Understanding the cryptic introgression and mixed ancestry of Red Junglefowl in India

19 Pages·2017·8.46 MB·English

RESEARCHARTICLE
Understanding the cryptic introgression and
mixed ancestry of Red Junglefowl in India
MukeshThakurID1,2¤a*,MerwynFernandes1¤b,SambandamSathyakumar1*,Sujeet
K.SinghID1,RameshKumarVijh3,JianlinHan4,5,Dong-DongWu2,Ya-PingZhang2
1 WildlifeInstituteofIndia,Chandrabani,Dehradun,Uttarakhand,India,2 StateKeyLaboratoryofGenetic
ResourcesandEvolutionandYunnanLaboratoryofMolecularBiologyofDomesticAnimals,Kunming
InstituteofZoology,ChineseAcademyofSciences,Kunming,Yunnan,P.R.China,3 ICAR-NationalBureau
ofAnimalGeneticResources(NBAGR),G.T.RoadByePass,NearBasantVihar,Karnal,Haryana,India,
4 CAAS—ILRIJointLaboratoryonLivestockandForageGeneticResources,InstituteofAnimalScience,
ChineseAcademyofAgriculturalSciences(CAAS),Beijing,P.R.China,5 InternationalLivestockResearch
Institute(ILRI),Nairobi,Kenya
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¤a Currentaddress:ZoologicalSurveyofIndia,NewAlipore,Kolkata,WestBengal,India.
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¤b Currentaddress:TRAFFIC,C/oWWFIndiaSecretariat,172-B,LodhiEstate,NewDelhi,India.
a1111111111 *[email protected](MT);[email protected](SS)
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Abstract
RedJunglefowls(RJFs),thewildprogenitorofmoderndaychickens(DCs),arebelievedto
OPENACCESS
beingeneticendangermentduetointrogressionofdomesticgenesthroughopportunistic
Citation:ThakurM,FernandesM,SathyakumarS,
matingswithdomesticorferalchickens.PreviousstudiesfromIndiareportedrarehybridiza-
SinghSK,VijhRK,HanJ,etal.(2018)
tionofRJFsinthewild.However,RJFpopulationgeneticstructure,patternofgeneflowand
Understandingthecrypticintrogressionandmixed
ancestryofRedJunglefowlinIndia.PLoSONE13 theiradmixturewithDCpopulationsarepoorlyunderstoodatthelandscapelevel.Wecon-
(10):e0204351.https://doi.org/10.1371/journal. ductedthisstudywithalargesamplesize,coveringthepredictednaturaldistributionrange
pone.0204351
ofRJFsinIndia.WedocumentedstrongevidenceofdirectionalgeneflowfromDCstofree-
Editor:GyaneshwerChaubey,BanarasHindu rangingwildRJFs,withtheNortheasternRJFpopulationexhibitingthemostgeneticvari-
University,INDIA
antsintheirnuclearandmitochondrialgenomes,indicatingittobetheancestralpopulation
Received:April20,2018 fromwhichearlyradiationmayhaveoccurred.Theresultsprovideevidencethatlandscape
Accepted:September6,2018 featuresdonotactasabarriertogeneflowandthedistributionpatterncouldnotbe
exploredduetophysicalsharingorexchangeofwildbirdsinthepastwhenforestswere
Published:October11,2018
continuousacrossRJFrangeinIndia.
Copyright:©2018Thakuretal.Thisisanopen
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited.
Introduction
DataAvailabilityStatement:Dataavailablefrom
theDryadDigitalRepository:https://doi.org/10. ThepolyphyleticoriginsofDomesticChickens(DCs,Gallusgallusdomesticus)isareasonto
5061/dryad.rv38cp4. speculatethatgeneflowbetweenRJFs(Gallusgallusmurghi)andDCsiswidespreadandmore
frequentthansupposedbypreviousstudies[1,2,3]whiledomesticationmayhaveoccurredat
Funding:Theprojectwasfundedthroughthegrant
multiplelocationsinSouthandSouth-EastAsia[4,5,6,7].However,crypticintrogressionfrom
inaidschemeofWildlifeInstituteofIndia,
Dehradun.FundswereraisedbyDr.S. domesticorferalDCstoRJFsorviceversaandthetransportofDCsamongstdifferentregions
Sathyakumar. obscurethehistoryofthesetwospecies.Severalstudieshavesuggestedphysicalmixingand
geneflowbetweenRJFinthewildandDCpopulations[4,8,9,10,11].Interestingly,Gering
Competinginterests:Theauthorshavedeclared
thatnocompetinginterestsexist. etal.[12]reportedferalisationofKauaichickenthroughinvasivegeneticsandfurtherraised
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GeneticintrogressionofwildRedJunglefowl
theissueof'domesticationinreverse'.Ingeneral,hybridizationintheabsenceofreproductive
isolationisaninevitablephenomenonandcannotbeavoidedincaseswheredomesticand
wildcongenericsaresympatric[13,14,15,16].Thesituationgetscomplexwhenhybridoff-
springsarereproductivelyviableandparticipatesubsequentmatingacrossthespecies.Allen-
dorfetal.[17]statedthat5%orlessproportionofhybridizationinRJFsisaneffectof
admixtureornaturalselectionwhereasanotherstudy,basedlargelyonbirdsrearedincaptivity
andreleasedintothewild,reportedrarehybridizationbetweenRJFsandDCsinthewildin
India[5].Berthoulyetal.[6]postulatedthattheirobservationoflowgeneticexchangemight
beduetosamplingbiasandreportedafairgeneflowfromRJFtolocalVietnamesechicken
populations.
RJFsinIndiaarewidelydistributedacross51x105km2in21States[18].Further,basedon
ourfieldobservationsandmonitoringonRJFsinthewild,weoftenencounteredRJFsand
DCsfeedinginthesameflocksinthevicinityofforesthabitats[19].Webelievethatthethreat
ofhybridizationtoRJFswithDCshasnotbeenaddressedappropriatelyatalandscapelevel.In
addition,theextentofhybridizationbetweenwildRJFsandDCsisstressedtobeofimpor-
tanceintheInternationalUnionforConservationofNature(IUCN)ActionPlanforPheas-
ants(2000).However,IUCNlistingofRJFsas“LeastConcern”,thenon-listingofRJFsonthe
ConventionofInternationalTradeinEndangeredSpeciesofWildFaunaandFlora(CITES)
[20],andthepresentinclusionofRJFsintheWildlife(Protection)Act,1972ofIndiahasno
provisionstoassessthehybridizationthreattothisspeciesdespiteamulti-billiondollarpoultry
industryhasevolvedthroughwildRJF.Recentpoultryepidemics,suchastheoneinHong
Kongin1998andthe‘birdflu’inIndiaandotherpartsofS.E.Asia,couldspelldoomtothe
poultryindustryandtheonlyfallbackoptionthepoultryfarmerswouldeventuallybethe
‘wild’RJF[18].
Further,oneoftheprimarypremisesinpresent-dayconservationprogramsistomaximize
theconservationofgeneticdiversityavailableforpotentialfutureuse.IfhybridizationofRJFs
withandDCsoccurrsandcontinues,itwouldproducepopulationswhichmaynotbevalued
forfuturebreedingandconservationpurposesundertheIUCNguidelines.Consideringthe
importanceofconservationconcerntosafeguardthewildancestorofDCs,weundertookthis
studytoanswertwoimportantquestions:
1. Whetherornot,thethreatofhybridizationandgeneticexchangebetweenRJFandDCin
Indiaissignificantorrareasdocumentedbyearlierstudies.
2. IfsuchhybridizationoccursorhasoccurredinIndiainthepast,whetheritislocalizedwith
specificdistributionpatternandhowdoesitaffectthecurrentpopulationgeneticstructure
ofRJF?
Materialsandmethods
SamplecollectionandDNAextraction
Forsampling,wedividedIndiaintofivezonesbasedontheavailabilityofcontinuoushabitats
forRJFviz.,NorththeStatesofJammuandKashmir,HimachalPradesh,Uttarakhand,Hary-
ana,PunjabandUttarPradesh,CentraltheStatesofMadhyaPradeshandChhattisgarh,East
theStatesofBihar,Jharkhand,WestBengalandSikkim,SoutheasttheStatesofOdishaand
AndhraPradeshandNortheasttheStatesofAssam,ArunachalPradesh,Nagaland,Mizoram,
Manipur,TripuraandMeghalaya.Wecollectedbloodsamplesfrom57wildRJFand79DC
individualsacrossIndia(Fig1).ThewildRJFwerelivetrappedandapproximately500μl
bloodfromeachbirdwaswithdrawnfromthebrachialveinandstoredinDNAzolBD
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GeneticintrogressionofwildRedJunglefowl
Fig1.IndiageographicmapshowingsamplinglocationsofRJFs.
https://doi.org/10.1371/journal.pone.0204351.g001
(InvitrogenTM,Carlsbad,CA,USA).AllDCsampleswerecollectedinthevicinityofwild
RJFsadjacenttotheforests.ThegenomicDNAfromwholebloodwasextractedfollowing
Mackeyetal.[21].
MicrosatellitegenotypingandsequencingofD-loopregion
Wegenotypedsampleswith30microsatelliteloci(ADL0268,MCW0206,LEI0166,
MCW0020,MCW0037,ADL0112,MCW0295,MCW0067,MCW0104,MCW0111,
MCW0034,MCW0222,LEI0094,MCW0216,MCW0081,MCW0330,LEI0234,MCW0103,
MCW0098,MCW0069,MCW0016,MCW0078,MCW0123,MCW0165,MCW0248,
ADL0278,LEI0192,MCW0014,MCW0183andMCW0284).Wescrutinizedtheselocibased
ontheirwidecoverageonthegenomeandhighdegreeofpolymorphism[22,23,24].ThePCR
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GeneticintrogressionofwildRedJunglefowl
compositionandcyclingconditionsfollowedstandardproceduresoutlinedelsewhere[25,26].
Thefluorescent-basedgenotypingwasperformedthroughcapillaryelectrophoresisonanABI
3130GeneticAnalyzer(AppliedBiosystems,FosterCity,CA,USA).Theallelescoringwas
donemanuallyusingGeneMapper(ver.3.7)(AppliedBiosystems,FosterCity,CA,USA).
ToamplifymitochondrialDNAD-loophypervariableregion(HV1),specificprimers
L16750(50-AGGACTACGGCTTGAAAAGC-30)astheforwardprimerandH547(50-ATGTGC
CTGACCGAGGAACCAG-30)asreverseprimerwereused.Thisprimerpairamplifieda550bp
fragmentbetweensites16750(GenBankaccessionnumberNC_001323;[27])and547(Gen-
BankaccessionnumberAB098668;[28]).ThePCRswereconductedin10μlreaction—0.25
mMMgCl ,1μMeachofforwardandreverseprimers,0.25mMdNTPsand1unitTaq poly-
2
merase.ThermocyclingconditionsofPCRconsistedofaninitialdenaturationat94˚Cfor2
min,followedby35cyclesofdenaturationat94˚Cfor30seconds,anannealingstepat52˚C
for20seconds,andanextensionat72˚Cfor60secondsandfinishedbyafinalextensionat
72˚Cfor10min.PCRproductswerepurifiedusingtheQIAquickPCRpurificationkit(QIA-
GEN,GmbH,Germany)accordingtothemanufacturer’sprotocol.Directsequencingofa
HV1segmentoftheD-loopregionwasperformedusingtwointernalprimersCR-for(50-TC
TATATTCCACATTTCTC-30) andCR-rev(50-GCGAGCATAACCAAATGG-30).Sequencing
wasdoneusingtheBigDyeTerminator(ver.3.1)CycleSequencingKit(AppliedBiosystems,
FosterCity,CA,USA)andthepurifiedsequencingproductswereelectrophoresedontheABI
3130GeneticAnalyzer.
Statisticalanalysis
(a)Microsatellites. Geneticvariability,inbreedingandpopulationdifferentiation.
Geneticdiversityestimatesi.e.observed(Na)andeffectivenumbersofalleles(ne),observed
(Ho),expected(He)andunbiasedexpectedheterozygosity(UHe)andpair-wiseF wereesti-
ST
matedusingGENEALEX(ver.6.5)[29].Themeanallelicrichness(A ),whichisanestimate
R
ofanindependentsamplesizeforeachpopulationandthepolymorphicinformationcontent
(PIC)wereestimatedusingFSTAT(ver.2.9.3.2)[30]andCervus(ver.3.0)[31],respectively.
WetestedanydeviationfromHardy–Weinbergequilibrium(HWE)ateachlocusfollowing
probabilitytestapproachinvolving10,000dememorizations,500batchesand10,000iterations
perbatchinGENEPOP(ver.4.2)[32,33].TheF followingWeirandCockerham[34]
IS
methodandLinkagedisequilibrium(LD)weretestedusingthealoglikelihoodratiostatistic
inGENEPOP(ver.4.2)[33].
Populationgeneticstructure.PopulationgeneticstructureofRJFinIndiawasinferred
usingtheBayesianclusteringalgorithmimplementedinprogramSTRUCTURE(ver.2.3.3)
[35]followingamodelassumingKpopulations(e.g.1to10).Weselectedanadmixturemodel
withaburn-inperiodof50,000and500,000MarkovchainMonteCarlorepetitionsaswellas
amodelofcorrelatedallelefrequencieswithoutpriorsamplinglocationinformation.Twenty
independentrunsateachKvaluewereperformed.ThemostappropriateKvaluewasdeter-
minedbycalculatingadhocquantity(ΔK)asproposedbyEvannoetal.[36]andeachindivid-
ualwasassignedtotheinferredclustersusingathresholdproportionofmembership(q),i.e.
q(cid:21)0.80,followingMukeshetal.[26,37,38].TheclusteringresultsofSTRUCTUREwere
visualizedoverClumpaK(http://clumpak.tau.ac.il/index.html),awebserverthatprovidesa
fullpipelineforclustering,summarizingandvisualizingtheSTRUCTUREresults.
Spatialgeneticsandbarrierdetection.Assamplingwasdoneacrossthespeciesdistribution
inIndia,weperformedaspatialBayesianclusteringanalysisinGENELAND(ver.)4.0.3[39]
todetectbarriersinthegeneflowbetweenfree-rangingRJFpopulations.Unlikepopulation
assignmentinSTRUCTURE,thisanalysisalsotakesintoaccountthespatialcoordinates(i.e.
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GeneticintrogressionofwildRedJunglefowl
latitudeandlongitude)ofeachindividualalongwiththeirmulti-locusgenotypesforspecifi-
callyidentifyinggeneticbreakswhilegeneratingmapsofpopulationranges.Weemployedthe
spatialmodeltoinferthemostlikelynumberofclustersindicatedas‘K’witheithercorrelated
oruncorrelatedallelefrequencymodel.Intheseanalyses,weconducted30independentruns
foreachKrangingfrom1to10,with1,000,000iterationsand1,000thinning.Otherparame-
tersweresettodefaultvalues(maximumrateofthePoissonprocess,100;uncertaintyonspa-
tialcoordinates,0;maximumnumberofnuclei,300;nullallelemodel,FALSE).Thetopthree
runsshowingthehighestaveragelogarithmoftheposteriorprobabilitywereselectedand
post-processingwasconductedusing100x100pixelsinthespatialdomainwithaburnin
periodof200.
(b)MitochondrialD-loopregion. Geneticvariabilityanddemographichistory.We
checkedallrawsequencesusingSequencherver.4.7(www.genecode.com)andanyambiguity,
ifencountered,wasmanuallycorrected.ThecleanedsequenceswerealignedusingCLUSTAL
W-multiplesequencealignmentalgorithm,implementedinBioEditversion7.2.5(http://
www.mbio.ncsu.edu/BioEdit/bioedit.html).UsingDnaSP(ver.5.10)[40],diversityestimates
i.e.polymorphicsites(S),numberofhaplotypes(H),nucleotidediversity(π),haplotypediver-
sity(Hd),averagenumberofnucleotidedifferences(K)andmismatchdistributiontestfor
demographicexpansion,equilibriumorbottleneck[41]werecomputed.Neutralitytests,i.e.
Tajima’sD[42],Fu’sFs[43]andFuandLi’sFandD[44]werecarriedouttoevaluatethe
demographiceffects.
(c)GeneticintrogressionandclusteringofRJFswithDCs. ProgramSTRUCTUREwith
thesameparametersasdescribedabovewasusedwiththeinclusionofDCsamplescollected
inthevicinityofRJFstoassigntheadmixedindividuals.Toevaluatethedirectionofgene
flow,haplotypesshared,andtheirfrequenciesofoccurrencebetweenRJFsandDCswere
examinedusingthephylogeneticnetworkbasedonthemedian-joiningmethod[45]imple-
mentedinNETWORKver.4.5.1(http://www.fluxusengineering.com).InNETWORK,we
assignedequalweightstoallvariablesitesandapplieddefaultvaluesfortheepsilonparameter
(epsilon=0).Themicrosatelliteandsequencingdataphylogenetictreesconstructedwereused
toinvestigatetherecentandlong-termadmixtureandthegeneticrelationshipbetweenwild
RJFandDCpopulations.Formicrosatellitedata,ageneticdistancematrixwascalculated
usingProgramGENALEX(ver.6.5)[29],andaphylogenetictreewasconstructedfollowing
themaximumlikelihood(ML)methodinMEGAprogramversion7.0[46].Forsequencing
data,maximumlikelihoodmethodandtheTamura3parameter,themostfitsubstitution
model,wereconsideredtoreconstructthephylogenetictreeusingMEGAprogramversion7.0
program[46].
Results
Amongthe57RJFand79DCsamplescollected,therewas68(32RJFsand36DCs)samples
fromNorth,25(9RJFsand16DCs)fromEast,5(2RJFsand3DCs)fromCentral,5(4RJFs
and1DC)fromSouthEastand33(10RJFsand23DCs)fromNortheastofIndia.Since,we
cantraponlyafewwildRJFsinCentralandSoutheastzones;wepooledsamplesofthese
zonestocreateaCent-Southeastpopulationforfurtheranalysis.Duringanalysis,twobirds-
RJ1(Cent-Southeast)andRJ9(Northeast)wereexcludedduetouncertaintyintheGPSloca-
tionsoftheirsamplepoints.
Wegenotypedallsamplesthreetimesorrepeatedtheprocessuntilwegeneratedconsensus
genotypesforallsamples.However,fourlocii.e.LEI0192,MCW0014,MCW0183and
MCW0284exhibitedaconsiderableamountofmissingvaluesforfewasamplesevenafter
multiplerepetition,soweremovedthemfromfurtheranalysis.Wemanuallycheckedallelic
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GeneticintrogressionofwildRedJunglefowl
data,andfoundnoindicationofanygenotypingerror(DataavailableontheDryadDigital
Repositoryonhttps://doi.org/10.5061/dryad.rv38cp4).
Geneticdiversityindicesanddemographichistory
All26microsatellitelociwerepolymorphic,yieldingatotalof692allelesacrossthefourRJF
populations.TheaverageNaandnerangedfrom4.27±0.34(Cent-Southeast)to9.69±0.76
(North)andfrom3.11±0.27(Cent-Southeast)to5.02±0.35(North),respectively.TheHoran-
gedfrom0.52(±0.04North;±0.06Cent-Southeast)to0.58(±0.05East;±0.04Northeast)and
didnotdiffersignificantlyamongthefourpopulations.ThemeanPICvaluesweregreater
than0.5forallthefourRJFpopulations.TheUHerangedfrom0.67±0.04inCent-Southeastto
0.81±0.02inNortheastpopulation,andtheaverageA (rarefactionaftertwodiploidindividu-
R
alsperpopulation)rangedfrom2.59±0.13inCent-Southeastto3.04±0.07inNortheastpopu-
lation.TotalcountofN wasthehighestinNorth(85alleles)butthelowestinCent-Southeast
P
(12alleles)RJFpopulations.TheF estimatesinthefourRJFpopulationsweresignificantly
IS
positive(P<0.001),suggestingthatallthepopulationswereslightlyinbred.Thenumberof
locideviatedfromHWE(P<0.05)rangedfromfourinCent-Southeastpopulationto22in
Northpopulationwiththemajorityofthemshowingaheterozygotedeficit.Neverthelessonly
twoloci(MCW0295andMCW0123)deviatedfromHWEacrossthefourpopulations
(P<0.05;S1Table).SeveralpairsoflocishowedsignificantLD(P<0.05;S2Table),butnoneof
themwaspresentacrossthefourpopulations.
ForD-loopdata,weanalysed100samples,i.e.38wildRJF(15North,7East,6Cent-South-
eastand10Northeast)and62DCcollectedinthevicinityoftheRJF.WildRJFyielded37
polymorphicsiteswhichformed31haplotypeswithanaverageof6.48nucleotidedifferences.
Allanalyzedsamplesyielded68haplotypes,amongwhich37werefoundinDCs.Mosthaplo-
typeswerepresentinonesampleeach.Haplotypes#1,12and13weresharedamongtheindi-
vidualsofthesamepopulationwhilehaplotype3wassharedbetweenNorthandNortheast
populations(S1Fig).NovelhaplotypesweredepositedintheGenBankdatabase(Accession
no.MG053424toMG053491).Northeastpopulationrepresentedthehighestdiversityesti-
mateswith26polymorphicsites,10haplotypesand8.71nucleotidedifferences.Theoverall
haplotype(Hd)andnucleotidediversity(π)were0.98±0.014and0.01±0.001,respectively
(Table1).AllfourRJFpopulationsshowedamultimodalpatternofmismatchdistribution,
supportingthesepopulationstobeunderdemographicequilibriumandwithoutanybottle-
neck(S2Fig).Theestimatesofneutralitytests,ingeneral,showedasimilarpatternrevealedby
mismatchdistributioncurvewhilenegativeestimatesofTajima’sDandFu’sFsstatistictests
impliedthatrareallelesweremorecommonthanexpected.However,theobservedestimates
werenotsignificant(P>0.10),suggestingthatpopulationshavenotundergoneanyexpansion.
FuandLi’sDandFtestsalsoindicatednosignificantdeparturefromneutrality(P>0.10).
Pair-wiseF valuesbetweenallfourRJFpopulationsweresignificant(P<0.01;Table2)for
ST
boththemicrosatelliteandD-loopdata.ThehighestdifferentiationwasfoundbetweenNorth
andCent-Southeastpopulations(0.10formicrosatellitesand0.30forDloopdata).However,
similarestimateswereobservedbetweenNorthandNortheastpopulationsformicrosatellites
(0.05)andD-loopsequences(0.06).
Inferenceofpopulationgeneticstructureandclusteringpatternsonthe
spatialscale
TheadhocquantityvaluewasthehighestatK=2(ΔK-139.8081;Fig2B;Table3).Allindivid-
ualsweresuccessfullyassignedtotwoclusters,cluster1–54.38%andcluster2–45.61%basedon
theirposteriorprobabilityvaluesi.e.q(cid:21)0.80.AfurtherincreaseintheKvaluedidnotsplit
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GeneticintrogressionofwildRedJunglefowl
Table1. SummaryofmoleculargeneticdiversityindicesofwildRJFpopulations.
RJF Diversityestimates Diversityestimates Neutralitytests
population (Microsatellites) (mt-DNAD-loop)
(Noofmsat/ Average Average A N Average Average S H K Hd P Tajima’sD(cid:3) Fu’s Fuand Fuand
mt)† numberof Heterogygosity R P PIC F Fs(cid:3) Li’sD(cid:3) Li’sF(cid:3)
IS
alleles
Na Ne Ho He UHe
North 9.69 5.02 0.52 0.77 0.79 2.96 85 0.74 0.34 19 11 5.31 0.93 0.01 -0.56 -3.07 -0.81 -0.85
(32/15) ±0.76 ±0.35 ±0.04 ±0.02 ±0.02 ±0.08 ±0.05 ±0.001
East 6.04 4.32 0.58 0.73 0.78 2.93 27 0.69 0.25 17 5 7.00 0.91 0.02 -0.26 0.94 -0.27 -0.30
(9/7) ±0.44 ±0.35 ±0.05 ±0.02 ±0.02 ±0.09 ±0.10 ±0.002
Cent- 4.27 3.11 0.52 0.61 0.67 2.59 12 0.56 0.25 6 6 2.40 1.0 0.01 -0.50 -3.85 -0.42 -0.46
Southeast ±.34 ±0.27 ±0.06 ±0.04 ±0.04 ±0.13 ±0.09 ±
(6/6) 0.0008
Northeast 6.62 4.74 0.58 0.76 0.81 3.04 22 0.73 0.29 26 10 8.71 1.0 0.02 -0.25 -3.89 -0.28 -0.31
(10/10) ±0.36 ±0.30 ±0.04 ±0.02 ±0.02 ±0.07 ±0.04 ±0.002
Pooled 6.65 4.30 0.55 0.72 0.76 3.09 0.79 0.23 37 31 6.48 0.98 0.01 -1.07 -23.14 -1.25 -1.41
(57/38) ±0.31 ±0.17 ±0.02 ±0.01 ±0.01 ±0.06 ± ±0.001
0.014
Na,-Observednumberofalleles;Ne,-Effectivenumberofalleles;Ho,-Observedheterozygosity;He,-Expectedheterozygosity;UHe,-Unbiasedexpectedheterozygosity;
AR,-Allelicrichness;NP,-Numberofprivatealleles;PIC,-Polymorphicinformationcontent;FIS,-Inbreedingcoefficientindex;S,-Polymorphicsites;H,-Numberof
haplotypes;K,-Averagenumberofnucleotidedifferences;Hd,-Haplotypesdiversity;P,-Nucleotidediversity.
†N,-samplesize(msat-formicrosatelliteanalysisand/mt-mitochondrialDloopanalysis
(cid:3)P>0.10(notsignificant).
https://doi.org/10.1371/journal.pone.0204351.t001
thesamplesintoadditionalclusters(Fig3).Interestingly,birdsfromNorthandNortheastpop-
ulationswereassignedtobothclusters,indicatingtheirsharedancestryinthepast.However,
nearlyallbirdsofEastandCent-Southeastpopulationswereassignedtocluster1.Theglobal
performanceofSTRUCTUREinassigningallindividualsatK=2was100%(Table3).
InaGENELANDanalysisassuminguncorrelatedallelefrequencymodel,fourgeneticclus-
ters(K=4)wereinferredin20outof30runs(66.66%;S4Table,thefirstthreecolumns,)
basedonthehighestaveragelogarithmoftheposteriorprobability(S3Fig).Incontrast,when
assumingcorrelatedallelefrequencymodel,nearlyallruns(29/30)indicated10clusters(S4
Table,thelastthreecolumns)whichwerenotinterpretable.Therefore,weconsideredonlythe
resultsoftheanalysisassuminguncorrelatedfrequencymodel.Theresultsofthetopthree
runsshowingthehighestaveragelogarithmoftheposteriorprobabilitywerelargelyconsis-
tent.Thespatialdistributionofthesefourinferredclustersshowedthattheboundariesof
theseclustersweregenerallycoincidedwithmultiplelandscapefeaturesandindividualswere
assignedtothesefourclustersascluster-I(green;36.36%),cluster-II(lightgreen;7.27%),clus-
ter-III(orange;5.45%)andcluster-IV(white;50.90%)(Fig4E).Interestingly,allthesamples
collectedfromtheStateUttarPradesh(PilibhitTigerReserve,DudhwaNationalPark,
Table2. Comparisonofpair-wiseF estimatesbetweenfourwildRJFandfourDCpopulations.
ST
Population North East Cent-Southeast Northeast
North 0.06 0.30 0.06
East 0.05 0.11 0.07
Cent-Southeast 0.10 0.06 0.27
Northeast 0.05 0.07 0.09
(Fstvaluesbelowdiagonal-microsatellitesandFstvaluesabovediagonalmt-DNA-Dloop)
https://doi.org/10.1371/journal.pone.0204351.t002
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GeneticintrogressionofwildRedJunglefowl
Fig2.GraphicalrepresentationofthetruenumberoftheclusterinwildRJFpopulations.(A)MeanL(K)(±SD)over20independentrunsforeachK-value.(B)The
adhocquantity(ΔK).
https://doi.org/10.1371/journal.pone.0204351.g002
Katerniaghat&KishanpurWildlifeSanctuary)wereassignedtocluster-IVwhilesamplescol-
lectedfromtheStateUttarakhand(TeraiForestDivision,CorbettNationalPark,Rajaji
NationalPark&WildlifeInstituteofIndiacampus)wereassignedtocluster-I.Allthesamples
collectedfromManipurweregroupedincluster-III(S4Table).Onlythreeindividuals(one
eachfromKhelmaForestDivision-Nagaland,HailakandiForestDivision-AssamandNameri
NationalPark-Assam)weregroupedincluster-II.Samplescollectedfromseverallocations
wereassignedtocluster-IV,andthiscanplausiblybeinterpretedbasedonthepossiblehistoric
geneflowandphysicalexchangeofthebirdsinthepast.Eachindividual’sestimatedpropor-
tionsofmembershipobtainedfromGENELANDanalysis(around0.50atmaximum,S5
Table)werelowerthanthoseobtainedusingtheSTRUCTUREanalysis.Theresultsprovided
indicationsthatthepresentlandscapefeaturesdidnotactasabarriertogeneflowortheactual
geo-spatialclusteringtrendinthelandscaperemainedmaskedbythefactofphysicalsharing/
exchangeofwildbirdsinthepastastheforestedareasandmostpreferredhabitatsforthespe-
ciesmaybeconnected.
Geneticintrogressionandidentificationofadmixedindividuals
Theresultsindicatedamulti-modalvalueofdeltaKaseachpeakbreakingoutquiteclearlyat
K=2andK=4(Fig5B).Individualassignmentwas>98%atK=2(Fig6;S3Table)and
~82%atK=4(Fig6;Table4)consideringaposteriorprobabilityvalueq(cid:21)0.80.AtK=2,all
theRJFsbutonebirdofEastpopulation(RJ-110_SK)wereassignedtoanyofthesetwo
Table3. GeneticassignmentofwildRJF(n=57)populationsthroughBayesianclusteringanalysis.
Population(No.) Cluster1 Cluster2
North(32) 0.375(12) 0.625(20)
East(9) 0.888(8) 0.111(1)
Cent-Southeast(6) 1(6) 0
Northeast(10) 0.5(5) 0.5(5)
https://doi.org/10.1371/journal.pone.0204351.t003
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GeneticintrogressionofwildRedJunglefowl
Fig3.BayesianclusteringpatternsofwildRJFpopulations(n=57).
https://doi.org/10.1371/journal.pone.0204351.g003
clusterswhileatK=4nineRJFswereadmixed(RJ-76_UK,RJ-84_UK,RJ-116_UP,RJ-55_UP,
RJ-58_UP,RJ-63_UP,RJ-105_BH,RJ-110_SKandRJ-113_AS).
AMaximumLikelihood(ML)treeobtainedfromthemicrosatellitedataformedseveral
groupsofpossiblehybridsbetweentheRJFsandDCs(representedbyaredtrianglewithpink
shadowinFig7.Therewere13RJFs(22.8%oftotalRJFsamples;RJ-123,RJ-116,RJ-66,RJ-98,
RJ-99,RJ-115,RJ-105,RJ-108,RJ-112,RJ-6andRJ-70,RJ-9andRJ-11)beinggroupedwith
DCsrepresentingsharingofsimilarallelesbetweenRJFsandDCs.Thisclusteringpatternas
revealedbyMLtreebasedonadistancematrixofmicrosatellitedataexhibitedtherecent
eventofadmixture.WhileMLtreebasedonD-loopsequencesidentifiedseveralRJFsi.e.
RJ123,RJ101,RJ104andRJ1sharingidenticalhaplotypespresentinDCsandseveralother
RJFsbeinggroupedwithDCs(Fig8).
Themedian-joiningnetworkanalysisof68haplotypessuggestedastronggeospatialstruc-
tureinthewildRJFsandDCs(Fig9).Tounderstandthedirectionofgeneflow,weprioritize
sharedhaplotypesoverthehaplotypesuniquetoindividualpopulations.Weassignedshared
haplotypesintothreecategoriesviz.shared—betweenRJFandDCpopulations(Hap#10,14
and19),betweenRJFpopulations(Hap#6)andbetweenDCpopulations(Hap#37,40,41,45,
50and53).AmongthreehaplotypessharedbetweenRJFsandDCs,Hap#14and19werepre-
dominantinDCsat60%and75%,respectively.WhileHap#10wassharedby50%inRJFsor
DCs.Inthesecondgroup,Hap#6whichwassharedbetweenRJFpopulationsofNorth(Utta-
rakhand)andNortheast(Assam)mightbetheancestralhaplotypereflectinghistoricgeneflow
betweenthesetwopopulations.Interestingly,itwaswellsupportedbytheresultsofSTRUC-
TURE(50%ofassignmentofNortheastRJFswithNorthRJFs;Table3)andGENELAND
(OneRJFsamplecollectedfromAssambeinggroupedincluster-IValongwithRJFscollected
fromUttarakhand;S5Table)analyses.However,wedidnotgivemuchattentiontothehaplo-
typessharedbetweenDCsasthiscouldbeduetothetransportofDCsacrossthecountryfor
thelocalbenefitorcommercialpurposes.
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GeneticintrogressionofwildRedJunglefowl
Fig4.ResultsofBayesianmodelbasedclusteringinGENELANDusingwildRJFindividuals(n=57;K=4asthebestrunshowingthehighestaveragelogarithm
oftheposteriorprobability).(a—d)MapsofposteriorprobabilityforeachinferredclustershowthespatiallocationofgeneticdiscontinuitiesofclustersI,II,IIIandIV;
(e)Amapofestimatedclustermembershipshowsspatialdistributionofthefourinferredgeneticclustersacrossthestudyareas.Samplelocationsarerepresentedby
blackdots.XandYspacesindicatelongitude(E)andlatitude(N).Lightyellowcoloringcorrespondstohighposteriorprobabilityandredcorrespondstolowposterior
probability.
https://doi.org/10.1371/journal.pone.0204351.g004
Fig5.GraphicalrepresentationofthetruenumberoftheclusterinwildRJFandDCpopulations.(A)MeanL(K)(±SD)over20independentrunsforeachKvalue.
(B)Theadhocquantity(ΔK).
https://doi.org/10.1371/journal.pone.0204351.g005
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