Species delimitation, genetic diversity and population historical

ORIGINALRESEARCHpublished:08September2015doi:10.3389/fpls.2015.00696Speciesdelimitation,geneticdiversityandpopulationhistoricaldynamicsofCycasdiannanensis(Cycadaceae)occurringsympatricallyintheRedRiverregionofChinaJianLiu1,2,3,WeiZhou1,2andXunGong1,2,4*KeyLaboratoryforPlantDiversityandBiogeographyofEastAsia,KunmingInstituteofBotany,ChineseAcademyofSciences,Kunming,China,2KeyLaboratoryofEconomicPlantsandBiotechnology,KunmingInstituteofBotany,ChineseAcademyofSciences,Kunming,China,3UniversityofChineseAcademyofSciences,Beijing,China,4YunnanKeyLaboratoryforWildPlantResources,Kunming,China1Editedby:MiguelArenas,InstituteofMolecularPathologyandImmunologyoftheUniversityofPorto,PortugalReviewedby:YangLiu,SchoolofLifeSciences/SunYat-senUniversity,ChinaXiaozengYang,DowChemical,USA*Correspondence:XunGong,KeyLaboratoryforPlantDiversityandBiogeographyofEastAsia,KunmingInstituteofBotany,ChineseAcademyofSciences,Heilongtan,LanheiRoad132,Kunming650201,Chinagongxun@ialtysection:ThisarticlewassubmittedtoEvolutionaryandPopulationGenetics,asectionofthejournalFrontiersinPlantScienceReceived:18June2015Accepted:21August2015Published:08September2015Citation:LiuJ,ZhouWandGongX(2015)Speciesdelimitation,geneticdiversityandpopulationhistoricaldynamicsofCycasdiannanensis(Cycadaceae)ci.6::10.3389/fpls.2015.00696Delimitatingspeciesboundariescouldbeofcriticalimportancewhenevaluatingthespecies’,speciesdelimitationwascarriedoutonthreeendemicandendangeredCycasspecieswithresemblingmorphologyandoverlappeddistributionrangealongtheRedRiver(Yuanjiang)inChina:,of137individualsfrom15populationsweregenotypedbyusingthreechloroplastic(psbA-trnH,atpI-atpH,andtrnL-rps4)andtwosinglecopynuclear(RPB1andSmHP)onthecarefullymorphologicalcomparisonandcladistichaplotypeaggregation(CHA)analysis,weproposeallthepopulationsasonespecies,cdiversityandstructureanalysisoftheconflnensisrevealedthisspeciespossessehergeneticdiversityamongpopulationsandrelativelowergeneticdiversitywithinpopulations,aswellasobviousgeneticdifferentiationamongpopulationsinferredfromchloroplasticDNA(cpDNA)onally,nensiscorrespondingwithgeographywasdetectedbasedoncpDNA,dividingitspopulationrangesinto“Yuanjiang-Nanhun”basinand“Ejia-Jiepai”aphicalhistoryanalysesbasedoncombinedcpDNAandonenuclearDNA(nDNA)nensisbegantodecreaseinQuaternaryglaciationwithnosubsequentexpansion,whileanothernDNARPB1revealedamorerecentsuddenexpansionafterlong-termpopulationsizecontraction,firationalguidelines,thedownstreampopulationswhichoccuption,ex-situconservationandreintroductionmeasuresfordecadesofgenerationsaresupplementedforimprovingthepods:Cycas,speciesdelimitation,tree-based,sympatry,populationdynamics,conservationgenetics,RedRiverregion,ChinaFrontiersinPlantScience|1September2015|Volume6|Article696

vationgeneticsofCycasdiannanensisIntroductionTheconceptualizationandboundaryofspeciesarecriticallyimportantandofgreatsignificancefortaxonomists,ecologistsandconservationbiologir,theissue“whataspeciesis”thathaslongbeendebatedsinceDarwin’stimeisstillcontroversial(DobzhanskyandDobzhansky,1937;Mayr,1942;Mallet,1995;deQueiroz,2005;DeQueiroz,2007),withnonesuchaunifieddefiaisingconcernsonthetopicofspeciation(Turellietal.,2001;Wu,2001;CoyneandOrr,2004;LexerandWidmer,2008;Butlinetal.,2012)inrecentdecades,speciesdelimitationagainattractsevolutionbiologists’attention(WiensandPenkrot,2002;Wiens,2007;PetitandExcoffier,2009;CarstensandDewey,2010;Fujitaetal.,2012)andspecificmodelsandmethodologieswereputforwardbyemployingmorphologicalcharacters(WiensandPenkrot,2002),geneticdatasets(O’meara,2009;YangandRannala,2010;BarrettandFreudenstein,2011;EnceandCarstens,2011;HarringtonandNear,2012;Niemilleretal.,2012),orgeographicaldata(RisslerandApodaca,2007)toclarifythelineage’.(Cycadaceae),whichisconsideredasthebasallineageoftheCycadales,andalsothesistergrouptotheothergymnosperms(Burleighetal.,2012;Luetal.,2014;WangandRan,2014),containsapproximately105extantspeciesaroundtheworld(Haynes,2011),mainlydistributedinthetropicandsub-tropicareasaroundthePacifidangeredbutquiterecent(mid-Miocene)radiantgymnospermgenus(Nagalingumetal.,2011),phenotult,somemorphology-resembledorcharacter-equivocalspeciesduetointerspecifichybridizationwereoftenputforwardbyablendednameof“complex”or“group”(seeHill,1994a,b;YangandMeerow,1996;Liu,2004;XiaoandGong,2006),makingthedefiRiveroriginsinnorthwestYunnanofChina,andisnamedas“Yuanjiang”inthebasinofYunnan,thenflowsinoftheRedRivercorrespondstoageologicalfaultzone(RedRiverfaultzone,RRFZ)thatisresultedfromtheupliftingofHimalayaandthebasinexpansionofSouthChinaSea(Harisonetal.,1992;Leloupetal.,1995).TheRRFZstretchesformorethan1000kmonlandwhichstandsoutadiscontinuityinthegeologyofYunnan(Tapponnieretal.,1990),andharborsanabundantCycasdiversitywithmorethan14species,inwhich10areendemictothisregion(Hill,2008).Withinthesespecies,nensis,a,vulaarethreesympatricandmorphologicalrelatedspecieswhichareallclassifiedintotheSectionStangeriodesbysharingglabrousovules,eespeciesalsodisplaysimilarun-subterraneousstemhabitandlongcataphyllswhichmadeitdiffiphologicaldiffaareintheshapeofmegasporophyllterminallamina,withthefovula,theyonlydifferinthenumberofovulesandthesizeofmegasporophyll,ssificationofCycasinChinaisconfusedespeciallyafternumerousdisputablenewspeciesbeingdescribedsincethe1990s(Wangetal.,1996;WuandRaven,1999;Hill,2008).NoneoftheabovethreespeciesislistedinFloraofChina(WuandRaven,1999),inwhichtheyaretreatedassynonymsofotherspecies,nensisisacceptedbytheworldlistofcycads(Haynes,2011).Previousstudieshelddifferentopinionswhendealingwiththeissue(2004)nenr,Nongetal.(2011)ashouldbeindependentspeciesaccordingtotheirRAPDresults,herresultsbasedonpalynology(Wang,2000)andISSRdata(XiaoandGong,2006)ashouldbeagoodspecies,er,thesamplesandgeneticmarkersinthesestudieswerelimited,sincethesefactorshavegreatimpactsontheresultswhendelimitingspecies(KnowlesandCarstens,2007).Therefore,subsequenttaxonomicalrevisionandphylogeneticanalysisarerequiredile,underthecircumstanceofwildpopulations’severesituationoftheCycasspeciesthroughtheinvestigationsalongtheRedRiver,aswellastheurgentthreateningstatusofCycasspeciesinChina,itshouldbeimpendingtodeterminetheactualspeciesboundariesandevaluatethegeneticdiversitybasingoncomprehensivesamplinganddifferentmolgraphicaldistributionofplantspecieshadbeenprofoundlyinfluencedbytheclimaticoscillationsintheQuaternary(Hewitt,2000),andspeciescolonizationorcontractiontriggeredbysuchclimatefluctuationsmayleadtounexpectedgeneticsubdivisionandmixtureofpopulations(ComesandKadereit,1998;Hewitt,2004).Thegeneticst,iceage),especiallyforthoselong-evolvedandsessileorganisms(Fengetal.,2014).Geneticdatacanprovideinsightsintoadaptivepotentialforparticularspeciesinpostglacialcolonizationrefugiaaswellasvaluableinformationandsuggestionsforthespeciesdelimitation,demographichistoryandconservationcategories(Gongetal.,2011;ZhaoandGong,2012;Jiaetal.,2014).Inthisstudy,wesequencedthreematernallyinheritednensis,a,vula,whichsharedanoverlappeddistributionintheRedRiverbasin,gso,weaimtodemarcatetheboundariesamongthesesympatricspecies,thenevaluatethegeneticdiversity,geneticstructureanddemographichistoryoftheexistingpopulations,andultimatelyprovideFrontiersinPlantScience|2September2015|Volume6|Article696

vationgeneticsofCycasdiannanenalsandMethodsTaxonSamplingAtotalof137individualswereselectedforsubsequentanalysisfromthe15populationscollectedalongtheRedRiverregioninChina,nensis,vula(sampling10individualsforeachpopulationandallindividualsforpopulationslessthan10).Amongthem,vulawereobtainedfromcultivatedindividualsinthevillageaftertheverifivulapopulationZSDweresampledfromthesameplace(Zhongshan,Chuxiong).Informationofsamplingsitesandthenumberofitsubunit,RPB1andSelaginellamoellendorffihypotheticalprotein,SmHP(Chiang,Y.C.,unpublished)forcompleteanalysis(forprimerinformation,seeTable2).PCRamplificationwascarriedoutin40µNA,thePCRreactionscontained20ngDNA,2.0µLMgCl2(25mM),2.0µLdNTPs(10mM),4.0µL10×PCRbuffer,0.6µLofeachprimer,0.6µLTaqDNApolymerase(5U/µL)(Takara,Shiga,Japan)and26µA,thePCRreactionscontained40ngDNA,2.4µLMgCl2(25mM),2.0µLdNTPs(10mM),2.0µLDMSO,4.0µL10×PCRbuffer,0.7µLofeachprimer,0.7µLTaqDNApolymerase(5U/µL)(Takara,Shiga,Japan)and24.6µlificationswereperformedinathermocyclerunderthefollowingconditions:aninitial5mindenaturationat80◦C,followedby34cyclesof1minat95◦C,1minannealingat50◦C,anda1.5minextensionat65◦C,andafinalextensionfor10minat65◦ADNAExtraction,PCRAmplification,SequencingandCloningMaterialsforDNAextractionwerefromyoungacDNAwasextractedfromdriedleavesusingthemodifiedCTABmethod(Doyle,1991).Approximately2–3individualsfromeachpopulationwereselectedforoffivemarkerswereselectedandsequencedwithinthetotal137individuals,includingthreecpDNAintergenicspacers:psbA-trnH,rps4-trnL,andatpI-atpH(Shawetal.,2005),andtwosinglecopynucleargenes:CycasrevolutaRNApolymeraseIITABLE2|psbA–trnH(cpDNA)rps4–trnL(cpDNA)atpI–atpH(cpDNA)RPB1(nDNA)SmHP(nDNA)Primersequences(5′-sequence-3′)ReferencespsbA:GTTATGCATGAACGTAATGCTCShawetal.,trnH:CGCGCATGGTGGATTCACAATCC2005rps4:CTGTNAGWCCRTAATGAAAACGtrnL:TCTACCGATTTCGCCATATCatpI:TATTTACAAGYGGTATTCAAGCTatpH:CCAAYCCAGCAGCAATAACF010:GTACCCCAGTCATTTGAGACR1142:AGCCAGCAGTAACCATTGCCF004:CAAAACTATGCTGTCAATCCR745:TTAGCATCACCAGTAATCCCShawetal.,2005Shawetal.,2005InthisstudyInthisstudyTABLE1|sSamplinglocationPopulationcodeLatitue(N0)Longtitude(E0)Altitude(m)Selected(andcollected)aHuashiban,YuanjiangMajie,YuanyangDong’e,vulaZhongshan,ChuxiongGejiu,(indowntown)nensisDutian,ChuxiongE’jia,ChuxiongWotuodi,ShuangbaiHongtupo,NanhuaXinqiao,XinpingManhao,GejiuGasa,XinpingDa’me,ChuxiongYisha,ChuxiongZhongshan,ChuxiongYJHYYMDEYZSDGJDDTXEJTEJWHTPJPXMHGXPGXSDXSYZSM23.55223.25923.70324.80723.35924.53224.50524.54624.94522.86523.01924.04424.72124.64324.807101.926102.662101.782101.987103.160101.465101.243101.207101.892103.571103.413101.530101.017101.085101.98711(11)7(7)10(28)5(5)5(5)10(10)10(28)10(26)10(27)10(22)10(28)10(22)10(15)10(26)10(15)AlllocationsaresituatedalongtheRedRiverinYunnan,ersinPlantScience|3September2015|Volume6|Article696

vationgeneticsofCycasdiannanensissequences,anprocedureofinitial4mindenaturationat94◦C,whichwasfollowedby34cyclesof50sat94◦C,1minannealingat50◦C(forSmHP)or55◦C(forRPB1),anda1.5minextensionat72◦C,andafinalextensionfor10minat72◦productsweresequencedinbothdirectionswiththesameprimersfortheamplificationreactions,usinganABI3770automatedsequenceratShanghaiSangonBiologicalEngineeringTechnology&ividualswithnDNAsequenceswhichhadoneormoreheterozygoussitesinthefiductswerepurifiedusingtheTIANgelMidiPurificationKit(Tiangen).Purifi5αencloneswererandomasetsoftheDNAsequencinginthisstudyweredepositedinGenBank(accessionnumbersfromKT334601–KT334653).DataAnalysisThecpDNAandnDNAsequenceswereeditedandgeneratedbySeqMan(SwindellandPlasterer,1997).MultiplealignmentsoftheDNAsequencesweremanuallyrefinedwithClustalXv1.83(Thompsonetal.,1997),withsubsequentadjustmentinBioeditv7.0.4.1(Hall,1999).AlthoughthecongruencytestforthethreecombinedcpDNAregionsinthisstudyshowedanon-significantrateofhomogeneity(P=0.4,<0.5)byPAUP∗4.0b10(Swofford,2002),suggestingindistinctivedegreeofhomogeneitybetweenthecpDNAregions,westillcombinedthesethreeregionstogainenoughvariablesitesinthesubsequentanalysisassomeformerstudiessuggested(Yoderetal.,2001;Quickeetal.,2007).HaplotypesfromfivemarkersforallthethreespecieswerecalculatedfromalignedDNAsequencesbyDnaSPv5.0(LibradoandRozas,2009).Thegeneticdiversitywithin-andamong-populationswereestimatedbycalculatingNei’snucleotidediversity(Pi)andhaplotypediversity(Hd)hin-populationgenediversity(HS),genediversityintotalpopulations(HT)andtwocoefficientofpopulationdifferentiation,GSTandNSTwerecalculatedbyPermutv1.0(/genetics/labo/Software/Permut).TheDnaSPv5.0softwarewasalsousedtoinvestigatethedemograima’sDandFuandLi’sF*valuewerecalculatedtodetectdeparturesfrompopulationequilibrium,andtheusedArlequinv3.0(Excoffieretal.,2005)tocalculatetheraggednessindexanditssignifi-of-squareddeviations(SSD)betweentheobservedandexpectedmismatchdistributionswerecomputed,andP-valueswerecalculatedasatednessdegreeamongcpDNAandamongnDNAhaplotypeswereestimatedbyusingNetworkv4.2.0.1(Forsteretal.,2007).Inthenetworkanalysis,equinv3.0(Excoffieretal.,2005)wasusedtoconductananalysisofmolecularvariance(AMOVA)anionbydistance(IBD)modelwastestedbetweenallpairsofpopulationsbycomputingManteltestsinGenAlExpackageversion6.3(PeakallandSmouse,2006)eneticrelationshipsamongcpDNAandnDNAhaplotypesgeneratedfromthethreespecieswereinferredusingmaximumlikelihoodmethodbyonlinePhyML(/phyml/)(Guindonetal.,2010)andBayesianinferencebyMrBayesv3.2(Ronquistetal.,2012),rredatree-basedspeciesdelimitationmethodofcladistichaplotypeaggregation(CHA,Brower,1999)whichtabulatedthetestinghaplotypestodeterminethepopulationprofiles,andaggregatedhaplotypesthatsharingidenticalprofiles,thenestimatedthephylogenyoftheunaggregatedgroupsofhaplotypes,anddivergencetimebetweenlineageswithinpopulationswereestimatedbyBEASTv1.7(Drummondetal.,2012)withastrictmolecularclockandtheevolutionaryratessetas1.01×10−9and5.1–7.1×10−9(6×10−9inthisstudy)mutationpersiteperyearforcpDNAandnDNArespectively,whichhadpreviouslybeenestimatedinseedplantsforsynonymoussites(GraurandLi,2000).ThetimeofthebasalnodeinferredfromtheaverageevolulogenicrelationshipofallsampleswasalsoconstructedbyMrBayesv3.2(Ronquistetal.,2012)toinfertheindividuals’clusteringforspeciesdelimitation,inwhichfoursimultaneousrunswithfourchainseachwererunforcombineddatafor107generationsandtreesweresampledevery1000generations,withthefirst25%vesamplingdataafterBayesiananalysiswasexaminedanddeterminedbyTracerv1.6(Rambautetal.,2014).Beforethephylogeneticanalysis,thebestevolutionmodelswerechosenbyjModeltest1.7(Posada,2008;Darribaetal.,2012)forbothcombinedcpDNA(F81+GforAIC,F81forBIC)andnDNA(bothHKY+Ifortwosequences).ABayesianSkylineplotwasalsocalculatedbytheBEASTv1.7(Drummondetal.,2012)iorestimatesofthemutationrateandtimeofdivergencewereobtainedbyMarkovChainMonteCarlo(MCMC)lysiswasrunfor107iterationswithaburn-inof106andastrictclockmodelundertheHKY+genceofparametersandmixingofchainswerefollowedbyvisualinspectionofparametertrendlinesandcheckingofeffectivesamplingsize(ESS)parameterwasexpectedtosurpass200,whichsuggestedacceptablemixingandsuffitesamplingandconvergencetothestationarydistributionwerecheckedusingTracerv1.6(Rambautetal.,2014).WealsoconductedananalysisonbothpopulationstructureandspeciesdelimitationbythesequencedatausingSTRUCTUREv2.2(Evannoetal.,2005),asstrategyemployedbySTRUCTUREisstraightforwardandmatchesreasonablyFrontiersinPlantScience|4September2015|Volume6|Article696

vationgeneticsofCycasdiannanensiswellthepropertiesofmetapopulationlineages(ShafferandThomson,2007).Sequencedatawerefiependentrunswereperformedforeachset,withvaluesofKrangingfrom1to15,aburn-inof1×105iterationsand1×binationofanadmit-fitnumberofgroupingwasevaluatedusing