Post on 24-Apr-2022
Mathematisch-Naturwissenschaftliche Fakultät
Renata F. Martins | Anke Schmidt | Dorina LenzAndreas Wilting | Joerns Fickel
Human-mediated introduction ofintrogressed deer across Wallace’s line
historical biogeography of Rusa unicolor and R. timorensis
Postprint archived at the Institutional Repository of the Potsdam University in:Postprints der Universität PotsdamMathematisch-Naturwissenschaftliche Reihe ; 617ISSN 1866-8372https://nbn-resolving.org/urn:nbn:de:kobv:517-opus4-423843DOI https://doi.org/10.25932/publishup-42384
Suggested citation referring to the original publication:Ecology and Evolution 8 (2018), pp. 1465–1479 DOI https://doi.org/10.1002/ece3.3754ISSN 2045-7758
Ecology and Evolution. 2018;8:1465–1479. | 1465www.ecolevol.org
Received:21August2017 | Revised:23November2017 | Accepted:27November2017DOI:10.1002/ece3.3754
O R I G I N A L R E S E A R C H
Human- mediated introduction of introgressed deer across Wallace’s line: Historical biogeography of Rusa unicolor and R. timorensis
Renata F. Martins1 | Anke Schmidt1 | Dorina Lenz1 | Andreas Wilting1 | Joerns Fickel1,2
ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse,distributionandreproductioninanymedium,providedtheoriginalworkisproperlycited.© 2017 The Authors. Ecology and EvolutionpublishedbyJohnWiley&SonsLtd.
1DepartmentofEvolutionaryGenetics,LeibnizInstituteforZooandWildlifeResearch,Berlin,Germany2InstituteforBiochemistryandBiology,UniversityofPotsdam,Potsdam,Germany
CorrespondenceRenataF.Martins,DepartmentofEvolutionaryGenetics,LeibnizInstituteforZooandWildlifeResearch,Berlin,Germany.Email:martins@izw-berlin.de
Funding informationLeibniz-Gemeinschaft,Grant/AwardNumber:SAW-2013-IZW-2
AbstractInthisstudywecomparedthephylogeographicpatternsoftwoRusaspecies,Rusa unicolor and Rusa timorensis,inordertounderstandwhatdroveandmaintaineddifferentiationbetweenthesetwogeographicallyandgeneticallyclosespeciesand investigatedtherouteof introductionofindividualstotheislandsoutsideoftheSundaShelf.Weanalyzedfullmitogenomesfrom56ar-chivalsamplesfromthedistributionareasofthetwospeciesand18microsatellitelociinasubsetof16individualstogeneratethephylogeographicpatternsofbothspecies.Bayesianinferencewithfossilcalibrationwasusedtoestimatetheageofeachspeciesandmajordivergenceevents.OurresultsindicatedthatthesplitbetweenthetwospeciestookplaceduringthePleistocene,~1.8Mya,possiblydrivenbyadaptationsofR. timorensistothedrierclimatefoundonJavacom-paredtotheotherislandsofSundaland.Althoughbothmarkersidentifiedtwowell-differentiatedclades,therewasalargelydiscrepantpatternbetweenmitochondrialandnuclearmarkers.WhilenDNAseparatedtheindividuals intothetwospecies, largelyinagreementwiththeirmuseumlabel,mtDNArevealedthatallR. timorensissampledtotheeastoftheSundashelfcarriedhaplo-typesfromR. unicolor and one Rusa unicolorfromSouthSumatracarriedaR. timorensishaplotype.OurresultsshowthathybridizationoccurredbetweenthesetwosisterspeciesinSundalanddur-ingtheLatePleistoceneandresultedinhuman-mediatedintroductionofhybriddescendantsin
allislandsoutsideSundaland.
K E Y W O R D S
Cervidae,humanintroduction,hybridization,Phylogeography,Sundaland,Wallace’sline
1 | INTRODUCTION
Biogeographic barriers interruptmigration and reproduction amongpopulations and thus are an important force responsible fordrivingandmaintaininggeneticdifferentiation,potentiallyleadingtospecia-tion.Sundaland,aSoutheastAsianbiodiversityhotspot,isborderedintheEastbyoneofthebestknownfaunalboundaries—theWallaceline(Bacon,Henderson,Mckenna,Milroy,&Simmons,2013).ThisbarrierisresponsibleforasharpbreakbetweenfaunalcompositionsofSundaandWallacea.Atthesouthernborder,theWallacelinerunsbetweenthe islandsofBaliandLombok,andat thenorthernedge, itdividesfaunaandfloraofBorneoandthePhilippinesfromthatofSulawesi
(Figure1).Althoughsomespeciesandpopulationshavenaturallydis-persedacross thisbarrier (squirrel sps.;MercerandRoth2003), thepresenceof themajorityofSundaicmammal speciesoccurringpasttheWallacelineintotheeasternislandsofWallaceaisassociatedwithhuman transportation (Groves, 1983; Heinsohn, 2003;Veron etal.,2014).
Sundaland’s dynamic geologic and climatic history, especiallyduringthePlio-Pleistoceneepochs,resultedinsealevelchangesthatrepeatedlyexposedthecontinentalshelfconnectingthemajorislandsofthisarchipelago(Voris,2000).Itisbelievedthattheseavailablelandbridgeswouldallowpopulationspreviouslyisolatedonsingleislandsto disperse, creating a largepanmictic populationwithin thiswhole
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system(Latinneetal.,2015;Demosetal.,2016).However,deepgeo-graphical barriers like theWallace linewould remain through evenlowsealevelperiods,thuscreatingpatternsofgeneticdivergencebe-tweentaxaonbothsidesofthesebarriers.
Here,weinvestigatedthephylogeographicpatternsoftwoRusaspecies:thesambar,Rusa unicolor,andtheJavandeer,Rusa timorensis. WhileR. unicoloriswidespreadthroughoutSouthandSoutheastAsia(fromIndiaandSriLanka,SouthernChinaandmostof IndochinatoBorneoandSumatra,thetwolargestSundaIslands),R. timorensis has itsnativerangeonJavaandBalionly(Figure1).ThepresenceofJavandeer on islands east of theWallace line (e.g., Lesser Sunda Islands,Sulawesi,andtheMoluccas)isdescribedtobetheresultofprehistorictohistorichuman-mediatedintroductionsduringtheHolocene.Thesehuman-mediated introductionswere attributed, for example, to theAustronesian-speaking peoples’ migrations, ca. 4,000years ago, re-sponsibleaswellfortheintroductionofotherdeerspecies(e.g.,munt-jacs),pigs,macaques,andcivets; (Heinsohn,2003;Groves&Grubb,2011).
Theaimofthisstudywastocomparephylogeographicpatterns,geneticdiversity,andevolutionaryhistoryofthesetworelatedspeciesinordertoanswerthefollowingthreequestions:(1)InthepresenceoflandbridgesconnectingislandsoftheSundaShelf,whatgeograph-icalorclimaticbarrierswereresponsibleforspeciationbetweenthetwospeciesandisthereevidenceofadmixturebetweenthem?(2)DopopulationsofthewidelydistributedR. unicolorshowsignsofgenetic
structuringcorrespondingtoknowngeographicalbarriers?(3)DoesR. timorensisshowageneticsignatureofnon-naturaldispersalandwhatisthemostlikelysourcepopulationoftheintroducedpopulationsEastoftheWallaceline?
2 | MATERIALS AND METHODS
2.1 | Sampling and DNA extraction
Wesampled110individualslabeledasRusa unicolor(RUN)andRusa timorensis (RTI) from European museums, aged between 180 and61yearsold.Wecollectedeitherturbinalbonesfromthenasalcav-ity,skin,drytissuefromskeletons,andantlerdrillsexclusivelyfromindividuals with known locality. All molecular work, including DNAextraction and sequencing library preparation, was conducted in alaboratorydedicatedtoworkwitharchivalsamplestoreducetheriskof contamination.DNAextraction followed theDNeasyTissue andBloodkitprotocol (Qiagen,Hilden,Germany),withovernightdiges-tionofsamples inLysisbufferandProteinaseKat56°Candapre-elutionincubationfor20minat37°C.
2.2 | Mitochondrial genome
All extractions, includingnegative controls,werebuilt into individualsequencinglibrarieswithsingle8-ntindexes(Fortes&Paijmans,2015),
F IGURE 1 Distributionmapofbothspeciesandsamplinglocation.LightgrayindicatesthedistributionrangeofRusa unicolor(a).DarkgrayindicatesthenativedistributionofRusa timorensis,(b)anddasheddarkgrayareasindicateintroductionrangeofR. timorensis(C).FilledcirclesanddiamondsindicateR. unicolor and R. timorensis,respectively,andsizeisproportionaltothenumberofsamples.FordetailedinformationaboutallsamplesseeTableA1
North India
India
Sri Lanka
Myanmar
Indochina
Thailand
South Thailand
West Sumatra
MentawaiSumatra
North Borno
Borneo
West Ja va
Java
Bal i
Sulawesi
South Sulawesi
Timorand LSI
Moluccas
B C
(a) (b)
(c)
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whichwerethensequencedonanIlluminaMiSeqtoassesssampleDNAquantityandquality(150cyclesv3kit,Illumina,CA,USA),asdescribedbelow. Samples with low-quality DNA were consequently enrichedfortheirmitochondrialDNA,usinganin-solutiontargethybridizationcapturetechnique(Maricic,Whitten,&Pääbo,2010).Baitsforhybridi-zationwere obtained by amplifying three overlappingmitochondrialfragments fromonefresh tissuesampleofR. unicolor (fromthe IZWarchive)whichwereconsequentlypreparedintocapturebaits(Maricicetal.,2010;primersandPCRconditionsasdescribedinMartinsetal.,2017).Afterhybridizationcapture,librarieswereamplifiedfornomorethan18cyclesandsequencedagainontheIlluminaMiSeqplatform.
2.3 | Bioinformatics
Sequencing readswere first demultiplexed into respective sampleswithBCL2FASTQv2.17(Illumina,CA,USA).CUTADAPTv1.3(Martin,2011)wasusedtofindandremoveadaptersequencesfromthese-quenced reads. Adapter-clipped reads were then quality trimmedthroughaslidingwindowapproachof10bpforaphredscoreofatleastQ20.Finally,readsshorterthan20bpwereremovedfromfur-ther analyses.Mapping of quality-filtered reads was carried out intwophases:AfirstmappingrunwasperformedwithBWAv.0.7.10(Li&Durbin, 2009), using a genome reference fromR. unicolor de-jeani (NCBI accessionno.NC_031835).Clonal readswere removedfrom the mapped reads using MARKDUPLICATES v1.106 (http://picard.sourceforge.net/picard-tools).SAMTOOLSMPILEUPv1.1andBCFTOOLS v1.2 (http://github.com/samtools/bcftools) were usedforvariantcalling(SNPsandInDels).Aconsensussequencewasthengeneratedforeachsampleusingathresholdofminimum3×cover-age andmajority rule (>50%) for base calling. The secondmappingstepusedthenewlygeneratedconsensussequenceasreferenceforeach sample, in order to increasemapping quality and base cover-age.BWAandMARKDUPLICATESwereusedasbefore,butGATKv1.6(McKennaetal.,2010)wasappliedforvariantcallingofthefinalconsensussequence.Positionswithcoveragelowerthan5×wereN-masked, aswere ambiguous heterozygous positions. A final qualityfilteringstepwasperformedtoremoveallsampleswithlessthan80%oftheirmitogenomecoveredatleastwith5×depth.Duetothelimita-tionsofthesequencingmethod(readsnolongerthan75bp),wewerenotabletoclearlyresolvetherepeatregionofthed-loop.Therefore,wetrimmedallsequences,byremoving460bpofthed-loopregion.Mitogenomes obtained were deposited in Genbank (for accessionnumbersseeTableA1).
2.4 | Microsatellite DNA
Microsatellitegenotypingwasachievedbyamplificationof18locionall56samplesforwhichmitochondrialDNAwasobtainedasdescribedabove (microsatellite loci and references in TableA2). All sampleswereamplifiedthroughPCRwiththeType-itMicrosatellitePCRkit(Qiagen,Hilden,Germany),with1μmol/Lofeachprimer.Annealingtemperaturesfollowedagradientfrom63°Cto55°Cin2°Cstepsandfinal amplificationoccurred for40cyclesat55°C.Allele sizeswere
determinedonanABI3130xlGeneticAnalyserusingGeneScan™ 500 ROX(bothThermoFischerScientific,Darmstadt,Germany)asinter-nalsizestandard.AlleleswerescoredwiththesoftwareGeneMapperv.4.0(AppliedBiosystems,Germany).
2.5 | Genetic diversity, phylogeography, and differentiation times
All mitochondrial sequences obtained were aligned using the autosettingasimplementedinMAFFTv7.245(Katoh&Standley,2013).The relationship among all haplotypes was reconstructed by amedian-joining(MJ)networkusingthesoftwareNETWORKv.4.6.1.4(Bandelt,Forster,&Röhl,1999).Haplotypesweregeneratedby re-movingnoninformativesitesandpositionswithgapsormissingdata.Haplotypicandnucleotidediversitiesforthefulldatasetandforeachspecieswere assessedwith the softwareDNASPv.5.10 (Librado&Rozas, 2009).We estimated genetic differentiation through FST as implemented in ARLEQUIN v.3.5.12 (Excoffier, Laval, & Schneider,2005).Forthisanalysis,wecreatedtwodatasets:(1)twopopulationscorrespondingtospeciesasdeterminedbythemuseumidentificationand(2)populationscorrespondingtomajorhaplotypeclades.
Thebestfittingsubstitutionmodelforthefullmitogenomedata-set(GTR+G+I)wasobtainedbythehierarchicallikelihoodratiotestasimplementedinJMODELTESTv2.1.7(Darriba,Taboada,Doallo,&Posada,2015).Wereconstructedphylogeneticrelationshipsthroughmaximumlikelihood(ML)withRAXMLGUIv1.5(Silvestro&Michalak,2012)andBayesianinference(BI)asimplementedinMRBAYESv3.2.6(Ronquist&Huelsenbeck, 2003), applying the determined substitu-tion model. Both approaches were congruent with the haplotypicnetworkandwitheachother.WedatedtheBItreebasedonfossilin-formationfortheCervidaestemoftheArctiodactylfamily(18.4Mya;Bibi,2013).Forthat,wefirstdetermineddivergencedatesforadata-set containing Bos javanicusasanoutgroup(JN632606.1),Muntiacus reevesi (AF527537.1), Axis axis (NC_020680.1) and A. porcinus (JN632600.1),D. dama (NC_020700.1),Cervus nippon (JN389444.1)and C. elaphus(AB245427.2),andthetwoRusaspeciesinvestigatedhere,usingthesoftwareBEAST(Drummondetal.2012).WerantwoMCMC chainswith 50million,with a lognormal uncorrelated clockandaYulespeciationtreeasfurtherestimationpriors.WeusedthecalibratedtimeofthesplitbetweenCervusandRusa(2.1My;HighestPosteriorDensity[HPD]=1–2.8My)tobesetasapriorfortherootheightofthegenetreeoftheRusasamplesobtainedinthisstudy,asbefore.TraceresultswereanalyzedwithTRACERv.1.6(implementedinBEASTv.1.8),forparameterconvergenceandESSvaluesabove200.TREEANNOTATORv.1.8.1(BEASTsoftwarepackage)wasusedtoan-notatealltrees,afteraburn-inof10%ofthetrees.AlltopologieswerevisualizedandeditedwiththesoftwareFIGTREEv.1.4.2(http://tree.bio.ed.ac.uk/software/figtree/).
2.6 | Population structure analyses
Analyses of microsatellite data proceeded by removing all indi-vidualswithmissingdata atmore than two loci.Weestimated the
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probability for the presence of null alleles on our datasetwith thesoftwareFREENA(Chapuis&Estoup,2007).TestsforthepresenceoflinkagedisequilibriaandanexacttestfordeviationsfromHardy–WeinbergEquilibrium (HWE)wereperformedwithARLEQUIN, ap-plyingtheBonferronicorrectionformultipletests.Levelsofobserved(HO)andexpected(HE)heterozygosityandinbreedingcoefficient(FIS)werecalculatedinGENETIXv.4.05.2(Belkhir,Borsa,Chikki,Raufaste,&Bonhomme,1996–2004).
A Bayesian approach was used to test for population struc-ture with the software STRUCTURE 2.3.4 (Pritchard, Stephens, &Donnelly,2000).TheλvaluewasestimatedbyrunningaprospectiverunofK=1with10 iterationsandaburn-inof10%after15×104 generations.A secondMCMC simulationwas run for 20×104 gen-erations,witha10%burn-in.The likelihoodswereestimated forKvalues from1to6,because6washigher thanthemaximumnum-ber of mitochondrial clades obtained in our analyses. The admix-turemodelwasappliedwithcorrelatedallelefrequenciesandλ=3.STRUCTURE HARVESTER v.0.6.94 (http://taylor0.biology.ucla.edu/structureHarvester/;Earl&vonHoldt,2012)wasused toestimatethemostlikelynumberofKusingthe∆Kmethod(Evanno,Regnaut,&Goudet,2005).PopulationdifferentiationwascalculatedwiththesoftwareARLEQUINbyestimatingFSTbothamongtheclustersiden-tifiedby∆Kandbyspecies.
3 | RESULTS
3.1 | Mitochondrial genome analyses
Thefinaldatasetconsistedof56individualsforwhichamitogenomeof16,064bpwasobtained.Ofthese,23sampleswerelabeledinthemuseumcollectionsasR. timorensisand33individualswerelabeledasR. unicolor(TableA1).TheoriginofthreespecimenslabeledasR. uni-color(RUN37Moluccas,RUN39Timor,andRUN61Java)suggeststhatthesespecimensareactually Javandeer,R. timorensis.Allothermu-seumlabelsmatchedthegeographicaldistributionrangesofthetwospecies.The56deershared46haplotypeswithanoverallhaplotypediversityofH=0.983(SD0.010)andaverylownucleotidediversityofπ=0.00941(SD0.00118).WithinindividualslabeledasRTI,wefound18haplotypeswithH=0.972(SD0.026)andπ=0.0085(SD0.0024).WithinRUN-labeledindividuals,therewere28uniquehaplotypes,andonehaplotypewassharedduetoadmixturewithRTI (H_3).Overall,the29haplotypeshadH=0.991(SD0.011)andπ=0.011(SD0.0012).
ThefullmtDNAhaplotypenetworkseparatedtwomajorcladesbyaminimumof168mutationalsteps(Figure2).Thesmallercladecom-prised six individuals from Java, Bali, Moluccas, and South Sumatra(Figure2)ofwhichfourhadbeenlabeledinthemuseumcollectionsasR. timorensis(Javandeer,RTI)andtwoasR. unicolor(sambar,RUN;RUN41:SouthSumatra;RUN37:Moluccas,seeabove).Thesecondmajorcladecomprisedallothersamples,includingsampleslabeledasRTIfromJavaand fromthe introduction rangeofJavandeer (LesserSunda Islands,Sulawesi,andtheMoluccas).BothMLandBItreetopologieswerecon-cordantwiththeoverallpatternrecoveredbythehaplotypicnetwork,alsorevealingtheexistenceoftwowell-differentiatedclades(Figure3).
Generally,geneticdiversityofhaplotypesshowedgeographicalstruc-tureonlyinsomepartsofthetree(subcladesA,B,andC).SamplesfromtheMoluccas,Sulawesi,andTimor(outsideSundaland)werepresentinmorethanonebranchofthephylogenetictree(subcladeD).
TheresultingnodeagesfortheestimatedageofCervidaespe-ciesweresimilartothosereportedinotherstudies(Table1).Usingthose,wethenestimatedthetimingofdivergencebetweenthetwomaincladestohavestartedintheearlyPleistocene,about1.8mil-lion years ago (Mya) (HPD=0.95–3.1). The position of all cladeswassimilartothetopologyrecoveredbyML,withtheexceptionoftheSriLankancladeposition (cladeB,Figures3and4),whichdi-vergedmorerecentlyintheBItree.Thissubcladewasalsoaccom-panied by lowBayesian Posterior Probability values (BPP=0.34).Nevertheless, ourdivergenceestimates indicated that subcladeAdiverged first within the RUNmitogenome clade about 1.4Mya(HPD=0.7–2.3); subclade C diverged 1.185 Mya (HPD=0.6–2)andsubcladeDataround1.13Mya (HPD=0.6–2) (Figure4).FST showed significant population differentiation only when popula-tionswerebasedonmtDNAcladeassignment,butnotwhentheywere based on species assignment from the museum collections(Table2).
3.2 | Microsatellite analyses
Ofallarchivalsamplesforwhichmitogenomeswereobtained,16in-dividuals (~29%) could be successfully genotyped at 18 loci. Theseindividualsweredistributedrelativelywellacrossthecladesofthemi-togenometree(Figure3).Linkagedisequilibriawerefoundat10%ofallpairwiselocicombinations,yetwithoutanyconsistency,andper-centageofnullalleleswas0.18.Therefore,weretainedalllociforfur-theranalyses.WedetectedsignificantdeviationsfromHHWE,whichindicatedprobablepopulationstructurewithinourdataset.Expectedandobservedheterozygositiesateachlocusrangedfrom0.23(locusMu_4D)to0.92(locusMu_1_51)andfrom0(locusMu_4D)to0.61(locusRoe09),respectively(TableA2).Numberofallelesvariedamongloci,withthehighestnumberfoundatlociMu_1_51andNVHRT48(16alleles)andthelowestfoundatlocusMu_4Dwithonlytwoalleles(TableA2).
According to the∆Kapproach, themost likelynumberofgeno-typicnDNAclusterswasK=2(Figure5).Thesetwomainclusterscor-respondedwelltothetwospecies,sambar(R. unicolor,greencluster)andJavandeer (R. timorensis, redcluster).Populationdifferentiation(FST)wasalwayssignificantbetweenthetwospecies,independentofthegroupingmethod(museumassignmentsorSTRUCTUREanalyses,Table2).
4 | DISCUSSION
ThemitochondrialgenomesandthenDNAlociofsambarandJavandeer investigatedhere revealedan intriguingandsurprisingpatternofgeneticdiversityandpopulationdifferentiationbetweenthetwospecies.AlthoughmonophylyofR. timorensis and R. unicolor remain
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undisputed,our resultspoint toamorecomplexhistoryofhybridi-zationbetweenspeciesandmultiplehuman-mediated introductionsoutsidetheSundaShelf.
Thepresenceoftwodivergentmatrilineagesclearlyindicatesmo-leculardifferentiationbetweentwogroupsofRusadeer,whichwein-terpretasthehistoricalcladogenesisofbothR. timorensis and R. unicolor. Ourfossil-calibratedestimatesarecorroboratedbyrecentstudies(Bibi,2013;Escobedo-Morales,Mandujano,Eguiarte,Rodriguez-Rodriguez,&Maldonado,2016;Table1)andareinaccordancewiththedatessug-gestedbyotherauthorsfortheageofthegenusRusa(e.g.,2–2.5Mya;DiStefano&Petronio,2002).Theseparationofthetwodeerspeciesinvestigatedherehadbeenchallengedinthepast (mentionedinVanBemmel,1949),yetsubsequentstudiesfoundrobustsupportfortheirdistinctiveness(morphological:vanBemmel,1994;Meijaard&Groves,2004; andmolecular: Emerson&Tate, 1993; Pitra, Fickel,Meijaard,& Groves, 2004). Our comprehensive molecular study corroboratedthese findings,butalsoprovidesevidence foramuchmorecomplexevolutionaryhistoryoftheRusadeer.IthasbeenproposedthatRusa-like deer have appeared in Northern India, around 2.5Mya, wheretheyadaptedtodenseforesthabitatswithsomeopengrassvegeta-tion(Geist,1998).However,duringthePleistocene,subtropicalforest
shiftedsouthwards, completelydisappearing fromChina (Meijaard&Groves, 2004).Thiswould have also shifted the distribution area ofsubtropicalforest-adaptedRusa(orRusa-like)speciessouthwardstoo.When low sea level allowed,Rusadeer couldhave reachedSundaicislands,includingJava.Sealevelremainedlowuntil1.4Mya,maintain-ingconnectionsbetweenlandmassesthroughtheemergedcontinentalshelf(VanDenBergh,DeVos,&Sondaar,2001).By1Mya,sealevelhadrisenagainandhadreachedahighstandat+5mcomparedtopresentday(Zazo,1999),therebyinterruptinglandbridgesbetweenislands.Atthistime,RusapopulationsofJavaandSumatra(cladeD)wouldhavebecomeisolatedandhabitatavailabilityforforest-dependentspecieswouldhavebeenreduced.
Our data indicate that therewas also a secondwaveof colo-nization to Sundaic islands by Rusa unicolor, likely from Thailand(Mainland). This second wave would have likely occurred duringtheLatePleistocene,withdropsinsealevelsandonceagaincoolerand drier climates. This southward expansion brought previouslyisolated Rusa unicolor in contact with Rusa timorensisfromJava,fa-cilitatedbythepresenceoftheemergedSundaStrait,astraitthatsubmerged just ~10kya (Sathiamurthy & Voris, 2006). This factraisestheobviousquestionofwhatthenmaintaineddifferentiation
F IGURE 2 Haplotypicnetworkforall46haplotypessharedamongthetwospecies.Circlesizeisinaccordancewithfrequencyandcolorrepresentssamplinglocation.Smallblackcirclesrepresentmedianvectors.Allbranchesrepresentonemutationstep,exceptwhenindicatedotherwisebynumbersonbranches.Twomajorcladeswererecoveredandareindicatedbythespeciesnames.HaplotypesaredescribedinTableA1
H_39
H_16H_14
H_46 H_21
H_44
H_40H_45
H_31
H_20
H_7H_2
H_43
H_28 H_29
H_25
H_17
H_19H_13
H_23 H_30H_41
H_35
H_34 H_5
H_6
H_33
H_9
H_42
H_18
H_32
H_8H_4 H_15
H_38
H_26
H_22
H_24
H_27
H_1
H_37
H_10
H_36
H_12
H_11
H_3
4
4 5
168
35
4
104 26 4
12
3 8
10
5511
6
2
33512
3
2
2
10
3
26
7 3
4
2
4
51
71
5147
28
2
3
36
18
9
420
IndochinaMentawai
SumatraBorneo
The Moluccas
JavaBaliSulawesiTimor and LSI
IndiaSri Lanka
R. timorensis R. unicolor
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betweenthetwospeciesand/orrestrictedhybridizationtoasmallsecondarycontact region.Wehypothesize that thedifferenteco-logicalnichesonJavaandSumatramighthavehadacentral role.Speciation may have resulted from the ecological adaptation ofJavandeerRusa timorensistotheprevailingvegetationtypeonJava,separatingitfromitssisterspecies,thesubtropicalforest-adaptedsambar Rusa unicolor. Java and Bali, although part of Sundaland,had(andstillhave)differentclimaticconditionsthanSumatraand
Borneoandthusalloweddifferentiationbetweenspeciesbasedonevolvedecological adaptations (Leonardetal., 2015).The climateonJavaischaracterizedbyaWest–Eastgradient,atransitionfromaslightlyseasonalclimate in theWest toastronglyseasonaloneintheEast.CentralandEastJavaarecharacterizedbydrier,coolerclimate(climate-data.org),andthevegetationhasmoregrassareasthan on the surrounding islands (Heany, 1991; Mishra, Gaillard,Hertler,Moigne,& Simanjuntak, 2010).Therefore, it is likely that
F IGURE 3 Mitogenomemaximumlikelihoodtreeofbothspecies.Colorsontipsrepresentsamplinglocation(asinFigure2)andstarsrepresentspliteventswithbootstrapvalues/Bayesianposteriorprobabilitieslowerthan90/0.95(butbiggerthan50/0.5).RedandgreendotsrepresentsamplesforwhichweobtainednDNA;reddot:assignedtotheRusa unicolorgenotypicclusterandgreendot:assignedtotheRusa timorensisgenotypiccluster.MajormtDNAcladesandsubcladesarelabeledwithcurvedbrackets.Scalebarindicatesnumberofsubstitutionsperposition
0.004
RTI27_Sulawesi
RTI8_Java
RUN61_Java
RUN21_Borneo
RUN3_SriLankaRUN45_ESumatra
RUN41_Sumatra
RUN37_Seram
RTI31_Sulawesi
RTI20_Sulawesi
RUN35_SriLanka
RTI30_Java
RUN55_India
RUN12_Borneo
RTI19_SSulawesi
RUN10_SriLanka
RTI17_Sulawesi
RTI3_Moluccas
RUN42_Sumatra
RTI44_Bali
RUN28_Thailand
RUN19_NBorneo
RTI18_SSulawesi
RUN65_Sumatra
RUN38_Sumatra
RTI38_WJava
RTI22_Lombok
RUN6_NBorneo
RTI33_Moluccas
RUN5_NBorneo
RUN58_Myanmar
RUN56_SriLanka
RUN20_Mentawai
RUN34_Java
RTI9_Java
RUN66_Sumatra
Run33_India
RTI39_Timor
RUN62_India
RTI43_Timor
RUN26_Borneo
RUN2_Sumatra
RTI32_JavaRUN32_Sumatra
RUN43_Indochina
RUN47_SThailandRUN44_Thailand
RTI2_Dompoe
RTI37_WJava
RUN54_NIndia
RUN39_Timor
RTI6_Timor
RUN51_SThailand
RTI21_Timor
RTI13_Java
RTI45_Bali
Rusa unicolor
mtD
NA clade
Subclade A
Subclade B
Subclade C
Rusa timorensismtDNA clade
Subclade D
Split
Age
Other studiesMedian Minimum Maximum
Bovidae/Cervidae 16.8 10.7 23.3 18.4(Bibi,2013)
Muntiacus/Cervus 7.2 4.1 10.2 7.5(Martinsetal.2017)
Axis/Cervus/Dama 4.6 2.6 5.4 6 (DiStefanio&Petronio,2002)
Cervus/Rusa 2.1 1 2.8 3.4(Pitraetal.2004)
R. unicolor/timorensis 1.4 0.7 2.2 –
Ages(inmillionyears[My])representthemedianobtainedforeachofthedescribedsplit.Valuesinboldrepresentfossil-basedcalibrations.
TABLE 1 CalibrateddivergencedatesestimatedfortheCervidaetree
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R. timorensis, being better adapted to drier climate, would havecrossed the dry central Sundaland during Pleistocene glacials tocolonizeeastJava (Sheldon,Lim,&Moyle,2015),where itstayedisolatedfromitssisterspecies.Afterthisinitialseparation,wefindevidenceofrangeexpansion,likelyduringconsequentdropsinsealevels,demonstratedbytheintrogressioninJavaandpossiblySouthSumatra.
One sambar individual from South Sumatrawas found to carryRTImtDNA.This indicatesthepossibilitythat individualsofR. timo-rensisalsomigratedtoatleastSouthSumatra,wheretheyhybridizedwith R. unicolor.SucharangeexpansionwouldhavebeenenabledbythedrierandcoolerclimatesandtheemergedlandcorridorbetweenSumatraandJavaduringtheLatePleistocene,asatthetimeofLGM,WestJavapresenteddrierandcoolerclimates in the lowlands (Sun,Li,Luo,&Chen,2000).Becausewedidnotobtain thegenotypeof
F IGURE 4 MitochondrialDNAdatedtreeaccordingtoBEASTanalyses,from3Myatopresent.Bluebarsrepresentassociateddeviationsforthemostimportantsplits.Atimescaleinmillionsofyearsandaroughestimateofsealevelchangesthroughtime(adaptedfromPatouetal.2010)arepresentedbelow
0.511.522.53
RTI20_Sulawesi
RTI13_Java
RUN5_NBorneo
RTI3_Mollucca
RTI38_WJava
RTI6_Timor
RTI19_SSulawesi
RTI17_Sulawesi
RTI32_Java
RTI27_Sulawesi
RTI2_Dompoe
RUN28_Thailand
RUN66_Sumatra
RUN2_Sumatra
RTI9_Java
RUN10_SriLanka
RUN55_India
C_elaphus
RUN26_Borneo
RUN56_SriLanka
RTI22_Lombok
RUN32_Sumatra
RTI33_Mollucas
RTI8_Java
RUN43_Indochina
RUN44_Thailand
RUN35_SriLanka
RUN51_SThailand
RTI39_Timor
RTI45_Bali
RTI37_WJava
RUN3_SriLanka
RUN62_India
RUN20_Mentawai
RUN45_ESumatra
RUN6_NBorneo
RUN54_NIndia
RUN42_Sumatra
RTI21_Timor
RUN38_Sumatra
RUN19_NBorneo
RTI31_Sulawesi
RUN41_Sumatra
RUN39_Timor
RUN12_Borneo
Run33_India
RTI43_Timor
RTI18_SSulawesi
RUN37_Seram
RUN58_Myanmar
RTI30_Java
RUN34_Java
RUN21_Borneo
RUN61_Java
RUN65_Sumatra
RUN47_SThailand
RTI44_Bali
C
A
B
+50
– 50
0 m
D
TABLE 2 Populationdifferentiationestimates(FST)accordingtomarkerandgrouping
FST p- value
mtDNA
MuseumID 0.085 >.001
Results(2clades) 0.75 <.001
nDNA
MuseumID 0.12 <.001
Results(K=2) 0.14 <.001
EstimationswereperformedbothforthemtDNAandnDNA.PopulationsweregeneratedbyeithermuseumID(bothformtDNAandnDNA)andbyassignmentof individualstooneofthetwomajorclades (mtDNA)ortooneofthegenotypicclusters(nDNA).Statisticallysignificantcomparisonsareindicatedbyboldp-values.
1472 | MARTINS eT Al.
thissample,moreintensivesamplingofSouthSumatranpopulationswouldberequiredtoconcludethattheseresultsreflectevidenceofreciprocal hybridizationbetween two sister speciesofdeer.On theotherhand,wefoundastrongevidencethatthesambar,likelyduringwetterinterglacialperiods,expandeditsrangefromSumatraatleasttowesternJavawhereithybridizedwiththeJavandeer.Thoseintro-gressedJavandeermostlikelyconstitutedthesourcepopulationfortheintroductionseastoftheWallaceline.
4.1 | Phylogeography and taxonomy of Rusa timorensis
WithinR. timorensis,individualsfromBaliandWestJavashowedgeneticdivergenceatmtDNA.ThissubstructurecouldindicatelimitedgeneflowduringpartsoftheLatePleistocene,whichmightcorroboratetheclas-sificationofBalipopulationsasR. t. renschi,withgeneticisolationmostlikelybeingtheresultofa“smallpopulationeffect”andalimited/inter-ruptedgeneflowtoJavanpopulations.However,becauseweonlyhadtwosamplesfromBaliandnosamplesfromEastJava,ourassessmenthastobeviewedcautiously.Itdoeshoweverindicatetheespeciallyur-gentneedtoassessifhybridizationoccurredaswellinthesepopulations,throughmoreextensivesamplingandinclusionofnuclearmarkers.
ThemtDNAofRTI-labeledsamplesfromislandsbeyondtheWallaceline clustered with R. unicolormtDNAbutdidnotshowanycleargeo-graphical distribution pattern. These RTI hybrids shared haplotypeswithRUN-labeledsamplesfromSumatraandBorneo(Figures2and3).GeneticdistancesamongRTIhybridhaplotypesandtootherRUNhap-lotypeswereverylow,indicatingarecent,thushuman-mediatedintro-ductiontotheseWallaceaislands.Quiterecently,ithadbeensuggestedto splitR. timorensis into seven subspecies according to their occur-renceonislandswithinandoutsideoftheSundashelf(Mattioli,2011;Hedges,Duckworth,Timmins,Semiadi,&Dryden,2015).However,ourdatadonotsupportsuchasuggestion,asallsamplesfromtheintro-duction range shared haplotypes, indicating a lack of differentiationamongindividualsfromtheseWallaceanislands.Furthermore,thefewsamplesfromJavaandTimorthatcouldbegenotypedshowedgeneticsimilarity,againindicatingthelackofdifferentiation.
4.2 | Phylogeography and taxonomy of Rusa unicolor
Sambar is currently subdivided into five subspecies: R. u. unicolor (India,Nepal,Bangladesh,andSriLanka),R. u. brookei (Borneo),R. u. cambojensis(mainlandSoutheastAsia,fromSouthChina/HainanandMyanmartoPeninsulaMalaysia),R. u. equine(SumatraandMentawai),and R. u. swinhoei(Taiwan;Mattioli,2011).Themitogenomestructurerecoveredhere,however,didnotsupportanyofthedescribedsub-species,asitindicatedgeneflowbetweenallpopulations.Especiallyamong populations of Sundaic islands, we found a lack of geneticstructurethatwouldcorrespondtoisolatedislands,evidencedbythepresenceofindividualsdistributedthroughoutthetreetopologiesandhaplotypicinferences.
Amongpopulationsofsambar,wefoundevidenceofatleastthreedeepsplit (>1My)subcladeswhichwerenot inaccordancewith thecurrentsubspeciesassignment.SubcladeAcomprisedhaplotypesfromMyanmarandIndia,withanageofabout1.36My;thesecondcladeincluded Sundaic populations from Sumatra, Mentawai, and Borneo(cladeC)andwasdatedtobe~1.18Myold;andthethirdoneincludedall haplotypes fromSri Lanka (cladeB) and split from the remainingpopulations ~1.13Mya. Subclade D encompassed all remaining in-dividuals of sambar, both fromMainland South and Southeast Asiaand theSundaic islands.Despite the ancient split of cladeA furthersampling ofmainland southeastAsia, particular India,Myanmar, andBangladesh isneededtorevealwhethercladeA is indeedgeograph-icallyseparatedfromtheothermainlandAsianpopulations,particularthoserepresentedinsubcladeD.Thus,albeithistoricallycladeAandDwereisolated,ourdataindicaterecentgeneflowbetweenthetwoclades,andthus,itremainsuncertainwhetherourdatawouldsupportasubspecificstatusofindividualsfromcladeA.Verysimilarly,SumatranindividualswerepresentinthedistinctmitochondrialcladesCandD,andthus,ourdatadidnotsupportseparatingthesecladesindistincttaxonomicunits.
These subclades could then rather represent centers of sambardistribution, which would have remained in place during times ofwarmerandwetterconditions,whichcontractedsambarpopulationsto subtropical refugia. From these centers, we observed waves of
F IGURE 5 Genotypingresultsfrom16individualsgenotypedfor18loci,showingastructureplotforK=2,withR. timorensis samplesingreenandR. unicolor individuals inred.Eachcolumnrepresentsasingleindividual,asidentifiedbelow
0.80
0.60
1.00
0.40
0.20
0.00
I45_
Bali
RTI4
3_Ti
mor
RTI3
9_Ti
mor
RUN
61_J
ava
RTI4
4_Ba
liRT
I6_T
imor
RUN
58_M
yanm
arRU
N56
_Sri
Lank
aRU
N43
_Ind
ochi
naRU
N3_
Sri L
anka
RUN
12_B
orne
oRU
N19
_Bor
neo
RUN
20_M
enta
wai
RUN
66_S
umat
raRU
N5_
Born
eoRU
N32
_Sum
atra
| 1473MARTINS eT Al.
expansion.ThebranchingorderofthesethreeoldsubcladesindicatedcolonizationfromnorthernIndochinasouthwardstoSriLankaandtotheSundaShelf, respectively.The “younger” individualswithin sub-cladeDfromIndia,aswellasfromSumatraandBorneo,appearthentobedescendantsfromasecond/thirdnaturaldispersionwave(pos-sibly fromThailand) during glacial periods of thePleistocene,whenlow sea level again exposed the shallow Sunda shelf connecting allmajor islands (Voris, 2000; Bird, Taylor, &Hunt, 2005). During gla-cialperiods, climatewasdrierandcooler in tropical regions (Gorog,Sinaga,&Engstrom, 2004).However, species that retained a broadecological niche such as Rusa unicolorwouldhavebeenabletoutilizethenewly emergedhabitats Such a scenariowould likely cause thehaplotypicdistributionpatternweobservedhere.Duringglacialperi-ods,SundalandwasalsoconnectedtoSoutheastAsianmainland,al-lowingsecondaryadmixturebetweenformerlyseparatedpopulations,thus generating the patternswe observe between haplotypes fromThailand,India,andSundaland.
Incontrast, thedistinctSri LankancladeBprovidesadditionalev-idence for the recognitionofSriLankansambarasbeingdistinct.Thissupportcomesbothfrommorphologicalassessments(Groves&Grubb,2011)andkaryotypedifferences(2n=56inSriLankansambarvs.2n=58inIndianand2n=62inChineseandMalaysiansambar;Leslie,2011).SriLankanpopulationsareoftenmoregenetically related to theWesternGhatsthantootherIndianregions.Veryrecently,a40bpinsertionwasdetectedinthecontrolregionofthemitochondrialDNAinsamplesfromtheWesternGhats(Gupta,Kumar,Gaur,&Hussain,2015),whosepres-encewe,however,wereunabletoverifyduetomethodlimitations.
Thus,furtherstudiesonthemainlandpopulationsthatcouldcon-firmthepresenceofoldsplitswithinR. unicolorareofurgency.Increasedsamplingand,especially,theinclusionofnuclearmarkerscouldconfirmtheextentofgeneflowbetweenthesepopulationsor,conversely,truegeneticdivergencebetweenthesubcladesrecoveredhere.Ifconfirmed,thesepopulationsmight represent subspeciesof sambaroccurring inhighlydisturbedregionsofMainlandSoutheastAsia.
4.3 | Introductions past the Wallace line
It is generally accepted that the presence of Rusa deer on islandsbeyond Sundaland (excluding Philippines) was the result of humaninterference (Long, 2003; Groves & Grubb, 2011; Hedges etal.,2015).However,untilnow,theseindividualswereassumedtohavebeenpureR. timorensis,collected,andtransportedforvenisonandasgamespeciesbyhumansfromtheislandsofJavaandBaliduringtheHolocene(Heinsohn,2003).WhilethenDNAdataclearlyseparatedthetwospecies—beinghighlyconcordantwiththeirdescriptionfromthemuseumcollections—themitochondrialgenomespointtoamoresurprisingpatternofpastPleistocenehybridizations.Human-mediatedintroductionsofthesedeeroccurredca.3,000yearsago(Heinsohn,2003),and,toourknowledge,theseresultscouldthereforeconstituteevidencefortheearliesttransportofintrogresseddeer.
AllmtDNAhaplotypesofsampleslabeledinthemuseumcollec-tions as R. timorensisandsampledfromWallacean islands (Sulawesi,Lesser Sunda Islands and TheMoluccas) were of R. unicolor origin,
renderingtheseindividualshybriddescendants.Themostparsimoni-ousexplanation for themolecularpatternsobtained in this study isthathybridizationoccurredonJava(centerofstar-likepatterninthehaplotypicnetwork),withnaturaldispersionoffemalesambar.Theseimmigrated individuals were likely from Sumatra and/or the Thai-MalayPeninsula,asindicatedbythebasalpositionofthetwoSouthernThailandindividuals(RUN51andRUN57),andtheywouldhaveusedtheconnectinglandbridges.AftertheintrogressionofSambarhaplo-types intoJavanpopulations, humanswould thenhave transportedthe introgressed descendants of Pleistocene hybrids from Java toTimor,theMoluccasandSulawesi.Despiteevidenceformultiple in-dependentintroductions(Section4),almostallintroducedindividualscarriedsambarhaplotypes(exceptRUN37fromSeram,theMoluccas).Thisindicatesthateitherhumansselectedforindividualstobeintro-duced(e.g.,carryingaparticulartraitonlyfoundinintrogressedJavandeer);thattheintrogressedindividualshadahighersurvivingprobabil-ityaftertheirintroduction;orthatmostintroductionsoccurredfromasingleregion(e.g.,WestJava)whereRUNhaplotypesgotfixed,possi-blethroughmitochondrialcaptureofsambarhaplotypes.
Introduction of Javan deer to Timor seems to have occurredonlyonceand,presumably,withveryfewfoundersbecauseofthelackofmtDNAdiversity foundamongall individuals. In fact,pop-ulations recently introduced toAustralia andNewCaledonia froma low,knownnumberof individualsfromTimor,havebeenshowntohavevery lowgenomicdiversity,whichwouldbe theexpectedresultafteranintroductionofindividualsthathadcomefromanal-readygeneticallyimpoverishedpopulation(Webley,Zenger,English,&Cooper,2004;deGarine-Wichatitskyetal.,2009).AlthoughweobtainedonlyveryfewsamplesfromtheMoluccasandotherLesserSundaIslands(DompoeandLombok),theydidnotsharehaplotypes,indicatingeithermultipleintroductionsorahighernumberoffound-ers.SulawesihadbyfarthemostgeneticallydiverseJavandeerpop-ulationofalltheWallaceanislands.ItshaplotypeswerepresentinalmostallyoungercladesofthemtDNAtree.ThispatternindicatedthatRusadeerreachedSulawesimultipletimes.Onesample(RTI18)was closer related to Bornean populations than the other haplo-types from Sulawesi. Although natural dispersal from Borneo toSulawesiovertheMakassarStraitisconceivable,itishighlyunlikelyasthelastpossibleconnectionbetweenthesetwolandmasseswasduringthelatePliocene/earlyPleistocene~2.5Mya,adatethatbyfarpredatestheemergenceofthismtDNAlineage.Themostlikelyscenario is a human-mediated introduction of Bornean sambar toSulawesiwhereithybridizedwiththeintroducedJavandeer.Iftrue,this represents a second hybridization event on Sulawesi (com-paredtotheLatePleistocenehybridization inJavaandpotentiallySouthSumatra)and further studiesonSulawesiJavandeerwouldberequiredtotestthishypothesis.TheremaininghaplotypesfromSulawesiindividualswerecloselyrelatedtothehaplotypesfromin-dividuals introducedtotheMoluccasandTimor Islands, indicatingeitherthatallofthemhavebeenintroducedinonewaveoratleastfromasimilarsourcepopulationfromJava.
This is the first reportofhistoricalhybridizationsbetweensam-bar (R. unicolor) and Javan deer (R. timorensis). Occurrence of such
1474 | MARTINS eT Al.
hybridizationhadbeendocumentedbefore,namelybetweenR. timo-rensisindividualsintroducedtoBorneowiththelocalBorneanR. uni-color(WestKalimantan,nowpossiblyextinct,Hedgesetal.,2015)andattainedthroughhusbandrybefore(Leslie,2011).Hybridizationswithfertileoffspringhavealsobeenreportedtooccurbetweenotherdeerspecies,amongothersbetweensambarandreddeerCervus elaphus (Muiretal.,1997)andreddeerandSikaCervus nippon(Smith,Carden,Coad,Birkitt,&Pemberton,2014).
This fact has potentially important conservation implications forthetwoRusaspeciesanalyzedinthisstudy.DespitebeingoneofthemostwidespreaddeerspeciesinsouthernAsia,R. unicoloristodaynolonger abundant throughoutmost of its native range (Timmins etal.,2015).Likewise,R. timorensis iscurrentlyconsideredapestspecies inareaswhere it has recently been introduced (e.g., Australia) but has,however,decreasedlargelyinpopulationnumbersinnativeandhistor-icalintroductionregions(Java,LesserSundaIslands,Sulawesi,andtheMoluccas).BothRusaspeciesstudiedherearenowconsideredvulner-ablebytheIUCN/RedListofThreatenedSpecies(Hedgesetal.,2015;Timminsetal.,2015).Therefore,geneticmonitoringofindividuals,bothatmtDNAandparticularlyalsonucleargenomes,isnecessarytoassesswhetherpureRTIandRUNindividualsarebeingintroduced(orrepro-ductivelyassistedintheirnativeranges)andnotintrogressedindividu-als.Moreover,moreintensiveandextensivesamplingofR. timorensis on theirnativerangeisnecessarytodiscernwhetherpureRTIpopulationsstillremaininJavaandBaliorwhethertheyarecomposedintheirma-joritybyhybridindividuals.
5 | CONCLUSION
In addition to representing the first comprehensive phylogeo-graphicalstudyonR. unicolor and R. timorensis,thisstudyrevealedsurprising evolutionary histories of these two sister species.Answering the questions poised before, we hypothesized thatwhile climateadaptationswere likely responsible formaintainingspecies monophyly, Pleistocene climate changes were responsi-ble forsecondarycontactandconsequenthybridizationbetweensambar and Javan deer.We recovered a pattern of (possibly re-ciprocal) introgressions between the two species, facilitated bythepresenceof landcorridorsduringperiodsof lowsealevels inSundaland. The introgressed populations of Javan deer on Javawere then the source of all human-mediated introductionwavestotheislandseastoftheWallaceline,aswefoundthatallR. timo-rensis individuals carried R. unicolorhaplotypes.Additionally,thesedramaticclimatechangeswerealsolikelyresponsibleforthediver-genceofpopulationswithinR. unicolor.
ACKNOWLEDGMENTS
The authors would like to thank all curators listed in TableA1 forprovidingaccesstotheircollections.ThisprojectwasfundedbytheLeibnizcompetitivefundSAW-2013-IZW-2.
CONFLICT OF INTEREST
Nonedeclared.
AUTHOR CONTRIBUTIONS
RFM,AW,andJFdesignedthestudy;RFMandASperformedthelab-oratoryprocedures;RFMandDLperformedthebioinformaticanaly-ses;RFM,AW,andJFwrotethemanuscript.Allauthorscontributedequallyforthefinalversionofthismanuscript.
ORCID
Renata F. Martins http://orcid.org/0000-0001-8145-6998
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How to cite this article:MartinsRF,SchmidtA,LenzD,WiltingA,FickelJ.Human-mediatedintroductionofintrogresseddeeracrossWallace’sline:HistoricalbiogeographyofRusa unicolor and R. timorensis. Ecol Evol. 2018;8:1465–1479. https://doi.org/10.1002/ece3.3754
| 1477MARTINS eT Al.
TABLE A1 Completedatasetdetails
Spec
ies
Sam
ple
IDO
rigin
MtD
NA
hap
loty
peA
cces
sion
Num
ber
nDN
A a
ssig
nmen
tM
useu
m C
olle
ctio
nM
useu
m ID
Cura
tor
Rusa
uni
colo
rRUN2
Sumatra
H_13
MF176993
BerlinNHM
7515
0F.Mayer
Rusa
uni
colo
rRUN3
SriLanka
H_14
MF1
7701
8RUN
BerlinNHM
75133
F.Mayer
Rusa
uni
colo
rRUN5
NorthBorneo
H_37
MF1
7701
9RUN
BerlinNHM
1125
9F.Mayer
Rusa
uni
colo
rRUN6
NorthBorneo
H_38
MF1
7702
0BerlinNHM
1126
1F.Mayer
Rusa
uni
colo
rRUN10
SriLanka
H_14
MF1
7699
4BerlinNHM
12368
F.Mayer
Rusa
uni
colo
rRUN12
Sarawak,Borneo
H_15
MF1
7699
5RUN
BerlinNHM
1470
2F.Mayer
Rusa
uni
colo
rRUN19
NorthBorneo
H_37
MF1
7702
1RUN
BerlinNHM
103292
F.Mayer
Rusa
uni
colo
rRUN20
Men
taw
aiH_16
MF1
7699
6RUN
Naturalis
139–50
P.Kamminga
Rusa
uni
colo
rRUN21
Borneo
H_37
MF1
7702
2StuttgartNHM
1690
0S.Merker
Rusa
uni
colo
rRUN26
Borneo
H_39
MF177023
StuttgartNHM
1590
0S.Merker
Rusa
uni
colo
rRUN28
Thai
land
H_17
MF1
7699
7BonnNHM
ZFMK_
MAM_2013.618
R.Hutterer
Rusa
uni
colo
rRUN32
Sumatra
H_18
MF1
7699
8RUN
ViennaNHM
7333
F.Zachos
Rusa
uni
colo
rRUN33
Indi
aH_19
MF1
7699
9ViennaNHM
4086
7F.Zachos
Rusa
uni
colo
rRUN34
Java
H_20
MF1
7700
0SenckenbergFrankfurt
15937
I.Ruf
Rusa
uni
colo
rRUN35
SriLanka
H_21
MF1
7700
1ParisNHM
1877-10
G.Veron
Rusa
uni
colo
rRUN37
Burum,Moluccas
H_22
MF2
7925
0MunichNHM
1906/3021
M.Hiermeyer
Rusa
uni
colo
rRUN38
Sumatra
H_23
MF1
7700
2MunichNHM
1905
/46
M.Hiermeyer
Rusa
uni
colo
rRUN39
Timor
H_3
MF177003
MunichNHM
1911
/214
7M.Hiermeyer
Rusa
uni
colo
rRUN41
SouthSumatra
H_24
MF2
7925
1MunichNHM
1908
/465
M.Hiermeyer
Rusa
uni
colo
rRUN42
Sumatra
H_25
MF1
7700
4MunichNHM
1908
/466
M.Hiermeyer
Rusa
uni
colo
rRUN43
Indo
chin
aH_26
MF1
7700
5RUN
MunichNHM
1962/235
M.Hiermeyer
Rusa
uni
colo
rRUN44
SouthThailand
H_27
MF1
7700
6CopenhagenNHM
M15
94D.K.Johansson
Rusa
uni
colo
rRUN45
EastSumatra
H_40
MF1
7702
4CopenhagenNHM
M16
50D.K.Johansson
Rusa
uni
colo
rRUN47
SouthThailand
H_41
MF1
7702
5CopenhagenNHM
M1593
D.K.Johansson
Rusa
uni
colo
rRUN51
SouthThailand
H_42
MF1
7702
6CopenhagenNHM
M1583
D.K.Johansson
Rusa
uni
colo
rRUN54
BhutanDooars,India
H_43
MF1
7702
7CopenhagenNHM
M534
D.K.Johansson
Rusa
uni
colo
rRUN55
BhutanDooars,India
H_28
MF1
7700
7CopenhagenNHM
M533
D.K.Johansson
Rusa
uni
colo
rRUN56
SriLanka
H_44
MF1
7702
8RUN
CopenhagenNHM
M1236
D.K.Johansson
Rusa
uni
colo
rRUN58
SouthMyanmar
H_29
MF1
7700
8RUN
Naturalis
RMNH.MAM.693.a
P.Kamminga
Rusa
uni
colo
rRUN61
CentralJava
H_3
MF1
7700
9RT
INaturalis
RMNH.MAM.33832
P.Kamminga
(Continued)
APP
END
IX
1478 | MARTINS eT Al.
Spec
ies
Sam
ple
IDO
rigin
MtD
NA
hap
loty
peA
cces
sion
Num
ber
nDN
A a
ssig
nmen
tM
useu
m C
olle
ctio
nM
useu
m ID
Cura
tor
Rusa
uni
colo
rRUN62
Bengala,India
H_30
MF1
7701
0Naturalis
RMNH.MAM.51460
P.Kamminga
Rusa
uni
colo
rRUN65
SouthSumatra
H_45
MF1
7702
9Naturalis
RMNH.MAM.51453
P.Kamminga
Rusa
uni
colo
rRUN66
WestSumatra
H_46
MF177030
RUN
Naturalis
RMNH.
MAM.1033.b
P.Kamminga
Rusa
tim
oren
sisRT
I2LesserSundaIslands
H_31
MF1
7701
1BerlinNHM
92305
F.Mayer
Rusa
tim
oren
sisRTI3
Mol
ucca
sH_1
MF1
7698
1BerlinNHM
30840
F.Mayer
Rusa
tim
oren
sisRT
I6Timor
H_2
MF1
7698
2RT
IStuttgartNHM
1587
8S.Merker
Rusa
tim
oren
sisRT
I8Java
H_3
MF176983
StuttgartNHM
1589
2S.Merker
Rusa
tim
oren
sisRT
I9Java
H_4
MF1
7698
4StuttgartNHM
1589
7S.Merker
Rusa
tim
oren
sisRTI13
Java
H_1
MF1
7698
5StuttgartNHM
1589
1S.Merker
Rusa
tim
oren
sisRT
I17
SouthSulawesi
H_5
MF1
7698
6BonnNHM
58.1
09R.Hutterer
Rusa
tim
oren
sisRT
I18
SouthSulawesi
H_32
MF1
7701
2BonnNHM
58.7
5R.Hutterer
Rusa
tim
oren
sisRT
I19
SouthSulawesi
H_33
MF177013
BonnNHM
58.1
05R.Hutterer
Rusa
tim
oren
sisRT
I20
SouthSulawesi
H_6
MF1
7698
7BonnNHM
58.1
12R.Hutterer
Rusa
tim
oren
sisRT
I21
Timor
H_3
MF1
7701
4ViennaNHM
231
F.Zachos
Rusa
tim
oren
sisRT
I22
LesserSundaIslands
H_34
MF1
7701
5ViennaNHM
2049
F.Zachos
Rusa
tim
oren
sisRT
I27
Sulawesi
H_35
MF1
7701
6ViennaNHM
7334
F.Zachos
Rusa
tim
oren
sisRTI30
Java
H_7
MF1
7698
8DresdenNHM
B730
C.Stefen
Rusa
tim
oren
sisRTI31
Sulawesi
H_3
MF1
7698
9DresdenNHM
B2686
C.Stefen
Rusa
tim
oren
sisRTI32
Java
H_8
MF1
7699
0SenckenbergFrankfurt
1546
2I.Ruf
Rusa
tim
oren
sisRTI33
Mol
ucca
sH_9
MF1
7699
1SenckenbergFrankfurt
5562
I.Ruf
Rusa
tim
oren
sisRTI37
WestJava
H_10
MF2
7924
7Naturalis
RMNH.MAM.5679
P.Kamminga
Rusa
tim
oren
sisRTI38
WestJava
H_11
MF2
7924
8Naturalis
RMNH.MAM.5680
P.Kamminga
Rusa
tim
oren
sisRTI39
Timor
H_3
MF1
7699
2RT
INaturalis
RMNH.MAM.51419
P.Kamminga
Rusa
tim
oren
sisRTI43
Timor
H_3
MF1
7701
7RT
INaturalis
RMNH.MAM.51417
P.Kamminga
Rusa
tim
oren
sisRT
I44
Bali
H_12
MF2
7924
9RT
INaturalis
RMNH.MAM.33828
P.Kamminga
Rusa
tim
oren
sisRT
I45
Bali
H_36
MF2
7925
2RT
INaturalis
RMNH.MAM.33827
P.Kamminga
Samplesareindicatedaccordingtothespeciesassignmentfromthemuseumcollections.mtDNAhaplotypeandgenotypicclusterassignmentareprovidedaccordingtotheresultsobtained.
TABLE A1 (Continued)
| 1479MARTINS eT Al.
Locus Allelic range Na HE HO FIS (W&C) Reference
Haut14 116–130 6 0.66 0.25 0.628 Kühn,Anastassiadis,andPirchner(1996)
VH110 101–153 12 0.89 0.44 0.516 Talbotetal.(1996)
NVHRT48 71–120 12 0.8 0.31 0.615 RøedandMidthjell(1998)
BM757 167–199 12 0.92 0.5 0.424 Slateetal.(1998)
CSSM39 158–200 12 0.87 0.37 0.575 Slateetal.(1998)
CSSM41 120–146 11 0.85 0.37 0.566 Slateetal.(1998)
FSHB 171–206 13 0.92 0.5 0.426 Slateetal.(1998)
Roe09 167–199 12 0.9 0.63 0.31 FickelandReinsch(2000)
CSSM14 131–161 10 0.82 0.13 0.851 Kühn,Schröder,Pirchner,andRottmann(2003)
T115 139–184 9 0.81 0.44 0.467 Meredith,Rodzen,Levine,andBanks(2005)
C143 154–174 4 0.71 0.19 0.741 Meredithetal.(2005)
C180 138–160 6 0.67 0.37 0.448 Meredithetal.(2005)
INRA6 103–141 10 0.78 0.37 0.529 SennandPemberton(2009)
Mu_4D 111–113 2 0.23 0 1.000 Schröderetal.(2016)
Mu_1_51 114–152 16 0.95 0.75 0.216 Schröderetal.(2016)
C183 119–133 5 0.65 0.25 0.62 Schröderetal.(2016)
Mu_1_25 98–110 6 0.78 0.37 0.529 Schröderetal.(2016)
Mu_1_550 139–173 14 0.94 0.37 0.608 Schröderetal.(2016)
Allelicrange,numberofalleles(Na),expectedandobservedheterozygosity(HE and HO),andinbreedingcoefficient(FIS)aregivenforeachloci,ascalculatedforthe16individualsgenotyped.
TABLE A2 Microsatellite loci details and results