Autosomal and mtDNA Markers Affirm the Distinctiveness of Lions in West and Central Africa

15 Pages·2015·1.44 MB·English

RESEARCHARTICLE
Autosomal and mtDNA Markers Affirm the
Distinctiveness of Lions in West and Central
Africa
LauraD.Bertola1,2*,LauraTensen3,PimvanHooft4,PaulaA.White5,CarlosA.Driscoll6,
PhilippHenschel7,AnthonyCaragiulo8,IsabelaDias-Freedman8,Etotépé
A.Sogbohossou1,9,PriceliaN.Tumenta1,10,TuqaH.Jirmo1,GeertR.deSnoo1,HansH.de
Iongh1,11☯,KlaasVrieling2☯
1 LeidenUniversity,InstituteofEnvironmentalSciences(CML),POBox9518,2300,RALeiden,The
Netherlands,2 LeidenUniversity,InstituteofBiologyLeiden(IBL),POBox9505,2300,RALeiden,
TheNetherlands,3 UniversityofJohannesburg,DepartmentofZoology,POBox524,Johannesburg,
Johannesburg,RepublicofSouthAfrica,4 WageningenUniversity,ResourceEcologyGroup,
Droevendaalsesteeg3a,6708,PBWageningen,TheNetherlands,5 CenterforTropicalResearch,Institute
oftheEnvironmentandSustainability,LaKretzHallSuite300,619CharlesE.YoungDr.East,Universityof
CaliforniaLosAngeles,LosAngeles,CA,90095–1496,UnitedStatesofAmerica,6 WildlifeInstituteofIndia,
Dehradun,248001,Uttarakhand,India,7 Panthera,8West40thStreet,18thFloor,NewYork,NY,10018,
UnitedStatesofAmerica,8 SacklerInstituteforComparativeGenomics,AmericanMuseumofNatural
History,79thStreetatCentralParkWest,NewYork,NY,10024,UnitedStatesofAmerica,9 Laboratoire
d’EcologieAppliquée,Universitéd’Abomey-Calavi,ChampdeFoire03BP1974,Cotonou,Benin,10 Centre
OPENACCESS
forEnvironmentandDevelopmentStudiesinCameroon,UniversityofDschang,BP410,Maroua,
Citation:BertolaLD,TensenL,vanHooftP,White Cameroon,11 UniversityofAntwerp,DepartmentBiology,EvolutionaryEcologyGroup,Groenenborgerlaan
171,2020,Antwerpen,Belgium
PA,DriscollCA,HenschelP,etal.(2015)Autosomal
andmtDNAMarkersAffirmtheDistinctivenessof
☯Theseauthorscontributedequallytothiswork.
LionsinWestandCentralAfrica.PLoSONE10(10): *[email protected]
e0137975.doi:10.1371/journal.pone.0137975
Editor:MichaelHofreiter,UniversityofYork,UNITED
KINGDOM Abstract
Received:June12,2014
Theevolutionaryhistoryofaspeciesiskeyforunderstandingthetaxonomyandforthe
Accepted:August25,2015
designofeffectivemanagementstrategiesforspeciesconservation.Theknowledgeabout
Published:October14,2015
thephylogeneticpositionofthelion(Pantheraleo)inWest/CentralAfricaislargelybased
Copyright:©2015Bertolaetal.Thisisanopen onmitochondrialmarkers.PreviousstudiesusingmtDNAonlyhaveshownthisregionto
accessarticledistributedunderthetermsofthe
holdadistinctevolutionarylineage.Inaddition,anthropogenicfactorshaveledtoastrong
CreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany declineinWest/CentralAfricanlionnumbers,thus,theconservationvalueofthesepopula-
medium,providedtheoriginalauthorandsourceare tionsisparticularlyhigh.Here,weinvestigatewhetherautosomalmarkersareconcordant
credited.
withpreviouslydescribedphylogeographicpatterns,andconfirmtheuniquepositionofthe
DataAvailabilityStatement:DNAsequenceshave West/CentralAfricanlion.Analysisof20microsatellitesand1,454bpofthemitochondrial
beendepositedinGenbankwithaccessionnumbers
DNAin16lionpopulationsrepresentingtheentiregeographicrangeofthespeciesfound
KJ652247-KJ652251andfragmentlengthdatafor
congruenceinbothtypesofmarkers,identifyingfourclusters:1)West/CentralAfrica,2)
microsatellitesareincludedinSupplementalS3
Table. EastAfrica,3)SouthernAfricaand4)India.Thisisnotinlinewiththecurrenttaxonomy,as
definedbytheIUCN,whichonlyrecognizesanAfricanandanAsiaticsubspecies.There
Funding:Theinvestigationsweresupportedbythe
DivisionforEarthandLifeSciences(ALW)with arenoindicationsthatgeneticdiversityinWest/CentralAfricalionsislowerthanineither
financialaidfromtheNetherlandsOrganizationfor EastorSouthernAfrica,however,giventhisgeneticdistinctionandtherecentdeclinesof
ScientificResearch(NWO)(projectno.820.01.002).
lionnumbersinthisregion,westronglyrecommendprioritizationofconservationprojectsin
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist.
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PhylogeographyandGeneticDiversityintheLion
West/CentralAfrica.Asthecurrenttaxonomicnomenclaturedoesnotreflecttheevolution-
aryhistoryofthelion,wesuggestthatataxonomicrevisionofthelioniswarranted.
Introduction
Identifyinganddescribingpatternsofmitochondrial(mtDNA)andnucleargeneticvariationis
acrucialcomponenttofullyunderstandingtheevolutionaryhistoryofaspecies.Highquality
phylogeographicdatathatrepresenttheunderlyinggeneticcomplexityareimportantfortax-
onomyandcontributetodesigningeffectiveconservationstrategies.Thisisofparticular
importanceforspeciessuchasthelion(Pantheraleo)thatoccupylargegeographicranges
withinwhichdisjunctpopulationsmaynotallowfornaturaldispersalandgeneflow.Increas-
inghabitatfragmentationandvariableanthropogenicfactorshavecreatedagrowingneedto
managelionsatthepopulationlevel[1].Inaddition,severalrecentpublicationshavesparked
thediscussionwhetherthecurrenttaxonomicnomenclatureforthelionisjustified[2–4].
TwosubspeciesoflionareofficiallyrecognizedbytheIUCN,basedongeneticdata[5,6]:
theAfricanlion(Pantheraleoleo),rangingthroughoutsub-SaharanAfricawiththeexception
ofdenserainforest,andtheAsiaticlion(Pantheraleopersica),whichexistsasasinglepopula-
tionintheGirforest,India.AlthoughallAfricanlionpopulationsareconsideredasbelonging
totheAfricansubspecies(P.l.leo),distinctsubgroupshavebeenrecognizedbasedonmor-
phology[7,8]andgenetics[2–5,9–12].Analysesofmorphometricdatahasledtothedistinc-
tionofatleastthreeextantclades(“subspecies”)ontheAfricancontinent[7].Lionsfromthe
northernpartoftheirrangefurthershowedarelativelycloserelationshiptotheAsiaticsubspe-
cies[7,8].Thispatternwasconfirmedbyphylogeneticanalysisofmitochondrialhaplotypes
only,basedonwhichlionsinWest/CentralAfricaweredescribedasageneticallydistinct
groupwitharelativelyclosegeneticrelationshiptotheAsiaticsubspecies[2–4](regiondefini-
tionsfrom[13,14],seeFig1).ThegeneticdichotomythatseparatestheWest/CentralAfrican
lionpopulationsfromEastandSouthernAfricanpopulationshasalsobeenfoundinother
largemammalspeciesandisoftenreflectedintheirtaxonomyincludingAfricanbuffalo(Syn-
ceruscaffer)[15,16],roanantelope(Hippotragusequinus)[17],hartebeest(Alcelaphusbusela-
phus)[18,19],giraffe(Giraffacamelopardalis)[20,21]andcheetah(Acinonyxjubatus)[22,23].
DuetothegeneticdifferentiationwithintheAfricanlionandthenestedpositionoftheAsi-
aticlionsubspecieswithintheWest/CentralAfricacladebasedonmtDNA,thecurrenttaxo-
nomicdivisionischallenged[2–4].However,mtDNAisasingle,nonrecombininglocusinthe
maternallineageanddoesnotpermitthedetectionofadmixtureeventsandsortingatmultiple
lociasmayoccurinautosomalmarkers.Therefore,theobservedpatterninmtDNAdatamay
notadequatelydepicttheunderlyinggeneticcomplexity.Autosomaldataareneededtocorrob-
oratethetopologybasedonmtDNA,sinceconflictingpatternsbetweenphylogeniesbasedon
mtDNAandphylogeniesbasedonautosomalmarkershavebeendescribedinseveralother
species[24–29].MostcommonlyamonophyleticpatternisdetectedinthemtDNA,butisnot
supported,oriscontradicted,byphylogeniesbasedonautosomalloci.Thisisoftenexplained
byincompletelineagesorting,ascoalescencetimeinmtDNAisfourtimesshorterthanin
autosomalmarkers.Sincelineagesortingduringtheprocessofcoalescencehasarandom
nature,thiscouldalsoleadtoan‘incorrect’genetreebymtDNAmarkersifpopulations’diver-
genceswerecloselyspacedintime.Femalephilopatryisanotherstrongcontributingfactorin
mtDNAtrees.Asgeneflowinlionsisbiasedtowardsthemalesex[30,31],genetreesbasedon
autosomalmarkersmayshowlessdiscretegroups.ThisargumenthasbeenusedbyAntunes
etal.(2008)toexplainincongruentpatternsintheirliondatabasedonmtDNAandautosomal
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markers.Taxonomicrevisionshavepotentiallyfar-reachingramificationswithregardtoman-
agement(e.g.,CITES,USFWS,IUCN)and,therefore,shouldbeapproachedcautiously.Ideally,
proposedrevisionsshouldbesupportedbyacombinationofbiogeographic,mtDNAandauto-
somalDNA,andmorphologicaldata.
Inthisstudy,weanalyzed20microsatellitelociforlionsfromthirteenwildpopulations,
oneofwhichislocatedinWestAfrica(Benin)andfourinCentralAfrica(Chad,DRCandtwo
fromCameroon).Furthermore,weincludedmicrosatellitedatafromanotherWestAfrican
populationinSenegalandfromtwodistinctzoopopulationsofEthiopianlionsrepresenting
theregionwherethetwomajorgeneticlineages(i.e.,West/CentralAfricaandEast/Southern
Africa)mayconnect.Tocomparethephylogeneticclustersderivedfromthemicrosatellite
dataandtocheckforcongruencewithpreviouslypublishedpatterns,weincludeddata
from1,454basepairs(bp)ofthemitochondrialDNAforeachsamplinglocation.Usingthis
approach,weareaimingtocontributetotheongoingdiscussionaboutliontaxonomyby
answeringfourquestions:1)Doautosomaldatasupportpreviouslydescribedphylogenetic
groupingsinthelioningeneralandthedistinctpositionoftheWest/CentralAfricanlionin
particular?,2)Cananeffectofsex-biasedgeneflowbedetected?,3)Howgeneticallydistinct
arethesampledpopulations,atboththecontinentalandregionalscales,anddolevelsof
geneticdiversityvaryamongstregionalsubdivisions,withaspecialfocusonWest/Central
Africa?and4)Aretheresignsforreducedgeneticdiversityinparticularlionpopulationswith
Fig1.Mapshowingthelocationofthe16lionpopulationsincludedintheanalysis.Inthelegend,the
compositionofthedatasetsandthenumberofincludedmicrosatellitelociisindicated.Lionrangedatafrom
IUCN(2014).RegiondefinitionsfromIUCNSSCCatSpecialistGroup(2006a;b).
doi:10.1371/journal.pone.0137975.g001
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anemphasisonWest/CentralAfrica?Ourstudyisthefirsttoincludemultiplelionpopulations
fromWest/CentralAfrica,usingbothautosomalandmtDNAmarkersinaphylogeneticcon-
textcoveringtheentirecurrentgeographicrangeofthelion.
MaterialandMethods
Weprocessedatotalof48samplesfromeightpopulations,includingonepopulationfrom
WestAfrica(Benin),fourpopulationsfromCentralAfrica(twofromCameroon,onefrom
ChadandonefromDRC),twopopulationsfromEastAfrica(Ethiopia2(captive)andKenya)
andonepopulationfromSouthernAfrica(Zambia).ExceptforEthiopia2,allsamplesorigi-
natedfromfree-ranginglions,withnoknownhistoryofanthropogenicintroductionsoflions
fromotherpopulations.Sampleswerecollectedinfullcompliancewithspecificpermits
(CITESandpermitsrelatedtonationallegislationinthecountriesoforigin).Detailsonper-
mits,samplestorage,DNAextraction,polymerasechainreaction(PCR)amplification,frag-
mentanalysisandqualitycontrolaregiveninS1File.SeeS1TableandS2Tableforusedloci
andprimerinformation.AllmicrosatelliteallelelengthdataaregiveninS2File.
Generatedmicrosatellitedataweresupplementedbypublisheddataforthesame20loci
fromanothersixpopulations[32],togethersummarizedasDataset1.Dataset2[12]consists
ofall15samplesfromEthiopia1(captive)withtenanalyzedloci,ofwhichsixareoverlapping
withourdataset.FortwosamplesfromEthiopia1,all20microsatelliteswereanalyzedand
addedtoDataset1.Dataset3(Panthera/AMNH)containsmicrosatellitedatafrom12loci
forsevenlionsfromSenegal,whichcouldnotberesizedtoDataset1andwerethereforeonly
includedforcalculationofdiversityindicesandbottleneckstatistics(fordetailsonpermitsand
theprocessingofSenegalsamples,seeS3File).Anoverviewofdatasetsusedineachanalysisis
providedinFig1andTable1.
Table1. Overviewoflionpopulationsincludedinthisstudy.
Set Population Area GeographicRegion PopSize Nmsat NmtDNA Sourcemsatdata
Benin PendjariNP WestAfrica 100 5 5 thisdataset
Cameroon1 WazaNP CentralAfrica 20 9 9 thisdataset
Cameroon2 BénouéEcosystem CentralAfrica 200 3 3 thisdataset
Chad ZakoumaNP CentralAfrica 140 4 4 thisdataset
DRC GarambaNP CentralAfrica 175 7 6 thisdataset
Ethiopia2 YemenZoo EastAfrica (captive) 4 4 thisdataset
1 Kenya AmboseliNP EastAfrica 60 7 7 thisdataset
Tanzania1 SerengetiNP EastAfrica 3465 10 3 Driscolletal.,2002
Tanzania2 NgorongoroCA EastAfrica 53 10 1 Driscolletal.,2002
Zambia LuangwaValley SouthernAfrica 750 9 9 Driscolletal.,2002
Namibia EtoshaNP SouthernAfrica 455 10 2 Driscolletal.,2002
RSA1 Kalahari-GemsbokNP SouthernAfrica 350 10 2 Driscolletal.,2002
RSA2 KrugerNP SouthernAfrica 1684 10 10* Driscolletal.,2002
India GirforestNP India 411 10 6 Driscolletal.,2002
2 Ethiopia1 AddisAbabaZoo EastAfrica (captive) 15 5 Brucheetal.,2012
3 Senegal NiokoloKobaNP WestAfrica 15 7 7 Panthera/AMNH
PopSize:populationsizeaccordingtothemostrecentestimateinRiggioetal.(2012)fortheAfricanpopulations,exceptforZambia:PaulaWhite
(personalcommunication);estimatefortheIndianpopulationfrom[56]
Nmsat:numberofsampledindividualsformicrosatelliteanalysis
NmtDNA:numberofsampledindividualsformtDNAanalysis.
*mtDNAandmicrosatellitedataarenotfromthesamesamples.
doi:10.1371/journal.pone.0137975.t001
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STRUCTURE2.3.3[33]wasusedforassessingpopulationstructureinDataset1with
unknownlociscoredasmissingdata.Simulationswererunassumingtheadmixturemodel
withcorrelatedallelefrequencies.TenrunswereperformedforK=1toK=8,using
10,000,000permutationsandaburn-inperiodof1,000,000.TochecktheassignmentofEthio-
pia1toanyoftheclustersidentifiedbySTRUCTURE,weincludedthetwoEthiopiansamples
forall20microsatellites.StructureHarvester[34]wasusedtodeterminethemostlikelynum-
berofclusters,followingtheΔKmethod[35].CLUMPPwasusedtocombinereplicateruns
andavoidlabelswitching[36].Clusteringofindividualswasfurtherassessedbyperforming
PrincipalComponentAnalysis(PCA)inGenAlEx6.501[37].Aneighbour-joiningtreewas
createdbasedonD distanceinPOPTREE2using1,000bootstraps[38].
A
Foreachsamplinglocation,amitochondrialregionof1,454bpthatencompassedcyto-
chromeB(cytB),tRNAThr,tRNAProandpartofthecontrolregionwasincludedforanumber
ofindividuals(Table1).Detailsonpolymerasechainreaction(PCR)amplificationand
sequencingaregiveninS1File.SequencesweredepositedinGenBankandsupplementedby
sequencespreviouslypublishedbyBertolaetal.(2011)(seeS4FileforsequencedataandS3
Tableforaccessionnumbers).Variablesitesandnucleotidediversitywerecalculatedusing
ARLEQUIN3.5[39].Forphylogeneticanalysis,ahaplotypenetworkwascreatedusingthe
median-joiningalgorithminNetwork4.6.1.1(www.fluxus-engineering.com).Arepeatregion
ofcytosinesofvariablelengthwasexcludedduetounknownhomology(positions1382–1393)
andallremainingcharacterswereincludedwithequalweighting.
ForAMOVAofDataset1,individualsforwhichall20lociwereanalyzedwereincludedas
either1)withoutanindicatedsubstructure(asall1group),2)followingIUCNclassification
(Africa;Asia),3)followingaNorth/Southdivisionaswasindicatedfromthehaplotypenet-
work,or4)usingthefourgroupsidentifiedbySTRUCTURE(West/CentralAfrica;EastAfrica;
SouthernAfrica;India).IsolationByDistance(IBD)wasassessedbycorrelatinggeographicto
geneticdistancesandusingaMantel’spermutationtestwith999permutations,asimple-
mentedinGenAlEx6.501[37].Inaddition,AMOVAandIBDanalysiswereperformedona
regionallevel,usingtheregionsasindicatedabove(Africa;North;South;West/CentralAfrica;
EastAfrica;SouthernAfrica).PairwiseF andNei’sgeneticdistanceswerecomputedwith
ST
GenAlEx6.501[37]formicrosatellitedataandwithARLEEQUIN3.5formtDNAdata[39].
Theaveragenumberofallelesperlocus(Na)wascalculatedusingARLEQUIN3.5[39].Pri-
vateallelicrichness(A )wascalculatedwithHP-Rare1.1[40]includingstatisticalrarefaction
p
tocompensatefordifferentsamplesizes.GenAlEx6.501[37]wasusedtocalculateobserved
(Ho)andunbiasedexpectedheterozygosity(uHe)[41].Toobtaininsightsintotheriskof
emergentinbreeding,F perpopulationwascalculatedinFSTAT[42]andtheoccurrenceof
IS
recentbottleneckswasevaluatedbyusingtheprogramBottleneck[43,44].TheBottlenecktest
isbasedonthetheorythatduringabottlenecktheallelenumbersarereducedfasterthanthe
heterozygosity,leadingtoanexcessofheterozygositycomparedtotheexpectedheterozygosity
underthemutation-driftequilibrium.Theprogramwasrunfor10,000iterations,usingthe
stepwisemutationmodel(SMM).Significant(P<0.05)resultsfromtheWilcoxonsigned-rank
testwerescored,asthistestprovedtobethemostpowerfulandrobustwhenusedwithfew
(<20)polymorphicloci[44].
Results
BasedontheSTRUCTUREresultsofDataset1,StructureHarvesteridentifiedthatthe
observedgeneticstructureisbestdescribedbyfourclustersrepresentingthefollowinggeo-
graphicareas:1)West/CentralAfrica,2)EastAfrica,3)SouthernAfricaand4)India(Fig2).
Thereisnoindicationforahierarchicalstructure,andforcingtheprogramtoidentifya
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PhylogeographyandGeneticDiversityintheLion
Fig2.ResultsofSTRUCTUREanalysisbasedon20microsatellitelociof15lionpopulations(Dataset
1+2individualsfromEthiopia1).A:representationofassignmentvaluesfoundbySTRUCTURE,using
K=4;B:Overviewofincludedpopulations;C:PlotindicatingmeanloglikelihoodLn(P(X|K);D:plot
indicatingΔKvaluesasafunctionofthenumberofgeneticclusters(K),inwhichΔK=mean(|L”(K)|)/sd(L(K));
E:RepresentationofassignmentvaluesfoundbySTRUCTURE,usingK=2.K=3,K=5andK=6.
doi:10.1371/journal.pone.0137975.g002
differentnumberofclustersleadstoanartificialclusteringcharacterizedbyheavyadmixture
andresultsnotinlinewithsuggestedevolutionaryhistoryderivedfromotherdata.Individuals
fromChadarepartoftheWest/CentralAfricacluster.TheEthiopianlionsshowaffiliation
eithertoWest/CentralAfrica,admixedwithSouthernAfrica(Ethiopia1)ortoEastAfrica,
admixedwithSouthernAfrica(Ethiopia2).TheZambiapopulationshowsasubstructureasa
resultofadmixture.AllZambianindividualsarepartiallyassignedtotheSouthernAfricaclus-
ter,anddependingontheindividual,eithertoWest/CentralAfrica,ortoEastAfrica.The
admixedsignaloftheZambiapopulationisalsovisiblebythecentralpositionintheplotofthe
firsttwoaxesofthePCAwhenIndiaisexcluded(Fig3B).STRUCTURErunswererepeated
excludingIndiangenotypes,sincePCAillustratedtheeffectofIndia(Fig3A)anditisknown
thatSTRUCTUREhasthetendencytoforceclusteringininappropriatelysmallnumberof
clustersundercertaincircumstances[45].Thismaybethecaseifasinglepopulationcontains
markedlylessgeneticdiversitywhichdrivestheprogramtoplaceallremainingpopulations
intoasingleclustertherebyprovidingaresultwhichdoesnotproperlyreflecttheevolutionary
history[45].Theseanalysesdidnotleadtoadifferenceinclusteringoftheremainingindividu-
alsandthesamethreegroupswereidentifiedwithinAfrica(datanotshown).
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Fig3.ResultsofPCAbasedon20microsatellitelocioflionpopulations.A:resultsofPCAof12
populations(Dataset1,excludingChadandEthiopia2),showninatwo-dimensionalplotandatable
indicatingthepercentageandthecumulativepercentageexplainedbythefirstthreeaxes;B:ResultsofPCA
of11populations,excludingIndia.
doi:10.1371/journal.pone.0137975.g003
Atotalof87sequencesof1,454bpwereanalyzed.Nucleotidediversity(π)was0.102.Based
on43polymorphicsites,15differenthaplotypesweredistinguished.Thehaplotypenetwork
(Fig4A)andtheneighbour-joiningtree(Fig4B)basedonthemicrosatellitedatashowasimi-
lartopologyinwhichWest/CentralAfricanlionsaregroupedtogetheronasupportedbranch
(bootstrapvalue>70)andEastandSouthernAfricanlionsarerepresentedontwodifferent
supportedbranches(Fig4B).AbasalsplitintoaNorthgroup(West/CentralAfricaandIndia)
andaSouthgroup(EastAfricaandSouthernAfrica)ismostclearlyvisibleinthehaplotype
network,astheclusteringofEastAfricaandSouthernAfricaonaSouthbranchinthephenetic
treehasonlylimitedsupport.Furthermore,KenyaandIndiabothhaveabasalandunresolved
positioninthetree.
ResultsfromAMOVAofthemicrosatellitedatashowthatusingtheclustersidentifiedby
STRUCTUREtoassignpopulationstofourgroupsresultedinarelativelyhighpercentageof
themolecularvariancebeingattributedtoamong-groupsformicrosatellitedata(17.4%)and
mtDNAdata(52.3%)(S4Table).Whileinthemicrosatellitedatathehighestpercentage
(29.6%)ofmolecularvarianceinamong-groupsvarianceisattributedtothesplitbetween
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Fig4.RelationshipbetweenpopulationsoflionsbasedonmtDNAdataandon20microsatelliteloci.
A:Haplotypenetworkbasedonmedian-joiningalgorithminNetwork;B:PhenetictreebasedonD genetic
A
distanceofmicrosatellitedataof12lionpopulations.
doi:10.1371/journal.pone.0137975.g004
AfricaandAsia,i.e.betweenthetwosubspecies,nomolecularvarianceamong-groupsforthe
Africa/AsiadivisionisfoundinthemtDNAdata.Inaddition,followingthebasalsplitina
NorthgroupandaSouthgroup,AMOVAattributes54.6%ofmolecularvariancetoamong-
groupsvarianceformtDNAdata,butonlyfinds7.5%inamong-groupsvariancewhenusing
microsatellitedata.However,absolutepercentagesmaybemisleading,aswithin-population
varianceisverydifferentamongsttheusedmarkers.
Manteltestsshowedthattheeffectofisolationbydistanceisevident,bothonthecontinen-
talandtheregionalscale(summaryandgraphsinS5Table).Inregionalanalyses,thehighest
valuesforamong-groupsvarianceaccordingtoAMOVAandthehighestnumbersforthe
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slopeofthetrendlineinIBDarefoundinWest/CentralAfrica(comparedtotheSouthgroup,
EastAfricaorSouthernAfrica)suggestingstrongisolationbetweenthesepopulations.Pairwise
F valuesrangedfrom0.064to0.736andweresignificantforallpairwisecomparisons
ST
(50,000permutations,P<0.05)(S6Table).WithinAfrica,pairwiseF valuesrangedfrom
ST
0.064to0.396.Nei’sgeneticdistancerangedfrom0.196to2.193foralllionpopulationsand
withinAfricaitrangedfrom0.196to2.018(S6Table).
Diversityindices(S7Table)showthattheIndianpopulationcomprisesthelowestnumber
ofmicrosatelliteallelesperlocus,smallestallelicrangeandthehighestnumberoffixedalleles.
IntheIndianpopulation,75%ofthelociarefixedwhileinallotherpopulationsatmaximum
17%ofthelociarefixed.DiversityindiceswerefoundtoberelativelyconstantacrosstheAfri-
canpopulations;surprisinglyWest/CentralAfricashowednoclearsignsoflossofgenetic
diversity.FouroutofsevenpopulationsinWest/CentralAfricacontainedmorethanonehap-
lotype(Cameroon1,Cameroon2,Chad,DRC),whereasthiswasonlyobservedfortwooutof
eightpopulationsinEastandSouthernAfrica(ZambiaandRSA2).Observedandexpectedhet-
erozygosityvaluesfurtherconfirmedthelowgeneticdiversityoftheIndianpopulation.F val-
IS
uesillustratedasignificantheterozygosityexcessinBenin(P<0.01)andCameroon1(P<0.01)
andasignificantheterozygositydeficiencyinZambia(P<0.01),RSA1(P<0.05)andEthiopia1
(P<0.05).Resultsofthebottleneckanalysisshowedthattherewasasignificantexcessofhet-
erozygotesfoundinCameroon1(P<0.01),Kenya(P<0.05)andEthiopia1(P<0.05),possibly
indicatingarecentreductioninpopulationsize.
Discussion
HerewedescribethedistinctpositionoflionsinWest/CentralAfrica,comparedtootherpop-
ulationsacrossthelion’scurrentgeographicrange,basedonphylogeneticanalysesofmicrosat-
elliteandmtDNAdatasets.Moreover,weassessedlevelsofgeneticdiversityacrossdifferent
geographicscalestodetectsignsoflowgeneticdiversity.
Analysisofmicrosatellitedata(STRUCTURE)identifiedthreeclustersintheAfricanlion:
1)West/CentralAfrica,2)EastAfrica,and3)SouthernAfrica,inadditiontoaclustercompris-
ingtheAsiaticsubspecies.AlthoughthehighleveloffixationofallelesintheAsiaticlionis
likelytocontributetotheidentificationofthispopulationasadistinctcluster,geneticstructure
isfoundwithintheAfricansubspecies.Thissupportsthegeneticallydistinctpositionoflions
fromWest/CentralAfricareportedpreviouslyandfoundagainherebasedonmtDNAdata
[2,3,9].Inaddition,STRUCTUREalsoindicatesdivergencewithintheEastandSouthernAfri-
canlions.TheobservedsplitbetweenEastandSouthernAfrica,aswaspreviouslyshownby
Brucheetal.(2012),remainedafterinclusionofapopulationfromZambiawhichisgeographi-
callyintermediatebetweenTanzaniaandRSA[12].Brucheetal.(2012)concludedthatthe
Ethiopia1populationformsauniqueclade,asitshowedtobedistinctfromIndia,EastAfrica
andSouthernAfrica[12].Inthisstudy,wedescribethatEthiopia1showsstrongadmixture
withWest/CentralAfricabasedonmicrosatellitedata,whichisfurtherconfirmedbythemito-
chondrialhaplotype.Thisleadstotheconclusionthattheseindividualsdonotformaunique
group,butareinsteadassignedforasubstantialparttoaclusterthatwasnotrepresentedinthe
workbyBrucheetal.(2012)[12].AlthoughtheoriginoftheEthiopia1founderlionsisdis-
puted,itisclaimedthattheyoriginatefromthesouth-westernpartofEthiopia[46]westofthe
RiftValley,whichhaspreviouslybeensuggestedasabarrierforliondispersal[4,5,9,47,48].
TheothercaptiveEthiopianpopulation,Ethiopia2,containsahaplotypethatclusterswithin
theEastAfricagroup.AssessmentofthemicrosatellitedatashowedthatEthiopia2individuals
indeedcontainedastrongersignalfromEastAfrica,comparedtoEthiopia1.Theobserved
admixtureinbothcaptiveEthiopianlionpopulationsmaybeexplainedbythegeographical
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PhylogeographyandGeneticDiversityintheLion
locationofEthiopia,however,human-mediatedtranslocationsarenotuncommoninzooset-
tingsandmayhavecontributedtotheobservedpattern.InZambia,asubstructureinthepopu-
lationisinducedduetothetwodetectedtypesofadmixture:theSouthernAfricaclusteris
admixedeitherwiththeWest/CentralAfricacluster,orwiththeEastAfricacluster.Thesefind-
ingsareparsimoniouswiththegeographicisolationrepresentativeofZambia’sLuangwaValley
whichisanoffshootoftheRiftValleySystem.Theabsenceofamitochondrialhaplotypefrom
outsidetheEastAfricaclusterintheZambianindividualsindicatesthatthepatternofadmix-
tureislikelyduetomale-mediatedgeneflow.
ThemtDNAhaplotypenetworkshowsthesamefourgroupsasidentifiedintheSTRUC-
TUREanalysis:1)West/CentralAfrica,2)EastAfrica,3)SouthernAfricaand4)India.The
deepestsplitinthehaplotypenetworkdistinguishesaNorthgroupconsistingoftheWest/Cen-
tralAfricanliontogetherwiththeAsiaticsubspecies,andaSouthgroupconsistingoflions
fromEastandSouthernAfrica.Withinasinglecountry,onlyoneortwocloselyrelatedhaplo-
typesarefound,withtwoexceptionswheremoredivergenthaplotypesarepresent:1)Ethiopia,
whichcouldbeexplainedbythegeographiclocationofthecountryaspreviouslynoted,and2)
RSA2,likelyduetopasttranslocationstoandamongstsmallreservesinRSA[49].Theneigh-
bour-joiningtree,basedonmicrosatellitedataalsoshowsadistinctionbetweenlionsfrom
West/CentralAfrica,andpopulationsfromEastandSouthernAfrica.Thebasalpositionofthe
IndianandKenyanlionsprobablyresultsfromthelowergeneticdiversityinthesepopulations,
asisindicatedbytherelativelyhighnumberofmonomorphicloci.Elongationofbranchlength
resultingfromapopulationsizereductionhasbeenpreviouslydescribed,especiallyforD asa
A
measureofgeneticdistance[50].Despiteofthis,D iscommonlyacceptedasthemostsuitable
A
measureforinferringphylogeneticrelationships[51,52]and,therefore,hasbeenusedinour
analyses.STRUCTUREandPCAplotsshowthatallpopulationsfromNamibiaandRSAare
assignedtoSouthernAfrica,withamorecentralpositionfortheadmixedZambiapopulation,
whileEastAfricanhaplotypesarefoundinRSA.Thesamediscrepancywaspreviously
describedbyAntunesetal.(2008)andattributedtosex-biasedgeneflow.Tofurtherassess
congruencebetweenmtDNAandautosomalmarkers,aManteltestwasperformedbasedon
correctedNei’sgeneticdistancesforbothdatasets(S8Table).Thisillustratesasignificantrela-
tionship(999permutations,P<0.01)betweenbothmeasures,whichincreasesfurtherafterthe
exclusionofIndia.StrongestcongruenceinAMOVAresultsbetweentheautosomaland
mtDNAdataarefoundwhenusingthegroupsidentifiedbySTRUCTURE,indicatingarobust
phylogeneticpatternthatisreflectedbybothgeneticmarkers.
ThefourlineageswedescribebasedonautosomalandmtDNAdataarefurthercorrobo-
ratedbythedistinctionoffourgroupsbasedonmorphologicaldata[7].Uptoeight“subspe-
cies”havebeenusedbysomesources[53],withtheBarbarylion(P.l.leo)verylikelytobe
extinctandtheCapelion(P.l.melanochaita)apossiblecon(sub)specificwithP.l.krugeri[10].
Oftheremainingsixsubspecies,Hemmer(1974)suggeststonotincludeP.l.bleyenberghi
(SouthWestAfrica)andP.l.azandica(NorthEastDRC)asfullydifferentiatedlineages.The
remainingfoursubspecies,P.l.persicaandP.l.senegalensisinthenorthernpartoftherange,
andP.l.nubicaandP.l.krugeriinthesouthernpartoftherangecorrespondtoIndia,West/
CentralAfrica,EastAfrica,andSouthernAfricarespectively,andreflectthedeepestsplitinthe
haplotypenetwork.Althoughsamplesizewaslimited,theclosegeneticrelationshipofWest/
CentralAfricanlionstotheAsiaticsubspecies,waslaterreconfirmedbycraniometricdata[8].
IBDexplainsthegeneticdistancesonacontinentalscaleandonaregionalscale.Thestrong
slopeofthetrendlineinIBDanalysisforWest/CentralAfrica,comparedtoSouthernandEast
Africa,issuggestiveofnearcompleteisolationbetweenpopulationsintheWest/Central
region.Thisisalsosupportedbythehighamong-groupsvarianceintheAMOVA.Basedon
PLOSONE|DOI:10.1371/journal.pone.0137975 October14,2015 10/15