Why only some plants emit isoprene

Why only some plants emit isoprenepce_12015 503..516

RUSSELL K. MONSON1, RYAN T. JONES2, TODD N. ROSENSTIEL3 & JÖRG-PETER SCHNITZLER4

1School of Natural Resources and the Environment and Laboratory for Tree Ring Research, University of Arizona, Tucson,AZ 85721, USA, 2Department of Environmental Science, Faculty of Agriculture and Environment, University of Sydney,Sydney, New South Wales 2006, Australia, 3Department of Biology and Center for Life in Extreme Environments, PortlandState University, Portland, OR 97201, USA and 4Research Unit Environmental Simulation, Institute of Biochemical PlantPathology, Helmholtz Zentrum, 85764 Neuherberg, Germany

ABSTRACT

Isoprene (2-methyl-1,3-butadiene) is emitted from manyplants and it appears to have an adaptive role in protectingleaves from abiotic stress. However, only some species emitisoprene. Isoprene emission has appeared and been lostmany times independently during the evolution of plants.As an example, our phylogenetic analysis shows that iso-prene emission is likely ancestral within the familyFabaceae (= Leguminosae), but that it has been lost at least16 times and secondarily gained at least 10 times throughindependent evolutionary events. Within the division Pteri-dophyta (ferns), we conservatively estimate that isopreneemissions have been gained five times and lost two timesthrough independent evolutionary events. Within the genusQuercus (oaks), isoprene emissions have been lost from oneclade, but replaced by a novel type of light-dependentmonoterpene emissions that uses the same metabolic path-ways and substrates as isoprene emissions. This novel typeof monoterpene emissions has appeared at least twice inde-pendently within Quercus, and has been lost from 9% of theindividuals within a single population of Quercus suber.Gain and loss of gene function for isoprene synthase ispossible through relatively few mutations. Thus, this traitappears frequently in lineages; but, once it appears, the timeavailable for evolutionary radiation into environments thatselect for the trait is short relative to the time required formutations capable of producing a non-functional isoprenesynthase gene. The high frequency of gains and losses of thetrait and its heterogeneous taxonomic distribution in plantsmay be explained by the relatively few mutations necessaryto produce or lose the isoprene synthase gene combinedwith the assumption that isoprene emission is advantageousin a narrow range of environments and phenotypes.

Key-words: drought; ferns; fitness; phloem; phylogeny; sub-strate; temperature; thermotolerance.

INTRODUCTION

Isoprene (2-methyl-1,3-butadiene) is one of numerous vola-tile organic compounds emitted from leaves (Harley,Monson & Lerdau 1999; Sharkey & Yeh 2001; Owen &

Peñuelas 2005; Loreto & Schnitzler 2010). Many studieshave been published in the past few decades on the conse-quences of plant isoprene emission on atmospheric chem-istry (Fehsenfeld et al. 1992; Kesselmeier & Staudt 1999;Fuentes et al. 2000; Monson & Holland 2001; Monson 2002).One issue that has intrigued plant biologists is the adaptivesignificance of the trait. In 1995, Sharkey & Singsaas (1995)published a paper entitled, ‘Why plants emit isoprene’, inwhich evidence was presented for the enhanced toleranceof photosynthetic processes to high temperature in thepresence of isoprene emission. This thermotolerancehypothesis has been supported in subsequent studies fromseveral laboratories, and has arguably been the hypothesismost often cited to explain the adaptive benefit of isopreneemission (Sharkey, Wiberley & Donohue 2008). However,the enhancement of thermotolerance by isoprene has notalways been observed (Logan & Monson 1999; Loivamäkiet al. 2007; Sharkey et al. 2008; Vickers et al. 2009a), andsince that initial report by Sharkey and Singsaas severalother hypotheses have been offered to explain the adaptivesignificance of isoprene production (Loreto et al. 2001;Affek & Yakir 2003; Rosenstiel et al. 2004; Owen &Peñuelas 2005; Magel et al. 2006; Laothawornkitkul et al.2008; Loivamäki et al. 2008;Vickers et al. 2009b).There is noa priori reason to believe that these alternate hypothesesare mutually exclusive and that there cannot be multipleadaptive roles for isoprene emission.

While progress has been made in explaining why plantsemit isoprene, there has been little progress in explainingwhy only some plants emit isoprene. It has been assumedthat the capacity for enzyme-catalysed isoprene emissionhas evolved independently within distinct lineages of plants,and may have been lost from some lineages (Loreto et al.1998; Harley et al. 1999; Sharkey et al. 2005); but whetherthese transitions occur frequently or rarely, and the distri-bution of such transitions across broad taxonomic groups,has not been discussed. In one of the few examples ofindependent origins that have been analysed, the isoprenesynthase (ISPS) genes from aspen and kudzu share only65% similarity in coding sequence (Sharkey et al. 2008); thisresult led the authors to suggest independent origins, albeitthrough informal inference. A cursory examination of thedistribution of isoprene emission among different plant lin-eages also supports independent origins (Harley et al.2004). For example, taxonomic groups as distinct as mosses

Correspondence: R. Monson. E-mail: [email protected]

Plant, Cell and Environment (2013) 36, 503–516 doi: 10.1111/pce.12015

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© 2012 Blackwell Publishing Ltd 503

and oak trees emit isoprene, but in groups as closely alliedas mosses and hornworts or oak and maple trees we can findboth emitters (mosses and oaks) and non-emitters (horn-worts and maples) (Hanson et al. 1999; Harley et al. 1999;Lerdau & Gray 2003). In some groups with high taxonomicdiversity, such as the Fabaceae (= Leguminosae), numerousisoprene-emitting genera exist and the trait is distributedamong traditionally defined subfamilies. In other groupswith high taxonomic diversity, such as the Poaceae, isopreneemission is relatively rare. Within the genus Quercus, it hasbeen observed that trees of most species emit isoprene, butone lineage of trees does not emit isoprene, and in anotherlineage trees emit light-dependent monoterpenes (Loretoet al. 1998, 2002). Thus, there are complex patterns in thedistribution of this trait; in some cases isoprene emissionseems to be phylogenetically conserved, whereas in others,the trait seems randomly distributed.

The fact that isoprene emission is dispersed so heteroge-neously among plant taxa must, in and of itself, havemeaning as to the adaptive significance of the trait. That is,answers to the questions – ‘why do plants emit isoprene’and ‘why do only some plants emit isoprene’ – are likely toemerge from common explanations, all dealing with thecosts and benefits by which the trait influences fitness, andincluding accommodation for stochasticity. In this paper, wehave conducted a more comprehensive phylogenetic analy-sis than has been attempted in the past with the aim ofgaining a deeper perspective of the lability of isopreneemission.We assembled our emission trait data from a data-base on isoprene emissions that included approximately1700 species of plants and were collated from numerouspast ‘survey-type’ studies. To explore the evolutionaryhistory of isoprene emission across plant diversity, wemapped isoprene emission on plant phylogenies that weconstructed using DNA sequence data. Our data set waslimited to plants that had both DNA sequence data and hadbeen tested for isoprene emissions. Nevertheless, we foundenough quantitative data to provide at least some broadinsight into phylogenetic dispersion of the trait and theextent to which it has been gained or lost independentlywithin well-defined lineages. After describing patterns ofphylogenetic distribution of isoprene emission in plant lin-eages at several taxonomic scales, we present the hypothesisthat this trait has been lost and gained frequently and sto-chastically due to a requirement for relatively few muta-tions to gain or lose a functional form of the enzymeisoprene synthase, combined with adaptive advantage in anarrow range of environments and phenotypes.

METHODS

Constructing a broad taxonomic database ofisoprene emission capacity

We derived data on isoprene emission capacity usingseveral sources. Most of the data were obtained from a listof ~1300 species that was constructed and has been main-tained by researchers at Lancaster University (http://

www.es.lancs.ac.uk/cnhgroup/iso-emissions.pdf), hereaftercalled the Lancaster List. In this list, species are not alwaysclassified with regard to rates of emission, and many aresimply classified as ‘emitters’ or ‘non-emitters’. We onlyused data for which we had quantitative measurements ofemission rate. For those species in which quantitative datawere included, the units used for emission rate varied. Weused species emission rates that were based on leaf area asthey were reported, and converted those based on leaf massto an area basis using an estimated specific leaf area(cm2 g-1) of 150 for angiosperms and 75 for gymnosperms(from Reich, Wright & Lusk 2007). The choice of thesevalues, while supported as averages obtained from broadsurveys, is subjective, and we acknowledge that it introducesuncertainty into the analysis. In recognition of these uncer-tainties, as well as those due to differences in measurementmethod and seasonality that exist among the multiplestudies in the Lancaster List, we classified the isopreneemission rate for specific taxa according to emission ranges– what we call ‘emission bins’.We obtained data on emissionrates from several additional studies that were not repre-sented in the Lancaster List, including surveys conducted inCosta Rica by Geron et al. (2002), southern Africa byHarley et al. (2003), the Amazon Basin by Harley et al.(2004), the Biosphere 2 facility by Pegoraro et al. (2006),southern California by Benjamin et al. (1996), China byLoreto et al. (2002) and Geron et al. (2006), and SouthAfrica by Zunckel et al. (2007). The five emission bins thatwe used are designated by progressively greater numbers ofasterisks within the presented phylogenies: no asterisk(emissions < 0.2 nmol m-2 s-1), * (emissions �0.2 and<10 nmol m-2 s-1), ** (emissions �10 and <30 nmol m-2 s-1),*** (emissions �30 and <50 nmol m-2 s-1), **** (emissions�50 nmol m-2 s-1). We only used values that were refer-enced to a photosynthetic photon flux density (PPFD) of1000 mmol m-2 s-1 and leaf or air temperature of 30 °C.

Construction of phylogenies

We acquired DNA sequence data from GenBank (http://www.ncbi.nlm.nih.gov/GenBank/) for four groups of plants:the legumes (Fabaceae), the oaks (Quercus), the Fagales (anorder of plants that includes the beech,birch,oak and walnutfamilies), and the ferns (Pteridophyta). Multiple genes wereused for each plant group: rbcL, trnL and matK for thelegumes (Supporting Information Table S1); 5.8S, CLAW,matK, rbcL, the intergenic space (IGS) between psbA andnuclear internal transcribed spacer (ITS), and the IGSbetween rbcL and atpB for the oaks (Supporting Informa-tion Table S2); ITS, trnL/trnF and matK for the Fagales(Supporting Information Table S3), and rbcL, atpA, matK,rps4, and IGS between trnL and trnF for the ferns (Support-ing InformationTable S4).Sequence data were aligned usingthe program, Muscle (http://www.drive5.com/muscle/), andedited by eye using Seaview v4.3.3 (http://pbil.univ-lyon1.fr/software/seaview.html). We used MrBayes v3.2 (http://mrbayes.sourceforge.net/) to generate phylogenies for eachplant group. We used a general time reversible substitution

504 R. K. Monson et al.

© 2012 Blackwell Publishing Ltd, Plant, Cell and Environment, 36, 503–516

model with gamma-distributed rate variation and a propor-tion of invariable sites. Data sets were partitioned such thatmodel parameters for each gene were estimated separately.We ran two independent runs (each started with a randomlychosen tree) with one cold chain and three heated chains perrun. Each of the four analyses was initially set to run for2 000 000 generations. We used a 25% burn-in (i.e. the first25% of the cold chains were discarded) and used the averagestandard deviation of split frequencies between the two runsto determine if the phylogenies between the runs had con-verged. If the average deviation of split frequencies was notbelow 0.01 after 2 000 000 generations, additional 1 000 000generations were added to the run. For Figs 2–5, isoprenepresence or absence was depicted on the consensus tree andnode values indicate the posterior distribution of the con-sensus topology.The number of state changes (i.e. gains andlosses of isoprene emission) was determined using parsi-mony. If there were equally parsimonious reconstructions,we chose losses of emission over gains of emission.

RESULTS

Emissions among all orders withindivision Angiospermae

We have mapped the capacity for isoprene emissionobserved in past studies onto the complete tree ofangiosperm orders developed by Soltis et al. (2011) using 17genes and 640 taxa (Fig. 1). The distribution of isopreneemissions at this scale is not particularly informative as toindependent state changes. However, it does reveal phylo-genetic domains that have been understudied. For example,in the Malvideae high emission rates have been observed inthe Sapindales and Myrtales, but nothing is known of emis-sion potentials in the two orders that separate the Sapindalesand Myrtales; that is, the Geraniales or Crossosomatales.Studies within Crossosomatales would be particularly inter-esting as this order includes woody, temperate-latitude trees,which occupy niches similar to those in Sapindales. TheGeraniales, in contrast, are largely composed of herbaceousspecies,but they are closely related to those woody species inthe Myrtales that have exceptionally high isoprene emissionrates. Thus, in the first comparison (Crossosomatales versusSapindales) ecological and growth habit tendencies aresimilar,but in the second comparison (Myrtales versus Gera-niales) they are not; yet, phylogenetic relatedness among allfour is high.

In other groups, we have highlighted a need for morestudies because they occupy basal positions within theangiosperm tree. For example, the Austrobaileyales is anorder with good representation of tropical woody trees(which often emit isoprene) and is a critical link betweenmonospecific Amborellaceae, the acknowledged most basalfamily of angiosperms, and other groups with high frequen-cies of tropical woody trees, such as Piperales, Laurales andMagnoliales. Insight into the origins and diversification ofisoprene emissions in the most basal angiosperms is limitedby a lack of sampling in the Amborellaceae and the Aus-trobaileyales.

Isoprene emissions have been observed within the Acro-gymnospermae (= Gymnospermae), particularly withinPinaceae.The potential for isoprene emissions in even moremembers of the Acrogymnospermae, however, has not beenclarified. For example, within Ephedraceae, isoprene emis-sions are known to be present, and at significantly high rates(Geron et al. 2006), but in two other gymnosperm families,Gnetaceae and Welwitschiaceae, emission potentials havenot been validated. There is unpublished evidence thatthese groups emit isoprene (Rasmussen, personal commu-nication 1991), but quantitative validation is not available.Further studies into isoprene emission in these groups,which share phylogenetic affinities with emitting membersof the Pinaceae and Ephedraceae, would sharpen ourunderstanding of diversification within the angiosperms.

Emissions within family Fabaceae

We explored the phylogenetic distribution of isopreneemission in Fabaceae (= Leguminosae), which includes alarge number of isoprene-emitting tropical tree species(Fig. 2). We chose Populus as the outgroup for our analysisof Fabaceae because we wanted to use a group that wasbasal yet close in affinity to the order Fabales, and includedadequate DNA sequence data for analysis. Based on theSoltis tree, the closest basal orders would be Oxalidales,Malpighiales and Celastrales. We chose Populus (in Mal-pighiales) because it provided us with the largest source ofsequence data. Assuming that isoprene emission wascarried into the Fabaceae from ancestors, we estimate atleast 16 subsequent losses and 10 secondary gains (i.e. gainsthat followed losses).1 One of the more active nodes ofevolution with regard to isoprene emissions withinFabaceae occurs in genera that were formerly groupedtogether within the genus Acacia (Table 1). Using tradi-tional taxonomy, Acacia is the second largest genus withinFabaceae, with over 1400 species (Lewis et al. 2005). Morerecently, however, traditional Acacia has been partitionedinto five new and distinct clades: Acacia sensu stricto(Acacia s.s.), Vachellia, Senegalia, Acaciella and Mariosousa(Brown et al. 2008; Bouchenak-Khelladi et al. 2010). Thestate of isoprene emissions in Mariosousa, Acaciella andAcacia s.s. is uncertain. Vachellia, however, appears to lackisoprene emitters, a state that was likely ancestral. Senegaliaappears to have obtained isoprene emissions secondarily,after divergence from Vachellia, and independently fromthe emergence of isoprene emissions in allied species inAlbizia and the Inga-Zygia clade.

1The numbers of gains and losses that we report are dependent notonly on our inferred phylogenies, but also on the incomplete sam-pling within lineages for the presence or absence of isoprene emis-sions. However, given acceptance of the phylogenetic models, theestimated number of state transitions should be conservative. If, forexample, in the future, isoprene emissions are discovered in a genusof otherwise non-emitting species, and we have designated thatgenus as having undergone a transition from emitting to non-emitting, then the new observation can only lead to a greater orequal number of estimated state transitions, not a lesser number.

Phylogeny of isoprene 505


FagalesCucurbitales

gg

RosalesFabales

CelastralesMalpighialesOxalidales

Zygophyllales

Sapindales

MalvalesBrassicalesHuerteales

CrossosomatalesGeranialesMyrtalesVitaceaeVVSaxifragales

GentianalesSolanalesBoraginaceaeLamiales

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VahliaceaeVV

IcacinaceaeGarryalesOncothecaceae

Asterales

Dipsacales

ApialesEscalloniales

Paracryphiales

BrunialesAquifolialesEricalesCornales

SantalalesDilleniaceae

CaryophyllalesBerberidopsidales

Gunneraceae

TrochodendraceaeBuxaceae

SabiaceaeProtealesRanunculalesCeratophyllaceae

Magnoliales

CanellalesLaurales

gg

PiperalesChloranthaceae

Monocotyledoneae

TrimeniaceaeSchisandraceaeAustrobaileyaceaeNymphaeaceae

Amborellaceae

PinaceaePodocarpaceaePodocarpaceaeCupressaceaeCycadalesC

GnetaceaeWelwitschiaceaeWW

Ginkgoaceae

Fabidae

Malvidae

Lamiidae

Campanulidae

Magnoliidae

Acrogymnospermae

Austrobaileyales

eadi

renn

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odel

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Picramniaceaepp

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Rosidae

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****

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*****

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Figure 1. Isoprene emission across the spermatophytes. Taxonomic groups with isoprene emitters are in black, groups lacking emittersare in grey, and untested groups are in red. Asterisks indicate relative isoprene emission rates (see text). Traits were mapped across thephylogeny using parsimony. Phylogeny was reproduced with permission based on Soltis et al. (2011).



0.02

ParkiaProsopis

Pentaclethra****

Eysenhardtia*?Cladastris

Swartzia**

Lotus*?

Sophora

Pictetia****

Medicago

Indigofera

Albizia**

Robinia

Ulex**

Vicia

Stryphnodendron

Pueraria***

Pterocarpus****

Erythrina

Calliandra

Amburana***

Isoberlinia*

Lathyrus*?

Mariosousa

Maackia*?

Acaciella

Pericopsis

Cordyla***

Mimosa

Burkea**

Colutea

Detarium**

Bolusanthus

Pithecellobium

Poecilanthe

Dalbergia**

Anthyllis

Vachellia

Zygia****

Poincianella

Pisum

Gliricidia

Vigna*?

Baphia*?

Bauhinia**

Mucuna****

Adenocarpus*?

Ormocarpum*

Phaseolus

Clitoria

Wisteria

Lupinus

Senegalia***

Guibourtia*

Lonchocarpus*

Cassia

Daniellia*

Sclerolobium

Populus****

Dialium

Trifolium

Cercis

Caragana

Piliostigma

Olneya

Glycine

Colophospermum

Cercidium

Leucaena

Arachis

Cytisus*

Mundulea****

Delonix

Macrolobium

Templetonia*?

Gilbertiodendron

Spartium*

Gymnocladus

Millettia

Calpurnia

Piptadeniastrum

Hymenostegia

Berlinia

Gleditsia

Dalhousiea*

Derris*?

Dipteryx**

Pachyelasma

Genista*

Inga***

Schizolobium

Figure 2. Isoprene emission across the Fabaceae (= Leguminosae). Taxonomic groups with isoprene emitters are in black and groupswithout emitters are in grey. Asterisks indicate relative isoprene emission rates (see text). Question marks indicate groups with significantuncertainty about isoprene emission rates. Traits were mapped across the phylogeny using parsimony. The scale bar indicates the numberof expected substitutions per site. Posterior probabilities are excluded from the figure because of lack of space, but of 174 branches, 141have 100% support, 160 have >90% support, 168 have >80% support, 170 have >70% support, 171 have >60% support, and all 174 havegreater than 50% support.



Biogeographic patterns suggest that Vachellia evolved inopen woodlands in America during the Oligocene, approxi-mately 16 million years ago (mya), and then subsequentlymigrated to Africa (Bouchenak-Khelladi et al. 2010). OnceVachellia expanded within Africa, diversification producedSenegalia (Bouchenak-Khelladi et al. 2010). The evolutionof isoprene emission in Senegalia likely occurred during therelatively arid mid-to-late Miocene and subsequentPliocene, beginning approximately 10 mya. This is the sameperiod in which C4 savannas expanded within Africa,America and Australia, which fostered open, semi-aridhabitats (Beerling & Osborne 2006). The emergence of iso-prene emission may have contributed to persistence ofSenegalia in the semi-arid regions of Africa, perhapsthrough enhanced tolerance of drought and associatedabiotic stresses.

Emissions within order Fagales

We chose to focus part of our phylogenetic reconstructionon the order Fagales (Fig. 3) because it contains the oaks,which are known to exhibit isoprene emission rates amongthe highest recorded. Gene sequence data analysed in two

independent studies have confirmed that Nothofagus, anon-isoprene-emitting genus, which had previously beenincluded within Fagaceae, is in fact monophyletic and basalto the ‘higher’ hamamelid families including Fagaceae,Casuarinaceae, Betulaceae, Myricaceae and Juglandaceae(Manos & Steele 1997; Li et al. 2004). The basal position ofNothofagus was supported in our phylogeny. Our analysisplaced Quercus and Lithocarpus within the same clade, withthe potential for isoprene emission arising in both throughone event. Independent origins appear to have occurred atthe diversification that gave rise to Casuarina, and possibly

Table 1. Species of Acacia that have been assayed for isopreneemission and are organized according to three of the five cladesrecognized from chloroplast DNA analysis (followingBouchenak-Khelladi et al. 2010)

VachelliaAcacia borlaeAcacia niloticaAcacia robustaAcacia sieberianaAcacia tortilisAcacia xanthophloea

SenegaliaAcacia berlandieriAcacia burkei **Acacia galpinii *Acacia nigrescens **Acacia pentagonaAcacia polyacantha *Acacia senegal **

Acacia s.s.Acacia baileyana ?Acacia dealbata ?Acacia decurrens ?Acacia longifoliaAcacia melanoxylonAcacia pennata

Unknown affinitiesAcacia greggiiAcacia podalyriaefoliaAcacia sophoreae

Data on the presence or absence of isoprene emission were takenfrom Harley et al. (2003) or the Lancaster Table at http://www.es.lancs.ac.uk/cnhgroup/iso-emissions.pdf. The question marks fol-lowing some taxa in the Acacia s.s. indicate questionable affinity(see text). The isoprene emission capacity is indicated according toa quantitative scale indicated by the number of asterisks (see text).

0.03

Castanopsis

Nothofagus

Castanea

Casuarina**

Myrica*?

Pterocarya

Populus****

Carpinus

Alnus

Juglans

Quercus****

Betula

Lithocarpus*

Corylus

Fagus

Ostrya

Carya

1

1

1

1

0.92

1

1

1

1

1

1

0.71

1

1

0.99

Figure 3. Isoprene emission across the Fagales. Taxonomicgroups with isoprene emitters are in black and groups withoutemitters are in grey. Asterisks indicate relative isoprene emissionrates (see text). Question marks indicate groups with significantuncertainty about isoprene emission rates. Traits were mappedacross the phylogeny using parsimony. The scale bar indicates thenumber of expected substitutions per site. Support for branchesis indicated by posterior probability values.



0.002

Q.ilex

Q.trojana

Lithocarpus*

Q. lobata*

Q. rubra***

Q.baronii

Q.coccifera

Q.libani

Q.acutissima

Q.cerris

Q.chrysolepis

Q.variabilis

Q. agrifolia**

Q. robur**

Q. virginiana**

Q.suber

Q. alba***

Q. laevis*

1

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0.98

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0.55

0.74

0.58

1

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0.002

Q.ilex

Q.trojana

Lithocarpus*

Q. lobata*

Q. rubra***

Q.baronii

Q.coccifera

Q.libani

Q.acutissima

Q.cerris

Q. chrysolepis*

Q.variabilis

Q. agrifolia**

Q. robur**

Q. virginiana**

Q.suber

Q. alba***

Q. laevis*

1

0.68

0.98

1

0.55

0.74

0.58

1

0.77

1

0.99

1

0.76

0.99

(a) (b)

Figure 4. Isoprene emission across the genus Quercus. Isoprene-emitting species are in black, light-dependent monoterpene-emittingspecies are in dashed black, and non-emitting species are in grey. Asterisks indicate relative isoprene emission rates (see text). Traits weremapped across the phylogeny using parsimony. The scale bar indicates the number of expected substitutions per site. Support for branchesis indicated by posterior probability values. Two equally parsimonious scenarios are presented: (a) isoprene emission switched tolight-dependent monoterpene emission, which was subsequently lost in one lineage; and (b) isoprene emission was lost and a subsequentlineage gained light-dependent monoterpene emission.



0.2

Alsophila*?

Cyathea*

Blechnum

Coniogramme****

Marsilea

Adiantum****

Platycerium

Dicksonia*?

Angiopteris

Drynaria

Nephrolepis

Thelypteris*

Pellaea*

Psilotum

Dryopteris*?

Cibotium*?

Polypodium*

Rumohra

Cyclosorus

Pteris

Asplenium

Tectaria****

Pyrrosia

Cyrtomium

Equisetum

0.96

1

1

1

10.95

1

0.99

1

0.99

0.72

1

1

1

1

1

1

1

0.72

0.95

1

0.94

1

****

Figure 5. Isoprene emission across the Pteridophyta. Taxonomic groups with isoprene emitters are in black and groups without emittersare in grey. Asterisks indicate relative isoprene emission rates (see text). Question marks indicate groups with significant uncertaintyabout isoprene emission rates. Traits were mapped across the phylogeny using parsimony. The scale bar indicates the number of expectedsubstitutions per site. Support for branches is indicated by posterior probability values.



Myrica if subsequent studies confirm emissions in the lattergroup. Thus, the trait appears to have been lost early in, orprior to, diversification of the order and then gained sec-ondarily at least twice.

Emissions within genus Quercus

It is informative to take a closer look at diversificationwithin the genus Quercus (Fig. 4), not only because someof its species are those with the highest recorded isopreneemission rates, but also because some of its species exhibitlight-dependent monoterpene emissions, rather than iso-prene emissions, and still others do not emit either type ofcompound. The phylogenetic distribution of isopreneemissions within Quercus has been analysed previouslyin Loreto (2002). In that analysis, 61 species were placedinto phylogenetic context through a combination of tradi-tional morphological taxonomies and gene sequenceanalysis. However, since that study our knowledge of phy-logenetic affinities and terpene emission capacities withinQuercus has increased and there is need for renewedassessment.

Loreto (2002) concluded that isoprene emission wasancestral within Quercus, and that the loss of emissions insome species was organized around nodes of traditionalsubgeneric clades. Loreto (2002) rooted Quercus with genusCyclobalanopsis, which may actually exist as a subgenuswithin Quercus (Manos, Doyle & Nixon 1999; Denk &Grimm 2009, 2010). In our analysis, we rooted Quercus withgenus Lithocarpus, which has been justified using genemarkers (Denk & Grimm 2010). Both Cyclobalanopsis andLithocarpus have been shown to emit isoprene at low-to-moderate rates (Loreto et al. 2002), so that rooting the treein either group leads to similar conclusions with regard tothe ancestral state of isoprene emission within Quercus. Inthe analysis presented in Fig. 4, we only charted thosespecies for which we had both good phylogenetic contextand quantitative measurements of isoprene or light-dependent monoterpene emission rates. We drew onseveral studies to obtain quantitative isoprene emissionrates for Quercus species, including Benjamin et al. (1996),Steinbrecher et al. (1997), Csiky & Seufert (1999), Harleyet al. (1999), Loreto et al. (2002), Staudt et al. (2004) and Pioet al. (2005).

Consistent with the study of Loreto (2002), we concludethat isoprene emission is ancestral within Quercus, havingbeen carried into the group during diversification fromLithocarpus. Early radiation within the genus led to twowell-supported lineages – the Protobalanus-Lobatae-Quercus clade and the Cerris-Cyclobalanopsis-Ilex clade(Denk & Grimm 2010). In Fig. 4, the node at which thesetwo clades diverged is clearly marked by the presence oflow-to-high isoprene emission rates (black branches) in theProtobalanus-Lobatae-Quercus clade and lack of isopreneemissions (grey branches) in the Cerris-Cyclobalanopsis-Ilex clade. The trait appears to have been preserved in allspecies and amplified in some species during radiation ofthe Protobalanus-Lobatae-Quercus clade, but lost and not

restored, at least within the Cerris-Ilex portions of theCerris-Cyclobalanopsis-Ilex clade. (We did not includeCyclobalanopsis in the analysis shown in Fig. 4 although itsposition as sister group to the Cerroid and Ilicoid lines, assuggested by Denk & Grimm (2010), would not change theconclusion that loss of isoprene emissions must haveoccurred early during radiation of the Cerris and Ilexgroups.)

The loss of isoprene emissions in the Cerris-Cyclobalanopsis-Ilex clade was accompanied by the gain oflight-dependent monoterpene emissions in the Ilicoidgenera and at least one of the Cerroid genera (Loreto 2002).There are two possible paths by which these gains could haveoccurred. In the first, the gene for isoprene synthase,which istargeted through a leader peptide sequence to the chloro-plast, could have undergone (1) neo-functionalization toproduce a monoterpene synthase gene, also targeted to thechloroplast, in the common ancestor to the Ilicoid andCerroid lines; (2) subsequent amplification in expression ofthis novel gene as the Ilicoid oaks radiated into the warmerand drier environment of the Mediterranean region; and (3)subsequent non-functionalization of this novel gene throughgenetic drift to produce a lineage with neither isoprene norlight-dependent monoterpene emissions as most of theCerroid oaks (e.g. Quercus libani and Quercus cerris) radi-ated into the humid temperate and subtropical regionsof East Asia and Western Eurasia. This possible path isthus characterized by serial events going from neo-functionalization in the ancestor to all Ilicoid and Cerroidspecies, followed by non-functionalization in most of theCerroid species. In the one species from the Cerroid oaksthat radiated into the Mediterranean region (Quercus-suber), light-dependent monoterpene emissions have

apparently emerged secondarily (Staudt et al. 2004; Pio et al.2005). In the second possible path, the gene for isoprenesynthase underwent non-functionalization in the ancestor tothe Ilicoid and Cerroid oaks, to form a clade of non-emittingoaks, following divergence from Cyclobalanopsis. Thiswould have been followed by neo-functionalization of aduplicated chloroplastic monoterpene synthase gene toproduce a novel type of light-dependent monoterpene emis-sions in the Ilicoid line as it radiated into Mediterraneanregions (Loreto 2002). This possible path is thus character-ized by serial events going from non-functionalization in theancestor to all Ilicoid and Cerroid genera, followed by neo-functionalization in the ancestor to the Ilicoid genera.Similar to the first alternative path, Q. suber, allied with theCerroid oaks, presumably gained light-dependent monoter-pene emissions secondarily as it radiated into Mediterra-nean regions. Both alternative paths involve threeindependent events; they simply differ as to the sequence ofevents and whether the source for light-dependent monot-erpene emissions in the Ilicoid oaks was a modified isoprenesynthase gene (first path) or a duplicated monoterpene syn-thase gene (second path).

The high degree of lability in isoprene emissions as a traitthat is lost and gained frequently is strikingly demonstratedwithin Quercus in the results reported by Staudt et al.



(2004). In a Q. suber population from northern Spain, 9% ofthe trees that were sampled did not emit light-dependentmonoterpenes or isoprene, whereas 91% of the treesemitted light-dependent monoterpenes. The reason for lossof emissions in some of the trees is not known; although it ismore likely to be due to non-functionalization of the light-dependent monoterpene synthase gene than an inability togenerate adequate dimethylallyl diphosphate (DMADP)substrate, as mutations in the MEP pathway leading toDMADP tend to interfere with metabolic processes thatare crucial to plant survival (Estevez et al. 2001; Floss et al.2008; Rodriguez-Concepción 2010). The particular popula-tion that was studied is one that has experienced geneticintrogression from a related sympatric species, Quercus ilex;although Q. ilex is also a light-dependent monoterpeneemitter, genetic introgression cannot explain the lack ofmonoterpene emissions in some trees of Q. suber.However, the fact that trees within a single population havepersisted for decades without light-dependent monoter-pene emissions indicates that the trait can be easily lostfrom a population, and that its presence has not beenrequired as an adaptation promoting tree survival andgrowth, at least within recent decades.

The distribution of isoprene emission withinthe ferns (Pteridophyta)

Some fern species are capable of isoprene emission ratesas high as 60–70 nmol m-2 s-1, which places them withinthe highest category of emissions observed in vascularplants (Hanson et al. 1999; Geron et al. 2006). We con-structed a phylogenetic tree for those genera that havebeen examined for isoprene emission. This analysisincluded measurements reported in three sources: Hansonet al. (1999), Saito & Yokouchi (2006) and Geron et al.(2006). The report by Hanson et al. (1999) containedreports of emissions from earlier studies by Rasmussen(compiled in the Lancaster List), Tingey et al. (1987) andIsidorov, Zenkevich & Ioffe (1985). In all, we had access toreports of isoprene emission measurements for 36 fernspecies distributed across 24 genera.

Our model of fern phylogeny reveals that lack of the traitwas the likely ancestral condition with the potential for atleast five independent gains and two subsequent losses(Fig. 5). These events include a gain and amplification ofexpression in Adiantum and Coniogramme, with subse-quent loss in Pteris.A gain and amplification appear to haveoccurred within Tectaria, with subsequent loss in thePyrossia-Drynaria-Platycerium lineage. An independentgain appears to have occurred in the Thelypteris-Cyclosorus lineage, with amplification in Thelypteris. Atpresent, we have no hypothesis to explain why the traitappeared and became amplified to high levels of expressionin some branches of a lineage, while becoming non-functional in other branches. There appears to be no cleardistinction among genera with these divergent patterns interms of ecological niche assortment or phenotype.

Random mutation and the origins of isoprenesynthase: why isoprene emission has so manyindependent origins

Plant terpene synthases (TPSs) represent a highly diverseset of enzymes controlled by an equally diverse set of genes(Chen et al. 2011). Many of the TPSs catalyse reactionsleading to more than one type of biochemical product(Bohlmann, Meyer-Gauen & Croteau 1998), and manyhave more than one active site, often catalysing differentreactions. The TPS gene family is subdivided into sevensubfamilies, TPS-a to TPS-h (Chen et al. 2011), with iso-prene synthase protein (ISPS) showing affinities to theTPS-b subfamily. [Two of the original subfamilies, TPS-eand TPS-f, were reorganized into a single subfamily in theclassification of Chen et al. (2011)]. The TPS-b subfamilyalso includes monoterpene synthases from angiosperms(Miller, Oschinski & Zimmer 2001). The active site of ISPScontains an a-helical class I terpenoid fold (Köksal et al.2010), which is characteristic of eudicot monoterpene andsesquiterpene synthases (Christianson 2006; Cao et al.2010). The active site fold of poplar ISPS (Köksal et al.2010) is shallower than those described for monoterpenesynthases in the TPS-b subfamily (Hyatt et al. 2007), consis-tent with the binding of the C5 substrate DMADP ratherthan the C10 substrate geranyl diphosphate (GDP). Usingprotein modelling, Gray et al. (2011) showed that thevolume of the active site fold of ISPS may have beenreduced during evolution by replacement of smaller aminoacids, in one or a few positions, with larger amino acids (e.g.phenylalanines). If true, this model would suggest that rela-tively few mutations are required to produce the gene forISPS from the duplicated genes of existing TPS (Gray et al.2011). Isoprene emission from gymnosperms, such asspecies in Picea, is likely due to ISPS protein derived froman ancestor within the TPS-d subfamily and may sharemore similarities with gymnosperm methylbutenol syn-thases and linalool synthases than with angiosperm ISPS(Gray et al. 2011).

The genes for ISPS from both kudzu and poplar havebeen transferred into Arabidopsis, and isoprene emissionfrom transformed Arabidopsis was observed (Sharkey et al.2005; Loivamäki et al. 2007). However, the rates of isopreneemission were approximately 1/20th the rates observed inwild-type species, and DMADP substrate concentration inArabidopsis leaves was 6–10 times lower than in wild-typeemitters (Loivamäki et al. 2007). Thus, even in those caseswhere a novel ISPS enzyme has appeared (in this case by‘engineering’), isoprene emission rates are often low, mostlikely affected by limited DMADP substrate availability.Evidence obtained from kudzu indicates that while geneticmodification to the expression of MEP pathway genes inisoprene emitters may not be required to sustain isopreneemissions, once ISPS appears, some form of adjustment(genetic or otherwise) must occur in order to increase thesupply of DMADP substrate (Sharkey et al. 2005), and thisdoes not happen solely as a consequence of the appearanceof ISPS.



More recently, ISPS was transferred into non-emittingtobacco plants, and rates of isoprene emission wereobserved to be higher than in the previous Arabidopsisexperiments; in fact, rates of isoprene emission from plantsin one of the four transgenic tobacco lines were similar tothose observed for Populus alba leaves (a native isopreneemitter) (Vickers et al. 2009a, 2011). The capacity for highrates of emission in this one transgenic tobacco line wassupported by high rates of DMADP substrate generation(Vickers et al. 2011).These studies indicate that pre-existingphenotype matters to the potential for isoprene emissionsin a lineage once a novel ISPS gene appears. Amplificationof expression in the novel ISPS gene may be controlled bythe pre-existing potential to generate DMADP substrate atthe time of trait appearance, and this in turn may be depen-dent on pre-existing potential to synthesize chloroplastmetabolites other than isoprene, which depend on DMADPsubstrate (e.g. other terpenoids, carotenoids and abscisicacid).

Inferring from these past studies, it is reasonable to con-clude that the evolution of isoprene emission in plantsrequires either (1) mutations that affect both the appear-ance of ISPS and up-regulation of the capacity for leaves toproduce DMADP substrate; or (2) a pre-existing pheno-type that enables amplification of expression of ISPS onceit appears. As stated in the study by Sharkey et al. (2005):‘The trait of isoprene emission appears to be easily acquiredby plants, although the regulation of DMADP concentra-tion may have to be altered to support the high rates ofisoprene emission seen in some plants.’ We agree.

Non-random selection for isoprene emissions:why isoprene emissions only persist in someplant lineages

The evidence drawn from phylogenetic distributions of iso-prene emission suggests that it has disappeared frequentlyfrom lineages during past diversification. There are twounderlying evolutionary forces that might explain loss ofthe trait. Firstly, isoprene emission may incur a cost togrowth and associated fitness in certain phenotypes and/orenvironments. In the presence of a cost to fitness, andwithout a balanced adaptive benefit, any random mutationsthat cause non-functionalization of the ISPS gene wouldprovide a selective advantage, favouring loss of the trait.Secondly, if the trait is neutral in its effect on fitness, thenthe accumulation of mutations leading to ISPS non-functionalization may occur through genetic drift.

We propose a model whereby the mutations giving riseto ISPS occur relatively frequently, but the range of envi-ronments or phenotypes within which isoprene emissionsare adaptive or within which its expression is capable ofbeing amplified, is relatively narrow. Thus, the determinantas to whether the trait exists in a lineage reflects thebalance between a relatively high frequency of origin anda relatively low frequency of retention. Furthermore, theavailability of niches in which isoprene emissions areadaptive is likely to change over time, either due to a

changing environment (e.g. change in atmospheric CO2

concentration or climate), changes in potential plantmigration patterns (e.g. due to continental drift or upliftof topography), or evolutionary changes in plant pheno-type (e.g. greater or lesser capacity to produce DMADPsubstrate).

What are the environmental conditions or functionalphenotypes that could determine the persistence or loss ofisoprene emissions? There is clear evidence that isopreneemission fosters tolerance of abiotic stresses. The exactnature of the tolerance, and the relative importance of dif-ferent stresses, continues to be debated (Loreto & Schnit-zler 2010). In one study, tolerance of extremely high leaftemperatures and photon flux densities by isoprene-emitting poplar leaves was shown to be greatest whentrees were grown at a low (190 ppmv) atmospheric CO2

concentration (Way et al. 2011). This observation was usedto suggest that the adaptive advantage provided by iso-prene emission was enhanced during past epochs with rela-tively low atmospheric CO2 concentrations (e.g. postMiocene) (Way et al. 2011). When isoprene-emittingspecies are grown in an atmosphere of elevated CO2, emis-sion rates are suppressed, most likely due to limited avail-ability of chloroplastic pyruvate, one of the initialsubstrates required for DMADP biosynthesis (Rosenstielet al. 2003; Monson et al. 2007; Wilkinson et al. 2009; Possell& Hewitt 2011; Trowbridge et al. 2012). When grown atelevated atmospheric CO2 concentrations, the photosyn-thetic processes of C3 plant leaves are typically more tol-erant of abiotic stresses (Morgan, Ainsworth & Long 2003;Leakey et al. 2009), especially high-temperature stress(Wang et al. 2012), meaning that isoprene emissions mayhave less of an adaptive margin during epochs with higheratmospheric CO2 concentration. There are also certaintypes of pre-existing plant phenotypes that may amplify ormute selection for isoprene emission once it appears in alineage. For example, it has been suggested that plants withcertain phloem-loading phenotypes, which favour high leafsugar concentrations in the cytosol of leaf mesophyll cells,might be better able to support the high rates of DMADPsynthesis required to amplify the expression of isopreneemissions once it appears (Logan, Monson & Potosnak2000; Kerstiens & Possell 2001). This latter point may berelevant to the puzzle as to why isoprene emission occursso commonly in lineages of trees, but so uncommonly inlineages of herbs – these growth habits tend to differ intheir phloem-loading mechanisms. Thus, a complex set ofinteractions exists among changes in the earth’s climateand atmospheric CO2 concentration, pre-existing andnovel plant phenotypes, and geographic congruencebetween trajectories of lineage radiation and the locationof those abiotic stresses most likely to favour, or not, thepersistence of isoprene emissions, once the trait appears.

Given the complexity of these interactions, the mostintriguing question to be answered would appear to be not‘why isoprene emissions has appeared so many times inde-pendently within plant lineages’, but rather ‘why has itbeen lost so many times independently’. Is it because



random patterns of evolutionary radiation have causedsome clades to assort into niches where the trait loses itsadaptive premium and genetic drift leads to eventual loss?If so, then we would expect to discover patterns of nicheassortment that correlate with the presence or absence ofthe trait within lineages. This is not always the case. Ingroups such as the Fabaceae, where so many loss eventsappear to have occurred, many species occupy similartropical or subtropical forest niches, whether they areemitters or not. Alternatively, is it because the trait has apositive effect on fitness in a relatively narrow range ofniches and phenotypes; and in the absence of the traitradiating into those niches or arising in those phenotypes,genetic drift (or weak negative selection) causes the traitto disappear as frequently as it appears? If few mutationsare required to create the trait, and even fewer mutationsare required to lose the trait, there is a narrow window ofopportunity for the trait to ‘find’ a situation that favoursadaptive persistence, before genetic drift causes it to belost.

CONCLUDING STATEMENTS

Most investigations to date on the topic of isoprene emis-sion from plants have focused on its potential adaptivebenefit to the photosynthetic processes of the leaf; in otherwords, ‘why plants emit isoprene’. However, one questionhas remained in the background and has not been explic-itly addressed during decades of investigation: ‘why doonly some plants emit isoprene?’ In order to address thisquestion, investigators will need to move beyond descrip-tions of plant function in emitting species, and considerthe differences that exist between emitting and non-emitting species in phenotype, ecological niche and phylo-genetic history. We have shown that there is substantialevidence suggesting that isoprene emission has emerged,and been lost, many times independently within plant lin-eages. An explanation of these patterns requires consider-ation of several inferences: (1) few mutations are requiredfor the trait to evolve; (2) the adaptive benefit of the traitvaries depending on several factors, including climate,atmospheric CO2 regimes, phenotype and patterns of geo-graphic migration; and (3) adaptive benefits are likely tooccur in a relatively narrow range of combinations ofthese factors. Given these conditions, it is possible toexplain patterns of frequent trait emergence and loss inthe phylogenetic record. We hope that by bringing theseperspectives into the arena of debate we can stimulatefuture research that will further clarify the ‘why’ questionsunderlying the distribution of isoprene emissions withregard to taxonomy, plant growth habit, ecological nicheand geography.

ACKNOWLEDGMENTS

We are grateful to the helpful comments provided by DrPeter Harley, Dr Hardeep Rai and an anonymous reviewer.The authors have no conflicts of interest to declare.

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Received 24 October 2011; received in revised form 24 August 2012;accepted for publication 10 September 2012

NOTE ADDED IN PROOF

In a recent study (Welter et al., 2012, Tree Physiology,doi:10.1093/treephys/tps069), populations of the NorthAfrican oak species, Quercus afares, which have hybridorigins from ancestral isoprene-emitting and light-dependent,monoterpene-emitting species,have been shownto have secondarily evolved complete suppression of iso-prene emission and modification of monoterpene emissioncapacity as it radiated into cooler, less arid ecosystems.Theseresults are consistent with our hypothesis of high evolution-ary lability and frequent shifts that lead to gains or losses ofisoprene and light-dependent monoterpene emissions.

SUPPORTING INFORMATION

Additional Supporting Information may be found in theonline version of this article:

Table S1. Accession numbers of DNA sequence data usedfor legume phylogenetic analyses.Table S2. Accession numbers of DNA sequence data usedfor Fagales phylogenetic analyses.Table S3. Accession numbers of DNA sequence data usedfor oak (Quercus) phylogenetic analyses.Table S4. Accession numbers of DNA sequence data usedfor fern phylogenetic analyses.



Why only some plants emit isoprene

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