alewife and blueback herring collected from across the species’ ranges from
2005 to 2012. Specimens were initially classified to species at the time of collection using peritoneal coloration and were later con-
firmed (or adjusted) based on results of Bayesian clustering analysis of microsatellite data
Watershed Code Latitude 2005 2008 2009 2010 2011 2012 Unknown Total N
Alewife
Miramichi River MIR 47.1419 54 54
Richibucto River RIC 46.6914 63 63
Tidnish River TID 45.9900 63 63
River Phillip RPH 45.7928 57 57
River John RJO 45.7539 117 117
Hillsborough River HIL 46.2403 60 60
Tracadie Bay TRA 46.3828 60 60
Margaree River MAR 46.4300 51 51
Bras d’Or Lakes BRA 45.8536 156 156
West River WES 44.9269 51 51
Sullivan Pond Outlet SUL 44.9269 52 52
Sackville River SAK 44.7292 71 71
LaHave River LHV 44.2928 56 56
Medway River MED 44.1339 51 51
Mersey River MER 44.0419 57 57
Tusket River TUS 43.8756 155 155
Gaspereau River GAS 45.0894 58 58
Shubenacadie River SHU 45.2603 51 51
Petitcodiac River PET 45.9053 49 49
Saint John River SJR 45.2594 50 50
St. Croix River STC 45.1911 111 25 136
Dennys River DEN 44.9089 30 30
East Machias River EMA 44.5419 59 59
Narraguagus River NAR 44.4906 30 30
West Bay Pond WBP 44.3583 30 30
Mt. Desert Island MDI 44.5011 29 29
Union River UNI 44.4408 59 28 49 136
Penobscot River PEN 44.4408 213 232 230 675
St. George River STG 43.9592 50 132 51 233
Damariscotta River DAM 44.0294 60 85 53 198
Sheepscot River SHE 44.0219 28 28
Kennebec River KEN 43.7519 44 323 321 311 999
Androscoggin River AND 43.9147 59 86 51 196
Presumpscot River PRE 43.7153 28 28
Saco River SAC 43.4669 29 29
Piscataqua River PIS 43.0664 47 74 121
Mystic River MYS 42.3439 20 49 69
Monument River MON 41.7244 46 46
Town Brook TOW 41.9625 49 49
Taunton River TAU 41.6094 89 89
Gilbert-Stuart GIL 41.4472 44 44
Thames River THA 41.3256 36 30 66
Bride Brook BRI 41.3006 34 27 61
Connecticut River CON 41.2828 7 26 33
Quinnipiac River QUI 41.2722 1 25 35 61
Housatonic River HOU 41.1708 13 25 38
Pequonnock River PEQ 41.1736 25 25
Mianus River MIA 41.0156 25 32 57
Hudson River HUD 40.6958 13 48 61
Delaware River DEL 39.1122 45 45
Nanticoke River NAN 38.1689 58 58
Rappahannock River RAP 37.4933 62 62
James River JAM 36.9686 1 1
© 2014 John Wiley & Sons Ltd
1140 D. J . HASSELMAN ET AL.
Thompson (2002) and as implemented in the ‘flexmix’
package in R (R Development Core Team 2010). We also
estimated a multilocus global measure of FST (h; Weir &
Cockerham 1984) between the species using FSTAT ver-
sion 2.9.3.2 (Goudet 2001).
To maximize the accuracy of delineating purebred
and hybrid individuals, we conducted admixture analy-
ses using two Bayesian clustering methods imple-
mented in STRUCTURE version 2.3.3 (Pritchard et al. 2000;
Falush et al. 2003) and NEWHYBRIDS version 1.0 (Anderson
& Thompson 2002). Each method probabilistically
assigns multilocus genotypes to clusters using a Markov
chain Monte Carlo (MCMC) simulation procedure that
provides estimates from the posterior distribution that
reflects the membership of each individual. For STRUC-
TURE, the posterior probability (q) describes the propor-
tion of an individual genotype originating from each of
K clusters. For NEWHYBRIDS, q describes the probability
that an individual belongs to each of six different geno-
type frequency classes (i.e. parental purebreds, F1
hybrid, F2 hybrid and backcrosses). For both programs,
we ran 10 iterations of a parameter set that included a
burn-in of 50 000 steps followed by 250 000 replicates
of the MCMC simulation. For STRUCTURE, we set K = 2
(i.e. assuming two species contributed to the gene pool
of the sample) and employed the admixture model and
correlated allele frequencies. For NEWHYBRIDS, we used
uninformative priors for both the allele frequency and
admixture distributions.
An important consideration when using Bayesian
methods to identify admixed individuals is the choice of
optimal threshold value (Tq) for the q associated with
their assignment as purebred or hybrid (V€ah€a & Primmer
2006). For both methods, we initially used q ≥ 0.9
Table 1 Continued
Watershed Code Latitude 2005 2008 2009 2010 2011 2012 Unknown Total N
Chowan River CHO 36.0811 55 55
Roanoke River ROA 35.9228 50 50
Kerr Lake KER 36.5702 76 76
Alligator River ALL 35.9125 49 49
Watershed Code Latitude 2008 2009 2010 2011 2012 Unknown Total N
Blueback Herring
Miramichi River MIR 47.1419 1 1
River John RJO 45.7539 8 8
Margaree River MAR 46.4300 47 47
Petitcodiac River PET 45.9053 48 48
Saint John River SJR 45.2594 50 50
East Machias River EMA 44.5419 58 58
Penobscot River PEN 44.4408 36 36
St. George River STG 43.9592 46 46
Kennebec River KEN 43.7519 6 2 8 16
Exeter River EXE 43.0664 41 41
Mystic River MYS 42.3439 10 58 68
Monument River MON 41.7244 51 51
Gilbert-Stuart River GIL 41.4472 38 38
Connecticut River CON 41.2828 34 62 46 142
Quinnipiac River QUI 41.2722 30 25 55
Hudson River HUD 40.6958 48 31 79
Delaware River DEL 39.1122 51 51
Nanticoke River NAN 38.1689 24 33 57
Rappahannock River RAP 37.4933 58 58
James River JAM 36.9686 97 97
Chowan River CHO 36.0811 12 59 71
Roanoke River ROA 35.9228 50 50
Kerr Lake KER 36.5702 44 44
Neuse River NEU 35.0764 65 65
Cape Fear River CFE 33.9286 57 57
Santee-Cooper River SAN 33.1833 62 62
Savannah River SAV 32.0481 52 52
Altamaha River ALT 31.3147 53 53
St .John’s River STJ 30.4081 37 34 71
© 2014 John Wiley & Sons Ltd
HYBRIDIZATION AND INTROGRESSION IN ALOSINES 1141
(Pritchard et al. 2000) to designate purebreds (i.e. crite-
rion #3 for NEWHYBRIDS; Burgarella et al. 2009). We then
randomly chose 100 individuals identified as purebred
alewife and blueback herring from across their range,
and simulated five data sets of parental and various
hybrid classes using HYBRIDLAB version 1.0 (Nielsen et al.
2006) to determine Tq. For each of the five simulated data
sets, we generated profiles for 200 alewife and blueback
herring. From these 400 simulated parental genotypes,
we generated 100 F1 hybrids that were used to generate
100 F2 hybrids, and 100 F1 backcrossed alewife and blue-
back herring.
Simulated data sets were analysed using STRUCTURE
and NEWHYBRIDS (as described above), and results were
summarized across iterations using CLUMPP version 1.1.2
(Jakobsson & Rosenberg 2007) and visualized with DI-
STRUCT version 1.1 (Rosenberg 2004). These results were
used to determine Tq and to develop classification rules
for assigning individuals from the empirical data set
as purebred or hybrid. For STRUCTURE, we assessed the
q value of each K cluster for every simulated parental
and hybrid class. In NEWHYBRIDS, assignment to a specific
hybrid class may be uncertain, with q split among geno-
type frequency classes (Burgarella et al. 2009). There-
fore, we summed q across hybrid genotype frequency
classes, because we were only concerned with the iden-
tification of hybrids, not distinguishing among hybrid
classes per se. We then assessed the q value for every
simulated parental and hybrid class.
To determine Tq, we used the simulated data set to
examine how the proportion of misassigned parental
and hybrid individuals using STRUCTURE and NEWHYBRIDS
changed in response to a shifting q (0.005 increments)
from 0.85 to 1.00. The resulting intersection between the
proportion of misassigned parental and hybrid individ-
uals minimizes overall misassignments and was taken
as Tq. We then used this optimal threshold to determine
the error rate for parental and hybrid classification and
to establish confidence bounds on our assignments for
the empirical data set.
The empirical data set was then analysed using STRUC-
TURE and NEWHYBRIDS (as described above). Results were
summarized across iterations using CLUMPP (Jakobsson
& Rosenberg 2007) and visualized with DISTRUCT (Rosen-
berg 2004). Hybrids were identified based on the classi-
fication rules established through the determination of
Tq (see Results; Table S1, Supporting Information), and
the frequency and extent of hybridization across the
ranges of both species were examined. Factorial corre-
spondence analysis (FCA) was used to visualize the
relative similarity of multilocus allele frequencies
among purebred alewife and blueback herring, and
anadromous and landlocked hybrids using GENETIX 4.05
(Belkhir et al. 2004).
Analysis of genomic clines. To examine whether patterns
of introgression differed among landlocked and anadro-
mous hybrids, we used the genomic clines method
(Gompert & Buerkle 2009) implemented in the R pack-
age ‘introgress’ (Gompert & Buerkle 2010; R Develop-
ment Core Team 2010). For this analysis, we only
selected specimens that were identified by both STRUC-
TURE and NEWHYBRIDS as hybrids. The proportion of blue-
back herring ancestry among hybrids was assessed
using a maximum-likelihood estimator of hybrid index
that calculates genome-wide admixture based on the
Fig. 1 Map of the Atlantic coast of North
America displaying sampling locations
for alewife and blueback herring. River
names associated with each sampling
location code are provided in Table 1.
© 2014 John Wiley & Sons Ltd
1142 D. J . HASSELMAN ET AL.
proportion of alleles inherited from each parental spe-
cies (Gompert & Buerkle 2009). Parental species were
represented by the 100 purebred alewife and blueback
herring used in prior HYBRIDLAB simulations. Multiallelic
microsatellite data were reduced to biallelic classifica-
tion, without the loss of information or distortion of the
relationship between the parental species, using ‘intro-
gress’. Hybrids were then recognized as interallelic
class (interspecific) heterozygotes (Aa/Ab) or homozyg-
otes (Aa/Aa, Ab/Ab) (i.e. both alleles from the same alle-
lic class). The probabilities of observing each of these
possible allelic classes at each locus (i.e. genomic clines;
Gompert & Buerkle 2009) were then predicted using
multinomial regression of the observed genotypes on
hybrid index.
We identified loci that deviated from expectations of
neutral introgression by comparing the likelihoods of
the regression model to that of a neutral model, given
the observed data. Expected genomic clines under neu-
tral introgression were generated using 10 000 runs of a
parametric procedure implemented in ‘introgress’.
Genomic clines for the landlocked and anadromous
hybrid zones were then fitted with a logistic regression
using the observed data to estimate probabilities of
observing homozygote and heterozygote interallelic clas-
ses as a function of hybrid index (Gompert & Buerkle
2009). Significant deviations from neutral expectations
for genomic clines were adjusted for multiple compari-
sons using the false discovery rate (FDR; Benjamini &
Hochberg 1995). We then summarized deviations from
neutrality on the basis of whether interallelic class ho-
mozygotes and heterozygotes were more or less com-
mon than expected under neutrality, and determined
whether locus-specific patterns of introgression differed
among landlocked and anadromous hybrids.
Results
Data conformance to model assumptions
Evidence for null alleles resulted in the exclusion of loci
for both alewife (Aa082, Ap037, Ap047, Ap070) and blue-
back herring (Aa081, Ap058) prior to further analyses (Mi-
crochecker). Remaining loci were retained as evidence
for null alleles was sporadic among loci and rivers. Exact
tests revealed that genotypic frequencies were largely in
accordance with HWE for both alewife (P > 0.05; sequen-
tial Bonferroni correction for 55 comparisons) and blue-
back herring (P > 0.05; sequential Bonferroni correction
for 25 comparisons). HWE departures for alewife and
blueback herring remained for 25 and 11 locus–river
comparisons, respectively, and were due to heterozygote
deficiencies from sporadic null alleles. Exact tests of
LD revealed that loci were physically unlinked and
statistically independent (P > 0.05; sequential Bonferroni
correction for 2762 and 1270 comparisons for alewife and
blueback herring, respectively).
Simulations and hybrid identification
Global FST between alewife and blueback herring was
0.352. Although no locus was diagnostic, the Kullback–
Leibler divergence revealed several loci that would be
informative in species discrimination when used
together (e.g. KL ≥ 6.0; Fig. S1, Supporting Information).
In Bayesian analyses, q-values for randomly chosen ale-
wife and blueback herring used in simulations ranged
from 0.94–1.00 (STRUCTURE) and 0.99–1.00 (NEWHYBRIDS),
exceeding Tq (see below) and confirming that we had
correctly selected pure strain individuals for simulating
parental and hybrid classes. Further, allele frequency
distributions for the randomly chosen individuals were
representative of each species [P > 0.05; two-sample
Kolmogorov–Smirnov test; SYSTAT version 11 (SPSS, Inc.
2004)].
Across the five simulated data sets, the assignment of
purebred individuals to their correct parental class
using either Bayesian method was highly accurate
(Fig. 2). STRUCTURE unambiguously discriminated F1 and
F2 hybrids from purebreds (Fig. S2a, Supporting Infor-
mation), but the increased variance (by an order of
magnitude) of F2 hybrids reduced assignment accuracy
using NEWHYBRIDS (Fig. S2b, Supporting Information).
Both Bayesian methods had difficulty with advanced
introgression and could not unambiguously discrimi-
nate all backcrossed alewife and blueback herring from
purebred individuals and F2 hybrids (Fig. S2, Support-
ing Information). Similar results have been observed
even using diagnostic markers (Gow et al. 2007).
Using STRUCTURE, the lowest proportion of misas-
signed purebreds (0.007) and hybrids (0.006) across the
five simulated data sets occurred when Tq = 0.935. For
NEWHYBRIDS, the lowest proportion of misassigned pure-
breds (0.004) and hybrids (0.004) occurred when
Tq = 0.870. Using these optimal thresholds, we estab-
lished a set of classification rules for delineating pure-
breds and hybrids from the empirical data set (Table
S1, Supporting Information). Using these classification
rules, we detected hybrids from across the range of
river herring. Across the species’ ranges, 162 anadro-
mous specimens (2.4%) were identified as hybrids, with
Bayesian methods producing consistent results for 90
specimens. Every specimen from Kerr Reservoir
(n = 119) was identified as a hybrid (Figs 3 and 4). Dis-
crepancies between Bayesian methods occurred pre-
dominantly where individuals identified as purebreds
by NEWHYBRIDS were identified as hybrids by STRUCTURE
(n = 67), and more infrequently vice versa (n = 5). The
© 2014 John Wiley & Sons Ltd
HYBRIDIZATION AND INTROGRESSION IN ALOSINES 1143
proportion of hybrids in anadromous populations gen-
erally ranged from 0.00 to 0.08, but there were notable
exceptions (Fig. 4). The greatest proportions of anadro-
mous hybrids were observed for the Petitcodiac River
(0.227, 0.216) and Margaree River (0.133, 0.112) using
STRUCTURE and NEWHYBRIDS, respectively (Table S2, Sup-
porting Information). Both Bayesian methods identified
identical proportions of hybrids from the St. John’s
River (0.056), Altamaha River (0.019) and Savannah
River (0.019), despite these rivers being beyond the
southern range limit for alewife. No purebred alewives
were detected south of the range limit.
Two factors in FCA explained 85.6% of the genetic
variation among purebred and hybrid river herring and
demonstrated the clear separation of landlocked
hybrids (Fig. 5). While Axis 1 (44.35%) delineated pure-
bred anadromous alewife and blueback herring and
revealed an intermediate genetic composition of anadro-
mous hybrids, Axis 2 (41.25%) isolated landlocked
hybrids from anadromous hybrids and purebred river
herrings. Interestingly, landlocked hybrids did not exhi-
bit Axis 1 scores similar to anadromous hybrids, but
values that exceeded those of purebred alewife (Fig. 5).
This prompted an examination of the distribution of
probability values (q) among genotype frequency classes
in NEWHYBRIDS for hybrids and revealed a signifi-
cantly (P < 0.001; two-sample KS test) higher frequency
of F1 and F2 individuals among anadromous than
among landlocked hybrids. Landlocked hybrids were
more deeply introgressed with alewife, consistent with
FCA results (data not shown) and genomic clines analy-
sis (see below).
Analysis of genomic clines
The genomic clines method revealed striking differences
in patterns of blueback herring ancestry and patterns of
introgression between anadromous and landlocked
hybrids (Fig. S3, Supporting Information). While the
hybrid index for anadromous hybrids ranged from 0.00
to 1.00, the fraction of the landlocked hybrid genome
that was derived from blueback herring never exceeded
0.78. Further, an approximately equal proportion of ale-
wife and blueback herring ancestry was observed
among anadromous hybrids, but there was little blue-
back herring ancestry among landlocked hybrids; only
two individuals exhibited blueback herring ancestry
≥0.5 (Fig. S3b, Supporting Information). Cumulatively,
these results support neutral introgression of blueback
herring genotypes among anadromous hybrids, but
directional introgression leading to increased prevalence
of alewife genotypes in Kerr Reservoir.
The genomic clines method also revealed marked het-
erogeneity in locus-specific patterns of introgression for
anadromous and landlocked hybrids. For anadromous
hybrids, variation at four loci (Aa046, Aa070, Aa093,
Ap033) deviated from neutral introgression based on
genome-wide admixture and remained significant after
FDR correction (P < 0.015) (Fig. 6a). For three of these
markers (Aa046, Aa070, Aa093), the total probability for
the heterozygous genotype was increased relative to
neutral expectations (Fig. 6a). For landlocked hybrids,
eight of the nine loci (except Aa046) deviated from neu-
tral introgression and remained significant after FDR
correction (P < 0.039) (Fig. 6b), with highly variable pat-
terns of deviation from neutral introgression among
markers. Nonetheless, for several of the deviant loci (i.e.
Ap071, Ap010, Aa070), the total probability for heterozy-
gous genotype was decreased relative to neutral expec-
tations (Fig. 6b). We also observed significant
differences in the patterns of introgression between
anadromous and landlocked hybrids for two of five
markers that could be directly compared (i.e. Aa074,
Ap033).
Fig. 2 Results of Bayesian clustering
analyses for five simulated data sets (ale-
wife, blueback herring, F1, F2, F1 back-
crosses) using STRUCTURE (K = 2; number
of species) and NEWHYBRIDS (K = 6; num-
ber of genotype frequency classes). Indi-
viduals are represented by a thin vertical
line which is partitioned into K-coloured
segments representing an individual’s
estimated membership fractions from
each of the identified clusters. Black lines
separate individuals from different geno-
type frequency classes (labelled below).
© 2014 John Wiley & Sons Ltd
1144 D. J . HASSELMAN ET AL.
Discussion
Most evidence for the formation of hybrid swarms
resulting from anthropogenic habitat disturbance comes
from studies of either the breakdown of reproductive
isolation between incipient species that have recently
diverged in sympatry (e.g. Todd & Stedman 1989;
Seehausen et al. 1997; Behm et al. 2010; Vonlanthen et al.
2012), or introgression between allopatric species fol-
lowing secondary contact (e.g. Echelle & Connor 1989;
Walters et al. 2008; McDevitt et al. 2009). Our rangewide
study of hybridization between alewife and blueback
herring demonstrates that human activities can also
impact the formation of hybrid swarms between diver-
gent species that naturally occur in sympatry, providing
empirical evidence that reinforcement does not always
sustain reproductive isolation under such circum-
stances.
Spatial extent of hybridization
Despite varying frequencies of hybridization across
their range (Figs 3 and 4), our study reveals that species
integrity is maintained in drainages where anadromous
alewife and blueback herring occur sympatrically. This
is consistent with hybridization studies of European
alosines (A. fallax and A. alosa; Alexandrino et al. 2006;
Coscia et al. 2010; Jolly et al. 2011) with similar diver-
gence times (Bentzen et al. 1993; Faria et al. 2006), and
with studies of other hybridizing species (e.g. Helianthus
sp.; Strasburg & Rieseberg 2008). The frequency of
hybridization among anadromous populations generally
ranged from 0 to 8% (Table S2, Supporting Informa-
tion). Whether this approximates the expected fre-
quency of hybridization under pristine environmental
conditions is uncertain. Many of the rivers examined
have dams that may impact the spatiotemporal distri-
bution of spawning migrations through restricted
Fig. 3 Results of Bayesian clustering analyses for rangewide empirical data for alewife and blueback herring using STRUCTURE (K = 2;
number of species) and NEWHYBRIDS (K = 6; number of genotype frequency classes). Individuals are represented by a thin vertical line
which is partitioned into K-coloured segments representing an individual’s estimated membership fractions from each of the identi-
fied clusters. Kerr Reservoir (KER) is denoted because it showed a substantially greater number of hybrids than other sampling loca-
tions.
Fig. 4 Spatial distribution of the proportion of hybrids identi-
fied using two Bayesian clustering analyses. MAR, PET and
KER (see Table 1) are denoted because they exhibited a sub-
stantially greater proportion of hybrids than other sampling
locations.
Fig. 5 Factorial correspondence analysis revealed two factors
that explained 85.6% of the genetic variation among purebred
and hybrid river herring and demonstrated the clear separation
of landlocked hybrids in Kerr Reservoir from alewife, blueback
herring and anadromous hybrids.
© 2014 John Wiley & Sons Ltd
HYBRIDIZATION AND INTROGRESSION IN ALOSINES 1145
(a)
(b)
Fig. 6 Genomic clines plots generated in
‘introgress’ for (a) anadromous (n = 90)
and (b) landlocked (Kerr Reservoir)
(n = 119) river herring hybrids for nine
microsatellite loci common to both data
sets. The name of each locus and P-value
for the test of departure from neutral
introgression is provided [P < 0.015 and
P < 0.039 indicate significance for anad-
romous and landlocked hybrids, respec-
tively, after FDR correction]. Solid
coloured clines represent the 95% confi-
dence intervals for hybrids with homozy-
gous (Aa/Aa or Ab/Ab; dark green) and
heterozygous (Aa/Ab; light green) geno-
mic clines given neutral introgression.
The solid and dashed lines give the esti-
mated genomic cline based on the
observed homozygous and heterozygous
allelic classes, respectively. Circles indi-
cate the raw allelic class data (Aa/Aa
along the top, Aa/Ab in the centre and
Ab/Ab along the bottom), with counts of
each allelic class along the right vertical
axis. The hybrid index quantifies the
fraction of alleles derived from blueback
herring across the nine microsatellite loci.
© 2014 John Wiley & Sons Ltd
1146 D. J . HASSELMAN ET AL.
habitat access (Hall et al. 2011) and altered spawning
cues (i.e. water temperature; Loesch 1987), and may
facilitate hybridization (Boisneau et al. 1992; Maitland &
Lyle 2005). Spawning alewife and blueback herring are
known to co-occur where upstream migration is
blocked by dams (Loesch 1987), and this may provide
opportunities for interspecific matings. However, the
Delaware River has no mainstem dams and exhibited
5–6% hybridization, suggesting that some level of
hybridization may be natural.
Although we do not know the extent to which pre-
and postzygotic reproductive isolating mechanisms
maintain species integrity, the general absence of deeply
introgressed (i.e. backcrossed) individuals in anadro-
mous populations may indicate an important role for
selection against hybrids under natural conditions.
In contrast to the maintenance of species integrity
among anadromous populations, our study reveals that a
hybrid swarm has become established in Kerr Reservoir
following dam construction that has prevented immigra-
tion of purebred alewife or blueback herring since 1953.
All individuals in Kerr Reservoir were identified as
hybrids (Table S2, Supporting Information; Fig. 4) and
were deeply introgressed – likely via multiple genera-
tions of backcrossing. This finding suggests that both
pre- and postzygotic reproductive isolating mechanisms
may have broken down and that there may not be selec-
tion against hybrids in this altered environment. In unal-
tered drainages, differential spawning habitat selection
exhibited by these species may limit opportunities for
interspecific matings (Loesch 1987). However, the lotic
spawning habitats preferred by blueback herring are not
available in Kerr Reservoir, potentially leading to
increased interspecific matings. Decreased spatial segre-
gation may be accompanied by altered (temperature-
dependent) migratory and spawning cues, possibly
increasing temporal spawning overlap within Kerr Reser-
voir as well. The exact mechanisms at play require fur-
ther investigation. Nonetheless, our study demonstrates
that anthropogenic habitat alterations can breakdown
reproductive isolation between distinct evolutionary lin-
eages that naturally occur in sympatry.
Although hybridization in temperate fishes as a con-
sequence of habitat disturbance is not a new concept
(Hubbs 1955), prior examinations were largely limited
to young evolutionary lineages of freshwater species, or
intraspecific comparisons of anadromous and resident
salmonids (Utter 2001). While hybridization between
anadromous species placed in novel freshwater envi-
ronments has been previously reported (Rosenfield et al.
2000), their collapse as a hybrid swarm has not been
documented. Our study reinforces the contention that
reproductive isolating mechanisms may be incomplete
during the first 2–5 million years after speciation
(Coyne & Orr 2004) and that a substantial portion of
global biodiversity may be susceptible to anthropogeni-
cally induced hybridization (Rhymer & Simberloff 1996;
Seehausen et al. 2008).
Elevated frequencies of hybridization in the Petitcodi-
ac River (~22%) and Margaree River (~12%) (Fig. 4)
were unexpected, but may be explained by a combina-
tion of migratory barriers, decreased abundances and
range-edge effects. A causeway with ineffective fish
passage was constructed near the head of tide in the
Petitcodiac River in 1968. This barrier completely
blocked the access of migratory fishes to upstream
spawning habitat and dramatically reduced the abun-
dance of alewife and blueback herring in the drainage
(Locke et al. 2003). Increased spatiotemporal overlap of
spawning adults below the causeway coupled with
population declines may have increased the chances of
interspecific matings (Reyer 2008). Although dams are
not present on the mainstem Margaree River, river her-
ring have been heavily exploited in this drainage (R.G.
Bradford, personal communication), and low popula-
tion abundances may have increased opportunities for
hybridization. Further, this drainage approximates the
northern range edge of blueback herring where popula-
tion density is low and heterospecific matings may be
more likely (Reyer 2008). However, we did not observe
elevated levels of hybridization for other northern
populations (i.e. Miramichi River, River John). Elevated
levels of hybridization reported for European alosines
(Jolly et al. 2011, 2012) have been attributed to similar
factors (Boisneau et al. 1992; Maitland & Lyle 2005),
but the role of shifting phenologies (Ellis & Vokoun
2009) on the frequency of hybridization requires
attention.
Hybrids detected in rivers from South Carolina to
Florida are beyond the southern range limit for alewife
and could be strays; our sampling of adult specimens
does not preclude their origin in more northerly drain-
ages. However, there is a paucity of evidence for
increased straying rates of hybrid fishes, especially
anadromous species (Scribner et al. 2001; but see Gilk
et al. 2004). Alternatively, these hybrids could be the
progeny of stray purebred alewife that reproduced with
blueback herring in southern rivers. These remain
untested hypotheses that require attention.
Detecting hybrids
Accurate identification of hybrids using molecular
methods depends on the markers used and the degree
of differentiation between parental species (Anderson &
Thompson 2002; V€ah€a & Primmer 2006). Nonetheless,
studies continue to employ a default Tq (0.90) that was
originally derived from simulations (V€ah€a & Primmer
© 2014 John Wiley & Sons Ltd
HYBRIDIZATION AND INTROGRESSION IN ALOSINES 1147
2006); rarely has Tq been assessed on an individual
study basis. Employing this arbitrary threshold in our
study would have inflated our estimates of hybridiza-
tion. Our simulations revealed that misclassifications
were minimized using a Tq of 0.87 (NEWHYBRIDS) and
0.935 (STRUCTURE). This value for STRUCTURE approximates
that used in hybridization studies of European alosines
(0.94; Jolly et al. 2012). We advocate a simulation
approach (as outlined herein) for setting the most
appropriate Tq for discriminating purebred and hybrid
specimens on an individual study basis.
Hybrid divergence in isolation
Although hybridization can threaten biodiversity (Rhymer
& Simberloff 1996), genetic admixture in novel and per-
turbed environments can generate diversity by increasing
the adaptive potential of admixed individuals with novel
genetic variation (e.g. Buerkle et al. 2000; Barton 2001;
Lexer et al. 2003). Our study demonstrates that landlocked
hybrids are genetically distinct from anadromous
hybrids, alewife and blueback herring (Fig. 5). Indeed,
landlocked hybrids are nearly as differentiated from ale-
wife (FST = 0.30) as alewife are from blueback herring
(FST = 0.38). This may have resulted from an initial popu-
lation bottleneck followed by hybridization, and the
effects of drift in isolation from anadromous congeners. A
number of landlocked European alosine populations have
become adapted to lacustrine habitats (Faria et al. 2006)
and are genetically distinct from their anadromous cong-
eners (e.g. A. f. killarensis; Jolly et al. 2012). Our data
suggest that Kerr Reservoir constitutes an admixed popu-
lation in a novel environment that is in the process of
divergence.
Contrasting patterns of introgression
The distribution of blueback herring ancestry observed
among anadromous hybrids (Fig. S3a, Supporting Infor-
mation) is consistent with neutral introgression. This
contrasts with landlocked hybrids, where the decreased
prevalence of blueback herring ancestry (Fig. S3b, Sup-
porting Information) suggests directional introgression
of alewife genotypes in Kerr Reservoir. Although the
proportion of the two species that became landlocked is
unknown, available evidence suggests that blueback
herring were more abundant in the Roanoke River than
alewife when the dam was constructed (Carnes 1965).
Thus, it is unlikely that our result simply reflects a
chance colonization event where alewife were numeri-
cally dominant and where the introgressed gene pool is
largely of alewife origin.
For hybrids, the extent of introgression at individ-
ual loci is a consequence of the fitness effects of
genotype combinations. Contrasts among markers per-
mit the identification of loci that lower hybrid fitness
and contribute to reproductive isolation, or that
increase fitness and promote adaptive introgression
(Gompert & Buerkle 2010). Although our analyses
revealed some concordance in locus-specific patterns
of introgression between anadromous and landlocked
hybrids, we also observed loci that exhibited mark-
edly different patterns of introgression and that may
be subject to varying selection in different settings
(i.e. environments and genetic backgrounds; Nolte
et al. 2009). Differential patterns of introgression have
been previously reported for sculpin (Cottus spp.)
hybrid zones, where discordance in locus-specific pat-
terns has been attributed to differing extrinsic factors
(i.e. local ecological conditions may impose different
selection pressures on admixed genotypes), differing
underlying genetic architecture of reproductive isola-
tion and adaptation and/or the influence of stochas-
ticity and drift in (small) hybrid populations (Nolte
et al. 2009). One or more of these factors may contrib-
ute to the discordance in patterns of introgression
that we observe.
Patterns of non-neutral introgression can be catego-
rized based on their correspondence with different
models of genotypic effects in hybrids (Nolte et al.
2009). For anadromous hybrids, loci that exhibited
increased probability for heterozygous (Aa/Ab) allelic
classes are consistent with overdominance, while
decreased probability for homozygous (Aa/Aa, Ab/Ab)
allelic classes is consistent with negative selection
(Fig. 6a). Conversely, for landlocked hybrids, loci that
exhibited decreased probability for heterozygous allelic
classes are consistent with positive selection, while
increased probability of homozygous allelic classes is
consistent with adaptive introgression (Fig. 6b).
Although different patterns of introgression are
apparent between anadromous and landlocked hybrids,
we hesitate to invoke any particular mode of selection
pending analyses of a larger suite of molecular markers.
The genomic clines method requires a sufficient number
of loci distributed broadly across the genome to ensure
that estimates of genome-wide admixture are represen-
tative of neutral introgression (Gompert & Buerkle
2009). Although the loci used in this study are not
linked, without a linkage map it will be unclear to what
extent loci demonstrating similar patterns of introgres-
sion are independent, and it will not be possible to
determine the proportion of the genome experiencing
different forms of selection (Gompert & Buerkle 2009).
The investigation of contrasting patterns of introgres-
sion between anadromous and landlocked hybrids
warrants further study using advanced genomic
approaches.
© 2014 John Wiley & Sons Ltd
1148 D. J . HASSELMAN ET AL.
Conclusions
Our study reveals that anthropogenic habitat changes
can breakdown reproductive isolation between diver-
gent evolutionary lineages that naturally occur in symp-
atry. While sympatric populations of anadromous
alewife and blueback herring maintain species integrity
across their range, reproductive isolation has broken
down in Kerr Reservoir, leading to the formation of a
hybrid swarm. The construction of dams constitutes a
dramatic alteration to habitat and may disrupt pro-
cesses that sustain reproductive isolation under natural
conditions. We posit that decreased opportunities for
spatiotemporal spawning segregation in Kerr Reservoir
have increased the likelihood of interspecific matings
and have lead to the breakdown of reproductive isola-
tion. The detection of deeply introgressed hybrids in
Kerr Reservoir indicates that hybrids are not disfa-
voured in this altered environment. This contrasts with
a general absence of deeply introgressed hybrids in
anadromous populations, suggesting an important role
for selection against hybrids under natural conditions.
Our results show that reinforcement may be insufficient
to sustain reproductive isolation in the face of some
types of human disturbance. The benefits of dam
removal for the restoration of anadromous fishes have
been largely discussed in the context of replenishing
historic spawning runs (e.g. Hasselman & Limburg
2012). Our study provides evidence that dam removal
could also help maintain species integrity for alewife
and blueback herring.
Acknowledgements
We are grateful to all those who contributed sampling effort to
our ongoing coast-wide genetic studies of river herring ecol-
ogy, evolution and conservation. B. Wynne (North Carolina
Wildlife Resources Commission), D. Michaelson (Virginia
Department of Game and Inland Fisheries) and Casey Seelig
(Dominion Environmental Biology) assisted with the sampling
of Kerr Reservoir. E. M. Labbe, S. Blienbry and L. Thornton
assisted with microsatellite data collection, and T. M. Apgar
assisted with Fig. 1. We also thank three anonymous reviewers
and the associate editor for constructive comments that
strengthened the quality of this paper. This research was
supported by an NSERC Discovery Grant to P. Bentzen, a
National Fish and Wildlife Foundation Grant to E.P. Palkovacs
and North Carolina SeaGrant funding to E.P. Palkovacs and
T.F. Schultz.
References
A’Hara SW, Amouroux P, Argo EE et al. (2012) Permanent
genetic resources added to Molecular Ecology Resources
Database 1(August), pp. 2011–30, September 2011. Molecular
Ecology Resources, 12, 185–189.
Alexandrino P, Faria R, Linhares D et al. (2006) Interspecific
differentiation and intraspecific substructure in to closely
related clupeids with extensive hybridization. Journal of Fish
Biology, 69(Supplement B), 242–259.Allendorf FW, Leary RF, Spruell P, Wenburg JK (2001) The
problems with hybrids: setting conservation guidelines.
Trends in Ecology & Evolution, 16, 613–622.
Anderson E (1948) Hybridization of the habitat. Evolution, 2,
1–9.
Anderson EC, Thompson EA (2002) A model-based method for
identifying species hybrids using multilocus genetic data.
Genetics, 160, 1217–1229.Atlantic States Marine Fisheries Commission (2012) River her-
ring benchmark stock assessment. Volume 1. Stock Assess-
ment Report No. 12-02 of the Atlantic States Marine
Fisheries Commission. Washington, DC.
Barton NH (2001) The role of hybridization in evolution. Molec-
ular Ecology, 10, 551–568.Behm JE, Ives AR, Boughman JW (2010) Breakdown in post-
mating isolation and the collapse of a species pair through
hybridization. The American Naturalist, 175, 11–26.
Belkhir K, Borsa P, Chikhi L, Raufaste N, Bonhomme F (2004)
GENETIX 4.05, logiciel sous Windows TM pour la genetique
des populations.
Benjamini Y, Hochberg Y (1995) Controlling the false disco-
very rate: a practical and powerful approach to multiple
testing. Journal of the Royal Statistical Society Series B, 57,
289–300.
Bentzen P, Paterson IG (2005) Genetic analyses of freshwater
and anadromous alewife (Alosa pseudoharengus) populations
from the St. Croix River, Maine/New Brunswick. Final
Report to Maine Rivers, 3 Wade Street, Augusta, ME 04330.
Bentzen P, Leggett WC, Brown GG (1993) Genetic relationships
among the shads (Alosa) revealed by mitochondrial DNA
analysis. Journal of Fish Biology, 43, 909–917.Boisneau P, Mennesson-Boisneau C, Guyomard R (1992) Electro-
phoretic identity between allis shad, Alosa alosa (L.), and twaite
shad, A. fallax (Lacepede). Journal of Fish Biology, 40, 731–738.
Buerkle CA, Morris RJ, Asmussen MA, Rieseberg LH (2000)
The likelihood of homoploid hybrid speciation. Heredity, 84
(Pt 4), 441–451.Burgarella C, Lorenzo Z, Jabbour-Zahab R et al. (2009) Detec-
tion of hybrids in nature: application to oaks (Quercus suber
and Q. ilex). Heredity, 102, 442–452.
Carnes WC (1965) Survey and Classification of the Roanoke River
Watershed North Carolina. North Carolina Wildlife Resources
Commission, Raleigh, North Carolina, 17p.
Chapman R, Patton J, Eleby B (1994) Comparison of mitochon-
drial DNA variation in four alosid species as revealed by the
total genome, the NADH dehydrogenase I and cytochrome b
regions. In: Genetics and Evolution of Aquatic Organisms (ed.
Beaumont A), pp. 29–263. Chapman and Hall, London, UK.
Coscia I, Rountree V, King JJ et al. (2010) A highly permeable
species boundary between two anadromous fishes. Journal of
Fish Biology, 77, 1137–1149.Coyne JA, Orr HA (2004) Speciation. Sinauer Associates, Sun-
derland, Massachusetts.
De Le�on LF, Raeymaekers JAM, Bermingham E et al. (2011)
Exploring possible human influences on the evolution of
Darwin’s finches. Evolution, 65, 2258–2272.
© 2014 John Wiley & Sons Ltd
HYBRIDIZATION AND INTROGRESSION IN ALOSINES 1149
Echelle AA, Connor PJ (1989) Geographically extensive genetic
introgression after secondary contact between two pupfish
species (Cyprinodon, cyprinodontidae). Evolution, 43, 717–727.
Ellis D, Vokoun JC (2009) Earlier spring warming of coastal
streams and implications for alewife migration timing. North
American Journal of Fisheries Management, 29, 1584–1589.Falush D, Stephens M, Pritchard JK (2003) Inference of popu-
lation structure using multilocus genotype data: Linked
loci and correlated allele frequencies. Genetics, 164, 1567–
1587.
Faria R, Weiss S, Alexandrino P (2006) A molecular phyloge-
netic perspective on the evolutionary history of Alosa spp.
(Clupeidae). Molecular Phylogenetics and Evolution, 40, 298–304.
Gilk SE, Wang IA, Hoover CL et al. (2004) Outbreeding depres-
sion in hybrids between spatially separated pink salmon,
Oncorhynchus gorbuscha, populations: marine survival, hom-
ing ability, and variability in family size. Environmental Biol-
ogy of Fishes, 69, 287–297.Gompert Z, Buerkle CA (2009) A powerful regression-based
method for admixture mapping of isolation across the gen-
ome of hybrids. Molecular Ecology, 18, 1207–1224.
Gompert Z, Buerkle CA (2010) Introgress: a software package
for mapping components of isolation in hybrids. Molecular
Ecology Resources, 10, 378–384.Goudet J (2001) FSTAT, a program to estimate and test gene
diversities and fixation indices (version 2.9.3).
Gow JL, Peichel CL, Taylor EB (2006) Contrasting hybridiza-
tion rates between sympatric three-spined sticklebacks high-
light the fragility of reproductive barriers between
evolutionarily young species. Molecular Ecology, 15, 739–752.
Gow JL, Piechel CL, Taylor EB (2007) Ecological selection
against hybrids in natural populations of sympatric three-
spine sticklebacks. Journal of Evolutionary Biology, 20, 2173–2180.
Guest WC, Drenner R (1991) Relationship between feeding of
blueback herring and the zooplankton community of a Texas
reservoir. Hydrobiologia, 209, 1–6.Hall CJ, Jordaan A, Frisk MG (2011) The historic influence of
dams on diadromous fish habitat with a focus on river her-
ring and hydrologic longitudinal connectivity. Landscape Ecol-
ogy, 26, 95–107.Hall CJ, Jordaan A, Frisk MG (2012) Centuries of anadromous
forage fish loss: consequences for ecosystem connectivity
and productivity. BioScience, 62, 723–731.
Hasselman DJ, Limburg KE (2012) Alosine restoration in the
21st century: challenging the status quo. Marine and Coast
Fisheries: Dynamics, Management, and Ecosystem Science, 4,
174–187.
Hendry AP, Grant PR, Rosemary Grant B et al. (2006) Possible
human impacts on adaptive radiation: beak size bimodality
in Darwin’s finches. Proceedings of the Royal Society B-Biologi-
cal Sciences, 273, 1887–1894.
Holm S (1979) A simple sequentially rejective multiple test pro-
cedure. Scandinavian Journal of Statistics, 6, 65–70.
Hubbs C (1955) Hybridization between fish species in nature.
Systematic Zoology, 4, 1–20.
Jakobsson M, Rosenberg NA (2007) CLUMPP: a cluster match-
ing and permutation program for dealing with label switch-
ing and multimodality in analysis of population structure.
Bioinformatics, 23, 1801–1806.
Jolly MT, Maitland PS, Genner MJ (2011) Genetic monitoring
of two decades of hybridization between allis shad (Alosa
alosa) and twaite shad (Alosa fallax). Conservation Genetics, 12,
1087–1100.Jolly MT, Aprahamian MW, Hawkins SJ et al. (2012) Popula-
tion genetic structure of protected allis shad (Alosa alosa) and
twaite shad (Alosa fallax). Marine Biology, 159, 675–687.
Jordan DS, Evermann BW (1896) The fishes of North and Mid-
dle America. Bulletin of the US National Museum, 47, 1–1240.
Julian SE, Bartron ML (2007) Microsatellite DNA markers for
American shad (Alosa sapidissima) and cross-species amplifi-
cation within the family Clupeidae. Molecular Ecology Notes,
7, 805–807.
Kullback S, Leibler RA (1951) On information and sufficiency.
The Annals of Mathematical Statistics, 22, 79–86.
Labbe EM (2012) Influence of stocking history and geography
on the population genetics of alewife (Alosa pseudoharengus)
in Maine rivers. M.Sc. Thesis. University of Southern Maine.
Portland, ME.
Lehman N, Eisenhawer A, Hansen K et al. (1991) Introgression
of coyote mitochondrial DNA into sympatric North Ameri-
can gray wolf populations. Evolution, 45, 104–119.Lexer C, Welch ME, Durphy JL, Rieseberg LH (2003) Natural
selection for salt tolerance quantitative trait loci (QTLs) in
wild sunflower hybrids: implications for the origin of
Helianthus paradoxus, a diploid hybrid species. Molecular
Ecology, 12, 1225–1235.
Limburg KE, Waldman JR (2009) Dramatic declines in North
Atlantic diadromous fishes. BioScience, 59, 955–965.Locke A, Hanson JM, Klassen GJ et al. (2003) The damming of
the Petitcodiac River: species, populations, and habitats lost.
Northeastern Naturalist, 10, 39–54.
Loesch JG (1987) Overview of life history aspects of anadro-
mous alewife and blueback herring in freshwater habitats.
American Fisheries Society Symposium, 1, 89–103.Maitland PS, Lyle AA (2005) Ecology of Allis Shad Alosa alosa
and Twaite Shad Alosa fallax in the Solway Firth, Scotland.
Hydrobiologia, 534, 205–221.
McBride M (2013) Population structure of river herring (alewife,
Alosa pseudoharengus and blueback herring, A. aestivalis)
examined using neutral genetic markers. M.Sc. Thesis, Dalhousie
University, Halifax, Nova Scotia.
McDevitt AD, Mariani S, Hebblewhite M et al. (2009) Survival
in the Rockies of an endangered hybrid swarm from
diverged caribou (Rangifer tarandus) lineages. Molecular Ecol-
ogy, 18, 665–679.
Messieh S (1977) Population structure and biology of alewife
(Alosa pseudoharengus) and blueback herring (A. aestivalis) in the
St. John River, NB. Environmental Biology of Fishes, 2, 195–210.Nielsen EE, Bach LA, Kotlicki P (2006) Hybridlab (version 1.0):
a program for generating simulated hybrids from population
samples. Molecular Ecology Notes, 6, 971–973.
Nolte AW, Gompert Z, Buerkle CA (2009) Variable patterns of
introgression in two sculpin hybrid zones suggest that geno-
mic isolation differs among populations. Molecular Ecology,
18, 2615–2627.
Palkovacs EP, Dion KB, Post DM, Caccone A (2008) Indepen-
dent evolutionary origins of landlocked alewife populations
and rapid parallel evolution of phenotypic traits. Molecular
Ecology, 17, 582–597.
© 2014 John Wiley & Sons Ltd
1150 D. J . HASSELMAN ET AL.
Palkovacs EP, Hasselman DJ, Argo EE et al. (2013) Combining
genetic and demographic information to prioritize recovery
efforts for anadromous alewife and blueback herring. Evolu-
tionary Applications, 7, 212–226.Prince ED, Barwick DH (1981) Landlocked blueback herring in
two South Carolina reservoirs: reproduction and suitability
as stocked prey. North American Journal of Fisheries Manage-
ment, 1, 41–45.Pritchard JK, Stephens M, Donnelly P (2000) Inference of popu-
lation structure using multilocus genotype data. Genetics,
155, 945–959.
R Development Core Team (2010) R: A Language and Environ-
ment for Statistical Computing. R Foundation for Statistical
Computing, Vienna, Austria. ISBN 3-900051-07-0, URL
http://www.R-project.org.
Reyer HU (2008) Mating with the wrong species can be right.
Trends in Ecology & Evolution, 23, 289–292.
Rhymer M, Simberloff D (1996) Extinction by hybridization
and introgression. Annual Reviews of Ecology and Systematics,
27, 83–109.Rice WR (1989) Analyzing tables of statistical tests. Evolution,
43, 223–225.Roca AL, Georgiadis N, O’Brien SJ (2005) Cytonuclear genomic
dissociation in African elephant species. Nature Genetics, 37,
96–100.
Rosenberg NA (2004) DISTRUCT: a program for the graphical
display of population structure. Molecular Ecology Notes, 4,
137–138.
Rosenfield JA, Todd T, Greil R (2000) Asymmetric hybridiza-
tion and introgression between pink salmon and Chinook
salmon in the Laurentian Great Lakes. Transactions of the
American Fisheries Society, 129, 670–679.
Rousset F (2007) Genepop’007: a complete re-implementation
of the genepop software for Windows and Linux. Molecular
Ecology Notes, 8, 103–106.Rundle HD, Nosil P (2005) Ecological speciation. Ecology Let-
ters, 8, 336–352.Rundle HD, Schluter D (1998) Reinforcement of stickleback
mate preferences: sympatry breeds contempt. Evolution, 52,
200–208.
Scott WB, Crossman ED (1973) Freshwater fishes of Canada. J.
Fish. Res. Bd. Can. No. 184.
Scribner KT, Page KS, Bartron ML (2001) Hybridization in
freshwater fishes: a review and case studies and cytonuclear
methods of biological inference. Reviews in Fish Biology and
Fisheries, 10, 293–323.
Seehausen O (2006) Conservation: losing biodiversity by
reverse speciation. Current Biology, 16, 334–337.
Seehausen O, van Alphen J, Witte F (1997) Cichlid fish diver-
sity threatened by eutrophication that curbs sexual selection.
Science, 277, 1808–1811.Seehausen O, Takimoto G, Roy D, Jokela J (2008) Speciation
reversal and biodiversity dynamics with hybridization in
changing environments. Molecular Ecology, 17, 30–44.
Servedio MR, Noor MAF (2003) The role of reinforcement in
speciation: theory and data. Annual Review of Ecology, Evolu-
tion, and Systematics, 34, 339–364.SPSS, Inc. (2004) SYSTAT version 11.0. SPSS, Inc., Chicago, Illi-
nois.
Strasburg JL, Rieseberg LH (2008) Molecular demographic
history of the annual sunflowers Helianthus annuus and H.
petiolaris-large effective population sizes and rates of long-
term gene flow. Evolution, 62, 1936–1950.Taylor EB, Boughman JW, Groenenboom M et al. (2006) Specia-
tion in reverse: morphological and genetic evidence of the
collapse of a three-spined stickleback (Gasterosteus aculeatus)
species pair. Molecular Ecology, 15, 343–355.Todd N, Stedman RM (1989) Hybridization of ciscoes (Coreg-
onus spp.) in Lake Huron. Canadian Journal of Zoology, 67,
1679–1685.
Utter F (2001) Patterns of subspecific anthropogenic introgres-
sion in two salmonid genera. Reviews in Fish Biology and Fish-
eries, 10, 265–279.V€ah€a JP, Primmer CR (2006) Efficiency of model-based Bayes-
ian methods for detecting hybrid individuals under different
hybridization scenarios and with different numbers of loci.
Molecular Ecology, 15, 63–72.Van Oosterhout C, Hutchinson WF, Wills DPM, Shipley P
(2004) MICRO-CHECKER: software for identifying and cor-
recting genotyping errors in microsatellite data. Molecular
Ecology Notes, 4, 535–538.Vonlanthen P, Bittner D, Hudson AG et al. (2012) Eutrophica-
tion causes speciation reversal in whitefish adaptive radia-
tions. Nature, 482, 357–362.
Walters DM, Blum MJ, Rashleigh B et al. (2008) Red shiner
invasion and hybridization with blacktail shiner in the upper
Coosa River, USA. Biological Invasions, 10, 1229–1242.Waters JM, Epifanio JM, Gunter T, Brown BL (2000) Homing
behaviour facilitates subtle genetic differentiation among
river populations of Alosa sapidissima: microsatellites and
mtDNA. Journal of Fish Biology, 56, 622–636.
Weir BS, Cockerham CC (1984) Estimating F-statistics for
the analysis of population structure. Evolution, 38, 1358–
1370.
Wiegand K (1935) A taxonomist’s experience with hybrids in
the wild. Science, 81, 161–166.
D.J.H. designed the study with E.P.P., conducted the
analyses and wrote the manuscript. E.E.A. M.C.M. and
A.A.P.-U. collected data and contributed to manuscript
revisions. E.P.P., T.F.S. and P.B. funded the project and
contributed to manuscript revisions.
Data accessibility
Microsatellite data used in this manuscript: DRYAD
Digital Repository. doi:10.5061/dryad.8v0c3.
All necessary input files required to replicate our
genomic clines analysis using the R package ‘intro-
gress’: DRYAD Digital Repository doi:10.5061/dryad.
ft48k.
Supporting information
Additional supporting information may be found in the online ver-
sion of this article.
© 2014 John Wiley & Sons Ltd
HYBRIDIZATION AND INTROGRESSION IN ALOSINES 1151
Fig. S1 Allele frequency distributions for alewife ( ) and blue-
back herring ( ) for 15 loci examined in this study. Estimates
of the Kullback–Leibler divergence between the species for
each locus are located in the top-right of each panel.
Fig. S2 Mean (�SD) of q-values for five simulated data sets of
six hybrid categories (ALE: alewife, BBH: blueback herring, F1,
F2 and F1 backcrosses) analysed using (a) STRUCTURE (K = 2;
number of species) and (b) NEWHYBRIDS (K = 6; number of geno-
type frequency classes). The horizontal dashed line indicates
q = 0.90.
Fig. S3 Ancestry plots generated in ‘introgress’ for (a) anadro-
mous (n = 90) and (b) landlocked (Kerr Reservoir) (n = 119)
river herring hybrids for nine microsatellite loci common to
both data sets. Dark green blocks indicate hybrids that are
homozygous for alewife allelic classes (Aa/Aa), light green
blocks indicate hybrids that are homozygous for blueback her-
ring allelic classes (Ab/Ab), and intermediate green blocks cor-
respond to hybrids that are interclass heterozygotes (Aa/Ab).
White blocks indicate missing data. The plot to the right in
each panel indicates the proportion of each individual’s gen-
ome that has blueback herring ancestry; equivalent to the
hybrid index. Individuals are sorted, with those that have
genomic compositions resembling alewife at the bottom and
increasing similarity to blueback herring toward the top.
Table S1 Classification rules for delineating purebred and
hybrid specimens based on the optimal threshold (Tq) deter-
mined using Bayesian analyses of five simulated data sets.
Table S2 Proportion of hybrids detected by river using STRUC-
TURE and NEWHYBRIDS.
© 2014 John Wiley & Sons Ltd
1152 D. J . HASSELMAN ET AL.