Adaptive Molecular Evolution of PHYE in Primulina, a Karst Cave Plant


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Adaptive Molecular Evolution of PHYE in Primulina, a Karst Cave Plant

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Adaptive Molecular Evolution of PHYE in Primulina, a Karst Cave Plant
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PLOS One
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Tao, Junjie
Qi, Qingwen
Kang, Ming
Huang, Hongwen
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Limestone ( local )
Primulina ( local )
Karst Cave ( local )
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serial ( sobekcm )

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Limestone Karst areas possess high levels of biodiversity and endemism. Primulina is a typical component of Karst endemic floras. The high species richness and wide distribution in various Karst microenvironments make the genus an idea model for studying speciation and local adaptation. In this study, we obtained 10 full-length sequences of the phytochrome PHYE from available transcriptome resources of Primulina and amplified partial sequences of PHYE from the genomic DNA of 74 Primulina species. Then, we used maximum-likelihood approaches to explore molecular evolution of PHYE in this Karst cave plant. The results showed that PHYE was dominated by purifying selection in both data sets, and two sites were identified as potentially under positive selection. Furthermore, the ω ratio varies greatly among different functional domains of PHYE and among different species lineages. These results suggest that potential positive selection in PHYE might have played an important role in the adaption of Primulina to heterogeneous light environments in Karst regions, and different species lineages might have been subjected to different selective pressures.

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RESEARCHARTICLEAdaptiveMolecularEvolutionof PHYE in Primulina ,aKarstCavePlantJunjieTao1,2,QingwenQi1,2,MingKang1* ,HongwenHuang1*1 KeyLaboratoryofPlantResourcesConservationandSustainableUtilization,SouthChinaBotanical Garden,ChineseAcademyofSciences,Guangzhou,China, 2 UniversityofChineseAcademyofSciences, Beijing,China * mingkang@scbg.ac.cn (MK); huanghw@mail.scbg.ac.cn (HH)AbstractLimestoneKarstareaspossesshighlevelsofbiodiversityandendemism. Primulina isatypicalcomponentofKarstendemicfloras.Thehighspeciesrichnessandwidedistributionin variousKarstmicroenvironmentsmakethegenusanideamodelforstudyingspeciation andlocaladaptation.Inthisstudy,weobtained10full-lengthsequencesofthephytochrome PHYE fromavailabletranscriptomeresourcesof Primulina andamplifiedpartialsequencesof PHYE fromthegenomicDNAof74 Primulina species.Then,weused maximum-likelihoodapproachestoexploremolecularevolutionof PHYE inthisKarstcave plant.Theresultsshowedthat PHYE wasdominatedbypurifyingselectioninbothdata sets,andtwositeswereidentifiedaspotentiallyunderpositiveselection.Furthermore,the ratiovariesgreatlyamongdifferentfunctionaldomainsof PHYE andamongdifferentspecieslineages.Theseresultssuggestthatpotentialpositiveselectionin PHYE mighthave playedanimportantroleintheadaptionof Primulina toheterogeneouslightenvironmentsin Karstregions,anddifferentspecieslineagesmighthavebeensubjectedtodifferentselectivepressures.IntroductionLightisnotonlythesourceofenergy,butalsoaveryimportantenvironmentalfactorforplant growthandsurvival.Assessileorganisms,plantshaveevolvedsophisticatedphotosensorysystemstorespondappropriatelytotheirlightenvironments.Phytochromesarespecialized photosensorsthatperceiveandinterpretlightsignalsfromtheenvironmenttoregulateplant growthanddevelopmentthroughoutthewholelifecycle[ 1 ].Recentstudieshaverevealedthat phytochromesplayanimportantroleinmodulatingbothbioticandabioticstress[ 2 ].Inangiosperms,thephytochromeapoproteingeneshavebeenclassifiedintofourorfivegenesubfamiliesbasedonsequencesimilaritytothefivephytochromegenesof Arabidopsis : PHYA , PHYB , PHYC , PHYD ,and PHYE [ 3 ].The PHYB and PHYD subfamiliesareevolutionarilyrelatedto PHYE ,whereas PHYA and PHYC arerelatedtoeachotherandformedanancientevolutionary clade[ 3 , 4 ].Asthethreetypicalisoformsofphytochromesthatareexpressedwidelyinseed plants,thefunctionandevolutionof PHYA , PHYB and PHYC havebeenextensivelystudied PLOSONE|DOI:10.1371/journal.pone.0127821June1,2015 1/14 OPENACCESS Citation: TaoJ,QiQ,KangM,HuangH(2015) AdaptiveMolecularEvolutionof PHYE in Primulina ,a KarstCavePlant.PLoSONE10(6):e0127821. doi:10.1371/journal.pone.0127821 AcademicEditor: MariaAnisimova,SwissFederal InstituteofTechnology(ETHZurich),SWITZERLAND Received: September21,2014 Accepted: April19,2015 Published: June1,2015 Copyright: ©2015Taoetal.Thisisanopenaccess articledistributedunderthetermsofthe Creative CommonsAttributionLicense ,whichpermits unrestricteduse,distribution,andreproductioninany medium,providedtheoriginalauthorandsourceare credited. DataAvailabilityStatement: Allrelevantdataare withinthepaperanditsSupportingInformationfiles. Funding: ThisworkwassupportedbytheNatural ScienceFoundationofChina(31270427). CompetingInterests: Theauthorshavedeclared thatnocompetinginterestsexist.

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[ 5 , 6 ].Thecrystalstructureof Arabidopsis PHYBwasresolvedrecently[ 7 ],anditprovidesa helpfulscaffoldforunderstandingthesignalingandfunctionalmechanismofplant phytochromes. Previousstudieshavedemonstratedthattheevolutionaryadaptationofphytochromesisassociatedwithpolymorphismsinthephytochromegenesregulatingecologicallyimportant traits[ 8 – 12 ].Aphylogeneticanalysissuggestedthatpositiveselectionin PHYA hasplayeda majorroleintheadaptiveevolutionofearlyangiosperms[ 13 ].Molecularevolutionaryanalysis ofthephytochromegenesinSorghum[ 14 ]andBrassicaceae[ 15 , 16 ]haveshownthattheevolutionofphytochromesismainlyconstrainedbypurifyingselection.Morerecently,population geneticstudiesofalpineplantshaverevealedpositiveselectionin PHYE ,suggestingitsinvolvementinadaptationtolocalenvironments[ 16 , 17 ]. PHYE isbroadlydistributedinfloweringplants,expressedthroughoutthecourseofdevelopmentandpresentinvariousorgans[ 18 ].Atcoolertemperatures, PHYE playsaprominent roleinregulationofgermination[ 19 ]andflowering[ 20 , 21 ].Inaddition, PHYE isanimportantcontributortogermination[ 22 , 23 ]andshadeavoidance[ 24 ]underenvironmentswitha lowerratioofredlight/far-redlight(R/FR).However,thefunctionsof PHYE arehighlyredundantwithotherphytochromes,especiallywith PHYB [ 24 , 25 ].Inmostcases, PHYE functions intheformofheterodimerswithotherphytochromes[ 26 , 27 ],alsomainlywith PHYB ,and fine-tuning PHYB -mediatedphysiologicalresponses. PHYB isaprincipalmediatorthatresponsestoRandFR[ 5 , 6 ],andevolvesunderconstraintsbypurifyingselectionin Arabidopsis [ 15 ].As PHYE hasredundantfunctionandheterodimerizewith PHYB ,functionalconstraints mayberelaxedin PHYE,allowingtheaccumulationofaminoacidreplacements,thus PHYE mayaccumulatemoremutationsintheprocessofadaptiveevolution.Therefore, PHYE should beapromisingcandidategeneforexploringlocaladaptationtodifferentlightintensity environments. Duetothehighlydiverseanduniquebiota,limestoneKarstareasinSoutheastAsiahave longbeenregardedas “ naturallaboratories ” forecologicalandevolutionarystudiestounderstandnaturalselectionandspeciation[ 28 ].TheKarstareasinsouthernChinahavebeenrecognizedasoneoftheworld ’ scentersofplantdiversity[ 29 ].ThespeciesrichnessinChina ’ sKarst regionshasbeenattributedtoitslargediversityofedaphicandclimaticvariability. Primulina (Gesneriaceae),atypicalcaveplant,isamonophyleticgenuscomprisingmorethan140species ofperennialsthatarewidelydistributedthroughouttheKarstregionsofsouthernChinaand theadjacentcountriesofSoutheastAsia.Thegenusoccursinawidelatitudinalrange(18°N31°N)withremarkablydiverselightregime,fromsteepcliffsandcaveentrancestotwilight zones.Assessileorganisms,theheterogeneouslightenvironmentsexertaselectionpressureon Primulina tosurvivalinKarsthabitats.Identifyinggenestargetedbynaturalselectioncan greatlyimproveourknowledgeoftheroleofadaptationinspeciesevolution.DespiterecentadvancesinourunderstandingofecophysiologicaladaptationtotheKarstenvironment[ 30 , 31 ], themolecular-geneticmechanismsbywhich Primulina adaptstoheterogeneouslightconditionshaveneverbeenexplored. Althoughphytochromesplayanimportantroleinplantlifecycle,littleisknownaboutthe compositionandevolutionofthephytochromegenefamilyin Primulina todate.Inthisstudy, wechose PHYE asthetargettoexplorewhetherthephytochromeinvolvedinlocaladaptation of Primulina tothediverselightenvironmentsinKarstareas.Forthispurpose,weobtained10 full-lengthsequencesand74partialsequencesof PHYE from Primulina ,whichweresampled fromawidegeographicrangeofthegenus.Weusedmolecularevolutionaryapproachestotest whetherpositiveselectionorselectiveconstraintsarisenonthisgene. AdaptiveMolecularEvolutionof PHYE in Primulina PLOSONE|DOI:10.1371/journal.pone.0127821June1,2015 2/14

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MaterialsandMethods EthicsStatementP . tabacum islistedintheInventoryofRareandEndangeredPlantsofChinaandtheKeyProtectedInventoryofWildPlantsofChina( http://db.kib.ac.cn/eflora/View/plant/ZXBWSpecies. aspx ).Theleafsamplesofthisspecieswerecollectedwithpermissionfromthegreenhouseof SouthChinaBotanicalGarden.Allotherspeciesarenotrecognizedastheendangeredorprotectedspeciesatthemoment,theleafsamplesusedinthisstudywerecollectedfromopen areas,andthelocationisnotprivatelyownedorprotectedinanyway,sonospecificpermits wererequiredforthesampling.Plantmaterialsandamplificationof PHYE genesTheplantmaterialsusedinthisstudywerecollectedfromfieldsthroughoutthegeographic rangeof Primulina inChina,asspecifiedin S1Table .Thesespeciesarewidelydistributed acrossthephylogenyofthegenus.Oneindividualofeachspecieswasused,andatotalof74 Primulina speciesandtwooutgroups( Didymocarpushancei and Petrocodondealbatus )were included.TotalgenomicDNAwasextractedfromsilica-geldriedleavesusingmodifiedCTAB methods. Usingthefull-lengthcodingsequencesof ArabidopsisthalianaPHYE (AT4G18130)as query,weobtained10full-lengthcodingsequencesof PHYE (3369bp)fromtranscriptomeresourcesof Primulina species[ 32 ].Thequeriedsequenceswerecheckedcarefullybyeyesand madecertainthattherewerenoanyambiguitycharacters,noframe-shiftmutationsorprematurestopcodons.Thestopcodonswereexcludedinthefollowinganalysis.However,itisimpracticaltoamplifythefull-lengthsequencesof PHYE ( > 3369bp)fromgenomicDNAforthe remainingspecieswithouttranscriptomeresources.Thus,thisstudymainlyfocusedonthe conservedcoresignalingdomainsofthephytochrome,i.e.PASandGAF[ 33 ].Thisledtothe twodatasetsanalyzedinthisstudy:full-lengthsequencesfor10speciesandpartialsequences for74species. Thespecificprimersusedintheamplificationofthecoresignalingdomainofthe PHYE geneweredesignedaccordingtothealignmentoffulllengthsequencesusingPrimerPremier 5.0(PremierBiosoftInterpairs,PaloAlto,CA),withtheforwardprimerPHYE-F:5 ’ -CT GTTTTGTCATCCTCTGCTGC-3 ’ andthereverseprimerPHYE-R:5 ’ -TGTGGTGAACGT AGGGTAGAATTAA-3 ’ .Polymerasechainreaction(PCR)amplificationswereperformedas followsandreached50 lwithsteriledistilledwater:5 lTakara10× ExTaq buffer(Mg2+plus),4 ldNTPMix(2.5mMeach),0.25 lTakara ExTaq DNApolymerase(5U/ l),2 l 10 mprimersandabout20ngDNA.Reactionconditionswereasfollows:94°Cfor3min, then35cyclesof94°Cfor30s,55°Cfor30s,and72°Cfor1min;withafinalextensionof 72°Cfor10min.AllofthePCRproductswerecheckedforlengthandyieldbyelectrophoresis on1%agarosegelstainedwithGoldview.Oncepurified,thePCRproductsweredirectlysequencedinbothdirectionsusingthesameprimersasinamplification.Allofthe PHYE sequencesweredepositedinGenBankandaccessionnumberswerelistedin S1Table .RecombinationdetectionandphylogeneticreconstructionTheobtainedsequenceswerealignedusingClustalWequippedinMEGAv5.2.2[ 34 ]and proofreadmanuallytomakesurenoambiguitycharactersexisted( S1 and S2 Files).Asrecombinationcanmisleadphylogeneticandpositiveselectionanalyses[ 35 ],weusedthegeneticalgorithmforrecombinationdetection(GARD)[ 36 ]methodimplementedintheDatamonkey web-server( www.datamonkey.org )[ 37 ]todetectpotentialrecombinationbreakpointsbefore AdaptiveMolecularEvolutionof PHYE in Primulina PLOSONE|DOI:10.1371/journal.pone.0127821June1,2015 3/14

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analysis.Kishino-Hasegawatests[ 38 ]wereusedtoteststatisticaldifferenceswhenpotential breakpointsweredetected. ThephylogeneticrelationshipswerereconstructedbyMrBayesv3.1.2[ 39 ],andthebest-fit nucleotidesubstitutionmodelswereindicatedbytheAkaikeInformationCriterion(AIC)implementedinMrModeltestv2.3[ 40 ].Forthepartialfragmentof PHYE ,thesubstitutionmodelsoffirst-,second-andthird-codonpositionsiteswereF81,HKYandGTR+G,respectively. Forthe10full-lengthsequences,thesubstitutionmodelsoffirst-,second-andthird-codonpositionsiteswereHKY+G,GTR+IandGTR+G,respectively.FortheBayesiananalysisofthe partialsequences, Didymocarpushancei and Petrocodondealbatus weresetasoutgroupsaccordingtoourpreviousphylogeneticanalysisofthegenus[ 30 ],andtheMarkovChainMonte Carlo(MCMC)searchwasrun8,000,000generationsandsampledevery100generations.The first25%ofthegenerationswerediscardedasburn-in,andtheremainingtreeswere concatenatedtoconstructthemajorityruleconsensustree.FortheBayesiantreeconstructed forthefull-lengthsequences,thetreewasrootedat P . swinglei ,asthepositionwassupported bythelargersetoftaxaandlocatedatbasalpositionasshownin Fig1 .TheMCMCwasrun for100,000generationsandsampledevery100generations,andthefirst250treeswerediscardedasburn-in.PositiveselectionanalysesToevaluatetheinfluenceofnaturalselectionon PHYE andidentifyspecificsitessubjecttopositiveselection,weusedavarietyofcodon-basedsite-specificsubstitutionmodelsimplemented intheCODEMLprogramofthePAMLv4.5package[ 41 ].Thenonsynoymous/synonymous substitutionratio ( = dN/ dS)wasestimated,where < 1,=1and > 1indicatedpurifying selection,neutralevolutionandpositiveselection,respectively.Threepairsofmodelswithdifferentassumed distributionswerecomparedusingthelikelihoodratiotests(LRTs)frameworktoteststatisticaldifferences:oneratiomodelM0versusdiscretemodelM3,nearly neutralmodelM1aversuspositiveselectionmodelM2aandbetamodelM7versusbeta& modelM8.TheBayesempiricalBayes(BEB)analysiswasusedtocalculatetheposteriorprobabilities.Siteswithgreaterposteriorprobability(PP > 95%)andtheexpected > 1wereinferredtobeunderpositiveselection.Theanalyseswererunseveraltimeswithdifferentinitial valuestoevaluatetheconvergence. Inaddition,wealsousedotherfourdifferentmethodsfromtheDatamonkeyweb-serverto evaluatespecificsitesevolvingunderpositiveorpurifyingselection:thesinglelikelihoodancestorcounting(SLAC)method,thefixedeffectslikelihood(FEL)method,therandomeffects likelihood(REL)method[ 42 ]andthemixedeffectsmodelofevolution(MEME)[ 43 ].Forthe SLAC,FELandMEMEmethods,siteswith p -values < 0.1wereacceptedascandidatesforselection,whereasforREL,theBayesfactor > 50wasapplied. Inordertoexplorepossiblevariationsinselectivepressureamongdifferentbranches,we firsttestedwhetherthefree-ratiomodel(M1)fitsthedatabetterthantheone-ratiomodel (M0).M1assumesanindependent ratioforeachbranch,whereasM0assumesallbranches tohavethesame [ 44 ].Themodelsarecomparedthroughlikelihoodratiotests(degreesof freedom=totalnumberofbranches-1).Inordertodetectevolutionaryselectionpressuresactinguponindividualbranches,weemployedtheHyPhybranchsite-randomeffectslikelihood (BS-REL)method[ 45 ]aswellasthePAMLoptimizedbranch-sitemodelAmethod[ 46 ]. BS-RELdoesnotrequiretheidentificationofforegroundbranches(lineagesunderpositiveselection)andbackgroundbranches(lineageslackingpositiveselection) apriori ,whilethe branch-sitemodelsrequiretheforegroundandbackgroundbranchestobedefined apriori . Predefinedbiologicalhypothesesareunavailable,anditisdifficulttodefinetheforeground AdaptiveMolecularEvolutionof PHYE in Primulina PLOSONE|DOI:10.1371/journal.pone.0127821June1,2015 4/14

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branches.Therefore,whenperformingbranch-sitemodelA,wetreatedeachspeciesbranchin thephylogenytreealternatelyastheforegroundbranchwhiletherestbrancheswereconsideredasthebackgroundbranches.LRTwasconstructedtocompareanalternativemodelthat allows tobegreaterthan1intheforegroundbranchwithanullmodelthatrestricts inthe foregroundbranchequivalentto1.TheBonferronicorrectionwasemployedtoaccountforthe Fig1.Bayesianphylogenetictreeofpartial PHYE sequencesfor74 Primulina species. Bayesian posteriorprobabilitiesabove0.5werelabeledatthenods. Petrocodondealbatus and Didymocarpushancei wereusedasoutgroups.Thespecieswithfull-lengthsequencesareshaded. doi:10.1371/journal.pone.0127821.g001 AdaptiveMolecularEvolutionof PHYE in Primulina PLOSONE|DOI:10.1371/journal.pone.0127821June1,2015 5/14

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problemofmultiplehypothesestesting[ 47 ].TheBEBapproachwasalsousedtoidentifythe sitesthataremostlikelyunderpositiveselection(posteriorprobability > 95%).SlidingwindowanalysisTointuitivelyshowselectivevariationin alongthe PHYE sequences,wefurtherperformeda slidingwindowanalysisusingthesoftwareSWAAPv1.0.2[ 48 ],withwindowandstepsizeof 30bpand3bp,respectively.Thevaluesof wereestimatedusingNei-Gojobori[ 49 ].Results SequencedataWeobtained10full-length PHYE sequences(3369bp)fromthetranscriptomeresourcesofthe Primulina species.Thespecificprimeramplifiedpartialfragment(861bp)of PHYE from74 Primulina speciesandthetwooutgroupspecies( Didymocarpushancei and Petrocodondealbatus ).Thepartiallengthsequencescorrespondtopositions142-1002bpofthefull-lengthsequencesalignment,coveringthecompletePASdomainandpartofGAFdomain.Thepartial sequencesamplifiedfrom P . leiophylla and P . heterotricha areidenticalwithsequencesfrom P . napoensis and P . pterppoda ,respectively.Weonlykeptuniquesequencesandremovedthose obtainedfrom P . napoensis and P . pterppoda .Thus,atotalof10full-lengthand74partial PHYE sequenceswereusedinthefollowinganalysis.PhylogeneticandselectionanalysesForthe74incompletesequences,theGARDtestfoundnoevidenceofrecombination.Forthe 10full-lengthsequences,threebreakpointswereidentified,buttheywerenotsupportedbythe Kishino-Hasegawatest.Thus,thesequencescanbeuseddirectlyinphylogeneticreconstructionandevolutionaryanalysis.TheBayesianphylogenetictreesconstructedbythe74partial sequences( Fig1 )andthe10full-lengthsequences( Fig2 )of PHYE wereusedinthefollowing adaptiveevolutionaryanalysis,respectively. Testsforpositiveandpurifyingselectionwereconductedusingseveralcodon-basedmaximumlikelihoodmethods.Thesite-specificmodelsindicatedthatthe74partialsequenceswere understrongpurifyingselectionwith =0.128intheone-ratiomodel(modelM0).ThediscretemodelM3wassignificantlybetterthanM0(-2 lnL=41.078, p < 0.001),indicatingthat the ratiowasnothomogeneousamongthesitesalongthesequence.Positiveselectionmodel M2awasnotsignificantlybetterfittothedatathanthenullmodelM1a(-2 lnL=0.482, p =0.786).Althoughonesite(92)wasdetectedunderpositiveselectionwithposteriorprobabilitycriterionatthe95%cutoff,thepositiveselectionmodelM8wasnotsignificantlybetter thanthenullmodelM7(-2 lnL=5.89, p =0.053)( Table1 ).Similarresultswerealsoobtained usingdifferentinitial values.Wefurthertestedforevidenceofpositiveselectionusingother fourdifferentmethodsimplementinDatamonkeyweb-server.TheRELanalysispredictedfour sitesunderselection(42,92,93and162),MEMEfoundtwosites(77,92)whiletheSLACand FELeachonlypredictedonesite(92)underpositiveselection( Table2 ).Intotal,thesemethods identifiedfivesites(42,77,92,93and162)underpositiveselectioninthe74partial PHYE sequences,andthesite92wasdetectedbyalmostallofthemethods. Intheanalysisofthe10full-length PHYE sequences,noevidenceofpositiveselectionwas detectedinthefulllengthsequencesusingsitemodelsinCODEML.Similartothepartialsequences,thefull-lengthsequenceswereunderstrongpurifyingselectionwith =0.193inthe one-ratiomodelM0,andthe wasalsonothomogeneousamongsitesalongthefull-lengthsequences,asthediscretemodelM3wassignificantlybetterthanM0(-2 lnL=12.546, AdaptiveMolecularEvolutionof PHYE in Primulina PLOSONE|DOI:10.1371/journal.pone.0127821June1,2015 6/14

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p =0.014).ThepositivemodelsM2aandM8werenotsignificantlybetterthanthenullmodels M1aandM7,respectively,andnositeswerefoundtobeunderpositiveselectionusingtheBEB approachwithaposteriorprobabilityatthe95%level( Table1 ).Giventhemethodsfromthe Datamonkeyweb-server,1and10selectedsitesweredetectedunderpositiveselectionbyFEL andREL,respectively,andonlyonesite(28)wasdetectedbybothofthemethodswhereasno sitewasdetectedbySLACandMEME( Table2 ). Inordertoexplorepossiblevariationinselectivepressureamongdifferentlineagesandto identifysitessubjecttoepisodicselection,whichrepresentsselectionalongoneorafewlineages,wefirstcomparedthefree-ratiomodel(M1)withone-ratiomodel(M0).Theresults showedthatM1fitsthedatabettersignificantlythanM0forbothofthedatasets( Table3 ),indicatingthatdifferent Primulina speciesexperiencedvariablelevelsofselectivepressure.Then weusedthebranchsite-randomeffectslikelihood(BS-REL)methodandbranch-sitemodelto identifyspecificlineagesonwhichasubsetofsiteshaveevolvedunderpositiveselection.For thetwodatasets,BS-RELfoundnobranchestobeunderselectionat p < 0.05level.The branch-sitemodelalsofoundnobranchesunderpositiveselectioninthefull-lengthsequences, whileforthepartialsequences,the P . eburnea branchwasfoundtobeunderselectivepressure Fig2.Bayesianphylogenetictreeoffull-length PHYE sequencesfor10 Primulina species. Thetree wasrootedat P . swinglei .Posteriorprobabilitieswerelabeledatthenodes. doi:10.1371/journal.pone.0127821.g002 AdaptiveMolecularEvolutionof PHYE in Primulina PLOSONE|DOI:10.1371/journal.pone.0127821June1,2015 7/14

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(-2 lnL=9.656, p value=0.002),andonesite(77)wasidentifiedunderepisodicpositiveselection( Table4 ).However,theLRTtestwasnotstatisticallysignificantafterperformingBonferronicorrection(Bonferronicriticalvalue=0.0007)( Table4 ).Theepisodicpositiveselectionat thissitewasalsodetectedbytheMEMEmethod( Table2 ).MEMEmethodallowsthedistributionof tovaryfromsitetositeandfrombranchtobranchatasite,andiscapableofidentifyingbothpervasiveandepisodicpositiveselection[ 43 ]. Table1.Phylogenetictestsofpositiveselectionfor PHYE in Primulina usingsitemodels. DatasetsModelNplnLParametersModels compared -2 lnL pvaluePositivelyselectedsites (posteriorprobability) Partial sequences M0147-3166.733 =0.128None M3151-3146.194p0=0.353, 0=0M0-M341.078**2.6×10– 842T(0.989);73H(0.676);77C (0.585) p1=0.613, 1=0.14292L(1.000);93M(0.985);134T (0.512) p2=0.033, 2=1.196162A(0.907) M1a148-3146.796p0=0.946, 0=0.079Notallowed p1=0.054, 1=1 M2a150-3146.555p0=0.949, 0=0.081; p1=0.048 M1a-M2a0.4820.78642T(0.605);77C(0.531) 1=1;p2=0.003, 2= 3.052 92L(0.832);93M(0.569) M7148-3149.285p=0.259,q=1.627Notallowed M8150-3146.34p0=0.967,p=1.138M7-M85.890.05242T(0.828);73H(0.541);77C (0.613);92L(0.968) q=11.09,p1=0.033, =1.3 93M(0.792);134T(0.548);162A (0.662) Full-length sequences M019-6347.891 =0.193None M323-6341.618p0=0.62, 0=0;p1= 0.378 M0-M312.546 * 0.01428A(0.687) 1=0.494;p2=0.002, 2=5.409 M1a20-6341.718p0=0.879, 0=0.087Notallowed p1=0.121, 1=1 M2a22-6341.669p0=0.888, 0=0.093; p1=0.111 M1a-M2a0.0980.95227A(0.515);28A(0.647);597G (0.502) 1=1;p2=0.001, 2= 5.523 M720-6341.805p=0.177,q=0.719Notallowed M822-6341.635p0=0.998,p=0.245M7-M80.340.84419D(0.618);27A(0.655);28A (0.813) q=1.039,p1=0.002, =5.39 472M(0.566);516K(0.614);597G (0.639) 611E(0.608);694E(0.596);1025P (P.604) 1079T(0.576);1083T(0.56) Forthepartialsequences,theaminoacidsreferto P . tenuifolia; Forthefull-lengthsequences,theaminoacidsreferto P . eburnea Np:numberofestimatedparameters;lnL:loglikelihoodscore*Signi cantat p < 0.05**Signi cantat p < 0.01. doi:10.1371/journal.pone.0127821.t001 AdaptiveMolecularEvolutionof PHYE in Primulina PLOSONE|DOI:10.1371/journal.pone.0127821June1,2015 8/14

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SlidingwindowtestresultsTheresultsoftheslidingwindowanalysesof ( dN/ dS)variationacrossthepartialandfulllengthsequencesof PHYE arepresentedin Fig3 .Similartotheresultsofthesite-specificand Datamonkeymethods,the valueswerenothomogeneousacrossthegene,andtherewere dramaticvariationsalongthedomainstructure.Forthe74partialsequences,whichcontained thecompletePASdomainandaportionoftheGAFdomain,theanalysisrevealed3peaksthat exceed1,mainlylocatedinthePASandnearbyareas.The10full-lengthsequenceshad4peaks greaterthan1andweremainlylocatedinthe5 ’ -endregion,thePHY-PAS1domainandthe HATPase-cdomain.Itisworthnotingthatthe valuesofthe5 ’ -endandthe3 ’ regionwere morevariablethanotherdomainregions.However,consideringtheresultsofselectionanalyses,theslidingwindowresultsmaynotbestatisticallysignificant.Nevertheless,itreflectsthe discrete ( dN/ dS)variationamongthedifferentgenefunctionaldomains.DiscussionInthisstudy,weusedseveralmaximum-likelihoodmethodstoexploretheadaptiveevolution ofphytochrome PHYE inadiversecaveplant Primulina withtwodifferentdatasets:74partial sequencesand10full-lengthsequences.Theresultsshowedthattheevolutionof PHYE was mainlyconstrainedbypurifyingselectionwithin Primulina ,andtheselectivepressureisvariableamongdifferentspecieslineages.Twosites(77,92)subjecttoepisodicdiversifyingselectionwereidentifiedfrompartialsequences. Table2.PositiveselectionanalysisusingSLAC,FEL,RELandMEMEmethods. DatasetsPositiveselectionsites Mean dN/ dSSLACa( p -value)FELb( p -value)RELc(BayesFactor)MEMEd( p -value) 74partialsequences 0.16292(0.029)92(0.021)42(543.984);92(6825.22)77(0.012);92(0.025) 93(331.067);162(85.6759) 10full-lengthsequences 0.225 — 28(0.084)19(71.725);28(127.283) — 198(59.846);516(64.578) 597(76.37);611(63.669) 694(62.788);1025(62.025) 1079(60.866);1083(58.532) Forthepartialsequences,theaminoacidsreferto P . tenuifolia ;Forthefull-lengthsequences,theaminoacidsreferto P . eburnea Sitesidenti edbymorethanonemethodareshaded.aSLAC:singlelikelihoodancestorcounting;codonswith pvalues < 0.1bFEL: xed-effectlikelihood;codonswith pvalues < 0.1cREL:randomeffectlikelihood;codonswithBayesFactor > 50dMEME:mixedeffectsmodelofevolution;codonswith p -values < 0.1. doi:10.1371/journal.pone.0127821.t002 Table3.Likelihoodratiotest(LRT)stastictsformodelsofvariableselectivepressureamongbranches. DatasetsModel 2 lnLDegreeoffreedomp-value Partialsequences M0versusM1255.4621443.1×10– 8Full-lengthsequences M0versusM127.037160.041 M0andM1areoneratiomodelandfree-ratiomodel,respectively. lnL:loglikelihoodscores;-2 lnL:likelihoodratiotest(LRT)todetectpositiveselection. doi:10.1371/journal.pone.0127821.t003 AdaptiveMolecularEvolutionof PHYE in Primulina PLOSONE|DOI:10.1371/journal.pone.0127821June1,2015 9/14

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Forthe74partialsequences,site92wasidentifiedascandidatesiteunderpositiveselection withposteriorprobabilities > 0.95,whereastheadditionalrequiredLRTwiththenullmodel formodelM8wasnotsignificant(-2 lnL=5.89, p =0.053)( Table1 ).However,thefourmethods(SLAC,FEL,RELandMEME)implementedinDatamonkeyweb-serverdetectedfivecandidatesitesunderpositiveselectionandthesite92wasdetectedbyallofthemethods ( Table2 ).Theresultsofthe10full-lengthsequencesweresimilartothoseofthepartialsequences,butthesite-modelsfailedtodetectsignificantcandidatesitesunderpositiveselection ( Table1 ).ThemethodsimplementedinDatamonkeydetected10candidatesitesunderpositiveselectioninfull-lengthsequencesandonlysite28wasdetectedbytwomethodsofFELand REL( Table2 ).Thesite77wasidentifiedbybranch-sitemodelAinpartiallengthsequences butwithoutstatisticalsignificanceafterBonferronicorrection,thissitewasalsoidentifiedby MEMEmethod.CombinedtheresultsfromPAMLandDatamonkey,onesites(92)in74partialsequencesandonesite(28)in10full-lengthsequencesweredetectedunderselectionbyat leasttwomethods.Furthermore,MEMEidentifiedtwosites(77and92)frompartialsequences Table4.PAMLbranch-siemodelAanalysistoidentifybranchesunderepisodicpositiveselection. DatasetForeground branch Parameters undernull model lnL(null)Parameters under alternative model lnL (alternative) -2 lnL p value Degree of freedom Positively selected sites Bonferroni critical value Partial sequences P . eburnea p0=0.854;p1= 0.042;p2a= 0.098;p2b= 0.005; 0= 0.079; 1=1; 2=1 -3146.039p0=0.948;p1= 0.047;p2a= 0.004;p2b= 0.0002; 0= 0.08; 1=1; 2=299.076 -3141.2119.6560.002177C (0.967)85S (0.626) 0.0007 lnL:loglikelihoodscores;-2 lnL:likelihoodratiotest(LRT)todetectpositiveselection. doi:10.1371/journal.pone.0127821.t004 Fig3.Slidingwindowanalysisshowvariationof valuealongthe PHYE genefrom74partialsequencesand10full-lengthsequences. The estimateswerebasedontheNei-Gojoborimethod.Thewindowsizewassetat30bpandstepsizewassetat3bp.Beneaththeplotisaschematicof PHYE , whichillustratesthedistributionofthecharacteristicfunctionaldomains. doi:10.1371/journal.pone.0127821.g003 AdaptiveMolecularEvolutionof PHYE in Primulina PLOSONE|DOI:10.1371/journal.pone.0127821June1,2015 10/14

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aspotentiallyunderepisodicpositiveselection.Theseresultstogethersuggestpositiveselection atthesesitesmayhaveplayedamajorroleduringtheadaptiveevolutionof PHYE tolocal Karstenvironments.Theresultsofdifferentselectiontestbetweenthetwodatasetscouldbe explainedbythefactthatthefull-lengthdataset(10sequences)containedfewersequences thanthepartialdataset(74sequences),andthereforedecreasedthepowerofdetectingpositive selectionatindividualsites.Alternatively,theadaptiveimportanceof PHYE maybeheterogeneousacrossspecieslineages,asevidencedbyourbranch-sitetest( Table4 ),whichdemonstratedthatthe P . eburnea branchmightbeunderselectivepressurewhileotherssubjectto selectiveconstraints. P . eburnea isthemostwidespreadspeciesofthegenus,andisdistributed indiverselightenvironmentconditions,whilemostotherspeciesarenarrowendemicsand single-siteendemicsareverycommon.Althoughnoevidenceofwidespreadpositiveselection actingon PHYE in Primulina wasidentifiedinthepresentstudy,thesignatureofpotentialpositiveselectionatafewsitesmaysuggesttheinvolvementof PHYE inlocaladaptation.Nevertheless,sequencinglongerfragmentsof PHYE frommorespeciesshouldallowforamuch morerobusttestofnaturalselectiononthisgene. Thecrystalstructureof Arabidopsisthaliana PHYBwasresolvedrecently[ 7 ].ThephytochromemoleculeconsistsofaconservedN-terminalphotosensorycoredomainandaC-terminalregulatorydomain.Thephotosensorydomaincanbefurtherdividedintothreeconsecutive subdomains,PAS,GAFandPHY,whichareconservedamongphytochromes[ 33 ].Thecore signalingdomainofphytochromecomprisesthePASandGAFdomains.ThePASdomainis involvedintheincorporationofchromophoreandtheGAFdomainisconnectedtothebilin chromophoreandismuchconserved.ThePHYdomainisnecessaryforfine-tuningphytochromeactivity.TheregulatorydomainharborsconsecutivePAS1,PAS2andhistidinekinaserelateddomains.PreviousworksontheadaptiveevolutionofphytochromeshavemainlyfocusedontheGAFandPHYdomains.Forexample,twopositivelyselectedsiteswereidentified intheGAFdomainsofphytochromesinGymnosperm[ 50 ].Moreover,positiveselectionsdetectedintheGAFandPHYdomainsof PHYA wereassumedtobeinvolvedintheadaptive evolutionofearlyangiosperms[ 13 ].Thepositivelyselectedsitesinthephytochromesofangiospermsdemonstratedthatpositiveselectionmighthavedrivenfunctionaldivergenceafter geneduplication[ 46 ].Inastudyon Cardaminenipponica ,thenonsynonymoussubstitution detectedinthePHYdomainprovidedevidenceoftheinvolvementof PHYE inlocaladaption inalpineplants[ 16 ].Inthisstudy,themolecularevolutionaryanalysisof74partialsequences mainlyfocusedonthePASandGAFdomains,andfoundthatthehigher valuesfluctuate acrossthePASdomainandnearbyregions,whiletheGAFdomainwashighlyconserved.Althoughaccumulated wasdetectedinthePASdomainacrosspartialsequences,thisdidnot ruleoutotherregionsof PHYE astargetsofpositiveselectionin Primulina .Thetendencytoward accumulationacrossthe10entiresequenceswasnotthesameasthatforthepartialsequencesinthePASdomain( Fig3 ); valuesinthe5 Â’ -endandthe3 Â’ -endwasmorevariable thanotherdomainregions.Thedifferencesinthetwosliding-windowanalysesareprobably duetothelimitednumberofsequencessampledinthedatasetofentiresequences,sincethe 10specieswithentiresequencesmightsimplynotincludetheonesthathappenedtocontain thevariantsleadingtothe peaksfoundinthelargerdatasetofpartialsequences. Thesignatureofpositiveselectioninthe PHYE acrossthe Primulina phylogenysuggeststhatphytochromesmighthavebeeninvolvedinadaptationtolocallightenvironmentsfor Primulina speciesinKarstcavehabitats.ThisnovelexplanationforplantadaptionmayyieldinsightsintothespeciesrichnessandendemismofcaveplantsinKarstregions.However,to betterunderstandtheinvolvementofphytochromesinlocaladaptation,othermembersof phytochromegenefamilyshouldbeincludedinfutureworks. AdaptiveMolecularEvolutionof PHYE in Primulina PLOSONE|DOI:10.1371/journal.pone.0127821June1,2015 11/14

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SupportingInformationS1Table.Thespeciesname,samplingsitesandGenBankaccessionnumbersusedinthis study. (DOCX) S1File.Alignmentof74partialsequencesof PHYE . (PDF) S2File.Alignmentof10full-lengthsequencesof PHYE . (PDF)AuthorContributionsConceivedanddesignedtheexperiments:MKHH.Performedtheexperiments:JT.Analyzed thedata:JT.Contributedreagents/materials/analysistools:JTQQ.Wrotethepaper:MKJT.References1. KamiC,LorrainS,HornitschekP,FankhauserC.Light-regulatedplantgrowthanddevelopment.Curr TopDevBiol.2010;91:29 – 66.doi: 10.1016/s0070-2153(10)91002-8 PMID: 20705178 . 2. CarvalhoRF,CamposML,AzevedoRA.Theroleofphytochromeinstresstolerance.JIntegrPlant Biol.2011;53:920 – 929.doi: 10.1111/j.1744-7909.2011.01081.x PMID: 22040287 . 3. ClackT,MathewsS,SharrockRA.Thephytochromeapoproteinfamilyin Arabidopsis isencodedby fivegenes:thesequecesandexpressionof PHYD and PHYE .PlantMolBiol.1994;25:413 – 427.doi: 10.1007/BF00043870 PMID: 8049367 . 4. MathewsS.Phytochromeevolutioningreenandnongreenplants.JHered.2005;96:197 – 204.doi: 10.1093/jhered/esi032 PMID: 15695552 . 5. MathewsS.Evolutionarystudiesilluminatethestructural-functionalmodelofplantphytochromes. PlantCell.2010;22:4 – 16.doi: 10.1105/tpc.109.072280 PMID: 20118225 . 6. FranklinKA,QuailPH.Phytochromefunctionsin Arabidopsis development.JExpBot.2010;61:11 – 24.doi: 10.1093/jxb/erp304 PMID: 19815685 . 7. BurgieES,BussellAN,WalkerJM,DubielK,VierstraRD.Crystalstructureofthephotosensingmodule fromared/far-redlight-absorbingplantphytochrome.ProcNatlAcadSciUSA.2014;111:10179 – 10184.doi: 10.1073/pnas.1403096111 PMID: 24982198 . 8. BalasubramanianS,SureshkumarS,AgrawalM,MichaelTP,WessingerC,MaloofJN,etal.ThePHYTOCHROMECphotoreceptorgenemediatesnaturalvariationinfloweringandgrowthresponsesof Arabidopsisthaliana .NatGenet.2006;38:711 – 715.doi: 10.1038/ng1818 PMID: 16732287 . 9. SamisKE,HeathKD,StinchcombeJR.Discordantlongitudinalclinesinfloweringtimeand phytochromeC in Arabidopsisthaliana .Evolution;2008;62:2971 – 2983.doi: 10.1111/j.1558-5646.2008. 00484.x PMID: 18752603 . 10. IngvarssonPK,GarciaMV,HallD,LuquezV,JanssonS.Clinalvariationin phyB2 ,acandidategene forday-length-inducedgrowthcessationandbudset,acrossalatitudinalgradientinEuropeanaspen ( Populustremula ).Genetics.2006;172:1845– 1853.doi: 10.1534/genetics.105.047522 PMID: 16361240 . 11. IngvarssonPK,GarciaMV,LuquezV,HallD,JanssonS.Nucleotidepolymorphismandphenotypicassociationswithinandaroundthe phytochromeB2 locusinEuropeanaspen( Populustremula ,Salicaceae).Genetics.2008;178:2217 – 2226.doi: 10.1534/genetics.107.082354 PMID: 18245834 . 12. SaidouAA,MariacC,LuongV,PhamJL,BezanconG,VigourouxY.Associationstudiesidentifynaturalvariationat PHYC linkedtofloweringtimeandmorphologicalvariationinpearlmillet.Genetics. 2009;182:899 – 910.doi: 10.1534/genetics.109.102756 PMID: 19433627 . 13. MathewsS,BurleighJG,DonoghueMJ.AdaptiveevolutioninthephotosensorydomainofphytochromeAinearlyangiosperms.MolBiolEvol.2003;20:1087 – 1097.doi: 10.1093/molbev/msg123 PMID: 12777523 . 14. WhiteGM,HamblinMT,KresovichS.Molecularevolutionofthephytochromegenefamilyinsorghum: changingratesofsynonymousandreplacementevolution.MolBiolEvol.2004;21:716 – 723.doi: 10. 1093/molbev/msh067 PMID: 14963106 . AdaptiveMolecularEvolutionof PHYE in Primulina PLOSONE|DOI:10.1371/journal.pone.0127821June1,2015 12/14

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