Phylogeography and Genetic Ancestry of Tigers (Panthera tigris)

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Nova Southeastern University
NSUWorks
Biology Faculty Articles Department of Biological Sciences
12-2004
Phylogeography and Genetic Ancestry of Tigers
(Panthera tigris)
Shu-Jin Luo
National Cancer Institute at Frederick; University of Minnesota - St. Paul
Jae-Heup Kim
National Cancer Institute at Frederick
Warren E. Johnson
National Cancer Institute at Frederick
Joelle van der Walt
National Cancer Institute at Frederick
Janice S. Martenson
National Cancer Institute at Frederick
See next page for additional authors
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NSUWorks Citation
Luo, Shu-Jin; Jae-Heup Kim; Warren E. Johnson; Joelle van der Walt; Janice S. Martenson; Naoya Yuhki; Dale Miquelle; Olga
Uphyrkina; John M. Goodrich; Howard Quigley; R. Tilson; Gerald Brady; Paolo Martelli; Vellayan Subramaniam; Charles McDougal;
Sun Hean; Shi-Qiang Huang; Wenshi Pan; Ullas K. Karanth; Melvin Sunquist; James L. D. Smith; and Stephen J. O'Brien. 2004.
"Phylogeography and Genetic Ancestry of Tigers (Panthera tigris)."PLoS Biology2, (12 e442): 2275-2293.
https://nsuworks.nova.edu/cnso_bio_facarticles/728
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Authors
Shu-Jin Luo, Jae-Heup Kim, Warren E. Johnson, Joelle van der Walt, Janice S. Martenson, Naoya Yuhki, Dale
Miquelle, Olga Uphyrkina, John M. Goodrich, Howard Quigley, R. Tilson, Gerald Brady, Paolo Martelli,
Vellayan Subramaniam, Charles McDougal, Sun Hean, Shi-Qiang Huang, Wenshi Pan, Ullas K. Karanth,
Melvin Sunquist, James L. D. Smith, and Stephen J. O'Brien
This article is available at NSUWorks:https://nsuworks.nova.edu/cnso_bio_facarticles/728
PLoS
Openaccess,freelyavailableonline BIOLOGY
Phylogeography and Genetic Ancestry
of Tigers (Panthera tigris)
Shu-Jin Luo1,2[, Jae-Heup Kim1[¤1, Warren E. Johnson1, Joelle van der Walt1¤2, Janice Martenson1,
Naoya Yuhki1, Dale G. Miquelle3, Olga Uphyrkina1¤3, John M. Goodrich4, Howard B. Quigley3,4,
Ronald Tilson5, Gerald Brady6, Paolo Martelli7, Vellayan Subramaniam8, Charles McDougal9, Sun Hean10,
Shi-Qiang Huang11, Wenshi Pan12, Ullas K. Karanth13, Melvin Sunquist14, James L. D. Smith2,
Stephen J. O’Brien1[*
1Laboratory of Genomic Diversity, National Cancer Institute, Frederick, Maryland, United States of America, 2Conservation Biology Graduate Program, University of
Minnesota,St.Paul,Minnesota,UnitedStatesofAmerica,3WildlifeConservationSociety,RussianFarEastProgram,Bronx,NewYork,UnitedStatesofAmerica,4Wildlife
ConservationSociety,HornockerWildlifeInstitute,Bozeman,Montana,UnitedStatesofAmerica,5MinnesotaZoo,AppleValley,Minnesota,UnitedStatesofAmerica,
6PotterParkZoo,Lansing,Michigan,UnitedStatesofAmerica,7SingaporeZoologicalGardens,Singapore,8ZooNegara,HuluKelang,Selangor,Malaysia,9TigerTops,
Kathmandu,Nepal,10InternationalCooperationOffice,MinistryofAgricultureForestryandFisheries,PhnomPenh,Cambodia,11BeijingZoo,Beijing,China,12Collegeof
Life Sciences, Peking University, Beijing, China, 13Wildlife Conservation Society—India Program, Bangalore, Karnataka, India, 14Department of Wildlife Ecology and
Conservation,UniversityofFlorida,Gainesville,Florida,UnitedStatesofAmerica
Eighttraditionalsubspeciesoftiger(Pantheratigris),ofwhichthreerecentlybecameextinct,arecommonlyrecognized
onthebasisofgeographicisolationandmorphologicalcharacteristics.Toinvestigatethespecies’evolutionaryhistory
and to establish objective methods for subspecies recognition, voucher specimens of blood, skin, hair, and/or skin
biopsies from 134 tigers with verified geographic origins or heritage across the whole distribution range were
examined for three molecular markers: (1) 4.0 kb of mitochondrial DNA (mtDNA) sequence; (2) allele variation in the
nuclearmajorhistocompatibilitycomplexclassIIDRBgene;and(3)compositenuclearmicrosatellitegenotypesbased
on 30 loci. Relatively low genetic variation with mtDNA, DRB, and microsatellite loci was found, but significant
populationsubdivisionwasnonethelessapparentamongfivelivingsubspecies.Inaddition,adistinctpartitionofthe
Indochinese subspecies P. t. corbetti into northern Indochinese and Malayan Peninsula populations was discovered.
Population genetic structure would suggest recognition of six taxonomic units or subspecies: (1) Amur tiger P. t.
altaica;(2)northernIndochinesetigerP.t.corbetti;(3)SouthChinatigerP.t.amoyensis;(4)MalayantigerP.t.jacksoni,
namedforthetigerconservationistPeterJackson;(5)SumatrantigerP.t.sumatrae;and(6)BengaltigerP.t.tigris.The
proposedSouthChinatigerlineageistentativeduetolimitedsampling.Theageofthemostrecentcommonancestor
for tiger mtDNA was estimated to be 72,000–108,000 y, relatively younger than some other Panthera species. A
combinationofpopulationexpansions,reducedgeneflow,andgeneticdriftfollowingthelastgeneticdiminution,and
the recent anthropogenic range contraction, have led to the distinct genetic partitions. These results provide an
explicitbasisforsubspeciesrecognitionandwillleadtotheimprovedmanagementandconservationoftheserecently
isolated but distinct geographic populations of tigers.
Citation:LuoSJ,KimJH,JohnsonWE,vanderWaltJ,MartensonJ,etal.(2004)Phylogeographyandgeneticancestryoftigers(Pantheratigris).PLoSBiol2(12):e442.
Introduction ReceivedFebruary10,2004;AcceptedOctober21,2004;PublishedDecember
7,2004
The tiger (Panthera tigris) is the largest felid species and a DOI:10.1371/journal.pbio.0020442
widely recognized symbol of wildlife conservation. Histor- Copyright:(cid:1)2004Luoetal.Thisisanopen-accessarticledistributedunder
the terms of the Creative Commons Attribution License, which permits
ically tigers inhabited much of Asia, including the regions
unrestricteduse,distribution,andreproductioninanymedium,providedthe
betweentheCaspianandAralSeas,southeasternRussia,and originalworkisproperlycited.
the Sunda islands (Mazak 1981; Hemmer 1987; Herrington
Abbreviations: AMOVA, analysis of molecular variance; CI, confidence interval;
1987). Since the early 1900s, however, habitat loss, fragmen- Cymt, cytoplasmic mitochondrial; Dkf, kinship coefficient; Dps, proportion of
sharedalleles;ME, minimumevolution;MHC,majorhistocompatibilitycomplex;
tation, and human persecution have reduced tiger popula-
ML,maximumlikelihood;MP,maximumparsimony;mtDNA,mitochondrialDNA;
tionsfromprobablyover100,000in1900tofewerthan7,000 MY(A),millionyears(ago);NJ,neighborjoining;Numt,nuclearmitochondrial;SSCP,
free-rangingindividuals(NowellandJackson1996;Dinerstein singlestrandconformationpolymorphism
etal. 1997;Kitchener and Dugmore 2000). Most populations AcademicEditor:CraigMoritz,UniversityofCalifornia,Berkeley
consist of less than 120 animals, increasing the risk of local *Towhomcorrespondenceshouldbeaddressed.E-mail:[email protected]
extirpation due to demographic and genetic factors (Smith
[Theseauthorscontributedequallytothiswork.
andMcDougal 1991; Dinersteinetal. 1997).
¤1Currentaddress:BiochipProjectTeam,SamsungAdvancedInstituteof
There are eight generally accepted tiger subspecies in
Technology,Suwon,Korea
accordancewiththeirgeographicdistribution(Figure1).Bali
¤2Currentaddress:CenterforHumanGenetics,DukeUniversityMedicalCenter,
(P.t.balica),Caspian(P.t.virgata),andJavan(P.t.sondaica)tiger Durham,NorthCarolina,UnitedStatesofAmerica
subspecies were eradicated by the 1940s, 1970s, and 1980s
¤3Currentaddress:EvolutionaryZoologyandGeneticsLaboratory,Instituteof
respectively (Nowell and Jackson 1996). Today an estimated BiologyandSoilSciences,Vladivostok,Russia
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TigerPhylogeography
Figure1.HistoricandCurrentGeographicDistributionofTigersCorrespondingtotheEightTraditionalSubspeciesDesignation
Geographicoriginofsamplesandsamplesize(circlesorsquares)fromeachlocationareindicated(seeTable3forsources).Three-lettercodes
(TIG,ALT,etc.)areindicatedsubspeciesabbreviations.Dottedlinesareapproximateboundariesbetweentigersubspeciesstudiedhere.The
IsthmusofKradividesthetraditionalIndochinesetigersintothenorthernIndochinesetigersP.t.corbettiIandtheMalayantigersP.t.corbettiII
basedonthepresentstudy.WeproposetheMalayantigersubspecies,CORII,benamedP.t.jacksoni,tohonorPeterJackson,theformerChairof
theIUCN’sCatSpecialistGroupwhohascontributedsignificantlytoworldwidetigerconservation.
DOI:10.1371/journal.pbio.0020442.g001
3,200–4,500 Indian or Bengal tigers (P. t. tigris) exist in 1996).However,theadequacyofthesetraditionalsubspecies
Bangladesh,Bhutan,westernChina,India,westernMyanmar, designations is tentative at best, since morphological dis-
andNepal(Seidenstickeretal.1999).Fewerthan500Amuror tinctionsinmanycaseshavebeenbasedonafewspecimens,
Siberian tigers (P. t. altaica) survive in eastern Russia, north- and because subsequent studies have failed to affirm these
eastern China, and Korea (Matyushkin et al. 1999; Miquelle distinctions. Herrington (1987) and Kitchener (1999) have
and Pikunov 2003), while approximately 50 Amoy or South revealedawiderangeofmorphologicalvariationswithinthe
Chinatigers(P.t.amoyensis)nowexistincaptivityonly(Tilson subspecies and, to some extent, overlapping among the
et al. 2004). An estimated 400–500 Sumatran tigers (P. t. subspecies. A previous molecular genetic assessment of 28
sumatrae) occur in Sumatra (Seidensticker et al. 1999); and tigershasindicatedalowlevelofgeneticvariation,revealing
1,200–1,800Indochinesetigers(P.t.corbetti)liveinCambodia, little evidence for subspecies distinctiveness (Wentzel et al.
China,Laos,Malaysia,eastMyanmar,Thailand,andVietnam 1999). Moreover, ecological analyses of tiger habitat (Kitch-
(Seidenstickeret al.1999) (Figure 1). ener and Dugmore 2000) indicate that there have been few
Subspecies of tigers are traditionally defined by body size, geographic barriers (e.g., mountain ranges and deserts) to
skull characters, pelage coloration, and striping patterns migration and gene flow that would have been sufficient for
(Mazak 1981; Herrington 1987). It is generally believed that subspecies isolation. One ecology-based conservation ap-
the largest tigers occur in the Russian Far East, and the proach emphasizes protection of about 160 continuous
smallest are found in the Sunda Islands. The shape of the habitat patches or tiger conservation units regardless of
occiput in the skull is characteristically narrow in the Javan subspeciesdesignation(Dinersteinetal.1997).Althoughthis
and Bali tigers and much broader in Caspian tigers (Mazak strategy may be desirable, optimal tiger conservation may
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TigerPhylogeography
also require additional interventions such as establishing
corridorsandbufferzonesand/orimplementingreintroduc-
tionprograms(Tilsonetal.2001).Tothisend,anassessment
ofpopulationgeneticstructureoflivingtigersinterpretedin
the context of traditional intraspecific taxonomy and the
species’evolutionaryhistorywouldbenefitbothinsituandex
situconservation managementdesign.
Molecular genetic markers have been increasingly applied
to assess genetic partitions among geographically isolated
populations,todefinetheevolutionarysignificantunitbelow
thespecieslevelforconservationmanagementpurposes,and
to revise the traditional species and subspecies designations
(Avise and Ball 1990; Moritz 1994; Fraser and Bernatchez
2001). Subspecies recognition is particularly relevant for
tigers, because the current conservation strategy for this
species has been inextricably bound to knowledge of its
subspecific taxonomy. In this study we adhere to the
subspecies concept as defined by Avise and Ball (1990) and
O’Brien and Mayr (1991), to include populations below the
species level that share a distinct geographic distribution, a
group of phylogenetically concordant characters, and a
unique natural history relative to other subdivisions of the
species.
Here we attempt to overcome several factors that have Figure2.SchematicofP.tigrismtDNA
complicated previous efforts to fully describe patterns of ThepositionofPCRprimersusedforamplificationofCymtspecific
genetic variation in tigers. Foremost among these has been sequencesandalignmentofthehomologousNumtsequence(outer,
the limited sample size of ‘‘voucher specimens’’ (defined as dashedline)intigermitochondria.FifteenCymt-specificprimersets
spanning 6,026 bp of mtDNA were designed and screened for
individuals that were verified as wild-born from a specific
polymorphism in tigers (inner, solid line). Five indicated segments
geographic locale or captive-born from geographically showed no variation among fifteen tigers that represented five
verified wild-born parents). In addition, the presence of traditional subspecies and therefore were excluded from further
analysis.Thetenvariablesegments(4,078bp)wereamplifiedin100
Numt, a nuclear pseudogene insertion of cytoplasmic
tiger individuals. Primer sequences are listed in Table 1. Diamonds
mitochondrial DNA (mtDNA) in tiger autosomes (Lopez et indicate polymorphic mtDNA segments; brackets indicate mono-
al.1994;Johnsonetal.1996;Cracraftetal.1998;J.H.Kim,A. morphic mtDNA segments among tigers that were excluded from
phylogeneticanalysis.
Antunes, S.-J. Luo, J. Menninger, W. G. Nash, et al., personal
DOI:10.1371/journal.pbio.0020442.g002
communication) has made it difficult to utilize universal
mammalian primer sets for mitochondrial genes, because
(pelt or hair) by amplifying shorter fragments (less than 400
they will coamplify Numt. Furthermore, paucity of genetic
bp) targeting selected variable sites to determine their
diversity across tigers, especially in mtDNA (Wentzel et al.
similarity to the previously characterized haplotypes. Com-
1999), has made it necessary to sequence a large portion of
binedmtDNAsequenceswereobtainedfrom100tigersfrom
the mtDNA genome and to assess genetic variation in
Russian Far East (n = 13), south China (n = 4), northern
multiplerapidly evolvingmicrosatellite loci.
Indochina(n=30), Malayan Peninsula (n=22),Sumatra (n
To establish proper biological reference specimens, sam-
= 16), and the Indian subcontinent (n = 15). The mtDNA
ples from 134 tigers of known geographic origin were
sequences specified 54 variable sites defining 25 haplotypes
collected. Three genetic markers were examined: (1) 4 kb of
(Table 2). Thirty of the polymorphisms were observed in
mtDNA sequence derived from primer pairs that excluded
more than one individual and were thus phylogenetically
Numt amplification, (2) allele variation in the major
informative (Table 2), and 29 of the 30 changes were
histocompatibility complex (MHC) DRB gene; and (3) allele
transitions.
sizevariationof30hypervariableshorttandemrepeatlocior
Phylogenetic analyses of the mtDNA haplotypes using
microsatellites. Observed patterns of population genetic
maximum parsimony (MP), minimum evolution (ME), and
variation replicated with different gene families form the
maximum likelihood (ML) approaches produced congruent
basisofinterpretationofthetiger’sevolutionaryhistoryand
topologies that defined major geographic partitions (Figure
recommendations forits management.
3A). Eight haplotypes (SUM1 to SUM8) generated from 16
Sumatrantigers(P.t.sumatrae)formedamonophyleticgroup
Results
(80% MP, 70% ME, and 66% ML bootstrap support). A
Phylogenetic Analysis of mtDNA and Microsatellites secondmonophyleticclusterofsixhaplotypes(TIG1toTIG6)
Mitochondrial gene fragments were amplified and se- from15Bengaltigers(P.t.tigris)alsoreceivedhighbootstrap
quenced from DNA extracted from 72 blood or tissue support (93% MP, 82% ME, and 90% ML). The rest of the
specimensusing10cytoplasmicmitochondria(Cymt)-specific mainlandAsianhaplotypesgroupedtogetherandpartitioned
primer pairs (Figure 2 and Table 1). The fragments were into three distinct geographic groups: (1) a genetically
concatenated in a 4,078-bp contiguous sequence. Additional invariant Amur tiger lineage (P. t. altaica) represented by a
mtDNAsequencesweregeneratedfrom28historicalsamples singlehaplotypein13individuals,(2)anorthernIndochinese
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Table1. PCRPrimers Specific forCytoplasmic Mitochondrial DNASequences
Primer ID Mitochondrial Forwarda Reversea Size(bp)
Segments
C53F1/T598R ND5 CCCAGATCCCTATATTAACCAGT TATATCATTTTGTGTGAGGGCAC 546
C708F/T1300R ND5 CCTTGTCTTCCTGCATATCTG CCATTGGAAAGTACCCGAGGAGGT 593
C1494F/T1936R ND6 TCTCCTTCATAATCACCCTGA TGGCTGGTGGTGTTGGTTGCGG 443
C2339F/T2893R CytB TTGCCGCGACGTAAACCACG GTTGGCGGGGATGTAGTTATC 555
CR-UPF/CR-R2B CR TCAAAGCTTACACCAGTCTTGTAAACC CGTGTTGTGTGTTCTGTAT 250
C4979F/T5424Rb 12S GCACTGAAAATGCCTAGATGAGT CCAGTTTGGGTCTTAGCTATCG 446
C-12S-F/N/C-12S-R 12S AAAGCCACAGTTAACGTAA TACGACTTGTCTCCTCTTGTGG 577
T7812F/C8294Rb ND1 CGTCGTAGGACCATACGGCC CTCAGTCTCCTTCTGTTAAAT 483
C8276F/T8620R ND1 CGAAGCGAGCTCCATTTGATTTA GTGGAATGCTTGCTGTAATGATGGG 345
T8942F/C9384R ND2 CTTATAGTCTGAATCGGCTTCG AGCTATGATTTTTCGTACCT 443
C9366F/T9882R ND2 GGGGAGTTAACCAAACCGAG CAAGGACGGATAGTATTGGTG 517
C10525F/T11013Rb COI GGAGGATTCGGAAACTGGCGA CCAGAAGTCTATATCTTAATCCCG 489
C11020F/T11428R COI CCAGAAGTCTATATCTTAATCCCG GCTCCTATTGACAAGACGTAGTGGA 409
T11988F/C12414b COII GGCATACCCCTTCCAACTAGGT TGCACACTTCTATTGCTAGT 427
C12618F/T12920Rb ATP8 TTGTCCATGAACTAGTCCCATCAT GGAAACAGCTATGACCGGCG 303
aPrimersarelistedinthe59-to-39direction
bPCRproductsamplifiedusingtheseprimersetsshownovariationamongallsamples
DOI:10.1371/journal.pbio.0020442.t001
lineage (P. t. corbetti I) of individuals originating from south 3B). Haplotypes from the same geographic group tended to
China to the Indochinese countries north of the Isthmus of be interrelated, and intergroup distances among haplotypes
Kra,and(3)aparaphyleticassemblyofhaplotypesfromtigers weregenerallylargerthanbranchlengthswithineachgroup
from Malayan Peninsula (P. t. corbetti II). Support for (1–4 bp). The exceptions were two lineages within the
subdividingtheconventionalIndochinesesubspeciesoftigers Malayan P. t. corbetti II cluster that were separated by 7 bp,
P.t.corbettiintotwoclusterswashigh(bootstrapvaluesforP.t. whichmaybearesultoftheexistenceoffurtherpopulation
corbetti I were 94% MP, 96% ME, and 94% ML). The COR1/ substructure or, alternatively, of limited sampling in the
AMO3 haplotype, represented by 22 individuals from region.Eachofthesixtigersubspeciesgroupswasconnected
Vietnam (n = 2), Cambodia (n = 14), northeast Thailand (n to other groups in close but not exact correspondence to
=5),andsouthChina(n=1),wastheonlyhaplotypefound their geographic location. For instance, P. t. altaica was the
in two classical subspecies lineages (P. t. amoyensis and P. t. sistertaxontoP.t.corbettiIwhichwasconnectedtoP.t.corbetti
corbetti) (Table2). II. P. t. sumatrae haplotypes were linked to P. t. tigris by 7 bp
Voucher samples of five captive tigers collected in China, andtoP.t.amoyensisby10bp.Nonetheless,thephylogenetic
designatedSouthChinasubspeciesP.t.amoyensis,fellintotwo relationships among the subspecies were not resolved to a
very distinct phylogenetic origins. Two tigers from the robust hierarchy, and therefore were consistent with a
Suzhou Zoo (Pti-217 and Pti-218; Table 3) carried the contemporaneous divergence of extant phylogeographic
COR1/AMO3 haplotype, and the third (Pti-222) contained lineages.
haplotype AMO2, which differed by a single nucleotide Composite genotypes from 30 felid-specific microsatellite
substitution from COR1/AMO3 (Table 2). The two South loci(Menotti-Raymondetal.1999)wereobtainedin113tiger
China tiger haplotypes grouped phylogenetically with the samples. Neighbor joining (NJ) analyses of individual tiger
northernIndochineseP.t.corbettiIhaplotypes(COR1–COR3) genotypesbasedontheproportionofsharedallele(Dps)and
in all phylogenetic analyses (Figure 3A and 3B), and likely kinship coefficient (Dkf) genetic distances produced con-
indicate that the maternal (mitochondrial) lineages of these cordanttopologies(Figures4andS1)thatlendsupporttothe
tigers derived from individuals from the P. t. corbetti I same phylogeographic population subdivisions observed in
phylogenetic lineages. In contrast, two P. t. amoyensis tigers the mtDNA analysis. Tigers from Sumatra (P. t. sumatrae)
(Pti-219andPti-220)fromtheChongqingZoocollectionhad formed a monophyletic clade with 97% bootstrap support,
ahaplotype (AMO1) that formed aseparate lineage that was and Amur tigers (P. t. altaica) grouped with 76% bootstrap
ten nucleotide substitutions from its nearest sequence support.Theremainingtigergenotypespartitionedintotwo
(Sumatran; Figure 3B and Table 2). If affirmed by larger weakly supported monophyletic lineages (Indian Subconti-
sampling, this lineage would reflect a unique P. t. amoyensis nent P. t. tigris and Malayan Peninsula P. t. corbetti II) and a
genetichaplotype. paraphyleticassemblageofnorthernIndochineseP.t.corbetti
A statistical parsimony network of the tiger mtDNA I.Forexample,threeindividualsfromThailand(Pti-296,Pti-
sequences provided additional analytical support for the 297, and Pti-301) clustered with samples from the India
differentiation of P. t. sumatrae, P. t. tigris, P. t. altaica, P. t. subcontinent, blurring the distinction between P. t. corbetti I
corbettiI,P.t.corbettiII,andP.t.amoyensis(AMO1only)(Figure andP.t.tigris.ThethreeSouthChinatigersfromtheSuzhou
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TigerPhylogeography
FR 7304 G – –––– ––––– –––––AAA ––––––
08
22
O1 110114 7287 C T TTTT TTTTT TTTTTTTT TTTTTT
C CT
5737 C – –––– ––––– –––––––– T––––T
5728 G A –––– ––––– –––––––– ––––––
5674 T C –––– ––––– –––––––– ––––––
5608 C – –––– ––––– TTTTTTTT ––––––
5533 G – –––– ––––– –––––––– AAAAAA
FR 5518 G A –––– ––––– –––––––– ––––––
6682 5515 A – –––– ––GGG –––––––– ––––––
9398 5349 T C –––– ––––– –––––––– ––––––
CT 5332 C T TTTT TTTTT TTTTTTTT TTTTTT
5186 A – ––G– ––––– –––––––– ––––––
2F4R 5155 T C –––– ––––– CCCCCCCC CCCCCC
D2 894938 5050 C – –––– ––––– –––––––– TTTTTT
N TC 4963 G – –––– ––––– –––––––– A–––––
4442 T – –––– ––––– CCCCCCCC –CCCCC
6F0R 4406 A – –––– ––––– ––––––GG ––––––
D1 827862 4357 A G –––– ––––– –––––––– ––––––
N CT T – –––C ––––– –––––––– ––––––
1712 A – –––– GG––– –––––––– ––––––
R
FS- 1671 T C –––– CCCCC CCCCCCCC CCC–CC
S 12S-C-12 1577 A – –––– ––––– G––G–––– ––––––
s 12 C-N/ 1420 T – –––– ––––– C––––––– ––––––
e
c
quen UPFR2B 1166331999 TC –– –––––––– –T–TC–C–C– –––––––––––––––– ––––––––––––
Se R R-R-
A C CC 16315 C – TTTT ––––– –––––––– ––––––
N 15756 C T –––– TTTTT TTTTTTTT TTTTTT
mtD 1155679413 CA –– –––––––– T–T––––––– –G–G–G–G–G–G–G–G –––––––––––– 3).
gris) 1155569052 GG –– A–A–A–A– –A–––––––– –––––––––––––––– –––––––––––– able
Table2.HaplotypesandVariableSitesinCombinedAnalysisof4,078bpofTiger(P.ti GeneND5ND6CytB PrimersC53F1C708FC1494FC2339FT598RT1300RT1936RT2893R a11111111111111111111111Position333333333334444444555550113347999900156773334523717420788481911857908bcN98387728136688181069038SubspeciesHaplotype dACCCCCCTCACTTTGTP.t.altaicaALT13GCACCGT P.t.amoyensisAMO12A–G–––C––T–––TC–G–––––– P.t.corbettiIAMO21––––––C–––––––––G––––A–e22–T––––C–––––––––G––––A–COR1/AMO3COR22–T––––C–––––––––G––––A–COR37–T––––C–––––––––G––––A– P.t.corbettiIICOR411––G–––C–––––––––G––––––(P.t.jacksoni)COR51––G–––C–––––––––G––––––COR62––GT––CT––––––––G––––––COR75––GT––CT––––––––GT–––––COR83––G–––CT––––––––GT––C–– P.t.sumatraeSUM14A–G–––C––––––T––G––––––SUM24A–G–––C––––––T––G––––––SUM31A–G–––C––––––T––G–––––CSUM41––G–––C––––––T––G––––––SUM51A–G–––C––––––T––G––C–––SUM61A–G–––C––––––T––G–C––––SUM73A–G–––C––––––T––G–C––––SUM81A–G–––C–T––TTT––G–C–––– P.t.tigrisTIG11––G–T–C–––T––––TG––C–––TIG21––G––AC––––––––TG––C–––TIG31––G–––C––––––––TG––––––TIG41––G–––C––––––––TG––C–––TIG56––G–––C––––––––TG––C–––TIG65––G–––C––––––––TG––C––– aNucleotidepositionscorrespondtothecompletereferenceFeliscatusmtDNAsequence(Lopezetal.1996).bSubspeciesabbreviationcodeasinFigure1.BasepairsidenticaltohaplotypeALTareindicatedbyadash.cNumberofindividualswitheachhaplotype.IndividualtigermtDNAhaplotypesarelistedinTable3.dRednucleotidesaresubspecies-specificsites.eCOR1/AMO3isahaplotypesharedby21tigersthatareinitiallydesignatedasCORandoneAMO(textandTDOI:10.1371/journal.pbio.0020442.t002
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Figure3.PhylogeneticRelationshipsamongTigersfrommtDNAHaplotypes
(A)PhylogeneticrelationshipsbasedonMPamongthetigermtDNAhaplotypesfromthecombined4,078bpmitochondrialsequence(Table2).
Branchesofthesamecolorrepresenthaplotypesofthesamesubspecies.TreesderivedfromMEandMLanalyseshaveidenticaltopologies.
Numbersabovebranchesrepresentbootstrapsupportfrom100replicatesusingtheMPmethod,followedbybootstrapvaluesusingtheME-ML
analyses(onlythose over 70% are indicated). Numbersbelow branches show numberof MP steps pernumberof homoplasiesfrom a strict
consensustree.Numbersinparenthesesrepresentnumbersofindividualssharingthesamehaplotype.MPanalysisusingheuristicsearchand
tree-bisection-reconnectionbranch-swappingapproachresultsintwoequallymost-parsimonioustreesandtheoneresemblingtheMEandML
trees is shown here (tree length = 60 steps; CI = 0.900). The ME tree is constructed with PAUP using Kimura two-parameter distances
(transitiontotransversionratio=2)andNJalgorithmfollowedbybranch-swappingprocedure(ME=0.0142).TheMLapproachisperformed
usingaTrN(Tamura-Nei)þI(withproportionofinvariablesites)model,andallnodesoftheMLtreeweresignificant(aconsensusof100trees,–
Lnlikelihood=5987.09).
(B)StatisticalparsimonynetworkoftigermtDNAhaplotypesbasedon4,078mtDNAsequencesconstructedusingtheTCSprogram(Clementet
al.2000).Theareaofthecircleisapproximatelyproportionaltothehaplotypefrequency,andthelengthofconnectinglinesisproportionalto
theexactnucleotidedifferencesbetweenhaplotypeswitheachunitrepresentingonenucleotidesubstitution.Missinghaplotypesinthenetwork
arerepresentedbydots.Haplotypecodesandthenumberofindividuals(inparentheses)witheachhaplotypeareshown(seeTable2).
DOI:10.1371/journal.pbio.0020442.g003
Zoo, China, that had clustered with P. t. corbetti I by mtDNA very high (0.838), indicating that 84% of the variation was
(Pti-217, Pti-218, and Pti-222) also associated more closely partitionedamongthedifferentphylogeographicsubspecies.
withP.t.corbettiIfromnorthernIndochinabymicrosatellite Each of the five subspecies showed highly significant
analysis(Figure4).Thetwodistinct(bymtDNA)P.t.amoyensis population genetic differentiation (p , 0.0001) by pairwise
individuals (Pti-219 and Pti-220) from the Chongqing Zoo, F and R with 10,000 permutations (Table 5). The contrast
st st
China,likewiseformedadistinctlineageinthemicrosatellite between the mtDNA and microsatellite genetic variation
analysis(Figure 4). probably reflects the difference in the effective population
size assessed by these two different markers and/or, to some
Population Subdivision Analysis
extent, theintersexual differences indispersal.
To quantify the extent of population differentiation in
Analternativeanalysisofthecombinedmicrosatelliteand
modern tigers, we evaluated four different geographic
mitochondrial haplotype data using a Bayesian approach
subdivision scenarios and compared them on the basis of
(Figure S2 and Table S1) as implemented in the program
analysis of molecular variance (AMOVA) with both mtDNA STRUCTURE(Pritchardetal.2000)supportedthepartition-
haplotypes and microsatellite genotypes (Table 4). P. t. ingofP.t.altaica,P.t.sumatrae,P.t.tigris,andP.t.corbettiII,but
amoyensis individuals (Pti-219 and Pti-220) were excluded in further split the 33 P. t. corbetti I individuals into three
thissubdivision analysisdue toour small samplesize.In our
distinctivepopulationgroups:(1)fourtigersfromChinaand
firsthypothesis,twogroupswereconsidered:theP.t.sumatrae Vietnam; (2) nine tigers from Cambodia; and (3) 20 tigers
island population and all contemporary mainland popula- from Cambodia and northern Thailand (K = 7, Pr[K] =
tions(P.t.altaica, P.t.corbettiI,P.t.corbettiII,P.t.tigris).This 0.993). In this scenario, most individuals were assigned to a
recently proposed model (Cracraft et al. 1998; Kitchener cluster with high probability (q . 0.90), indicating very low
1999; Kitchener and Dugmore 2000) presumes continuous
levelofgeneflowbetweenthegroups.However,becausethis
habitat distribution on the mainland. The second scenario
additional substructure within P. t. corbetti I had little
considered tigers as three groups: the Sumatran population
geographicorecologicalbasis,andbecauseAMOVAanalysis
(P. t. sumatrae), the Amur tigers (P. t. altaica), which presently basedonthispopulationsubdivisionresultedinlowerF and
st
areisolatedfromothertigerpopulationsbymorethantheir
R values than that in the five-group scenario (unpublished
st
maximum known dispersal distance (Mazak 1996), and a
data), the distinction was not considered to be a consistent
group of the other mainland tigers subspecies. The third basis for subspecies classification and may reflect additional
hypothesis followed the division of the four traditional
populationdifferentiation withinasubspecies.
subspecies: (1) Amur tigers (P. t. altaica), (2) P. t. corbetti,
including Indochina and part of south China, (3) Bengal Genetic Variation in Tigers
tigers(P.t. tigris), and(4) Sumatrantigers(P.t. sumatrae). The Quantitative estimates of mtDNA diversity in tigers with
fourth scenario, based on the results of the mtDNA comparableestimatesfromselectedfelidspeciesdemonstra-
phylogenetic analyses (see Figure 3) and the hypothesis that ted that overall, tigers had moderate levels of mtDNA
the Isthmus of Kra may serve as a potential geographic diversity(Table6),substantiallylessthanleopards (P.pardus)
barrier (Kitchener 1999), further subdivided classical P. t. (Uphyrkina et al. 2001), Geoffroy’s cat (Oncifelis geoffroyi),
corbetti into the northern Indochina region P. t. corbetti I and Pampascat(O.colocolo),ortigrina(Leopardustigrinus)(Johnson
theMalayanPeninsulaP.t.corbettiII,resultinginfivegroups. etal.1999),butcomparabletopumas(Pumaconcolor)(Culver
The AMOVA results for each of the four scenarios are et al. 2000) in percent variable sites, mean pairwise distance
presented inTable 4. among individuals, and average nucleotide diversity. Four
Forbothmitochondrialhaplotypeandmicrosatellitedata, tigersubspecies(P.t.tigris,P.t.sumatrae,P.t.corbettiI,andP.t.
the five-group scenario yielded the highest F (for mtDNA, corbetti II) showed moderate nucleotide diversity (p), ranging
st
defined as the proportion of total genetic variation that is from0.0001to0.0070(Table6).TheP.t.altaiasamplingof13
attributabletogeneticdifferencesbetweenpopulations)and individuals showed no mtDNA haplotype variation. Of the
R (formicrosatellites, anF analogy suitedforthestepwise five individuals originally designated as P. t. amoyensis, three
st st
mutation model that applies to microsatellite data) values. weregeneticallyindistinguishablefromP.t.corbettiI,resulting
Under this model, 31% of the microsatellite variation in an inadequate sample size for a meaningful estimation of
discriminated between the five groups, while the balance, populationvariation.
69%, occurred within each group. For mtDNA the F was Parameters ofmicrosatellite variationhavebeenshownto
st
PLoSBiology|www.plosbiology.org 2281 December2004|Volume2|Issue12|e442
TigerPhylogeography
2)
2 R
., 2 O
bPtiCode(mtDNAcHaplotype)d,.MHC-DRB f,.111(ALT),112(ALT),113(ALT)AA,,.,114AA,115,117,118,120(ALT),121AA,.122,123AA,,.124,125AA,126,127,128(ALT),130(ALT),131(ALT),133(ALT),134,135,137(ALT),138,142,143,145,146,147148 151(ALT),152 153(ALT),154 156(ALT),158,161157,15965(ALT) 219(AMO1),220(AMO1) ,.,.217(AMO2)AA,218(AMO3/COR1)AA,2 315(COR2),316(COR3) 249(AMO3/COR1),250(AMO3/COR1) 290TH(AMO3/COR1),291(AMO3/COR1),292(C 296(AMO3/COR1) 297(AMO3/COR1)301(AMO3/COR1) 305(AMO3/COR1) 306(AMO3/COR1)307(COR3)
a
s
hu
tt
ra
BiSt W C W W CWU C C W W W W UU U WW
Source;Contact HornockerWildlifeResearchInstitute,USA;H.QuigleyandD.Miquelle MinnesotaZoo,USA;F.WrightandA.ShimaMoscowZoo,Russia;V.SpitsinTallinZoo,Estonia;V.FainsteinKievZoo,Ukraine;L.Korotkaya PhiladelphiaZoo,USA;K.Hinshaw ChongqingZoo,China;W.PanandS.Huang SuzhouZoo,China;W.PanandS.HuangKunmingInstituteofZoology,China;Y.ZhangHanoiZoologicalGardens,Vietnam;D.G.TungKhaoKheoOpenZoo,Thailand;W.TunwattanaChiangmaiZoo,Thailand;W.Tunwattana SongkalaZoo,Thailand;W.TunwattanaNhimVauda,Kamput,Cambodia;R.Marx PhnomTamao,Cambodia;R.Marx
e
c
r
u g
o 8 1 2 2 5 1 2 3 2 2 3 2 1 2 1
S 2
s
al
r u
eStudy NumbeofIndivid(Total) (39) (2) (33)
h
t
n
sedi gin st china
ntheratigrisU LocaleofOri RussianFarEa SouthChina NorthernIndo
a
P
Table3.Samplesof Subspecies(Common)Name P.t.altaica(Amurtiger) P.t.amoyensis(SouthChinatiger) P.t.corbettiI(Indochinesetiger)
PLoSBiology|www.plosbiology.org 2282 December2004|Volume2|Issue12|e442