Sexbiased flow and colonization in the Cynopterus sphinx inference.docx

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