A century of decline: loss of genetic diversity in a …5 85 number of transboundary conservation initiatives (Naidoo et al., 2012) such as the Kavango-Zambezi 86 transfrontier conservation

1

Acenturyofdecline:lossofgeneticdiversityinasouthernAfricanlion-conservationstronghold1

SimonG.Duresab*,ChrisCarbonea,AndrewJ.Loveridgec,GlynMauded,NeilMidlanee,Ortwin2

Aschenbornf,DadaGottellia3

4

aInstituteofZoology,ZoologicalSocietyofLondon,RegentsPark,London,NW14RY,UK.5

bDepartmentofLifeSciences,ImperialCollegeLondon,Ascot,SL57PY,UK6

cWildlifeConservationResearchUnit,DepartmentofZoology,UniversityofOxford,Recanati-Kaplan7

Centre,TubneyHouse,AbingdonRoad,Tubney,OX135QL,UK8

dKalahariResearchandConservation,Maun,Botswana9

eSingita,CapeTown,SouthAfrica10

fMinistryofEnvironmentandTourism,Windhoek,Namibia11

*Correspondingauthor:[email protected]

13

Acknowledgements14

WethankNHMLondonforaccesstohistoricsamples;theBotswanaDepartmentofWildlifeand15

NationalParksforprovidingresearchandcollectionpermits(EWT8/36/4XIII[35]);DebbiePeak,Rob16

Jackson,Kyleburger,RobynCoetzee,RobertRiggs&BotswanaPredatorConservationTrustfor17

contributingsamples;thestaffatWildernessSafarisBotswana,Chitabe,andMachaba;Crispin18

Sanderson,GrantHuskisson,DaneHawk,RickNelson,ErikVerreynne,AlanWilson,AnnaButterfield19

&JaquesVandeMerweofVisionInternational,WiltonRaats,DominikBauerandKristinaKeschfor20

logisticalandveterinarysupport;AntonvanSchalkwykandHanriEhlersforinvaluablesupportand21

funding;PneuDartforequipment;WildernessWildlifeTrustforfinancialsupport.Wealsothank22

.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted November 21, 2018. . https://doi.org/10.1101/474940doi: bioRxiv preprint

https://doi.org/10.1101/474940

http://creativecommons.org/licenses/by-nc-nd/4.0/

2

JinliangWangforhelpfulcommentsonpreviousdrafts.Allimportandexportpermitsweregranted.23

SGDwassupportedbyaBBSRCCASE-studentship(BB/F017324/1).24

25

Abstract26

Aim27

Thereisadearthofevidencethatdeterminesthegeneticdiversityofpopulationscontainedwithin28

present-dayprotectedareascomparedwiththeirhistoricstatepriortolarge-scalespeciesdeclines,29

makinginferencesaboutaspecies’conservationgeneticstatusdifficulttoassess.Theaimofthis30

paperistodemonstratetheuseofhistoricspecimenstoassessthechangeingeneticdiversityover31

adefinedspatialarea.32

Location33

Likeotherspecies,Africanlionpopulations(Pantheraleo)areundergoingdramaticcontractionsin34

rangeanddeclinesinnumbers,motivatingtheidentificationofanumberoflionconservation35

strongholdsacrossEastandsouthernAfrica.Wefocusononesuchstronghold,theKavango-Zambezi36

transfrontierconservationarea(KAZA)ofBotswana,Namibia,ZambiaandZimbabwe.37

Methods38

Wecomparegeneticdiversitybetweenhistoricalmuseumspecimens,collectedduringthelate19th39

andearly20thcentury,withsamplesfromthemodernextantpopulation.Weuse16microsatellite40

markersandsequence337basepairsofthehypervariablecontrolregion(HVR1)ofthe41

mitochondrialgenome.Weusebootstrapresamplingtoallowforcomparisonsbetweenthehistoric42

andmoderndata. 43

44


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Results45

Weshowthatthegeneticdiversityofthemodernpopulationwasreducedby12%to17%,witha46

reductioninallelicdiversityofapproximately15%,comparedtohistoricpopulations,inaddition47

tohavinglostanumberofmitochondrialhaplotypes.Wealsoidentifyreducedallelicdiversityand48

anumberof‘ghostalleles’inthehistoricalsamplesnolongerpresentintheextantpopulation.49

MainConclusions50

Wearguearapiddeclineinallelicrichnessafter1895suggeststheerosionofgeneticdiversity51

coincideswiththeriseofaEuropeancolonialpresenceandtheoutbreakofrinderpestinthe52

region.Ourresultssupporttheneedtoimprovedconnectivitybetweenprotectedareasinorderto53

preventfurtherlossofgeneticdiversityintheregion.54

55

56

Keywords57

Conservation,landscapegenetics,historicDNA,microsatellites,mitochondrialDNA,Pantheraleo.58

59

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Introduction61

Globally,mammalwildlifepopulationsarereportedtohaveundergonea52%declineinthepasthalf62

century(McRaeetal.,2014),butoverlongertimeperiodstherangesandpopulationdeclineshave63

beenfarmoresevere(Ceballos,2002;Janeckaetal.,2014;Creesetal.,2016).Whilesuchstudies64

focusonlosesinpopulationsizesandspecies’distributions,relativelyfewhaveexploredtemporal65

lossesingeneticdiversity(Leonard,2008),whichmayhavesignificantimpactsonaspecies’abilityto66

respondtoenvironmentalstochasticityandassociatedconservationinterventions(Spielmanetal.,67

2004).68

Severalreviewshighlightinsufficientgeneticdataavailabletodecisionmakersasamajorchallenge69

inconservationgeneticstoday(Frankham,2010;Hobanetal.,2014).Geneticmonitoringof70

individualsandpopulationsovertimewasidentifiedasoneofthemaintopicsinneedofurgent71

attention.Itiscrucialtoestablishbaselinegeneticdiversitymeasuresagainstwhichfuture72

comparisonscanbemadetodemonstratedeclineorrecovery(Jacksonetal.,2011).Tothiseffect73

theuseofancientmuseumsamplesprovideanimportantgenetictooltomeasurewithin-species74

geneticdiversity.Thisinformationinturnwillbeusedtothedevelopmentandimplementationof75

strategiesaimatminimizinggeneticerosionandsafeguardinggeneticdiversity.76

Oneimportantflagshipspeciesthathasundergoneamajordeclineinpopulationsizeand77

geographicrangeisthelion(Pantheraleo)(Baueretal.,2015a).Recentassessmentsofthelion78

populationinAfricaestimatebetweenonly16,500and35,000individualsremain(Riggioetal.,79

2013;Baueretal.,2015b),withanestimateddeclineof42%overthepast21years(Baueretal.,80

2015a;Baueretal.,2015b).Majordeclinesinwildlifepopulationsacrosstheregion,however,have81

alsobeennotedfurtherbackintime(Selous,1908).82

ThedramaticdeclineoftheAfricanlionhasmadetheprotectionoftheremainingpopulationsand83

theimprovementofgeneflowbetweenpopulationsoftheupmostimportanceandhasledtoa84


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numberoftransboundaryconservationinitiatives(Naidooetal.,2012)suchastheKavango-Zambezi85

transfrontierconservationarea(KAZA).ThesizeoftheKAZAregionanditsabilitytosupportalarge86

numberoflionprides,resultsitbeingconsideredoneofthefewremainingstrongholdsforthelion87

population(Cushmanetal.,2015).Whilethispopulation,andtheabilityoflionstodisperselong88

distancesintheregion,maybeenoughtosustainarobustpopulation(Björklund,2003),numbersdo89

notnecessarilyallowustounderstandallaspectsofpopulationstatus.Diminishedpopulationsare90

lesseffectiveateliminatingdeleteriousvariantsthroughselection(Xueetal.,2009;Spielmanetal.,91

2004)makingthemvulnerabletoreducedindividualfitness,thelossofspecies’evolutionary92

potential,anddiminishedecosystemfunctionandresilience.Thereisariskofoverestimatingthe93

potentialformodernpopulationstoresisttheeffectsofdemographicandgeneticstochasticevents94

onsmallpopulationsifgeneticfactorsarenotconsidered.Populationswhichmaybeconsidered95

stablebycontemporaryconservationmanagersmayinfactshowsignsofgeneticerosion,thus96

needinggreaterconservationattention.However,currentlythereisnobaselinegeneticdataforlion97

populationsotherthanfromthemodernpopulations,whicharelikelytohavealsosufferedmajor98

lossesingeneticdiversity(Björklund,2003).99

Attheendofthe19thandbeginningofthe20thcenturylargenumbersofanimalspecimenswere100

beingarchivedinnaturalhistorymuseumsaroundtheworld(e.g.Dollman,1921),includinglions101

shotacrosstheKAZAregion.WiththeadventofmethodstoextractandanalyseDNAfromhistoric102

archivalspecimens(Higuchietal,1984;Leonard,2008)thereistheopportunitytoassessthegenetic103

diversityofpopulationspre-datinganysignificantanthropogenicinfluence.Bycomparinggenetic104

datafrommuseumcollectionswithmodernwildpopulationsfromthesamearea,onecouldassess105

theextenttowhichcurrentlevelsofgeneticvariationhavebeenreduced(Wandeleretal.,2007;106

Gebremedhinetal.,2009).107

Todeterminewhethergeneticdiversityhasdeclinedovertime,wecomparedgeneticdiversity108

betweenhistoricalandmodernlionpopulationsfromtheKAZAregion.WeextractedDNAfrom109


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historicallionsamplestakenfrommuseumspecimensinordertocomparehistoriclevelsofgenetic110

diversityagainstmodernlevelsfromthesameregion.Weusedasuiteofmicrosatellitemarkersas111

wellassequencingofpartofthehypervariablecontrolregion(HVR1)ofthemitochondrialgenome112

(mtDNA)toassessthedegreetowhichgeneticdiversityinthispopulationhasbeenlostasaresult113

ofregionaldeclinesinlionnumbersanddistribution. 114

Methods115

Samples116

TheNaturalHistoryMuseumofLondon’scollectionscontainlargenumbersoflionskinsandskulls117

fromacrossthespeciesrange.Thelabellingofthecollectiondatawasofvaryingqualityso118

specimenswerecross-referencedwithcollectorcatalogueswhereverpossible.Twenty-sevenlion119

specimensweresampled,originallycollectedfromwithinthestudyregionbetween1879until1935120

(Table1,Fig.1).Scrapesofanytissueremainingontheskullsorskin,orfragmentsofdetached121

maxilloturbinalbone(thinbonesinsidethenasalcavities)werecollectedfromeachspecimen.122

Modernsampleswerecollectedfrom204freerangingwildlionsbetween2010until2013(Fig.1)in123

theformofblood(n=23),freshtissue(n=113),drytissue(n=13),faecal(n=14)andhair-pulls(n=41).124

Freshtissuesampleswerecollectedusingaremotebiopsydartsystem(Kareshetal.,1987).Hair125

pullsandbloodweretakenfromimmobilisedanimals.Drytissuesamplesweretakenfromanimals126

shotbythetrophyhuntingindustry.127

128

AncientDNAprecautions129

Allpre-PCRworkwasperformedinalaboratoryexclusivelydevotedtoancientDNA,situatedona130

differentfloorfromthePCRamplificationlaboratoryandwithanindependentairhandlingsystem.131

Toavoidsamplecross-contaminationadifferentsetofequipmentwasusedforeachextraction(e.g.132

mortarandpestle,scalpelbladesetc).Single-useequipmentwasimmersedinsodiumhypochlorite133


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andremovedfromtheworkingareaafteruse.Theworkingareawascleanedwithsodium134

hypochloritesolutionbeforeworkonthenextsamplecommenced.AllequipmentwasUV-irradiated135

overnightpriortofurtheruse.Filtertipswereusedtoreducecrosscontamination(Rohland&136

Hofreiter,2007).Twoblankextractionscontainingnotissueorbonewereincludedduringboth137

extractionprotocolstoserveasnegativeextractionandPCRcontrols.Eachfragmentwas138

independentlyamplifiedbyPCRatleastthreetimesfollowingthemulti-tubeapproach(Taberletet139

al.,1999)inanattempttodetectcontaminationandgenotypingerrors.140

141

DNAextraction142

TotalgenomicDNAwasextractedfromeachmuseumskinsampleusingapproximately25mgof143

tissueusingDNeasy®BloodandTissuekits(Qiagen).Wefollowedthemanufacturer’sinstructions144

butaddedasecondincubation.Toincreasetissuelysisthefirstincubationwasrunovernight,forthe145

seconddigestionweaddedafurther180µlBufferATLand20µlproteinaseK(600mAU/ml)andthen146

incubatedforafurther3hoursat56°C.147

DNAfrombonesampleswasextractedusingapproximately100mgofbonepowderpreviously148

groundinapestleandmortar.Amastermixwaspreparedwhich,foreachsample,comprisedof149

0.2ml10%SDS(Invitrogen),0.15mlproteinaseKat15mg/µl,a1x1mmpieceofDTTat10mMand150

1.65mlEDTAofpH8.0at0.5M.Thiswaswarmedat56°Cuntilallingredientsdissolvedandaddedto151

eachbone-powdersample.Sampleswereincubatedonarotatorat56°Cfor48hours.Following152

digestion,tubeswerecentrifugedforoneminuteat1300rpmandsupernatanttransferredtoan153

Amicon®MilliporeUltraCentrifugefilterwhichwascentrifugedfor30minutesat1300rpm.A154

MinElutepurificationkit(Qiagen)wasusedtopurify100µlofextractfollowingthemanufacturer’s155

instructions,washingthreetimeswithPEbuffer.156


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ModernDNAwasextractedusingapproximately25mgoftissue,100μlofrawbloodor5-6hair157

folliclesusingDNeasy®BloodandTissuekits(Qiagen)accordingtothemanufacturer’sinstructions.158

FaecalDNAwasextractedusingapproximately200mgofstoolusingQIAamp®DNAStoolkits159

(Qiagen)accordingtothemanufacturer’sinstructions.160

161

Microsatelliteamplification162

Weusedsixteenmicrosatellitelocipreviouslyidentifiedandamplifiedinthedomesticcat(Menotti-163

Raymondetal.,1999)(FCA1,FCA45,FCA69,FCA75,FCA77,FCA96,FCA97,F115,FCA126,FCA129,164

FCA133,FCA193,FCA205,FCA224,FCA247,FCA391)whichhavepreviouslybeensuccessfullyusedin165

lions(C.Driscoll,1992;C.A.Driscolletal.,2002;Dubachetal.,2013;Lykeetal.,2013;Spong&166

Creel,2001).Thenuclearmarkerprimersweredividedintomultiplexcombinationsandfluoro-167

labelledwithoneofVIC,6-FAM,PETorNEDdyes,accordingtoprimerannealingtemperaturesand168

non-overlappingallelesizerangecombinations(seeSupplementarymaterials).SeeSupplementary169

Materialforamplificationconditionsandsequencingdetails.Theallelesizesandgenotypeswere170

scoredinGENEMAPPERv4.1(AppliedBiosystems).171

172

Mitochondrialsequencing173

Weamplifieda337bphypervariableregion(HVR1)ofthePantheraleomitochondrialcontrolregion,174

usingpreviouslypublishedreverseandforwardprimers(Barnettetal.,2006).Toimprovethequality175

ofthesequencingandavoidtheproblemofdoublebandingduetothereverseprimerbeingableto176

bindtomultiplereversesequencerepeats,identifiedpreviouslywiththeseprimers,weuseda177

nestedreverseprimerdesignedfordirectsequencing(Barnettetal.,2006).SeeSupplementary178

InformationforPCRandsequencingconditions.Consensussequenceforeachindividualwas179


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obtainedthroughalignmentoftheforwardandreversesequencesinGENEIOUS(Kearseetal.,2012)180

toyieldaminimumof2xcoverageforeachbase.181

182

Estimationofchangeinnucleardiversity183

Todetectchangesinnucleardiversitybetweenthemodernandhistoricpopulations,usingthe184

microsatellitedata,wecalculatedNei’sunbiasedestimateofexpectedheterozygosity(HE),observed185

heterozygosity(HO),inbreedingcoefficient(FIS)andmeannumberofallelesperlocus(A).Thiswas186

performedusingGENEPOP(Rousset,2008)usingmethodsdocumentedinpreviousresearchon187

white-tailedeagles(Haileretal.,2006).GENEPOPwasalsousedtodetectsignificantdepartures188

fromHardy-Weinburgequilibrium(HWE)andevidenceoflinkagedisequilibriumwithinthesample189

groups.UniquealleleswereidentifiedforeachtimeperiodusingCONVERT(Glaubitz,2004).The190

meannumberofprivateallelesperlocusfoundineachpopulationwascalculatedusingararefaction191

approachtocontrolfordifferencesinsamplesize,implementedinADZEetal.,2008).DnaSpv.5192

(Librado&Rozas,2009)wasusedtocalculatemtDNAhaplotypediversity(H)andnucleotide193

diversity(π),aswellasTajima’sDtotestfordeviationsfromneutralevolutionforboththemodern194

andhistoricpopulations.195

196

Bootstrapresampling197

Thereisaninherentinabilitytocontrolthesamplingdesignwhenusingmuseumcollections,198

includingsamplesize,dateandlocationoftheircollection.Toallowcomparisonsbetweenmodern199

andhistoricnucleicdiversityweusedabootstrappingprocedure.Whenanalysingthemorerapidly200

mutatingnuclearmicrosatellitedata,weprogressivelyrestricted;i)thespatialextentofthehistoric201

samples,tomatchwithmorecertaintytheextentofthemodernsamples;ii)thetimeperiodover202

whichthehistoricsampleswerecollected,torestrictthepossibleinfluenceofgeneticdriftwithtime203


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withinthesampleset.Thus,wedividedourhistoricdataintothreespatialzonesrepresenting;I)the204

sampleswithinthemodernsamplingarea;II)thesampleslikelytobewithinmaledispersing205

distanceofthemodernsamplingarea,takenas200km;III)allremainingsamplesacrosstheregion206

(Table1).Wealsodividedthehistoricdataintotwotimeperiods,1874-1895(A)and1929-1935(B)207

(Table1).Theresultsfromthehistoricsamplessetswerecomparedagainstourmoderndataset208

usingabootstrappingprocedureimplementedinPOPTOOLS(Hood,2011).Wecreated100209

populationsofequalsizetothehistoricdatabeingused.Furthermore,toaccountforanapparent210

lackofhistoricsamplingfromwithintheOkavangoDeltabootstrapsamplingwasrepeatedbothwith211

andwithoutmodernOkavangoDeltasamples.Inaspeciessuchaslions,wherefemalesiblingstend212

toremaininthesameprideorformaneighbouringprideandmalesiblingscommonlyforgea213

coalition,thelikelihoodofcollectingdatafromcloserelativeswashigh.Totestfortheeffectsof214

closerelatives,wefollowedtherecommendationsofRodríguez-Ramilo&Wang(2012)and215

calculatedallpossiblefull-siblingandparent-offspringclustersintheprogrammeCOLONY(Wang&216

Scribner,2014).Wethenrandomlyselectedjustoneindividualfromeachclose-relativecluster,217

beforere-rerunningthebootstrapprocedureonthereduceddataset.218

219

Mitochondrial‘ghost’alleles220

Followingtheidentificationofallhaplotypespresentinthecombinedmodernandhistoricdataset,221

wewereabletoassessprivatehaplotypesonlypresentinoneorothertimeperiod.Duetothemuch222

poorerqualityofthemuseumsampledatamanysequenceswereconsiderablyshorterthanthe223

moderncounterparts,makingdirectcomparisonsofdiversitydifficultandlackingpower.However,224

wewereabletoidentifyhaplotypesonlypresentinthehistoricdata,likelytohavebeenlostfrom225

themodernpopulation(Leonardetal.,2005).226

227


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Results228

Weachievedsuccessfulmicrosatelliteamplificationofall27museumsamplesandobtainedusable229

mitochondrialsequencesfrom18ofthese.Anumberofmicrosatellitelocicouldnotbesuccessfully230

genotypedacrosseverysample,achievingameanof23.7(s.d.±3.5)completegenotypesperlocus231

(DataavailableonFigshare,DOI10.6084/m9.figshare.3514469).Nosinglelocusorwithingroup232

deviationsfromHWEweredetectedandtestsforlinkagedisequilibriumwerenotsignificantafter233

Bonferronicorrection.Mitochondrialconsensussequencelengthsvariedfrombetween204to234

322bp,acrossa337bpregion(GenBankAccessionno.KX661326-KX661331).235

Ineverybootstrapcombinationofourmicrosatellitedata,regardlessofhowmanysampleswere236

excluded,thehistoriclionpopulationexhibitedahigherheterozygosity,bothobserved(t=8.75,p=237

0.006)andexpected(t=14.80,p=0.002).Thesameresultsforreducedheterozygositywere238

returnedwhentheOkavangolionswereremovedfromtheanalysis(observed,t=8.75,p=0.006;239

expected,t=14.79,p=0.002).240

Ineveryiterationofthedatathemodernpopulationshowedamuchgreaterdeficiencyinthe241

observedheterozygositycomparedtotheexpected,representedbyasignificantlylargerinbreeding242

coefficient(FIS)forallmodernsamplesets(t=5.42,p=0.016;Table2).Thereductioninthe243

geographicextentofthehistoricdataresultedinalimitedchangeintheobservedheterozygosity244

from0.7565forthebroadestsampleset,upto0.7975forthemostlimited.Whenwecontrolfor245

differencesinsamplesize(n=27vs.12)using100bootstrapreplicationstheobservedheterozygosity246

forthefullsamplesetofzonesI-IIIincreasedfrom0.7565to0.7612,similartolevelsobserved247

amongthemorespatiallyrestricteddataencompassingjustzonesIandII.248

Acrossthedataweidentified29allelespresentonlyinthehistoricsamplesand54privatealleles249

onlyfoundinthemoderndata,howeverthelattercomefromamuchlargerdataset.Themean250

numberofprivateallelesisconsistentlyhigherinthehistoricdatathaninthemoderndatawhen251


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controllingforsamplesize(Fig.2).Such‘ghostalleles’(Bouzartetal.,1998;Groombridgeetal.,252

2000)wereidentifiedin14outofthe16microsatellitemarkers,onlyabsentfromFca126and253

Fca391.Evenwhenreducingthehistoricdatatoonlythosewithinthemostconservativespatialarea254

(n=13)westillfound18allelesnotpresentinthemodernsamples,spreadacrossallmicrosatellite255

markersexceptFca126,Fca129,Fca193andFca391.256

Removingsamplescollectedbetween1929-1935madenodifferencetoheterozygosityacrossthe257

data(seeSupplementarymaterials),however,itdidresultinareductionintheallelicrichnessfrom258

7.5to6.29,thelatterbeingsimilartothepresentdayvalues.Whenwereducedthedatatoinclude259

onlysamplescollectedbetween1929-1935,theallelicrichness(5.88)closelymatchesthatfound260

withinthemodernsamples.261

Removingcloserelativeshadanegligibleeffectonanyvalues.Inthefullmoderndatasetthe262

observedheterozygosityincreasedfrom0.6541to0.6570,expectedheterozygosityfrom0.6989to263

0.7039,theinbreedingcoefficientfrom0.064to0.066andthemeannumberofallelesfrom6.55to264

6.65.265

ThemtDNAdata(Table3)indicatessixhaplotypespresentwithinthehistoricdataset(H=0.6993,π266

=0.00065),butthreeoftheseappeartobemissingfromtheextantlions(H=0.3257,π=0.0007).267

Tajima’sDforboththehistoric(D=-1.09629;p<0.1)andmodern(D=-0.53568,p<0.1)population268

arenegativebutnotsignificant,suggestingnodeviationfromneutrality.Asidefromthethree‘ghost’269

haplotypesidentified,theremaybeotherspresentwithinthesamemtDNAregionthatduetothe270

degradationofthehistoricDNAremainunidentified.Sincetwoofthe‘ghost’haplotypeswere271

identifiedfromsingleindividuals,eachonlywithasinglenucleotideinsertion,wemustcautionthat272

theymaybefalsehaplotypescausedbyDNAdegradation(Wandeleretal.,2007).Evenfollowinga273

moreconservativeapproach,onepreviouslycommonhaplotyperemainsunrepresentedinthe274

modernsamples.275


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276

Discussion277

Thevalueofgeneticdiversityisincreasinglyrecognizedforcontributingtoindividualfitness,species’278

evolutionarypotential,andecosystemfunctionandresilience(Whithametal.2008).Thereis279

thereforeanurgentneedforpolicy-relevantstudiestohelpdefinesensitiveandrobustindicatorsof280

changesingeneticdiversity(Hobanetal.2013a).281

OuranalysisdemonstratesthatoverthepastcenturythelionpopulationoftheKavango-Zambezi282

regionhaslostgeneticdiversity.Contemporaryobservedheterozygosityhasbeenreducedby12%283

to17%comparedtohistoricpopulations.Despitehavinganumberofmissingallelesacrossthe284

samples,geneticdiversitywasstillhistoricallyhigherthaninthecontemporarylionpopulation.The285

declineinheterozygosityisnotasdramaticasthatseeninsomehighlythreatenedorbottlenecked286

species,forexample,57%intheMauritiuskestrel(Falcopunctatus)(Groombridgeetal.,2000)or287

43%inseaotters(Enhydralutris)(Larsonetal.,2002),itneverthelessrepresentsaworrying288

reductionindiversityconsideringthispopulationisoneofonlysixlionstrongholdsremainingin289

Africa.290

Whilethelowsamplesizeofthebootstrappingmeanscautionshouldbetakenbeforeextrapolating291

tothetrueFIS,itisclearthatthereducedheterozygosityexposeslionsoftheregiontoahigherrisk292

ofinbreedingdepressionthantheirhistoriccounterparts.Aswellascleardeclineinnuclear293

diversity,asassessedwiththemicrosatelliteanalysis,thereisalsoanindicationofalossin294

mitochondrialdiversity.Onehaplotypedetectedinmultiplehistoricsamples,andtwomore295

haplotypesdetectedinsinglesamples,remainentirelyundetectedinthemodernpopulation.The296

resultsareinagreementwithpreviousresearchwhichhasidentifiedbothdecliningpopulationsand297

increasingfragmentationintheregion(Elliotetal.,2014;Loveridgeetal.,2007).298


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Similartootherspecies,theglobaldeclineinlionnumbershaslargelybeendrivenbyhuman-wildlife299

conflictandhabitatloss(Keyghobadietal.,2005;Baueretal.,2015b).Giventherapidexpansionof300

humanactivitiesintheregioninthe20thcentury,thedownwardtrendingeneticdiversitywe301

observedisperhapsunsurprisingandseeminglyconfirmsthepessimisticobservationsmadeinthe302

late19thcentury.Forexample,oneaccountfromFrederickCourtneySelousrecords,“Duringthe303

twentyyearssincemyfirstarrivalin1871,I…hadseengameofallkindsgraduallydecreaseand304

dwindleinnumberstosuchanextentthatIthoughtthatnowheresouthoftheGreatLakescould305

therebeacornerofAfricaleftwherethewildanimalshadnotbeenverymuchthinnedout”(Selous,306

1908).Interestingly,allelicrichnessdidnotdifferbetweentheintermediatetemporal(1929-1935)307

andcontemporarypopulationsamples,suggestingthatallelicrichnesswaslostpriortothe308

intermediatesamplingperiod.Atemporaldeclineingeneticdiversityofthehistoricsampleswas309

notdetectedthroughmeasuresofheterozygosity,likelyduetochangesinallelicrichnessbeing310

detectablebeforepopulationdeclinesimpactuponheterozygosity(Athreyetal.,2011).Therapid311

declineobservedinallelicrichnessdoescoincidewiththearrivalofthefirstwesternsettlersin1890312

andthesubsequentriseofthecolonialpresenceintheregionaftertheendoftheMatabeleWarsin313

1897(Parsons,1993).Furthermore,modernfirearmsbecamemoreprevalentfollowingEuropean314

settlementandpredatorswereoftenpersecutedasvermin(Woodroffe,2000),whichlikely315

contributedtotheearlierdeclineoflionsinthestudyregion.Whilstthetimingofgeneticdecline316

andcolonialsettlementiscompellingenoughtosuggestcausation,theevidenceisnotconclusive.317

Theepizooticoftherinderpestvirusalsostruckduringthelate1890’sresultinginthedeathofvast318

populationsofbuffalo,giraffeandwildebeest,aswellasdomesticlivestock(VandenBosscheetal.319

2010).Suchanepidemicisverylikelytohavealsohadaconsiderableimpactonthepredatorsofthe320

region.321

Giventhelevelofhabitatlossandfragmentationobservedacrosssub-SaharanAfrica(Keyghobadiet322

al.,2005;Baueretal.,2015b),theincreasedthreatofepizooticsfacilitatedbyhumanmovements323

(Butleretal.,2004),aswellastheimpactsofachangingclimate(Thomasetal.2004),itis324


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imperativethateffortsaremadetoconservegeneticdiversity.Withoutsuchgeneticdiversity,a325

speciesresilienceandabilitytoadapttofuturestochasticeventsbecomesgreatlycompromised326

(Whithametal.2008).Thisstudyprovidesquantitativedataontemporalgeneticmonitoringthatis327

urgentlyneededtooptimizeconservationandmanagementefforts.SinceKAZAisconsideredoneof328

themorestablelionpopulationsinAfrica,theworkpresentedhereshouldprovidemotivationfor329

increasedconservationactiontosafeguardagainstfurtherlossofgeneticdiversityoflionsandother330

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populationinprotectedareasacrosstheregionandthusthemixingofgeneticmaterialshouldbe332

supported(Cushmanetal.,2015).333

334

335

336


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476

DataAccessibilityStatement477

MicrosatellitedataisavailableatFigshare,DOI10.6084/m9.figshare.3514469478

MitochondrialsequencedatahasbeensubmittedtotheGenBankdatabaseunderaccessionno.479

KX661326-KX661331.480

481


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Table1MuseumsamplesfromtheNaturalHistoryMuseumofLondongroupedaccordingtothreespatialzones.Sample482Numberreferstopositiononfigure1.Spatialzonesrepresent;I)sampleswithinthemodernsamplingarea;II)thesamples483likelytobewithinmaximummaledispersingdistanceofthemodernsamplingarea,takenas200km;III)allremaining484samplesacrosstheregion.Timeperiodsrepresentsamplescollectedbetween;A)1874to1895;B)1930-1935.Unclear485datesuseaccessionnumberasdatereference.Longitudeandlatitudeareestimatedbasedonlocationdataavailablefor486eachspecimen.487

Sample

Number

Accession

number

Collection

date

Time

period Collectionlocation

Approximate

longitude

Approximate

Latitude

Zone

I

1 19.7.15.21 1879 A Mababe 24.33 -19.12

2 19.7.15.22 1879 A Mababe 24.19 -18.99

3 19.7.15.23 1879 A Mababe 24.03 -19.14

4 19.7.15.24 1879 A Mababeplain 24.36 -18.84

5 19.7.15.25 1879 A Botetiriver 24.37 -20.80

6 19.7.15.27 1879 A Linyanti-ChobeNorthbank 23.76 -18.46

7 19.7.15.15 1884 A NorthernKalahari-Botswana 23.56 -20.43

8 31.2.1.4 1930 B Mababeflats/Mogogeloriver 23.96 -18.89

9 31.2.1.4a 1930 B Mababeflats/Mogogeloriver 23.74 -19.75

10 31.2.1.5 1930 B Mababeflats/Mogogeloriver 24.15 -18.62

11 31.2.1.5a 1930 B Mababeflats/Mogogeloriver 23.87 -19.55

12 31.2.14.6 approx.1930 B Kabulubula60milesWestof

Livingstone

24.88 -18.03

Zone

II

13 19.7.15.29 1874 A

Uppertatuiriver-Zimbabwe/Botswana

bordernearFrancestown 27.14 -20.81

14 19.7.15.31 1880 A Umfuliriver-North-centralZimbabwe 28.21 -17.46

15 19.7.15.26 1882 A Mashonaland-NorthZimbabwe

approx.200milesWestofHarare

27.97 -18.42

16 19.7.15.14 1883 A Mashonaland-approx200milesWest

ofHarare

28.23 -18.82

17 19.7.15.30 1886 A 20milesSouthofBulawayo 28.48 -20.76

18 35.3.16.1 unknown,

1935?

B NorthWestRhodesia-Solwezidistrict 25.84 -13.39

19 35.3.16.2 unknown,

1935?

B NorthWestRhodesia-Solwezidistrict 26.26 -12.86

Zone

III

20 19.7.15.17 1880 A

GwabiriverNorthernZimbabweon

Zambiaborder 27.94 -15.89

21 19.7.15.17a 1880 A



22 19.7.15.18 1880 A



23 93.5.21.1 1893 A Botswana poordata

24 79.2188 1895 A Botswana poordata

25 1887.5.16.2 1887 A SebakweRiverMashunaZimbabwe 30.95 -21.19

26 19.7.15.32 1891 A Hartleyhills,nearHarare 30.42 -18.07

27 35.9.1.129 1929 B KarakuwiriGrootfontain 18.42 -19.51aAccessionnumberusedasecondtimefortwodifferentsamples.488


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489

Table2GeneticdiversityfortheKavango-ZambziAfricanlionpopulationwithineachspatialscalefor16microsatelliteloci.490Modernsamplesrepresenttheaveragevaluefrom100bootstrapreplicationsincludingorexcludingtheOkavangosamples491respectively.N=samplesize;HE=expectedheterozygosityHO=observedheterozygosity;FIS=inbreedingcoefficient;A=492meannumberofallelesperlocus;s.d.=standarddeviationofbootstrapreplications.493

Sampleset N HE s.d. HO s.d. FIS A s.d.

Zone

I-III Historic 27 0.7813 - 0.7565 - 0.032 8.50 -

Modern 27 0.6989 (0.014) 0.6541 (0.025) 0.064 6.55 (0.37)

Modern-withoutOkavango 27 0.7186 (0.013) 0.6688 (0.020) 0.069 7.00 (0.37)

Zone

I-II

Historic 19 0.7807 - 0.7676 - 0.017 7.69 -

Modern 19 0.6928 (0.017) 0.6483 (0.025) 0.064 6.23 (0.35)


Zone

I

Historic 12 0.7945 - 0.7975 - -0.004 6.75 -

Modern 12 0.6946 (0.023) 0.6523 (0.034) 0.061 5.39 (0.30)


494

495

Table3MitochondrialDNAcontrolregionhaplotypesfromhistoricalspecimensandtheextantlionpopulationoftheKAZA496region.“-“and“N/A”representadeletionormissingsequencedata,respectively,atthespecifiednucleotideposition.497

Samplesize Haplotype Variablenucleotidepositiona

Modern Historic 221 343 367 368 378

31 5 i - - T A -

9 ii T - T A -

1 iii N/A - T A C

1 iv N/A C T A -

3 1 v - - C A -

4 1 vi - T T G -

a1correspondstoposition16,176inthecompletePatheraleoleomtDNAsequence(Ma&Wang,2014).498499

500

501

502


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Figure1MapofKavango-Zambeziregionshowingsamplingdistributionofmodernlionsamples(redcircles)andmuseum503

samples(bluetrianglesandnumbered)504

Figure2Meannumberofprivateallelesperlocusasafunctionofstandardisedsamplesizeforhistoricandmodern505

microsatellitedata.506

507

508Figure1509


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510Figure2511


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Documents

A century of decline: loss of genetic diversity in a …5 85 number of transboundary conservation initiatives (Naidoo et al., 2012) such as the Kavango-Zambezi 86 transfrontier conservation