High genetic structure and low mitochondrial diversity in bottlenose dolphins of the Archipelago of

22 Pages·2017·3.12 MB·English

RESEARCHARTICLE
High genetic structure and low mitochondrial
diversity in bottlenose dolphins of the
Archipelago of Bocas del Toro, Panama: A
population at risk?
DaliaC.Barraga´n-Barrera1,2☯*,LauraJ.May-Collado3,4☯,GabrielaTezanos-Pinto5‡,
ValentinaIslas-Villanueva6‡,CamiloA.Correa-Ca´rdenas7,8‡,SusanaCaballero1☯
a1111111111 1 LaboratoriodeEcolog´ıaMoleculardeVertebradosAcua´ticosLEMVA,DepartamentodeCiencias
Biolo´gicas,UniversidaddelosAndes,LaboratorioJ-202,Bogota´,Colombia,2 Fundacio´nMacua´ticos
a1111111111
Colombia,Medell´ın,Colombia,3 DepartmentofBiology,UniversityofVermont,Burlington,VT,UnitedStates
a1111111111
ofAmerica,4 CentrodeInvestigacionesdelMaryLimnolog´ıa,UniversidaddeCostaRica,SanJose,Costa
a1111111111
Rica,5 Coastal-MarineResearchGroup,INMS,MasseyUniversity,Auckland,NewZealand,6 CONACYT,
a1111111111 UniversidaddelMar,InstitutodeGene´tica,CiudadUniversitaria,PuertoA´ngel,DistritodeSanPedro
Pochutla,Oaxaca,Me´xico,7 FacultaddeIngenier´ıayCienciasBa´sicas,DepartamentodeCiencias
Naturales,UniversidadCentral,Bogota´,Colombia,8 DepartamentodeCienciasBa´sicas,UniversidaddeLa
Salle,Bogota´,Colombia
☯Theseauthorscontributedequallytothiswork.
OPENACCESS ‡Theseauthorsalsocontributedequallytothiswork.
*[email protected]
Citation:Barraga´n-BarreraDC,May-ColladoLJ,
Tezanos-PintoG,Islas-VillanuevaV,Correa-
Ca´rdenasCA,CaballeroS(2017)Highgenetic
structureandlowmitochondrialdiversityin Abstract
bottlenosedolphinsoftheArchipelagoofBocasdel
Toro,Panama:Apopulationatrisk?PLoSONE12 Thecurrentconservationstatusofthebottlenosedolphin(Tursiopstruncatus)underthe
(12):e0189370.https://doi.org/10.1371/journal.
IUCNis‘leastconcern’.However,intheCaribbean,smallandlocalizedpopulationsofthe
pone.0189370
‘inshoreform’maybeathigherriskofextinctionthanthe‘worldwidedistributedform’dueto
Editor:Tzen-YuhChiang,NationalChengKung
acombinationoffactorsincludingsmallpopulationsize,highsitefidelity,geneticisolation,
University,TAIWAN
andrangeoverlapwithhumanactivities.Here,westudythepopulationgeneticstructureof
Received:December5,2016
bottlenosedolphinsfromtheArchipelagoofBocasdelToroinPanama.Thisisasmallpopu-
Accepted:November26,2017 lationcharacterizedbyhighsitefidelityandiscurrentlyheavily-impactedbythelocaldol-
Published:December13,2017 phin-watchingindustry.Wecollectedskintissuesamplesfrom25dolphinstostudythe
geneticdiversityandstructureofthispopulation.Weamplifiedaportionofthemitochondrial
Copyright:©2017Barraga´n-Barreraetal.Thisis
anopenaccessarticledistributedundertheterms ControlRegion(mtDNA-CR)andninemicrosatelliteloci.ThemtDNA-CRanalysesrevealed
oftheCreativeCommonsAttributionLicense, thatdolphinsinBocasdelTorobelongtothe‘inshoreform’,groupedwiththeBahamas-
whichpermitsunrestricteduse,distribution,and
Colombia-Cuba-Mexicopopulationunit.Theyalsopossessauniquehaplotypenewforthe
reproductioninanymedium,providedtheoriginal
Caribbean.ThemicrosatellitedataindicatedthattheBocasdelTorodolphinpopulationis
authorandsourcearecredited.
highlystructured,likelyduetorestrictedmovementpatterns.Previousabundanceestimates
DataAvailabilityStatement:Allrelevantdataare
obtainedwithmark-recapturemethodsreportedasmallpopulationof80dolphins(95%CI=
withinthepaperanditsSupportingInformation
files. 72–87),whichissimilartothecontemporaryeffectivepopulationsizeestimatedinthisstudy
(Ne=73individuals;CI=18.0-1;0.05).Thecombinationofsmallpopulationsize,high
Funding:ThisworkwassupportedbytheSociety
forMarineMammalogyprovidedaSmall-in-Aid degreeofgeneticisolation,andintensedailyinteractionswithdolphin-watchingboatsputs
ResearchgrantbyUS$1000;TheSciencesFaculty theBocasdelTorodolphintoathighriskofextinction.Despitenationalguidelinestoregu-
ofUniversidaddelosAndesprovidedaResearch
latethedolphin-watchingindustryinBocasdelToroandongoingeducationalprogramsfor
Grant“ProyectoSemilla”inthe2012-1Callfor
FundingofResearchCategory:Masterand touroperators,onlyin2012sevenanimalshavediedduetoboatcollisions.Ourresults
PLOSONE|https://doi.org/10.1371/journal.pone.0189370 December13,2017 1/22
PopulationgeneticsofbottlenosedolphinsinBocasdelToro
Doctoralstudents,project“Geneticstructureand suggestthattheconservationstatusofbottlenosedolphinsinBocasdelToroshouldbeele-
diversityofbottlenosedolphinsTursiopstruncatus vatedto‘endangered’atthenationallevel,asaprecautionarymeasurewhilepopulation
(Montagu,1821)(Cetacea:Delphinidae)inBocas
andviabilityestimatesareconducted.
delToro,CaribbeanCoastofPanama”.They
provideCOP$3’213.600(~US$1700)andthe
RuffordSmallGrantFoundationprovidedtwo
grantsforthisresearch:theRuffordSmallGrantby
£6000,andtheSecondRuffordSmallGrantby£
6000.Fundsforthisstudyweregenerously Introduction
providedbytheRuffordFoundation,Societyfor
Thebottlenosedolphin(Tursiops truncatus)isacosmopolitanspecies[1]thatdespiteits
MarineMammalogy,andUniversidaddelosAndes
potentialabilityforlong-distancemovement,showsremarkablegeneticdifferentiationatvari-
(SciencesFaculty)toDCBB.Thefundershadno
roleinstudydesign,datacollectionandanalysis, ousgeographicscales(e.g.,[2,3]).Aswithotherdelphinids,habitatspecializationisconsid-
decisiontopublish,orpreparationofthe eredanimportantdriverforgeneticdifferentiation[4,5,6,7,8].Recentevidencesuggeststhat
manuscript. inthisspecies,habitatspecializationmayhaveoccurredindependentlyindifferentocean
Competinginterests:Theauthorshavedeclared basis,leadingtodifferentecotypes[2,9,10].Forexample,intheWesternNorthAtlantictwo
thatnocompetinginterestsexist. formsorecotypes,‘offshore’and‘inshore’showcleardifferencesintheirecology,morphology,
physiology,behavior,andgeneticstructure(e.g.,[3,4,6,9,11,12,13,14]).However,thesedif-
ferencesarenotevidentinbottlenosedolphinsfromotherregions[2,15].Itappearsthatthe
‘inshoreform’intheWesternNorthAtlanticistheonlyecotypeclearlydescribedsofarthatis
geneticallydifferenttotherest(namely,the‘offshoreform’)[2,9].
Somecoastalpopulationsofbottlenosedolphinsliveinsmallpopulationscharacterizedby
lowgeneticdiversityandlowgeneflowbetweenneighboringpopulations,suggestinglocal
foundereventsatleastinsomeareasorrecentisolation(e.g.,[3,6,8,9,13,16,17,18,19]).In
someofthesebottlenosedolphinpopulations’malesandfemaleshavedifferinghomeranges,
withfemalesremainingwithintheirnatalgroups,whilemalesrangefurtherandvisitdifferent
socialunits(e.g.,[8,20]).Thisfinescalegeneticstructuringincoastalbottlenosedolphinsis
evidencedintheremarkablevariationoftheirsocialstructureandfeedingbehaviors(e.g.,[21,
22,23,24,25])acrosscoastalpopulations.However,thehighlevelofsitefidelity,geneticisola-
tion,andlowabundanceobservedinsomelocalpopulations[19,26,27],mayimposesignifi-
cantchallengestotheseanimalsastheymaynotbeabletorespondtorapidchangesintheir
habitat,particularlythosecausedbyhumanactivities(e.g.noiseandchemicalpollution,over-
fishing,entanglements,boattraffic)[28,29,30,31].Inaddition,severalpopulationsofbottle-
nosedolphinsareaffectedbydirectexploitationforexhibitionpurposes[e.g.32],andothers
areregularlytargetedbycommercialdolphin-watchingactivities,whichtendtohavecumula-
tiveeffectsontheirpopulations[26,32,33,34].
Conservationprioritiesandultimately,fundingtomonitormarinemammalpopulationsis
largelydependentontheIUCNconservationstatusofthespecies.Giventheirworldwidedis-
tribution,thebottlenosedolphinisclassifiedbytheIUCNas‘leastconcern’[35],withthe
exceptionofthreepopulations:FiordlandinNewZealand(classifiedas‘criticallyendangered’)
[36],theMediterraneanSea(classifiedas‘vulnerable’)[37]andfinally,thesubspeciesT.trun-
catusspp.ponticusfromtheBlackSea(classifiedas‘endangered’)[38].However,therearesev-
eraldistinctbottlenosedolphincoastalpopulationsforwhichtheirconservationstatusis
underestimatedduetocountriesadoptingthe‘leastconcern’approach.Thismeansthatfund-
ingandresearchprioritiesarenotdirectedtothesepopulations.Forexample,theresident
coastalbottlenosedolphinpopulationofSanAntonioBayinArgentinaisgeneticallyisolated
fromneighboringpopulations[18]andpresentsandalarmingpopulationdeclinecaused
mainlybypollutionandoverfishing[19].However,theArgentiniangovernmentstillclassifies
thispopulationas‘leastconcern’[19].
Likeothercoastalbottlenosedolphins,thedolphinpopulationfoundintheArchipelagoof
BocasdelToroinPanamaissmall.Preliminaryestimatessuggestapopulationof80dolphins
PLOSONE|https://doi.org/10.1371/journal.pone.0189370 December13,2017 2/22
PopulationgeneticsofbottlenosedolphinsinBocasdelToro
(95%CI=72–87)[34]andbothsexesshowhighsitefidelity[34,39,40,41].Analysesofmark-
recaptureusingphoto-identification,indicatedthispopulationconsistsoftwointeractingdol-
phincommunities:oneconsistingofdolphinswithhighresidencyandasmallhomerange
locatedprimarilywithinDolphinBay,andasecondcommunityconsistingofhighlymobile
dolphinsthatshowlowresidency[34].Whilethetransitofwaterboattaxisandothertransport
boatsiscommoninthehabitatofbothcommunities,dolphinsintheDolphinBaycommunity
aretheoncedirectlytargetedbylocaldolphin-watchingboats[34,41,42].Thisdolphincom-
munityispreferredfordolphin-watching,duetotheirhighpredictabilitywithinthebay[34].
Since2004,researchershaveobservedanexponentialincreaseinthenumberofdolphin-
watchingboatsvisitingthebay[41].In2012,thehighestnumberofboatsinteractingwithdol-
phinswasreported,withupto39boatsinteractingwiththesamegroupdolphinwithina
periodofonehour[41].Thatsameyearsevendolphinswerereporteddeadduetoboatcolli-
sion[43].Previousstudieshaveshowedthatthisincreaseinthenumberofboatsisaccompa-
niedbyanincreaseinenginenoiselevels[42]andhasthepotentialtoimpacttheiracoustic
spaceforcommunication[41],especiallyduringforagingactivities[41,44],particularlyin
groupswithnursingmothers[45].
Here,weevaluatetheconservationstatusofthebottlenosedolphinsofBocasdelToro
byestimatingthegeneticdiversityandpopulationstructureusingninemicrosatellite
markersandaround750basepairs(bp)fragmentofthemitochondrialDNAControl
Region(mtDNA-CR).Wetestthehypothesisthatthereisahighdegreeofgeneticdistinc-
tivenessindolphinsfromBocasdelToroincomparisontootherbottlenosedolphinpop-
ulationsintheCaribbean,largelyduetothesmallpopulationsizeandhighindividual
philopatry.
Methods
TheArchipelagoofBocasdelToro(BDT)islocatedontheCaribbeancoastofPanama(Fig1).
Thisareahasseveralimportantmarineecosystems,includingmangroveforests,seagrasses
andcoralreefs[46,47,48].
Tissuesamplescollection
ThemethodologyforsamplecollectionwasapprovedbytheSmithsonianTropicalResearch
InstituteIACUC(InstitutionalAnimalCareandUseCommittee;permitnumber2011-1125-
2014-06).SampleswerecollectedwithpermissionfromtheAutoridadNacionaldel
Ambiente–Panama´(ANAM;permitsSC/A-11-12,SC/A-43-12,andSC/A-17-14).
FollowingKru¨tzenetal.[49],tissuesampleswereobtainedfromwilddolphinsbyfiring
remotebiopsydartswithamodified0.22veterinaryriflefromadistanceofapproximately10
mfromtheresearchboat[20,49,50,51].Thebiopsydartshaveahollowpolycarbonatebody
andasmallstainlesssteelbiopsytip(5mmdiameter,9mmlength)[49,52].Thissystemallows
thecollectionofskinsamplescausingonlyasmallwound,andshort-termbehavioralreactions
[4952].Additionally,photographsofthedolphinbiopsysampledwerecollectedtoallowindi-
vidualidentificationandlinkingdemographic(e.g.sitefidelity,residency)withgeneticdata
(sex,lineage).Thisstepwasalsoincludedtoavoidsamplingthesameindividualrepeatedly
[18,49].Atotalof24biopsieswerecollectedfromsevenlocationswithintheArchipelago
includingAlmiranteBay,DolphinBay,LomaPartida,PastoresIslands,Popa,SharkHole,and
TierraOscura(Fig1).Oneadditionalsamplewascollectedfromadeadstrandeddolphinin
Bocastorito.WealsoincludedfivesamplesfromGandoca-Manzanillo(CostaRica),abottle-
nosedolphinpopulationlocatedlessthan35kmfromBocasdelToro.Thesefivesampleswere
collectedbytheEarthwatchProject‘DolphinsofCostaRica’(authorizationgrantedby
PLOSONE|https://doi.org/10.1371/journal.pone.0189370 December13,2017 3/22
PopulationgeneticsofbottlenosedolphinsinBocasdelToro
Fig1.LocationofBocasdelToro(Panama)andthesamplingsitesincludinginthisstudy(ingreen).
ThesegeographiclocationsalsoincludeTheBahamas,Colombia,CostaRica,Cuba,Honduras,Mexico,and
PuertoRico.ThemapofBocasdelTorobelowshowsthepositionofcommonbottlenosedolphinssampledin
thesevenlocationswithintheArchipelago,whichincludeAlmiranteBay,DolphinBay,LomaPartida,
PastoresIslands,Popa,SharkHole,andTierraOscura.
https://doi.org/10.1371/journal.pone.0189370.g001
MinisteriodeAmbienteyEnerg´ıa–CostaRica,MINAE).Allsampleswereconservedin70%
ethanolandstoredat-20˚C[53]forsubsequentlaboratoryanalysis.
DNAExtraction,PCRanalysis,sequencing,andmolecularsexing
DNAwasextractedfromskinsamplesusingtheDNeasykit(QIAGEN,Valencia,CA,USA).
TheportionofthemtDNA-CR(approximately750bp)wasamplifiedbythepolymerasechain
reaction(PCR),usingtheprimerspairs,t-Pro-whaleM13Dlp1.5(50-TGTAAAACGACAGCCA
GTTCACCCAAAGCTGRARTTCTA-30)andDlp8G(50- GGAGTACTATG TCCTGTAACCA-
30).AmplificationfollowedtheprotocolfromBakeretal.[54]:aninitialstepof94˚Cfor2
min,with34cyclesof2–4repeattimesof30sat94˚C,followedby45sat55˚Candanexten-
sionat72˚C,withafinalextensionafterthelastcycleof10minat72˚C.EachPCRmixof
30μlreactioncontaineda1Xreactionbuffer(10mMTris–HCl,50mMKCl,pH8.3),1.5mM
MgCl ,200μMdNTP,2unitsBSA,0.5μMofeachprimer,and1UBiolaseDNApolymerase
2
(BiolineUSA).PCRproductswerepurifiedfollowingaPolietilenglicolprotocol(PEG20%),
PLOSONE|https://doi.org/10.1371/journal.pone.0189370 December13,2017 4/22
PopulationgeneticsofbottlenosedolphinsinBocasdelToro
andDNAwassequencedusingtheSangersequencingmethod[55].Toexaminethepossibility
ofsharedhaplotypesamongdifferentlocations(BocasdelToro,Gandoca-Manzanillo,and
WiderCaribbean),previouslypublishedsequencesfromCaballeroandIslas-Villanuevaetal.
[3]wereobtainedfromNCBI(Accessionnumbers:JN596281-JN596321).Finally,samples
weresexedfollowingtheprotocolproposedbyGilsonetal.[56],amplifyingthemale-specific
SRYgeneandZFY/ZFXgenesofmalesandfemalesaspositivecontrols.Thehaplotypes
sequencesidentifiedinthisstudyweresubmittedtoGenBankasaccessionnumberKX833116
forBocasdelToropopulation,andKY817220andKY817221forindividualsfromCostaRica.
ThesetwohaplotypesfromCostaRicaweresuccessfullyamplifiedinonlytwosamples.
AnalysisofelevenpolymorphicmicrosatellitelocifollowedCaballeroandIslas-Villanueva
etal.[3].Microsatellitelociincluded:D08,D22[57],TexVet7, TexVet5 [58],MK6,MK8,MK9
[59],EV1[60],Tur48,Tur91andTur117[61],co-loadedforsizinginthreelotsforamplifica-
tion.Eachmicrosatellitelocuswasamplifiedseparatelyin10μlmixPCRreactions.Thether-
mocyclingprofileforeachlocusfollowedCaballeroandIslas-Villanuevaetal.[3].Primers
werefluorescentlylabeledfordetectiononanABI3500automatedsequencerusingtheinter-
nalROX500sizestandard.Allindividualsweregenotypedforatleastnineloci.Inorderto
allowforcomparisonswithpreviousmicrosatellitedatafromsamplescollectedintheCarib-
bean[3],aninternalcontrolusingpreviousdataof128samplesfromsixlocationswasusedin
allPCRamplificationsandgenotypificationruns.Inthisdata,TexVet7 andEV1werenot
included;thereforethesemicrosatellitelociwereexcludedinouranalysesaswell.Alleleswere
visualizedandsubsequentlybinnedusingGeneMapperSoftware(LifeTechnologies).These
microsatellitedataoftheindividualdolphinsfromBocasdelToroandCostaRicaareavailable
inDryad(S1Data).
Dataanalyses
MitochondrialDNAdataanalyses. Toexaminepotentialsharedhaplotypes,weused40
mtDNA-CRsequencesofbottlenosedolphinsfromtheWiderCaribbeanobtainedfrom
CaballeroandIslas-Villanuevaetal.[3](availableatNCBIaccessionnumbersJN596281–
JN596321).FrequencydistributionofthesehaplotypesintheCaribbeanlocationsarefoundin
Table1.AllsequenceswereeditedandalignedmanuallywithsequencesobtainedfromBocas
delToroandCostaRica,usingtheprogramGENEIOUSversion4.8.5[62].Inordertounder-
standtherelationshipsamongBocasdelTorohaplotypeswithCostaRicanonesandprevi-
ouslydescribedhaplotypesfromtheCaribbean,aNeighbor-Joiningtreewasconstructed
usingthesoftwarePAUPversion4.0[63].
WeassessedgeneticsubdivisionamongCaribbeanpopulationswithananalysisofmolecu-
larvariance(AMOVA)[64]performedbyARLEQUINversion3.5[65],alongwiththepair-
wisecomparisonofpopulationdifferentiationindicesF [66]andφ betweenallthe
ST ST
populationsanalyzed.TheTamura-Neigeneticdistancemodel[67]wasusedtoobtainφ
ST
estimates.Toconducttheseanalysesweonlyconsideredsamplingregionswithn(cid:21)2.There-
fore,eightregionswereincluded(TheBahamas,Colombia,CostaRica,Cuba,Honduras,
Mexico,Panama-BDT,andPuertoRico),andsamplesfromJamaicaandtheUSVirginIslands
wereexcluded(n=1)(Table1).
Microsatellitedataanalysis. Todeterminethegeneticstructureofbottlenosedolphins
fromBocasdelTorointheCaribbean,wecomparedourmicrosatelliteresults(n=25)with
nucleardataofsamplesfromCostaRica(n=5)andpreviouslypublishednucleardatafrom
sixdifferentCaribbeangeographiclocations,includingTheBahamas(n=11),Colombia
(n=3),Cuba(n=53),Honduras(n=6),Mexico(n=30),andPuertoRico(n=20)[3].Over-
all,ninemicrosatellitelociwereanalyzedforatotalof153individuals.
PLOSONE|https://doi.org/10.1371/journal.pone.0189370 December13,2017 5/22
PopulationgeneticsofbottlenosedolphinsinBocasdelToro
Table1. D-loophaplotypefrequencydistributionamongCaribbeanbottlenosedolphinpopulationsaccordingtoCaballeroandIslas-Villanueva
etal.[3]. Newhaplotypesreportedinthisstudy(TruBOCfromBocasdelToro,andTtruCR1andTruCR2fromCostaRica)areinbold.
Location TheBahamas Colombia Cuba Honduras Jamaica Mexico PuertoRico VirginIslands Panama(BDT) CostaRica
N=15 N=4 N=65 N=6 N=1 N=40 N=26 N=1 N=25 N=2
Haplotypes Inshoreform
TtruCAR-A 6 36
TtruCAR-L 1
TtruCAR-D 1 9
TtruCAR-E 9 1
TtruCAR-JJ 1
TtruCAR-F 3
TtruCAR-AA 5
TtruCAR-K 2 2
TtruCAR-N 1
TtruCARITA02 2
TtruCAR-S 1
TtruCAR-X 1
TtruCAR-M 1
TtruCAR-B 12 2 4 1
TtruCAR-Z 1
TtruCAR-DD 1
TtruCAR-U 1
TtruCAR-EE 1
TtruCAR-FF 2
TtruCAR-CC 1
TtruCAR-V 2
TtruCAR-BB 1
TtruCAR-Q 1
Ttru-BOC 25
WorldwideDistributedForm(WDF)
TtruCAR-C 3 4 4 5
TtruCAR-MM 1
TtruCAR-P 1
TtruCAR-G 2
TtruCAR-J 1
TtruCAR-GG 1
TtruCARQR1 2
TtruCAR-H 9
TtruCAR-II 1
TtruCAR-KK 1
TtruCAR-I 2
TtruCAR-T 1
TtruCAR-O 1
TtruCAR-R 1
TtruCAR-Y 3
TtruCAR-W 1
TtruCAR-HH 1
TtruCAR-LL 1
Ttru-CR1 1
Ttru-CR2 1
https://doi.org/10.1371/journal.pone.0189370.t001
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PopulationgeneticsofbottlenosedolphinsinBocasdelToro
TheprogramTANDEMwasusedtoconductbinningofmicrosatellitedata.Thissoftware
isbasedonaheuristicsearchwiththeNelder-MeadDownhillSimplexalgorithm,andtocalcu-
latethenumberofallelesitappliesaleast-squareminimizationofroundingerrors[68].We
usedthesoftwareMICRO-CHECKERversion2.2.3withBonferronicorrectionformultiple
comparisonsinallloci,toevaluatethepresenceofnullalleles,alleledropouts,andpotential
scoringerrorsduetostutterpeaks[69].
ToexaminethegeneticstructureinBocasdelToroandtheWiderCaribbean,weusedthe
softwareSTRUCTUREversion2.3.4[70],withaburninperiodsetto10000iterations.We
determinedtheprobabilityestimatesusing100000MarkovchainMonteCarlo(MCMC)itera-
tions.ToinferthetrueKfromthelogprobabilityofthedataLnP(D)[71],wefirstconducted
therunswithKsetfrom1to10foreachvalueofKwiththeadmixturemodelandcorrelated
frequenciesinthesoftwareSTRUCTURE.After,usingtheprogramSTRUCTUREHAR-
VESTER[72],wecomparethelogprobabilityLnP(D)ofdifferentvaluesforKusinganadhoc
statisticΔK,whichcalculatesthesecondorderrateofchangeofLnP(D).Thecorresponding
valuesforeachKwereplottedtodeterminetheuppermostlevelofpopulationstructurefor
ourdataset.
Weassessedgeneticdifferentiationanddiversityamongpopulationunitsdefinedprevi-
ouslybySTRUCTUREusingthesoftwareARLEQUINversion3.5[65].Wecalculatedthe
observedheterozygosity(H ),theexpectedheterozygosity(H ),levelsofpolymorphism
o E
(100000Markovchain,100dememorizationsteps),andthelinkagedisequilibrium(LD)
(10000permutations).Weestimatedthenumberofalleles(N )andallelicrichness(AR)per
A
locususingtherarefactiontestimplementedinthesoftwareHP-RARE[73].Wetesteddevia-
tionfromHWequilibrium(HWE)usingtheMicrosoftExceladd-inGENALEXsoftwarever-
sion6.5[74].WeassessedsignificancevaluesusingasequentialBonferronicorrection[75]as
implementedinanEXCELcalculatorversion1.1[76].WecalculatedpairwiseF andR val-
ST ST
uesforeachpopulation’spairtoestimategeneflowusingthesoftwareGENALEXversion6.5.
(1000permutations)[74].WealsocalculatedglobalconventionalF andglobalcorrectedF
ST ST
usingtheHIERFSTATpackageinRversion3.3.3[77].Todeterminegeneticsubdivision
amongCaribbeanpopulations,andtocomparevariationsbetweenandwithingroups,weper-
formedanAMOVA[64]intheprogramARLEQUINversion3.5[65].
Toestimatethenumberofmigrantspergeneration(N )amongpairsofpopulationunits
m
ofbottlenosedolphinsintheCaribbean,weranthesoftwareMIGRATEversion3.0.3with
microsatellitedata.ThissoftwareusesthecoalescentapproachofMarkovchainMonteCarlo
(MCMC)torunthemaximumlikelihoodwithallpopulationunitssimultaneously[78,79].To
evaluatesolutionconvergenceoftheparametersobtainedbyMCMC,weranmultipletimes
tenshortchains(500usedtreesoutofasampled10000)bythreelongchains(5000usedtrees
outofasampled100000),andlastly,aburn-inof10000[3].
Finally,weassessedthecontemporaryeffectivepopulationsize(Ne)fortheBocasdelToro
population,usingthebias-correctedversionofthemethodoflinkagedisequilibrium[80,81],
asimplementedintheprogramNeESTIMATORversion2.01(usingpolymorphismthresh-
oldsof0.05and0.02)[82].Thissoftwareusesmulti-locusdiploidgenotypesfrompopulation
samples,andcanestimateareasonablecontemporaryNeinsmallpopulations[82].Tocon-
ductthisanalysis,onlysamplesfromadultswereconsidered(twosamplesfrommalejuveniles
wereomitted).
Results
Wecollected24biopsysamplesfrombottlenosedolphinswithintheArchipelagoofBocasdel
Toro,includingsamplesfromthefollowinglocations:AlmiranteBay(n=3),DolphinBay
PLOSONE|https://doi.org/10.1371/journal.pone.0189370 December13,2017 7/22
PopulationgeneticsofbottlenosedolphinsinBocasdelToro
(n=10),LomaPartida(n=3),PastoresIslands(n=2),Popa(n=2),SharkHole(n=2),and
TierraOscura(n=2)(Fig1).Additionally,onesamplewasobtainedfromadeadstrandeddol-
phinfoundinthemangroveinBocastorito.Overall,therewere14males(12adults,twojuve-
niles)and11females(alladults).
MtDNA-CRresults,populationstructureandecotypeclassification
Fromthe25samplescollectedintheArchipelagoofBocasdelToro,onlyonehaplotypewas
identified(760bp).Wecomparedthishaplotypetotwosequences(760bpand540bp)
obtainedfrombottlenosedolphinsofGandoca-ManzanilloinCostaRica,locatedonly35km
northofBocasdelToro,andwith40publishedbottlenosedolphinshaplotypesfoundinmulti-
plelocationsintheWiderCaribbean.OurresultsrevealedthatthehaplotypefoundinBocas
delToroisunique,asitisnotfoundinanyotherbottlenosedolphinpopulationintheCarib-
bean(Table1).Thedifferencesareduetoonetransition,inposition192,(C!T).
TheuniquehaplotypefromBocasdelTorowastrimmeddownto320bptocomparewith
previouslypublishedmtDNA-CRsequences.ComparisonswereconductingNeighbor-Joining
analysesthatgroupedthehaplotypefromBocasdelToroinacladewithotherhaplotypespre-
viouslydescribedasthe‘inshoreform’fromTheBahamas(TtruCAR-E),Cuba(TtruCAR-D,
TtruCAR-E,TtruCAR-K,TtruCAR-N,TtruCAR-S),Mexico(TtruCAR-AA,TtruCAR-D,Ttru-
CAR-F,TtruCAR-K,TtruCAR-X,TtruCAR1TA02),andPuertoRico(TtruCAR-JJ)(Fig2;
Table1).Particularly,thehaplotypeTtruCAR-D,sharedwithCubaaMexico,hasbeensug-
gestedpreviouslyasancestral[3].The‘inshoreform’wasincluding24haplotypes(seeTable1)
fromTheBahamas,Cuba,Mexico,PuertoRico,Panama-BDT,andVirginIslands.Mostof
thesedolphinpopulationssharedhaplotypes(Table1),suggestingsomedegreeofpastorpres-
entconnectivityamongthem.
ThehaplotypefromBocasdelTorowasnotsharedwithanyotherpopulation.Incontrast,
thetwohaplotypesfromGandoca-Manzanillo(CostaRica)weregroupedwith‘worldwidedis-
tributedform’(WDF)haplotypesfromtheCaribbean(seeFig2;Table1).Specifically,they
nestedwithWDFhaplotypesfromColombia(TtruCAR-MM),Cuba(TtruCAR-J,TtruCAR-P),
Honduras(TtruCAR-G),Mexico(TtruCAR-QR1),andPuertoRico(TtruCAR-GG).Inaddi-
tion,theCostaRicanhaplotypesnestedwiththehaplotypeTtCAR-C,anancestralWDF
Fig2.Neighbor-JoiningreconstructionshowinggroupingofWiderCaribbeanControlRegion
haplotypes.BlackarrowindicatesBocasdelTorohaplotype,whichisgroupedwithhaplotypesdefinedas
belongingtothe‘inshoreform’.TheseotherhaplotypeshavebeenreportedinTheBahamas(TtruCAR-E),
Cuba(TtruCAR-D,TtruCAR-E,TtruCAR-K,TtruCAR-N,TtruCAR-S),Mexico(TtruCAR-AA,TtruCAR-D,
TtruCAR-F,TtruCAR-K,TtruCAR-X,TtruCAR1TA02),andPuertoRico(TtruCAR-JJ).Greyarrowindicates
CostaRicanhaplotypes,whichisnestedwithWDFhaplotypes.
https://doi.org/10.1371/journal.pone.0189370.g002
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PopulationgeneticsofbottlenosedolphinsinBocasdelToro
haplotyperelatedtoWDFhaplotypesfromMadeira[3],whichisalsosharedwithbottlenose
dolphinsfromColombia,Cuba,Honduras,andPuertoRico(Fig2;Table1).
Intheanalysisofpopulationstructure,wedefinedtwopopulationunits,whereBocasdel
Toro(BDT)wasgroupedwithothersubpopulations:BDT-Bahamas-Colombia-Cuba-Mexico,
meanwhileCostaRicawasgroupedwithHondurasandPuertoRico(Table2).Significantpop-
ulationdifferentiation(P=0.000)wasfoundamongbothpopulationunitsattheF andF
ST ST
levels(Table2).Ingeneral,thesepopulationsunitsshowedadifferentiationlesssignificant
(P<0.1)sincewefoundavalueofФ =0.205(P=0.052),Ф =0.427(P=0.000),andthe
CT ST
Ф =0.279(P=0.000).
SC
Microsatelliteresults
Populationstructure. BasedontheassumptionthatCaribbeanbottlenosedolphinshave
acommonancestor,weperformedtheSTRUCTUREanalysisundertheadmixturemodel
withcorrelatedfrequencies,inordertoassessthenumberofunits(K)presentintheCarib-
bean.TheevaluationoftheKvaluesusingtheΔKmethodproducedaclearpeakatK=2(S1
Fig,S1Table)inwhichBocasdelTorowasidentifiedasonepopulationunit(Fig3A),and
therefore,thesoftwareidentifiedK=2asthemostlikelynumberofgroupspresentinthedata
(ΔK=267.317)(Fig3A).However,apreviousworkbyCaballeroandIslas-Villanuevaetal.[3]
reportedtoK=4asthenumberofpopulationsunitspresentintheCaribbeanaccordingto
thesamemicrosatellitedata(withoutincludingBocasdelToroandCostaRicansamples).
Basedonfourpopulationunitsidentifiedinthispreviouswork(Bahamas;Colombia-Hondu-
ras-PuertoRico;Cuba;andMexico),andinordertoidentifypopulationstructurebetweenthe
BocasdelToropopulationandindividualsfromCostaRica,weassessedthepopulationstruc-
tureanalysesinK=6,inwhichBocasdelToro(Panama)andGandoca-Manzanillo(Costa
Rica)belongtotwodistinctpopulationunits(Fig3B).InFig3B(K=6),wheretheXaxiscor-
respondstoeachindividual,itisnotablethattheBocasdelToroclusterconformsadiscrete
populationunit.ThisplotalsoshowsthattheBocasdelToropopulationunitishighlydiffer-
entiatedfromotherpopulationunitsandthatindividualsdonotappeartomaintainpresent
geneflowwithotherdolphinpopulationintheCaribbean,includingGandoca-Manzanillo,
andiftheydo,itisminimal.
TheresultsfromtheMIGRATEanalysisindicatedthattherewasonlyonemigrantpergen-
erationfromBocasdelTorototheneighborpopulationinGandoca-Manzanillo(N =1.070)
m
(Table3),andratesofreversemigrationarealmostnil(N =0.132),suggestingasymmetrical
m
migration.IndividualsfromGandoca-Manzanilloalsoshowedlowratesofmigrationtoward
otherlocationsintheCaribbean.WhilebothBocasdelToroandGandoca-Manzanillodol-
phinsshowlowmigrationrates,otherdolphinpopulationsintheCaribbeanseemtomaintain
connectivitywithatleastonemigrantpergeneration(Table3).Forinstance,theColombia-
Honduras-PuertoRicopopulationunitreceivesmigrantsfromTheBahamasandCuba(N =
m
Table2. PopulationdifferentiationofTursiopstruncatusbetweenpairwisepopulationsintheCaribbeanobtainedwithmtDNA-CR. Highandsignif-
icantvaluesareindicatedinboldandtheP-valueisshownbeloweachvalue(P-valueswereobtainedafter1000permutations).Ф valuesareindicated
ST
belowthediagonal.F valuesareabovediagonal.
ST
F BDT-Bahamas-Colombia-Cuba-Mexico CostaRica-Honduras-PuertoRico
ST
Ф N=149 N=34
ST
BDT-Bahamas-Colombia-Cuba-Mexico - 0.133
(0.000)
CostaRica-Honduras-PuertoRico 0.299 -
(0.000)
https://doi.org/10.1371/journal.pone.0189370.t002
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PopulationgeneticsofbottlenosedolphinsinBocasdelToro
Fig3.Barplotofthelikelihood(Y-axis)ofeachindividual’s(X-axis)assignmenttoaparticular
populationunitsaccordingtoΔKmethod[64](K=2,figurea).Basedonfourpopulationunitsidentifiedin
theCaribbeaninapreviouswork[3],andintheassumptionthatBocasdelToro(Panama)andGandoca-
Manzanillo(CostaRica)belongtotwodistinctpopulationunits,wealsoincludeK=6(figureb).Inbothfigures
BocasdelToroconsistsofonediscretepopulationunit.
https://doi.org/10.1371/journal.pone.0189370.g003
1.113andN =1.133,respectively).Similarly,Colombia-Honduras-PuertoRico,TheBahamas,
m
andMexicopopulationunitscontributeonemigrantpergenerationtowardsCuba(N =
m
1.526,N =1.605,andN =1.894,respectively),andtheMexicopopulationunitreceives
m m
migrantsfromCuba(N =1.598).Hence,allpopulationsunitsintheCaribbeanshowacer-
m
tainlevelofconnectivity,exceptforBocasdelToroandGandoca-Manzanillo.
Ingeneral,globalconventionalF (0.219,P=0.000)andcorrectedF (0.232,P=0.000)
ST ST
valuesshowedapopulationstructureamongbottlenosedolphinsintheWiderCaribbean.The
calculatedpairwisepopulationdifferentiationindicesF andR forallsixproposeddolphin
ST ST
populationsaredescribedinTable4,andshowthatforthemostpartR valuesarehigher
ST
thanF values.Thissuggestsanolderdifferentiationamongdolphinpopulationswithsome
ST
recentgeneticconnectivity[3].ParticularlyforBocasdelToro,F andR indicatethatthe
ST ST
BocasdelToropopulationisdifferentiatedfromtheotherCaribbeandolphinpopulations,
suggestingacertaindegreeofisolation.TheotherdolphinpopulationsintheCaribbeanshow
Table3. Numberofmigrantspergeneration(N )estimatedforCaribbeanTursiopstruncatuspopulationunits(credibilityintervalat95%). N >1
m m
areinbold.
Receivingpopulationn Panama(BDT) CostaRica Colombia-Honduras-PuertoRico TheBahamas Cuba Mexico
Sourcepopulation
Panama(BDT) - 1.070 0.216 0.161 0.145 0.153
(0.040–0.381) (0.026–0.092) (0.100–0.107) (0.100–0.107) (0.018–0.054)
CostaRica 0.132 - 0.212 0.195 0.158 0.157
(0.002–0.010) (0.026–0.090) (0.122–0.131) (0.109–0.117) (0.019–0.055)
Colombia-Honduras-PuertoRico 0.031 0.143 - 0.733 1.526 0.861
(0.0005–0.509) (0.005–0.051) (0.456–0.490) (1.054–1.129) (0.103–0.303)
TheBahamas 0.029 0.146 1.113 - 1.605 0.670
(0.0005–0.470) (0.006–0.052) (0.136–0.471) (1.110–1.188) (0.080–0.236)
Cuba 0.029 0.143 1.133 0.797 - 1.598
(0.0005–0.480) (0.005–0.051) (0.138–0.480) (0.496–0.532) (0.192–0.562)
Mexico 0.028 0.149 0.736 0.747 1.894 -
(0.0004–0.470) (0.006–0.053) (0.090–0.311) (0.465–0.499) (1.309–1.401)
https://doi.org/10.1371/journal.pone.0189370.t003
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