Sexbiased flow and colonization in the Cynopterus sphinx inference.docx
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Sexbiased flow and colonization in the Cynopterus sphinx inference.docx
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SexbiasedflowandcolonizationintheCynopterussphinxinference
Sex-biasedflowandcolonizationintheCynopterussphinx:
inferencefrommicrosatellitemarkersandmtDNAcytb
SunYH1,2,LiuXS2,ChenJP1
1GuangdongEntomologicalInstitute,Guangzhou,510260,China
2HuazhongNormalUniversity,Wuhan,430079,China
Abstract:
Thegreatershort-nosedfruitbat(Cynopterussphinx)isakindofabundantandwidelydistributedpteropodidsinsouthernAsia,toinferthegeneticdiversityandsexdifferencesingeneflow.Eightspecies-specificmicrosatellitelociandcytbwereusedtoexaminethegeneticdiversityandstructurewithinandamongsevencoloniesofthegreatershort-nosedfruitbat.Wefounddespitelarge-scalehunting,habitatdestructionandfragmentationinrecentyears,thepopulationsofC.sphinxcontinuetodisplayahighdegreeofgeneticdiversityinChinaandexhibitedsignificantdifferentiationamongthepopulations.Also,weconcludethatC.sphinxshowfemale-biaseddispersal,whichwasstronglysupportedbyevidencefromconcomitantecologicalstudies.
Keywords:
greatershort-nosedfruitbat,Cynopterussphinx,microsatellite,geneticdiversity,sex-biaseddispersal
Animalbehaviorcanaffectdispersal,whichhelpstopreventinbreeding,enhancegeneflowandextenddistributionarea(Greenwoodetal.,1980).Sex-biaseddispersalisverycommoninanimals,anddispersaltendstobemale-biasedinmammalsandfemale-biasedinbirds(Clarkeetal.,1997;Goudetetal.,2002).Mostevolutionarymodelsexplainingsex-biaseddispersalfallintothosebasedonavoidanceofinbreeding,competitionbyrelatedmalesformates,andalsocompetitionbyrelatedfemalesforresources(Greenwoodetal.,1980;Dobsonetal.,1982;MooreandAlietal.,1984).Dispersalisonekeydemographicforceshapingnaturalpopulations(Howard,1960;Proctoretal.,2004)andhasbecomeafocusfordemographicandecologicalresearch(Nathan,2001;KokkoandLopez-Sepulcre,2006;Nathan,2006).
Thegreatershort-nosedfruitbatstypicallynestinpalmtreesduringthebreedingseason,andarecharacterizedbyasingle-male,harem-formingsocialstructure(Storzetal.,2001).Thisfrugivorousbatplaysanimportantroleinplantpollinationandseeddispersal(Tangetal.,2005;Chenetal.,2007),buttheyalsodevourculturedfruitinfarms,whichhasledtolarge-scalekillingbypeopleandconsequentlyadramaticdecreaseintheirpopulationsize(Zengetal.,1996).Todate,however,thesespecieshavedrawnlittlescientificattentioninChina,andtherehasbeennoattempttodescribethepopulationgeneticstructureandrelationshipsatdifferentgeographicscales.
Inthisstudy,weusedmicrosatellitemarkersandcytochromebgenetoexaminethepopulationstructureandgeneticdiversityofC.sphinx,whichwillbehelpfulfordesigningandimplementingaconservationprogramforthisspecies.Theaimsofthestudyweretousemolecularevidencetostudythespatialdistributionofrelatedpopulations,andtoexplorethegeneraldispersalpatternamongthesexes.
MaterialandMethods
Samplescollection
Wingmembranesampleswerecollectedfrom224greatershort-nosedfruitbatsinsouthernChinaandIndia(Figure1).Batswerecapturedwithmistnetsatnight,recordtheirmorphologicalcharactersandtwo3mmdiameterskinbiopsiesweretakenfromeachbat(WorthingtonWilmeretal.,1996)andstoredin80%ethanol.Thebatswerethenreleased.
DNAextractionandmicrosatellitegenotyping
DNAwasextractedfromthebiopsieswiththeDNeasyTissuekit(QIAGEN,Shanghai,China).Polymerasechainreactions(PCR)wereconductedinaPTC-200thermalcycler(MJResearch),inatotalvolumeof10μl,containing50-100nggenomicDNA,0.25μMofeachprimer,and1×PCRbuffercontaining2mMMgCl2,0.2mMofeachdNTP,and0.25Uhot-startTaqDNApolymerase(QIAGEN).Forwardprimerswere5′-fluorescently-labelled(MedProbe).Thereactionswereperformedusingthefollowingconditions:
denaturationat95˚Cfor15min,followedby35cyclesof94˚Cfor30s,annealingtemperaturefor30s,72˚Cfor30s,withafinalextensionstepat72˚Cfor20min(Table1).
AllC.sphinxindividualsweregenotypedateightspecies-specificmicrosatelliteloci(Table1).Alloftheselocihavebeendescribedpreviously(Storzetal.,2000).
ThePCRproductsweregenotypedusinganABI3100DNAsequencer(AppliedBiosystems).FragmentlengthofthePCRproductswasdeterminedwithGenescansoftware(version2.1,AppliedBiosystems),andmarkergenotypeswereassignedtotheanimalsusingGenotypersoftware(Version2.5,AppliedBiosystems).
mtDNAsequencing
Sixtoeightindividualsfromeachpopulation(totaln=59)wereamplifiedatthecytochromebgene(~1100bp)usingthepublishedprimerscy1(5’-AAATCACCGTTGTACTTCAAC-3’)andcy2(5’-TAGAATATCAGCTTTGGGTG-3’)(Lietal.,2006).PCRswereperformedusingaPTC-220thermalcycler(MJResearch)in50μlreactionvolumes,eachcontaining25μlof2×ExTaqpolymerase(Takara),0.5μlofDNAtemplates(50μg/μl),and2μlofeachprimer(10μM).ForPCRconditionsseethemicrosatellitegenotypingsectionabove.PCRproductsweresequencedusingBigDyeTerminatorkits(AppliedBiosystems)onanABI3730automatedsequencerwithbothprimers.
Geneticdiversity
Ineachpopulation,themeannumberofallelesperlocus,observedheterozygosity(Ho),andexpectedheterozygosity(He)foreachlocuswerecomputedusingGENEPOPversion3.3(RaymondandRousset,1995)andtheGENETIXsoftwarepackage(Belkhiretal.,1996).Linkagedisequilibriumbetweenpairwisecombinationsofloci,estimatesofexpectedallelenumberperlocusandpopulationcorrectedforunequalsamplesize(allelicrichness,AR)werecomputedinFSTAT2.9.3.2(Goudet,2001).PotentialdeviationsfromHardy-Weinbergequilibrium(HWE)andgameticequilibriumweretestedforeachpopulationandlocususingtheMarkovchainmethod(10,000dememorizationsteps,10,000batchesand10,000iterationsperbatch)inGENEPOP(RaymondandRousset,1995).
GeneticdiversitywasmeasuredformtDNAbasedonhapoltypediversity(h)andnucletidediversity(π).Hapoltypediversitywasdefinedastheprobabilitythattworandomlychosensequencesfromasampleweredifferentandnucleotidediversitywastheaveragenumberofnucletiodedifferencepersitebetweentworandomchosensequences(Nei,1987).Valuesforthenumbersofpolymorphicsitesandthenumbersofpairwisedifferenceamongsequenceswerealsoestimated.ThesediversityindiceswerecomputedwiththesoftwareDNASP,version4.0(Rozasetal.,2003)
Geneticstructure
Geneticstructurewasanalyzedassuminganinfiniteallelemodel(IAM)theestimationofFSTvalues(KimuraandCrow,1964;KimuraandOhta,1978;Slatkin,1995).OverallandpairwisetestsbetweenpopulationswerecalculatedforFSTusingbothFSTAT(Goudet,2001)andGENEPOP(RaymondandRousset,1995),andthestatisticalsignificanceofbothestimateswasbasedon6,000permutations.FormtDNAstoverallandpairwisetestsbetweenpopulationswascalculatedusingArlequin3.1.
Analysisofmolecularvariance,AMOVAwasusedtoexaminegeneticvariationwithinandamongthedifferentpopulations,basedonallsamplesandforeachsexseparately.Totestforevidenceofgeneticsubdivision,weassessedwhetherthederivedindicesweresignificantlydifferentfromzerousingapermutationprocedure(5,000iterations)inthesoftwareArlequin3.1(Excoffieretal.,2005).
Sex-biaseddispersal
Weexploredpotentialdifferencesindispersalratesbetweenfemalesandmalesoverallpopulations.FSTATversion2.9.3.2wasusedtoquantifygeneticvarianceamongpopulations(FST),aswellasthemean(mAIc)andvariance(vAIc)ofanassignmentindexforbothsexes,using10,000permutations.FSTisastatisticexpressingtheproportionofthetotalgeneticvariancethatresidesamongpopulations(HartlandClark,1997).FSTforthemorephilopatricsex,therefore,isexpectedtobehigherthanthatofthemoredispersingsex(Goudetetal.,2002).Assignmentindex(AI)referstotheprobabilityofanindividualoccurringinalocality(Paetkauetal.,1995;Favreetal.,1997).Becausepopulationscancontainverydifferentlevelsofgenediversity,themulti-locusprobabilitiesofindividualsindifferentpopulationsarenotdirectlycomparable.Toremovethisproblem,acorrectedassignmentindex(AIc)isused.Ingeneral,apositivevalueindicatesaresidentindividualandanegativevalueindicatesapotentialdisperser.BecauseimmigrantstendtohavelowerAIcvaluesthanresidents,undersex-biaseddispersal,mAIcforthephilopatricsexshouldbehigherthanthatofthemoredispersingsex.Incontrast,vAIcshouldbelargerforthedispersingsexbecausemembersofthissexusuallyincludebothcommongenotypesfromresidentsandrareonesfromimmigrants(Goudetetal.,2002).
Results
LinkagedisequilibriumandHardy-Weinbergequilibrium
IntheeightmicrosatellitelociforC.sphinx,nolocishowedsignificantlinkagedisequilibrium,andtherewasnosignificantdeviationfromHardyWeinbergequilibriumdetectedineitherspeciesfollowingBonferronicorrectionformultipletests.
Geneticdiversity
AlleightmicrosatellitelociwerepolymorphicinC.sphinx.Thenumberofallelesperlocusrangedfrom7.1intheMandiyoorpopulationto12.6intheXishuangbanna/Yunnanpopulation.Themeanobservedheterozygosity(Ho)oftheeightpopulationswas0.78,andtheZhongshan/Guangdongpopulationshowedthehighestobservedheterozygosityacrossallloci(Ho=0.84).ThelowestobservedheterozygositywasfoundintheBeihai/Guangxipopulation(Ho=0.73).Theallelicrichness(AR)rangedfrom6.5(Mandiyoor/India)to8.9(Xishuangbanna/Y
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