Genomic Ancestry of the American Puma - Journal of Heredity

12 Pages·2000·2.28 MB·English

Genomic Ancestry of the American Puma
(Puma concolor)
M. Culver, W. E. Johnson, J. Pecon-Slattery, and S. J. O’Brien
Puma concolor, a large American cat species, occupies the most extensiverange
of any New World terrestrial mammal, spanning 110 degrees of latitude from the
Canadian Yukon to the Straits of Magellan. Until the recent Holocene, pumas co-
existed with a diverse array of carnivores including the American lion (Panthera
atrox), the North American cheetah (Miracynonyx trumani), and the saber toothed
tiger(Smilodonfatalis).GenomicDNAspecimensfrom315pumasofspecifiedgeo-
graphic origin (261 contemporary and 54 museum specimens) were collected for
moleculargeneticandphylogeneticanalysesofthreemitochondrialgenesequenc-
es(16SrRNA,ATPase-8,andNADH-5)pluscompositemicrosatellitegenotypes(10
feline loci). Six phylogeographic groupings or subspecies were resolved, and the
entire North American population (186 individuals from 15 previously namedsub-
species) was genetically homogeneous in overall variation relative to central and
South American populations. The marked uniformity of mtDNA and a reductionin
microsatellite allele size expansion indicates that North American pumas derive
from a recent (late Pleistocene circa 10,000 years ago) replacement and recoloni-
zation by a small number of founders who themselves originated from a centrum
of puma genetic diversity in eastern South America 200,000–300,000 years ago.
The recolonization of North American pumas was coincident with a massive late
Pleistoceneextinctioneventthateliminated80%oflargevertebratesinNorthAmer-
ica and may have extirpatedpumasfrom thatcontinentas well.
Pumas(alsocalledmountainlions,orcou- shall 1981; Webb and Rancy 1996). Al-
gars) occupy a vast range of ecological thoughtheeventwouldsuggestthatmod-
zones(Figure1A)asdiverseasdesert,sa- ern pumas originated from a North
vannah, tropical rain forest, and alpine Americanancestry,thepumafossilrecord
steppes (Anderson 1983; Hansen 1992). in South America is so poor that a more
Adult size varies from 50 to 70 kg at the recent neotropical origin and northward
equator to twice that size in the extreme dispersal cannot be ruled out (Kurten
reaches of the Canadian Yukon and Pata- 1976;Werdelin1989).
gonianpampas.Thepumafossilrecordis ThepeoplingofNorthandSouthAmer-
lessthanhalfamillionyearsold,butboth ica clearly diminished puma populations
From the Laboratory of Genomic Diversity, National molecular and morphologic studies sug- inrecentcenturies,reducingtherangeby
Cancer Institute, Frederick Cancer Research and De-
velopment Center, Frederick, MD 21702-1201. We are gest that the puma’s origin dates to the two-thirds in North America as a conse-
gratefultoacknowledgefullcollaborativecredittothe lateMiocene[5–8millionyearsbeforethe quence of habitat destruction and human
investigatorslistedinFigure1Awhosuppliedbiologi-
present (MYBP)], when pumas evolved depredation (Figure 1A). Except for the
cal specimens upon which this study is based. We
wouldliketothankallthecollaboratorsthatcollected fromacommonancestorwiththeAfrican small endangered population of Florida
oraidedinthecollectionofsamplesforthisstudy.We cheetah (Acinonyx jubatus) and American panther(Pumaconcolorcoryi)livingincy-
alsoappreciatetheexperttechnicalassistanceandin-
jaguarundi(Herpailurusyaguaroundi)(Jan- pressswampsofsouthFlorida,pumasare
sightful advice of Marilyn Raymond, Melody Roelke-
Parker,VictorDavid,ClayStephens,JaniceMartenson, czewski et al. 1995; Johnson and O’Brien extinct east of the Mississippi (Hansen
StanCevario,CarlosDriscoll,AlRoca,andEllenFrazier. 1997;Pecon-SlatteryandO’Brien1998;Van 1992; Maehr 1998; Roelke et al. 1993).
The content of this publication does notnecessarily
reflect the views or policies of the Department of Valkenburghetal.1990).Pumasmostcer- Nonetheless, elimination of bounties and
HealthandHumanServices,nordoesmentionoftrade tainlyarrivedinSouthAmerica2–4MYBP recent legislativeprotectionhasledtoan
names, commercial products, or organizationsimply
during the Great American Interchange, increase in puma numbers in many areas
endorsementbytheU.S.government.Addresscorre-
spondencetoStephenJ.O’Brienattheaddressabove. when eutherian carnivores first migrated since 1900. Some 32 separate geographic
Thispaperwasdeliveredatasymposiumentitled‘‘Ge- south from North America with the geo- subspecies of puma have been described
neticDiversityandEvolution’’sponsoredbytheAmer-
logic joining of the Panamanian land based on geographic and morphometric
icanGeneticAssociationatthePennsylvaniaStateUni-
versity,UniversityPark,PA,USA,June12–13,1999. bridge (Marshall et al. 1982; Stehli and criteria(Figure1A)(Neff1983;Youngand
(cid:1)2000TheAmericanGeneticAssociation91:186–197 Webb 1985; Webb 1978; Webb and Mar- Goldman 1946). The eastern cougar (P. c.
186
couguar) and the Wisconsin puma (P. c. cheetah, jaguarundi, Geoffroy’s cat (Onci- ed in GenBank (accession numbers
schorgeri)arepresumedextinct,andthree felis geoffroyi), and domestic cat(Felisca- AF241812–AF241820).
subspecies (P. c. coryi, P. c. costaricensis, tus)wereincludedasoutgroupspecies.
and P. c. brownii) are classified as endan- DNA was extracted from white blood PhylogeneticAnalyses of mtDNA
gered or threatened by the U.S. Fish and cells, wholeblood,primaryfibroblastcul- Haplotypes
WildlifeService(Hansen1992). turesfromskinbiopsies,ortissuesfollow- PhylogeneticanalysesofmtDNAvariation
Here we examine the extent of genetic ing a phenol-chloroform protocol (Sam- were conducted for samples which suc-
diversity using three groups of genetic brook et al. 1989). DNA from hide, hair, cessfully amplified for all three gene seg-
markers (mtDNA coding genes, Y-chro- and bone samples was extracted using a ments(n(cid:1)286).Sequencesforeachgene
mosome Zfy intron sequence, and 10 nu- silicabased,guanidiumextractionmethod were combined for a total evidence ap-
clearfelinemicrosatelliteloci)among315 (Ho¨ss and Pa¨a¨bo 1993; Johnson et al. proach(Huelsenbecketal.1996)covering
individual pumas: 261 contemporaryani- 1998;Pa¨a¨boetal.1988). 891 bp. The combined data were edited
mals and 54 museum specimens, eachof and aligned using the program SE-
known geographic origin representing31 QUENCHER (version 3.0, Gene Codes
mtDNAMarkers
of 32 named subspecies. The pattern of Corp.,AnnArbor,MI)andverifiedvisually.
Products for both single-strand confor-
variation within and among subspecies Geneticdistanceswereestimatedbothby
mation polymorphism (SSCP) and se-
was employed to verify phylogeographic absolute number of base pair differences
quencing of mtDNA regions were ob-
subdivision and to interpret the natural amonghaplotypes(pdistance)andbythe
tained by PCR amplification of genomic
history of the species. The cumulative Tajima-Nei model of substitution (Tajima
DNA(Saikietal.1985)usingprimersthat
data support the recognition of six oper- and Nei 1984) as implementedin thepro-
amplify portions of the 16S rRNA (16S),
ational taxonomic units (OTUs) or sub- gram PAUP* (used with permission of D.
NADH-5(ND5),andATPase-8(ATP8)genes
species based on their sharing of a Swofford). Phylogenetic relationships
(Johnsonetal.1998;JohnsonandO’Brien
‘‘unique geographic range or habitat, a among thehaplotypeswereestimatedus-
1997).PCRamplificationofmuseumsam-
group of phylogenetically concordant’’ ing three major methods—minimum evo-
ples was performed with two pairs of
phenotypiccharactersand‘‘auniquenat- lutionestimatedbyneighborjoining(NJ),
hemi-nested primers for 16S and ND5
ural history’’ (Avise and Ball 1990; maximumparsimony(MP),andmaximum
(Johnson et al. 1998) except16S-3F-5(cid:2)GA-
O’Brien andMayr1991).Molecularmark- likelihood (ML)—using the program
GACCCATTAATTTCAACCG-3(cid:2) is substitut-
ers show that individualsfromtheNorth PAUP*. Concordance among the resultant
ed for 16S-1NF. PCR reactions were per-
Americancontinentcomprisealargepan- topologies was considered to be strong
formedusing20ngofgenomicDNAinthe
mictic population and display reduced supportfortheobservedphylogeny.
presence of 10 mM Tris-HCl (pH 8.3), 50
genetic variation relative to South Amer- NJtreesweregeneratedwiththeTajima-
ican pumas, an indication of an historic mM KCl, 1.5 mM MgCl2, 200 (cid:3)M each of Nei distance estimatesanda generalheu-
founder effect in the North American dATP,dCTP,dGTP,dTTP,0.16mg/mlBSA, risticsearchwithtreebisection-reconnec-
pumaancestry.Fourofthesixrecognized 1(cid:3)Mofeachprimer,and1unitTaqpoly- tion branch swapping. The MP analysis
puma OTUs reflect geographic(potential merase enzyme in a volume of 10 (cid:3)l (2.5 was conducted by a general heuristic
faunal) boundaries, while the other two units in 25 (cid:3)l for museum specimens). search using simple sequence addition of
are likely hybrid (or intergrade) zones Thermocycling conditions consisted of sequences and tree bisection-reconnec-
fromgeographicallyadjacentsubspecies. 0.5 min denaturation at 94(cid:4)C, 1.5 min an- tionbranchswapping.MLtreesweregen-
Consistency of the genetic/phylogenetic nealing at 49(cid:4)C, and 1 min extension at erated using the HKY or F84 model (Has-
results with each category of genetic 72(cid:4)Cfor30cycles(35or40cyclesformu- egawa et al. 1985). The starting tree was
markers offers confidence in subspecies seum samples). Resulting products were obtained from the NJ analysis, the shape
classification and has implications for visualized on a 2% agarose gel in TBE parameter((cid:6))forthegammadistribution
naturalhistoryandpresentconservation buffer. ofrateheterogeneityamongsitesandthe
managementof the puma. SSCP analyses were performed on transition:transversion (Ts:Tv) ratio were
mtDNA amplification products from con- setatdefaultvalues,andbasefrequencies
temporary samples (Orita et al. 1989a;b; wereempiricallyderived.Aniterativepro-
Materials and Methods
Poduslo et al. 1991). PCR was performed cesswherebyeachsuccessiveMLtreein-
Samples asdescribedabovewiththeadditionof1 corporated parameters estimated from
Biological samples were collected from (cid:3)Ci of dCTP[(cid:5)-32P]. The PCR product was thepreceedingMLanalysiscontinuedun-
315 pumas and were obtained from the denaturedat95(cid:4)Candelectrophoresedon til an optimal tree was consistently de-
wild(n(cid:1)148),captivefacilities(n(cid:1)113), a 4.5% nondenaturing polyacrylamide gel rived. RelationshipsamongmtDNAhaplo-
and museums (n (cid:1) 54) (Figure 1A). Sam- with10% glycerol at60Wfor6h.Several types were estimated using a minimum
ples included 1–35 animals from each of individuals with identical banding pat- spanningnetwork,computedfromtheab-
32 subspecies. Captive animals were wild terns were sequenced to confirm that solutenumberofdifferencesbetweenhap-
born and of known geographic origin. each had the same allele. PCR products lotypesusingtheprogramMINSPNET(Ex-
Sampleswereassignedtothenearestsub- frommuseumspecimensweredirectlyse- coffieretal.1992).
speciesbasedongeographiclocation(Ca- quenced. The mtDNA PCR products were
brera1963;Jackson1955;Neff1983;Young sequencedinbothforwardandreversedi- ZfyIntron Sequence
and Goldman 1946). Population samples rectionsusingaPrismDyePrimerkitand The terminal intron of Zfy, located on the
wereselectedtoexcludeknownrelatedin- were analyzed by an ABI 373A automated Y chromosome, was examined in 14 rep-
dividuals,orinthecaseofFlorida,known DNA sequencer (Applied Biosystems Inc., resentative male pumas (and one female
hybrids (O’Brien et al. 1990). Samples of Foster City, CA). Sequences weredeposit- for a negative control). PCR was per-
Culveretal•GenomicAncestryoftheAmericanPuma 187
Figure1. (A)Geographicrangesof32currentlyrecognizedsubspeciesofpuma(Pumaconcolor)(Cabrera1963;Jackson1955;Neff1983;YoungandGoldman1946).The
subspeciesarelabeledwithathree-lettercode,andthetotalnumberofsamplesexaminedhereareindicated.Collaboratorsandinstitutionsthatsuppliedpumaspecimens
are(listedalphabeticallybyinstitution):AlbertoParas,AfricamSafari,Mexico;AgustinIriarte,AgricultureServiceSAG,Chile;CharlesLand,AlaskaDepartmentofFishand
Game;ErnestoBoede,AquariumJ.V.Seijas,Venezuela;DonBuckley,Arizona;LisaHaynes,StanCunningham,MattPeirce,andHarleyShaw,ArizonaGameandFishDepartment;
TroyBest,andNedGentz,AuburnUniversity,Alabama;LorenzaCalvo,AuroraNationalZoo,Guatemala;NiniN.DeBerger,AutoSafariChapin,Guatemala;SheilaSchmelling,
TonyGarel,andLaurieWilkins,BelizeZoo;DarylHebertandDanLay,BritishColumbiaMinistryofEnvironment,Canada;KnutAtkinson,RickDavies,BritishColumbiaWildlife
Branch,Canada;JuanRomero,BuenosAiresZoologicalPark,Argentina;SueMainka,WandaDwelle,LoriRogers,andBobCooper,CalgaryZooandBotanicalGardens,Canada;
Dr.Hunter,DaveJessup,TonyAtkinson,JimBanks,andKennethLevine,CaliforniaDepartmentofFishandGame;StanvanZylldeJong,CanadianMuseumofNature,Quebec;
Lilly and Werner Hagnauer, Canas, Las Pumas, Costa Rica; Ramon Quevedo Giorgiana, Dulce BroussetandEnriqueYanto,CentrodeConvivenciaInfantil,Mexico;Allen
Anderson,ColoradoDivisionofWildlife;SoniaAlcazarandVivianaQuse,CordobaZoologicalPark,Argentina;RosanaNogueiradeMorias,CuritibaZoo,Brazil;RicardoAyala,
DuraznoZoologicalPark,Paraguay;MelodyRoelke,FloridaGameandFreshwaterFishCommission;LaurieWilkins,FloridaMuseumofNaturalHistory,Gainesville;Helida
Ferreira,GoianaZoo,Brazil;RossAnderson,HogleZoo,SaltLakeCity,Utah;KerryMurphy,LindaSweanor,andKenLogan,HornockerWildlifeResearchInstitute;JoeFlanagan,
Houston Zoo,Texas;JohnLaundre,IdahoStateUniversity;RichardGreenwell,InternationalSocietyofCryptozoology,Tucson,Arizona;EmersonSuemitsu,Wanderleide
Moraes,Itaipu,BrazilandParaguay;MiriamLazo,JuigalpaZoo,Nicaragua;MelodyRoelke,KamloopsGameFarm,Canada;OttoCarlosJordan,LaPazZoo,Bolivia;Dr.Marinelli,
LaPlataZoologicalPark,Argentina;SenorMeneses,LaPonderosa,Chile;WarrenJohnsonandMelanieCulver,LaboratoryofGenomicDiversity,NationalCancerInstitute,
Maryland;MelodyRoelke,LimaZoo,Peru;BeverlyFronk,LycomingCountyHistoricalSocietyMuseum,Pennsylvania;VictorianoCorazon,ManaguaZoo,ZoologicoNacional,
Nicaragua;JorgeBustelo,MendozaZoologicalPark,Argentina;JoseOlazarri,MercedesZoologicalPark,Uruguay;ErnieStrahm,Mexico;WarrenJohnson,MinaElSoldado,
Chile;MariaRutzmoser,MuseumofComparativeZoology,Cambridge,Massachusetts;JamesPatton,MuseumofVertebrateZoology,Berkeley,California;PeterDratch,National
FishandWildlifeForensicsLab,Oregon;AlfredGardner,NationalMuseumofNaturalHistory,Washington,DC;MikeCox,NevadaDivisionofWildlife;WaltVanDyke,Oregon
DepartmentofFishandWildlife;BobGagliuso,OregonStateUniversity;FernandoManvelVictoria,ParqueCentenario,Merida,Mexico;MelodyRoelkeandWarrenJohnson,
ParquedeFaunaCONAF,Chile;PeterCrawshaw,ParqueNacionalFozdeIguazu,CENAP,IBAMA;GambinoVasquez,ParqueZoologicodeLeon,Mexico;VicenteMongrell,
ParqueZoologicodeTamatan,Mexico;KathyQuigley,PetencitaZoologicalPark,Guatemala;Jean-ChristopheVie,ProgrammeFauneSauvage,FrenchGuyana;SandroJerez,
RawsonZoologicalPark,Argentina;MattPeirceandHarleyShaw,ReidParkZoo,Tucson,Arizona;DaveGonzalez,RimCountyVeterinaryClinic,Nevada;HectorSeuanez,Rio
deJaneiroZoo,Brazil;RoyMcBride,SanAntonioZoologicalGardensandAquarium;DavidF.Johnson,SanDiegoMesaCollege,AnimalHealth,California;PaulBeier,Santa
AnaMountainsCougarStudy,California;OttoCarlosJordan,SantaCruzZoo,Bolivia;PatricioYa˜nez,SantiagoMuseumofNaturalHistory,Chile;JeffreyWyattandFaical
Simon,SaoPauloZoo,Brazil;YolandaMatamoros,SimonBolivarZoo,CostaRica;SamuelOcana,SonoranEcologicalCenter,Mexico;AduatoNunes,SorocabaZoo,Brazil;
RandallE.Junge,St.LouisZoologicalPark,Missouri;AnabelHerrera,SummitZoo,Panama;JanEnglundandOlaviGronwald,SwedishMuseumofNaturalHistory,Stockholm,
Sweden;BillFranklin,TorresdelPaineNationalPark,Chile;MikeSyvanen,UniversityofCalifornia,Davis;FrankIwenandAdrianWydeven,UniversityofWisconsinZoological
Museum, Madison, Wisconsin; Ken Elowe, Utah Division of Wildlife Resources;EsmeraldaMujica,ValenciaAquarium,Venezuela;MelodyRoelke,VancouverGameFarm,
Canada;BriggsHall,DonMagoon,JimByse,andWillieNation,WashingtonDepartmentofFishandGame;ScottCitino,WhiteOakPlantation,Florida;MelodyRoelke,Laurie
Marker,andMikeBriggs,WildlifeSafari,Oregon;MelodyRoelke,YosemiteNationalPark,California;Dr.Riveros,ZoologicoNacionaldeChile;MiguelAlvarez,Zoomat,Mexico.
(B) DistributionofmtDNAhaplotypesacrossthepuma’sranges.LettersandpiechartsrefertomtDNAhaplotypesspecifiedbyfifteensinglenucleotidepolymorphisms
definedinpumas(Table1).ThegeographicrangesofsixrevisedsubspeciesofpumasasdefinedherebymtDNAandmicrosatelliteanalysisareindicated.Dottedlines
demarcateformersubspeciesrange(Neff1983;YoungandGoldman1946).Nx4andMx3,4,5etc.,indicatesthenumberofindividualsinthedefinedlocationthathadtheN
orMhaplotype,respectively.Squaresaroundthehaplotypeletterindicatesamuseumsample.
formedusingfirst-roundprimersZF2Fand bothforwardandreversedirectionsusing edited and aligned using the program SE-
ZF1R,andsecondround‘‘nested’’primers the Dye Terminator Prism sequencing kit QUENCHER (version 3.0,GeneCodesCor-
ZFY2F and ZFY1R (Pecon-Slattery and and analyzed by an ABI 373A automated poration, Ann Arbor, MI). The sequence
O’Brien 1998). PCR products of 350 bp DNA sequencer [Applied Biosystems Inc. wasdepositedinGenBankusingaccession
from the Zfy intron were sequenced in (ABI),FosterCity,CA].Zfysequenceswere numberAF241870.
188 TheJournalofHeredity2000:91(3)
Table1. Geographiclocationandancestralinferencesof14mtDNAhaplotypesresultingfrom15polymorphicsitesincombinedanalysesofthe16S(382
bp),ATP8(191bp),andND5(318bp)genes,in286pumasa
16S ATP8 ND5
Haplotype 3063b3094 8630 8681 8700 8725 8756 1272312751 12809 12819 128341284012908 12909ANCc Nd Geographiclocation
A T G C C T T C T A A C C G T A 9 1 Paraguay
B . . . . . C . . . . . . A . . 9 1 Venezuela
C . . T . . . T . . . . . . . G 8 2 CostaRica,Panama
D . . . . . C . . . G . . . . . 7 4 Chile
E . . . . . C . . . . . T . . . 7 2 Bolivia,Peru
F . . . . . C . . . . . . . . . 8 46 nA,Bo,Br,CR,E,G,Pa,Pe,Ve
G . . . . . C . C . . . . . . . 8 1 Brazil
H . . . . . . T . . . . . . . . 10 9 Brazil,Paraguay
I . . . . . . T . . . . . A . G 10 4 Brazil
J . . . . C . T . . . . . . . . 9 19 Argentina,Chile
K C . . . . C . . . . T . . . . 8 1 Brazil
L . A . . . C . . . . . . . . . 9 2 Fr.Guyana,Brazil
M . A . T . . T . G . . . . . . 9 190 NorthAmerica,CR,Panama
N . A . T . . T . G . . . . C . 8 4 Washington,USA
Hya C A C C T T C T A A T C G T A 286 totalsamples
Aju T A C C T T T C A A C C A T A
Oge T A C C T T T T A A C C A T A
Fcab A A C C T T T C A A T C A T A
ancestralstatec A C C T T T A A C A T A
codonpositionn/a n/a n/a 3 1 2 3 3 1 2 3 3 3 2 3
aachange n/a n/a n/a — S(cid:7)P V(cid:7)A — — I(cid:7)V Q(cid:7)R M(cid:7)I — — V(cid:7)A —
aBasepairsthatareidenticaltohaplotypeAareindicatedbyaperiod.
bFromthecompleteFeliscatusmtDNAsequence(Lopezetal.1996).
cThenumberofsiteswithinferredancestralstatebasedonoccurrenceinthreeoffouroutgroupspecies’(Hya,Aju,OgeandFca)mtDNAhomologue.
dNumberofindividualswitheachhaplotype.
eAbbreviations:nA,northernArgentina;Bo,Bolivia;Br,Brazil;CR,CostaRica;E,Ecuador;G,Guyana;Pa,Paraguay;Pe,Peru;V,Venezuela;(Aju)cheetah,(Hya)jaguarundi,
(Oge)Geoffroyi’scat,and(Fca)domesticcat.
MicrosatellitePCR and Length groups of pumas were estimated using range of number of repeats were estimat-
Determination the kinship coefficient (Dkf) andpropor- ed from microsatellite datausing the pro-
The 10 most polymorphic microsatellite tion shared alleles (Dps) algorithms gram MICROSAT (version 1.5, Minch et
markers were selected (FCA008, FCA035, (Bowcock et al. 1994) asimplementedin al.). Deviations from Hardy–Weinberg
FCA043,FCA082,FCA090,FCA096,FCA117, the program MICROSAT (version 1.5, equilibrium (Guo and Thompson 1992)
FCA166, FCA249, FCA262) from 43 loci ex- Minch et al.). These distance estimators, were tested for each microsatellite locus
amined whichwere initiallycharacterized whicharebasedonsharedalleles,arehy- using the program ARLEQUIN (version
in the domestic cat (Menotti-Raymond et pothesized to be more appropriate for 1.1)(Schneideretal.1997).
al. 1997; Menotti-Raymond et al. 1999).Of comparisons among populations (Dris- Population subdivision was estimated
the 10 loci, 7 were unlinked on a genetic coll 1998; Goldstein and Pollock 1997). by FST (Reynolds et al. 1983) and number
recombinationmap,one(FCA166)wasun- Phylogenetic relationships were deter- ofmigrants(Slatkin1994)forbothmtDNA
sequence data and microsatellite length
mapped and two (FCA043 and FCA117) mined among individuals that amplified
were20cMapart(Menotti-Raymondetal. for eight or more loci (n (cid:1) 262) and variation data, using only the contempo-
rary samples (excluding museum speci-
1999).PCRamplificationwasperformedas among subspecies incorporating all sam-
mens). Estimates of F among mtDNA
described (Menotti-Raymond et al. 1997), plesthatamplifiedforatleastonemicro- ST
except for FCA116 which amplified using satellite locus (n (cid:1) 277). Phylogenetic haplotypes were obtained by analysis of
the following primers: FCA166F 5(cid:2)-AGGT- trees were constructed from the Dkf and molecularvariance(AMOVA)(Excoffieret
al.1992)usingTajima-Nei(1984)distances
ATTCTTCATCCCTAGGCA-3(cid:2) and FCA166R Dps distance matrixes using the NEIGH-
as implemented in the program ARLE-
5(cid:2)-TGTGCTGACAGCACCGAG-3(cid:2) (Culver BOR option of the program PHYLIP (ver-
QUIN. With microsatellite data, F esti-
1999). Products were electrophoresed on sion 3.572) (Felsenstein 1993). Trees ST
matesweregeneratedbyAMOVAwiththe
a 6% polyacrylamidegelusinganApplied weredrawnusingtheprogramTREEVIEW
‘‘number of different alleles’’ distance
Biosystems 373A automated sequencer. (version 1.5)(Page 1996).
methodusingARLEQUIN.Thesignificance
Microsatellite allele sizes were estimated
levelsofF wereassessedafteremploying
bycomparisonwithaGS350TAMRA(ABI) ST
Genetic Diversityand Population a Bonferroni adjustment (Weir 1996) for
internal size standard, and two pumas
StructureAnalysis multiplecomparisons.Alternativewaysof
were used as allele size controls on each
Measures of genetic diversity were esti- subdividing the individuals into groups
gel. Data were collected and analyzed us- mated for individuals, as well as within were compared to determinewhichparti-
ing the programs GENESCAN (version and between subspecies. Nucleotide di- tions best accounted for the genetic vari-
1.2.2-1) and GENOTYPER software (ver- versity((cid:8))(NeiandLi1979)wasestimat- ance. An AMOVA was also performed
sion 1.1)(ABI). among subspecies within groups (Excof-
edfrommtDNAhaplotype data,usingthe
fieret al.1992).
PhylogeneticAnalyses of Microsatellite program SENDBS (version 2, National In-
Data stitute of Genetics, Shizuoka, Japan). Av- Divergence Time Estimates
Pairwise genetic distances among indi- erage heterozygosity (H), average vari- Divergence dates among mtDNA haplo-
0
viduals, among subspecies, and among ance in number of repeats, and average types and outgroup specieswereestimat-
Culveretal•GenomicAncestryoftheAmericanPuma 189
Table2. MeasuresofgeneticvariationacrosscombinedanalysesofthreemtDNAgenesegments16S(382bp),ATP8(191bp),andND5(318bp),and10
microsatellitelociinpumasgroupedbysubspecies
Microsatelliteloci
Number Average
ofindividual Total number
Subspecies,continent, mtDNA/ number AverageH ofalleles Average Average Maximum
orgroupa (cid:3)satb mtDNAHaplotype ofallelesc %P (SE)e% o perlocus varianced ranged range
NorthAmericansubspecies:
P.c.missoulensis 23/22 M 34 100 48(0.40) 3.4 5.1 5.5 9
P.c.oregonensis 16/15 M 32 100 35(0.24) 3.2 4.0 5.2 9
P.c.vancouverensis 6 M 18 50 7(0.35) 1.8 1.1 1.9 5
P.c.olympus 4 N 18 50 31(0.18) 1.8 3.0 2.5 9
P.c.californica 25/24 M 32 90 27(0.03) 3.2 2.9 5.1 9
P.c.kaibabensis 5 M 32 90 53(0.35) 3.2 4.4 4.9 9
P.c.hippolestes 14 M 32 90 43(0.44) 3.2 3.4 4.5 9
P.c.couguar(cid:9)schorgeri(ancient) 3(cid:9)1 M 11 20 22(0.23) 1.2 2.1 0.8 6
P.c.browni 15 M 39 100 54(0.45) 3.9 3.8 5.4 9
P.c.browni(ancient) 4 M 13(3) 80 38(0.53) 1.6 9.2 4.1 14
P.c.azteca 35 M 46 100 50(0.27) 4.6 4.5 6.4 13
P.c.stanleyana 10 M 38 80 47(0.18) 3.8 4.1 4.5 9
P.c.coryi 6 M 12 20 5(0.0) 1.2 0.3 0.5 6
P.c.coryi(ancient) 6/4 M 9(6) 50 42(0.38) 2.3 9.6 4.8 10
P.c.improcera(ancient) 1/0 M
P.c.mayensis 12 M 49 100 60(0.0) 4.9 7.2 7.8 17
P.c.costaricensis 13 M F C 54 100 63(0.11) 5.4 11.1 8.4 20
SouthAmericansubspecies:
P.c.borbensis 2 F H 25 100 72(0.24) 2.8 10.2 5.6 12
P.c.borbensis(ancient) 1/0 L
P.c.greeni 1 H 15 50 50(0.0) 1.5 9.5 2.6 11
P.c.acrocodia 11 A F G H I K 72 100 68(0.27) 7.2 6.9 8.4 19
P.c.capricornensis 8 F H I 49 100 77(0.04) 4.9 8.5 7.9 19
P.c.bangsi 5 F 43 100 81(0.18) 4.3 6.9 6.2 18
P.c.concolor 3 F B 37 100 80(0.0) 3.7 6.5 5.8 12
P.c.concolor(ancient) 2 L F 19(11) 90 69(0.27) 2.4 5.7 3.3 5
P.c.soderstromi(ancient) 4 F 19 90 52(0.15) 1.9 8.9 3.2 12
P.c.incarum 5 F E 55 100 78(0.58) 5.5 8.7 5.5 15
P.c.incarum(ancient) 1 F 2(0)1 100 na na na na 7
P.c.osgoodi 5 F E 49 100 81(0.0) 4.9 10.3 7.0 18
P.c.cabrerae 10 J F 57 100 77(3.61) 5.7 9.6 8.4 21
P.c.hudsoni 7 F 51 100 74(2.90) 5.1 8.5 8.1 19
P.c.puma 6 J D 39 100 69(0.35) 3.9 8.0 5.8 11
P.c.araucanus 4 J D 30 100 43(0.53) 3.0 6.5 5.1 10
P.c.araucanus(ancient) 2 J 18(5) 100 83(0.35) 2.0 5.3 3.0 6
P.c.patagonica 6 J 32 100 63(0.35) 3.2 9.4 6.2 20
P.c.pearsoni 4 J F 34 100 78(0.24) 3.8 7.3 6.0 10
aSubspeciesorderedinageographicalclinefromnorthtosouth,ancientsamplescamefrommuseums.
bNumberofindividualstypedformtDNA/microsatelliteanalysis;onevalueindicatesthesamenumberforboth.
cAllelesfoundinmuseumbutnotinmodernsamplesofthesamepopulationareinparentheses.
dVarianceandrangeinthenumberofrepeatsaveragedacrossallloci.
eAverageobservedheterozygosityacrossalllociandstandarderror.
edbyaveragingallpairwise(p)distances of 315 pumas, collected from throughout and used to screen pumas in Figure 1A.
between haplotypes. Feline-specific theirrange(Figure1A).Thesequencesof The 15 polymorphic sites defined 14
mtDNA divergence rates of 1.39% (ATP8), three mitochondrial genes [382 bp of 16S mtDNAhaplotypes(Tables1and2,Figure
1.22% (ND5), and 0.97% (16S) per million rRNA(16S),191bpofATPase-8(ATP8)and 1B). All pumas north of Nicaragua (N (cid:1)
years (MY) were developed previously 318bpof(ND5)NADH-5]weredetermined 186), except four from the Pacific North-
(Lopez et al. 1997). A weighted average, from pumas using specific primers de- west, shared the M haplotype, including
based on the number of base pairs used signed from an alignment of available historicmuseumspecimensfromtwopos-
foreachgene,wasusedtoobtainthecom- mammalian mtDNA sequences (Lopez et sibly extinct North American subspecies
posite divergence rate of 1.15%/MY. A di- al. 1996). Sequence analysis revealed two (P. c. couguar and P. c. schorgeri). Three
vergencetimewasalsoestimatedfromZfy single nucleotide polymorphisms in the haplotypeswere presentinCentralAmer-
sequence data using a rate for felids of 16S gene, five in the ATP8 gene,andeight ican (C,F,M)pumasand11(A,B,D-L)were
0.11%/MY (Pecon-Slattery and O’Brien in the ND5 gene (Table 1). All polymor- found in South American pumas. Haplo-
1998). From this rate, the estimated time phisms were transitions and 6 of 13 mu- types M and F had wide distributions
to acquire one mutation, in the 350 bp of tations in protein coding genes werenon- throughout North and South America, re-
intronic sequence, was approximately 2.5 synonymous. RNA secondary structure spectively, while the other haplotypes
MY.
estimated by FoldRNA (GCG, Version 8 were geographically restricted (Table 2,
1994)indicatedthatthetwo16SrRNAvar- Figure1B).HaplotypeLwasuniquetomu-
Results
iantswerenoncompensatory. seum samples, one from French Guyana
DNAwasextractedandamplifiedbypoly- SSCPassays(Oritaetal.1989a;b;Podul- and a second from Brazil (located at op-
merase chain reaction (PCR) from a total so et al. 1991) were developed (Table 1) posite sides of the Amazon River, Figure
190 TheJournalofHeredity2000:91(3)
1B). Overall mtDNA diversity among all
haplotypes((cid:8)) was0.318%.
The phylogenetic relationship among
the 14 mtDNA haplotypes was examined
using maximum parsimony (MP), mini-
mum evolution (ME), and maximum like-
lihood (ML) algorithms. Each method
produced an identical topology which is
illustratedbyamaximumparsimonytree
(Figure 2A). Beyond continental distinc-
tions (M,N versus others; bootstrap pro-
portion 85% MP, 97% NJ) there was little
mtDNA defined phylogeographic struc-
tureamongpopulationsfromdifferentre-
gions of the continents. The average
amount of mtDNA sequence differences
amonghaplotypeswaslowamongallpu-
mas: 0.1–0.9% as compared to 9.0–11.0%
between homologous segments from the
cat species listed in Table 1 (http://lgd.
nci.nih.gov).
To explore the ancestry of thesehaplo-
types, nucleotide differences among pu-
mas were compared to homologous resi-
duesfromfouroutgroupspecies(cheetah,
jaguarundi, Geoffroy’s cat, and domestic
cat; Table 1). A nucleotide site polymor-
phisminpumaswasconsideredancestral
if at least three of four outgroup taxa
sharedthesamenucleotide.Usingthiscri-
terion,ancestralnucleotidestateswerein-
dicatedat12of15variablesites.Thetwo
haplotypeswiththemostancestralstates
(10of12),HandI(Table1),wererestrict-
edtoBrazilandParaguay(Figure1B).The
minimum spanning network (Figure 2B)
indicates that all haplotypes are one to
four mutational steps from the ancestral
centralH andFhaplotypes.
The mammalian Zfx and Zfy genes on
the respective X and Y chromosomes in-
cludeintronsthathavebeenshowntobe
powerfulnuclearindicatorsofphylogenet-
ic divergence in cats and other species
(Bianchi et al. 1992; Pecon-Slattery and
O’Brien 1998; Shimmin et al. 1993). DNA
sequenceofthemorerapidlyevolvingZfy
intron was determined in 14 puma speci-
Figure2. (A)Maximumparsimonytopologyofthe14haplotypesfromthecombined16S(382bp),ATP8(191
mensselectedfromawidegeographicdis-
bp),andND4(318bp)mitochondrialgenes(Table1).ThesingleMPtree(CI(cid:1)0.882,totaltreelength(cid:1)17)
rooted with the NA clade is depicted. Trees derived from MP, NJ, and ML analyses had identicaltopologies. tribution. Among seven North American
Numbersabovebranchesrepresentthenumberofsteps/numberofhomoplasies.Numbersbelowbranchesare and seven South American male pumas,
bootstrapvalues(from100replicates)fromNJanalysis/MPanalysis;dashesindicateabootstrapvalueofless
than50%.TheNJanalysisgeneratedasingletree(ME(cid:1)0.0194).AllnodesoftheMLtreeweresignificant(a no DNA sequencevariationwasobserved
consensusof100trees,-lnlikelihood(cid:1)1311.0909).Rootingbyoutgrouptaxaofjaguarundi,cheetah,andGeoffroyi’s in a 350bpregionof theintron.
catwasuninformativeduetoprohibitivelylargeinterspecificgeneticdistances(0.09–0.12)relativetointraspecific In contrast to limited variation in
values.Thustreesareunrootedbutthearrowindicatespositionofthemidpoint.(B)Minimumspanningnetwork
ofthe14mitochondrialhaplotypes(Table1),consistingof16S,ATP8,andND5genesusingMINSPNET(Oritaet mtDNA coding genes and Y chromosome
al.1989a)andthenumberofindividualswitheachhaplotype.Crossmarksonbranchesdefinethenumberof Zfy intron sequences, appreciable allelic
substitutionsbetweenconnectedhaplotypes.Ahypotheticalancestor(?)likelyconnectsI,C,andHasindicated.
diversity was apparent in a genotypic
screen of 10 unlinked nuclear microsatel-
liteloci.Tenlociselectedfromagroupof
253 markers that havebeenisolatedfrom
and mapped in domestic cat were typed
in 277 pumas (258 contemporary and 19
Culveretal•GenomicAncestryoftheAmericanPuma 191
Table3. Measuresofgeneticvariationacross10microsatellitelociandmtDNAgenesegments,inpumasgroupedbyphylogeographicgroup
Microsatellites
Average
Number mtDNA Number number
ofindividual Total of ofalleles
mtDNA/ Average number unique perlocus Average Average Maximum
Continentorgroup (cid:3)sat Haplotype (cid:8)((cid:10)102) H(SE)a% ofalleles alleles (SE) variance range range
o
Pumaconcolor 286/277 A–N 0.32 52( )b 121 — 12.1 6.9 12.5 21
NorthAmerica-NA(15subsp) 186/78 M N 0.02 42(0.16) 64 5 6.4c 4.8c 9.8 18
CentralAmerica-CA(1subsp) 13/13 M F C 0.40 63(0.11) 54 12 5.4 11.1 8.4 20
SouthAmerica-SA 87/86 A B D–L 0.30 71(0.33) 110 41 11.1 9.3 10.8 21
ESA(4subsp) 23/22 A F H G I K L 0.22 71(0.09) 86 5 8.6 8.0 9.3 19
NSA(5subsp) 25/25 B F E L 0.04 75(0.52) 91 5 9.1 8.2 10.0 19
CSA(2subsp) 17/17 F J 0.10 75(0.46) 67 1 6.7 9.6 9.2 21
SSA(4subsp) 22/22 F D J 0.19 64(1.16) 60 1 6.0 8.3 8.6 20
NA,NorthAmerica;CA,CentralAmerica;ESA,easternSouthAmerica;NSA,northernSouthAmerica;CSA,centralSouthAmerica;SSA,southernSouthAmerica,continent
(NA,NorthAmerica;SA,SouthAmerica),andspecies.
aAverageobservedpercentheterozygosityacrossalllociandstandarderror.
bSignificantlydifferentfromSouthAmericansubspecies(P(cid:11).05).
cSignificantlydifferentfromSouthAmericansubspecies(P(cid:11).01).
museum specimens). A summary of the small sample size for these subspecies .05);andaveragenumberofallelesperlo-
quantityandpatternofmicrosatellitevar- which were represented by few (two to cus(11.1versus6.4)(P(cid:11).01).Inaddition,
iability observed in each of 30 previously six)individuals. themolecularsizedistributionorvariance
named trinomial subspecies is presented A comparison of both mtDNA and mi- in allele size for microsatellite loci (i.e.,
in Table 2. The results indicate that all crosatellite allele variation revealed a the average number of repeat motifs be-
SouthAmericansubspeciesdisplayappre- marked difference between pumas from tween alleles) differed appreciably be-
ciable microsatellite diversity, with 90– NorthversusSouthAmerica(summarized tweenNorth(4.8)andSouthAmerica(9.3)
100% of loci polymorphic and an average in Table 3). South American subspecies (P (cid:11) .01). The lower variance likely re-
heterozygosity of 43–83%. Most North displayed greater overall variation than flectsthedisjunctpatternofmicrosatellite
American subspecies were also variable, the North Americanpopulation inseveral allele distribution of North American pu-
although four subspecies (P. c. coryi, P. c. measuresincludingthenumberofmtDNA mas compared to a more continuous dis-
couguar,P.c.olympus,andP.c.vancouver- haplotypes (11 versus 2, respectively); tribution among two South American lo-
ensis)showedreduced levelsofmicrosat- mtDNA genetic diversity ((cid:8) (cid:1) 0.3 versus cales illustrated in Figure 3. In North
ellite variation compared to other North 0.02, respectively); number of microsatel- American subspecies, one to threemicro-
American subspecies.Thisreductionmay lite alleles (110 versus 64, respectively); satellite alleles predominate for 7 of 10
reflect evidenceforhistoricinbreedingas numberofuniqueorcontinentspecifical- loci and for these loci, a single allele oc-
has been reported for P. c. coryi (Roelke leles (41 versus 5); average microsatellite curredat(cid:1)40%frequency(allexceptFCA-
et al. 1993), but could also result from heterozygosity (71% versus 42%) (P (cid:11) 043, -082, -096) (Figure 3). By contrast,
South American populations’ allele size
class distributions were more continuous
in that no predominant alleles ((cid:1)40%)
werepresentin anyofthe10loci.
The relationships among free-ranging
puma samples were examined in a phylo-
genetic analysis of composite microsatel-
lite genotypes of 262 individuals (Figure
4A,B). Minimum evolution topologies (es-
timatedbytheNJalgorithm)usingtwoge-
netic distance measures: percent allele
sharing, Dps, and mean kinship, Dkf, pre-
viously determined to be applicable for
closelyrelatedpopulations(Driscolletal.
in preparation; Goldstein and Pollock
1997)clusteredpumaindividualsfromthe
samegeographiclocales,althoughthesta-
tistical bootstrap support was low. Conti-
nental concordance had few exceptions,
although no phylogeographic structure
Figure3. Frequencydistributionofallelesat10microsatellitelociamongcombinedNorthAmericansubspecies
(N(cid:1)186individualsfrom15namedsubspecies,Figure1A)andtwocombinedphylogeographicgroups:northern wasapparentwithinNorthAmerican(NA)
SouthAmerica(NSA)(N(cid:1)25pumas)andeasternSouthAmerica(ESA)(N(cid:1)22pumas).Thesegroupswere populations. With a few exceptions (20of
selectedasasubsetofallSouthAmericanpopulationslikelytoreflectancestralgeographicregionbasedonmtDNA
262 genotypes), the genetic distance-
analysis(seetext).Horizontalaxisincludes2bp(CArepeat)incrementsoneithersizeofthemostcommonallele
inthegeographicpopulation.Verticalaxisisthecountofnumberofalleles. based trees indicate a consistent parti-
192 TheJournalofHeredity2000:91(3)
Table4. PopulationpairwiseF microsatelliteestimatesabovethediagonalandmitochondrialgenes andDps,respectively)asdidCSAandSSA
ST
belowthediagonal (bootstrap (cid:1) 98% and 91% for Dkf and
NA CA ESA NSA CSA SSA DPs, respectively), but therelationshipof
ESAandNSAdiffereddependingonthege-
NA — 0.110* 0.103* 0.059* 0.186* 0.367*
netic distance measure employed (Figure
CA 0.784* — 0.179* 0.126* 0.126* 0.228*
ESA 0.815* 0.057 — 0.031* 0.094* 0.330* 4E). Five of the groups (all except CSA)
NSA 0.958* 0.492* 0.384 — 0.077* 0.316* hadatleastoneuniquepopulationspecif-
CSA 0.935* 0.233 0.177* 0.107* — 0.172*
ic mtDNA haplotype (Table 3) and each
SSA 0.835* 0.240 0.186* 0.526* 0.281* —
had a distinct frequency distribution of
BelowdiagonalFSTforthecombinedmitochondrialgenes16S(382bp),ATP8(191bp),ND5(318bp),andTajima– mtDNAhaplotypefrequencies(Figure1B).
Neidistancemethod(TajimaandNei1984)inthesixgroups,NA(NorthAmerica),CA(CentralAmerica),ESA
Using previously calibrated divergence
(easternSouthAmerica),NSA(northernSouthAmerica),CSA(centralSouthAmerica),andSSA(southernSouth
America). ratesforthecombinedthreefelinemtDNA
Museumsampleswereexcluded.Toequalizesamplesizeamonggroups,eachNorthAmericansubspecieswas genes (Lopez et al.1997)—thecomposite
limitedtoonlythreeorfewerrandomlychosencontemporarysamples(n(cid:1)129).
mutation rate for this combinedregionof
*P(cid:11).0033(afterBonferroniadjustment),basedon100permutations.
mtDNA was estimatedas1.15%/MY.Using
thisdivergencerate,pumaandjaguarundi
tioning among South and Central Ameri- wereusedtotestF andM;however,the lastsharedacommonancestor4.2MYBP,
ST
can pumas, which correspond to five six groups described here best explained while extant puma lineages diverged ap-
geographic areas (Figures 1B and 4A,B): the observed genetic variation. Boundar- proximately 390,000 years ago and North
southern South America (SSA) including ies between some of these groups were Americanpumassharedacommonances-
Chile and southwestern Argentina (Pata- somewhat arbitrary due to either incom- torlessthan 20,000years ago.TheZfyin-
gonian/Andean region); eastern South pletesamplingin thearea orto slightdif- tronic sequence divergence rate in felids
America (ESA) including Brazil (south of ferences between results of the mtDNA hasbeenestimatedatonemutationevery
Amazon River) and Paraguay; northern and microsatellite analyses. The CSA 2.5MY(Pecon-SlatteryandO’Brien1998).
SouthAmerica(NSA)includingVenezuela, groupwasrecognizedbecauseindividuals From this rate, extant puma lineages are
Ecuador, Bolivia, Guyana, and Colombia; from this area shared affinities with the probably less than 2.5 MY old, since no
Central South America (CSA) including other South American groups, but could variation was detected among pumas in
northeast Argentina; and Central America not be readily assigned to either of these oursample.
(CA) including Costa Rica, Panama, and groups.BoundariesoftheCSAgroupwere
Nicaragua. defined as the region between Rio Negro
Discussion
Phylogenetic analyses of individual ge- and Rio Parana´ and included the P. c. ca-
notypes constrained in accordance with breraeandP.c.hudsonisubspecies. Distinguished by a hemispheric distribu-
previouslynamedtrinomialsubspeciesre- AsummaryofthedistributionofmtDNA tionfromtheArctictundraofthenorthto
inforcedthephylogeographicconclusions andmicrosatellitevariationamongpumas Patagonia in the south, pumas (P. conco-
(Figure4C,D).NorthAmericansubspecies considered as a species, as continental lor) exhibit bothbroadandfine-scalepat-
clustered with strong bootstrap support populations, and as the six phylogeo- terns of genetic differentiation. Concor-
(73%Dkfand60%Dps).Threesubspecies graphic groups is presented in Table 3. dance between microsatellite and
had unusually long branch lengths in the The most diverse group based on mtDNA mitochondrialDNAsequencevariationre-
Dkftree(Figure4d)duetoasmallsample haplotypes was ESA (7 haplotypes; (cid:8) (cid:1) vealedanevolutionaryhistorymarkedby
size (P. c. couguar, P. c. greeni, and P. c. 0.02), while NA had the fewest (2 haplo- ancient ancestry and a period of recent
borbensis), while others with long branch types; (cid:8) (cid:1) 0.22). For microsatellites the North American repopulation. Previous
lengths proved to be homozygous at five groups with the highest diversity (ESA classification into 32 subspecies (Neff
toeightloci(P.c.olympus,P.c.vancouver- andNSA)werecentraltothepumasrange 1983; Young and Goldman 1946) was not
ensis,andP.c.coryi). and,ofinterest,werethefocusofthemost affirmed; rather modern populations as-
Population distinctiveness among the ancestralmtDNAhaplotypes,HandI(Fig- sorted by molecular genetic criteria into
six puma groups was examined by ure 1B). Pumas from ESA or NSA had the sixbroadphylogeographicregions(Figure
Wright’s F values for both mtDNA hap- greatest number of alleles, the most 1B)withevidenceofhistoricinbreedingin
ST
lotypes and for composite microsatellite unique alleles, and the highest heterozy- a few isolated localities and admixture in
genotypes (Table 4). Significantdifferenc- gosity (Table 3). A broad continuous dis- others.
es were obtained for all pairwise group tribution of allele size classes was found Thesixregions(Figure1B)weredefined
comparisons with microsatellites and for among pumas from ESA and NSA (Figure based on phylogenetic partitions as indi-
11of15mtDNAcomparisons.Similarsub- 3), in contrast with other groups whose cated by composite individual and popu-
divisionwasaffirmedbycalculatingM,mi- allele sizes were largely a subset of the lation genotypes at 10 unlinked feline mi-
grant number (Slatkin 1994), and AMOVA ESA/NSAsizeclasses. crosatellite loci (Figure 4), by the
analysis (Excoffier et al. 1992; Schneider When the six puma groups were exam- distributionandfrequencyofmtDNAhap-
et al. 1997). The combined phylogeo- ined phylogenetically using microsatellite lotypesof15polymorphicnucleotidesites
graphic analyses of microsatellite geno- genotypes (Figure 4E), we observed within three genes (16S, ATP8, ND5;Table
types (Figure 4) and mtDNA coding gene strong support for group definition, but 1) and by the pattern of diversityindicat-
haplotypes (Figures 1B and 2) form the someinconsistencyinthehierarchicalre- edbytheseloci.Thepumagroupsroughly
basisforrecognition ofthesixtaxonomic lationshipsamongthegroups,particularly correlate with geographic barriers that
units or subspecies of modern pumas inSouthAmerica.CAandNAclusteredto- could restrict migration and gene flow.
(Figure 1B). Other geographic partitions gether (bootstrap (cid:1) 98%; 100% for Dkf NorthAmericanpumas(NA)aremadeup
Culveretal•GenomicAncestryoftheAmericanPuma 193
194 TheJournalofHeredity2000:91(3)
lelesbeingasubsetofthosefoundinCen-
tral and South America. Considered to-
gether, the reduction in mtDNA and
microsatellite variation is indicative of ei-
ther an historic founder effect or a popu-
lation bottleneck that effectively reduced
overallgenomicdiversity(Maruyamaand
Fuerst1985;Mayr1970;Neietal.1975).
The date for the postulated bottleneck
in the history of North American pumas
was estimated bycomparingthevariance
in microsatellite allele to that in other
populations that have also experienced
historicdemographicreductions(Driscoll
1998; Menotti-Raymond and O’Brien1993;
O’Brienetal.1985;O’Brienetal.1987).Mi-
crosatelliteallelevarianceisameasureof
thebreadthoftime-dependentmutational
expansion that microsatellites show in a
population (Driscoll 1998;Goldsteinetal.
1995; Reich and Goldstein 1998). Because
allele variance takes longer for reconsti-
tution after a bottleneck than overall het-
Figure4.Continued. (E)ME-NJtreeusingD andD geneticdistanceamongthesixphylogeographicsubspe- erozygosity or allele number (Driscoll
kf ps
ciesdefinedbythisstudy.Numbersonnodesarebootstrappercentages.
1998; Goldstein et al. 1995; Goldstein and
Pollock1997;ReichandGoldstein1998)it
of 15 previously named subspecies north genetic diversity (Table3)thatcanbein- is a valuable chronometer for founder
of Nicaragua. Central American pumas terpretedinthecontextofgeographyand events that are older than the period re-
(CA) inhabit mostly tropical rainforest. puma natural history. As ESA is the pop- quired for reconstitution of heterozygosi-
Bounded by two major rivers, Rio Negro ulation distinguished by the most ances- ty.
and Rio Parana´, central South American tral (Table 1) and central (Figure 2B) The average allele variance among the
(CSA) pumas include two previously mtDNA haplotypes, modern puma geno- entireNorthAmericanpopulationwas4.8,
named subspecies from the pampas de- mic diversity likely traces its origin to comparedto8.2–11.1forotherpumasub-
sert.EasternSouth America(ESA)pumas near or within the ESA locale. This infer- species(Table3).TheobservedNAallele
inhabit several biogeographic zones bor- enceissupportedbytheobservationthat varianceisequivalenttotherestrictedal-
deredbytheAmazon,RioParana´,andthe ESA has the highest number of mtDNA lelevarianceobservedinAfricancheetahs
Paraguayriversandincludefourprevious haplotypes (n (cid:1) 7), near maximal micro- (based on a larger screen of 88 microsat-
subspecies. Demarcated by the Amazon satellitediversity,andthebroadestdistri- ellite loci) (Driscoll 1998), a species doc-
and Paraguay rivers, the northern South bution of allele size classes. In contrast, umented by several measures to have ex-
American (NSA) region includes six NA had the lowest variation both in perienced a severe population bottleneck
namedsubspeciesdistributedacrossmul- mtDNAandinmicrosatellitemeasures.NA around 10,000–12,000 years ago, near the
tiple habitat types. Southern South Amer- pumas are predominately one mitochon- end of the Pleistocene (Menotti-Raymond
ica (SSA) includes four subspecies of drial haplotype (M) with an infrequent and O’Brien 1993; O’Brien et al. 1985;
puma from Patagonia and the diverse second haplotype (N) restricted to the O’Brienetal.1987).Ifmicrosatelliteallele
mountainous habitats of Argentina and Olympic peninsula. The entire NA puma variance reconstitution following demo-
Chile. sample (n (cid:1) 191) possessed less micro- graphic homogenization occurred at the
Patterns of mtDNA and microsatellite satellite diversity than any of the other same rate in cheetahs and pumas, the re-
variation revealed differences in relative phylogeographicgroupswithnearlyallal- sultsimplythatNApumasaredescended
←
Figure 4. Phylogeneticrelationshipsamong262individualpumasconstructedfrom10microsatelliteloci.UnrootedNJtreesestimatedusing(A)proportionofshared
alleles,Dps;and(B)meankinship,Dkf,geneticdistanceswitha1-pstransformation.Individuals,subspecies,orgroupsarecolorcodedasindicatedbygeographicarea
abbreviationsdefinedintext,Figure1B,andTable3.Numberedtaxaindicateexceptiontophylogeographicclustersandcorrespondto1,Brazil(Pco-194);2,Venezuela(Pco-
295);3,Peru(Pco-409);4,Panama(Pco-542);5,Brazil(Pco-696);6,Paraguay(Pco-699);7,Paraguay(Pco-701);8,Paraguay(Pco-714);9,Guyana(Pco-856);10,Ecuador(Pco-
867);11,Chile(Pco-215);12,Chile(Pco-212);13,Belize(Pco-271);14,Argentina(Pco-580);15,Argentina(Pco-574);16,Argentina(Pco-562).Numbers16–29ontheDpstree
(A)correspondto17,Bolivia(Pco-707);18,Mexico(Pco-757);19,Argentina(Pco-566);20,Argentina(Pco-564);21,Brazil(Pco-195);22,Brazil(Pco-196);23,CostaRica(Pco-
547);24,Arizona(Pco-604);25,Mexico(Pco-592);26,Arizona(Pco-561);27,Brazil(Pco-697);28,CostaRica(Pco-548);29,Argentina(Pco-576).Numbers16–35ontheDkf
tree(5B)correspondto17,Peru(Pco-406);18,Peru(Pco-407);19,Argentina(Pco-571);20,Nicaragua(Pco-551);21,Paraguay(Pco-710);22,Argentina(Pco-565);23,Chile
(Pco-597);24,CostaRica(Pco-545);25,Nicaragua(Pco-554);26,Nicaragua(Pco-549);27,Nicaragua(Pco-550);28,Mexico(Pco-593);29,Argentina(Pco-576);30,Argentina
(Pco-560);31,Argentina(Pco-568);32,Argentina(Pco-579);33,Argentina(Pco-573);34,Arizona(Pco-73);35,Arizona(Pco-163).Exceptionalindividuals(11%oftotal)insome
casescanbeexplainedbysizehomoplasyofmicrosatellitealleles,demonstratedtooccurforsomelocibasedonsequenceanalysisof85microsatellitehomozygoteswithin
pumas(Culveretal.,inpreparation).(C)NJ-MEanalysisusingcombinedpopulationgenotypesbasedonDpsdistancebetweenthe29namedsubspecies(Neff1983;Young
andGoldman1946)(Figure1A).P.c.shorgeriandP.c.couguarwerecombined.Bootstrapsvaluesover40%areindicatedondivergencenodes.(D)NJ-MEanalysisofcombined
subspeciesgenotypesusingtheD geneticdistanceasin(C).
kf
Culveretal•GenomicAncestryoftheAmericanPuma 195