The Ancestral Carnivore Karyotype As Substantiated by Comparative Chromosome Painting of

15 Pages·2016·3.45 MB·English

RESEARCHARTICLE
The Ancestral Carnivore Karyotype As
Substantiated by Comparative Chromosome
Painting of Three Pinnipeds, the Walrus, the
Steller Sea Lion and the Baikal Seal
(Pinnipedia, Carnivora)
ViolettaR.Beklemisheva1*,PolinaL.Perelman1,4,NatalyaA.Lemskaya1,Anastasia
I.Kulemzina1,AnastasiaA.Proskuryakova1,4,VladimirN.Burkanov2,3,Alexander
S.Graphodatsky1,4
1 DepartmentofComparativeGenomics,InstituteofMolecularandCellularBiology,SiberianBranchof
RussianAcademyofSciences,Novosibirsk,Russia,2 DepartmentofHigherVertebratesEcology,
KamchatkaBranchofPacificGeographicalInstituteofFarEastBranchofRussianAcademyofSciences,
Petropavlovsk-Kamchatski,Russia,3 NationalMarineMammalLaboratory,AlaskaFisheriesScience
Centre,NationalMarineFisheriesService,Seattle,Washington,UnitedStatesofAmerica,4 Novosibirsk
StateUniversity,Novosibirsk,Russia
OPENACCESS *[email protected]
Citation:BeklemishevaVR,PerelmanPL,Lemskaya
NA,KulemzinaAI,ProskuryakovaAA,BurkanovVN,
Abstract
etal.(2016)TheAncestralCarnivoreKaryotypeAs
SubstantiatedbyComparativeChromosomePainting
ofThreePinnipeds,theWalrus,theStellerSeaLion KaryotypeevolutioninCarnivoraisthoroughlystudiedbyclassicalandmolecularcytogenet-
andtheBaikalSeal(Pinnipedia,Carnivora).PLoS icsandsupplementedbyreconstructionsofAncestralCarnivoraKaryotype(ACK).However
ONE11(1):e0147647.doi:10.1371/journal.
chromosomepaintinginformationfromtwopinnipedfamilies(OdobenidaeandOtariidae)is
pone.0147647
noticeablymissing.Wereportontheconstructionofthecomparativechromosomemapfor
Editor:WilliamJ.Murphy,TexasA&MUniversity,
speciesfromeachofthethreepinnipedfamilies:thewalrus(Odobenusrosmarus,Odobeni-
UNITEDSTATES
dae–monotypicfamily),nearthreatenedStellersealion(Eumetopiasjubatus,Otariidae)and
Received:October26,2015
theendemicBaikalseal(Pusasibirica,Phocidae)usingcombinationofhuman,domestic
Accepted:January6,2016 dogandstonemartenwhole-chromosomepaintingprobes.Theearliestkaryologicalstudies
Published:January28,2016 ofPinnipediashowedthatpinnipedswerecharacterizedbyapronouncedkaryologicalcon-
servatismthatisconfirmedherewithspeciesfromPhocidae,OtariidaeandOdobenidae
Copyright:Thisisanopenaccessarticle,freeofall
copyright,andmaybefreelyreproduced,distributed, sharingsamelownumberofconservedhumanautosomalsegments(32).Chromosome
transmitted,modified,builtupon,orotherwiseused paintinginPinnipediaandcomparisonwithnon-pinnipedcarnivorekaryotypesprovide
byanyoneforanylawfulpurpose.Theworkismade
strongsupportforrefinedstructureofACKwith2n=38.Constructedcomparativechromo-
availableundertheCreativeCommonsCC0public
domaindedication. somemapsshowthatpinnipedkaryotypeevolutionwascharacterizedbyfewtandem
fusions,seeminglyabsentinversionsandslowrateofgenomerearrangements(lessthen
DataAvailabilityStatement:Allrelevantdataare
withinthepaper. onerearrangementper10millionyears).Integrativecomparativeanalyseswithpublished
chromosomepaintingofPhocavitulinarevealedcommoncytogeneticsignatureforPhoca/
Funding:ThisworkwassupportedbyRussian
FoundationforBasicResearch(https://kias.rfbr.ru): PusabranchandsupportsPhocidaeandOtaroidea(Otariidae/Odobenidae)assister
project14-04-00467toVRB;project15-04-00962to groups.Werevealedrearrangementsspecificforwalruskaryotypeandfoundthechromo-
ASG;project15-29-02384toASGandtheResearch
somalsignaturelinkingtogetherfamiliesOtariidaeandOdobenidae.TheStellersealionkar-
grantoftheMolecularandCellBiologyofRASto
yotypeisthemostconservedamongthreestudiedspeciesanddiffersfromtheACKby
ASG.
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ChromosomeEvolutioninPinnipeds
CompetingInterests:Theauthorshavedeclared singlefusion.ThestudyunderlinedthestrikinglyslowkaryotypeevolutionofthePinnipedia
thatnocompetinginterestsexist ingeneralandtheOtariidaeinparticular.
Introduction
Pinnipedsareremarkablegroupofmarineanimalswithuniqueadaptationstosemiaquatic
lifecomprisingseals,sealionsandwalrus.Thepinnipedswereoneofthefirstmammalianspe-
ciesgrouptobethoroughlyinvestigatedateachstageofcytogeneticsdevelopment.Animal
cytogeneticsmadegreatprogressoverthepastcenturystartingwithintroductionoftechniques
forefficientchromosomevisualization(likecolchicineapplication,hypotonictreatment,
advancesincellcultureandchromosomepreparation)allowingdetaileddescriptionofkaryo-
type[1].Developmentofdifferentialstainingmethods(G-bandinginparticular)allowediden-
tificationofhomologouselementsandchromosomalrearrangementsinkaryotypesofdifferent
species.Thisenabledkaryotypecomparisonofdifferentmammalianspeciesandgaveriseto
comparativemammaliangenomicsproducingvastdataaboutchromosomeevolution.The
introductionofchromosomespecificprobesensuredprecisedelineationofhomologoussynte-
nicregionsandidentificationofevolutionarychromosomerearrangementsevenamongdis-
tantspecies.
Thequestionabouttheroleofchromosomerearrangementsduringspeciationremainsone
ofthefewunsolvedandintriguingpuzzlesofcurrentevolutionarybiology.Evenmoreinterest-
ingarethegenome-shapingevolutionaryprocessesofspeciationinmarineenvironment.Fin-
footedpinnipedshavesplitfromterrestrialmembersofCarnivoraabout40MYA[2].This
groupissodistinctthattheywereonceconsideredtobeaseparatesuborderofCarnivoraor
separatemammalianorderPinnipedia[3,4].Therewasevenquestionwhetherpinnipedsare
monophyleticorthetruesealshadoriginatedfromweaselsandtheothertwofamiliesfrom
bears[5].Inrecentdecadesmolecularphylogeneticstudieshavesettledmanyquestionsshow-
ingthatPinnipedsaremonophyleticandaffinetoCaniformiabranchofCarnivorawithsome
remaininguncertaintyaboutgroupingwithmusteloidsorursids[2,3,6,7].
ThePinnipediaincludethreeextantfamilies,Odobenidae(walruses,monotypic),Otariidae
(sealionsandfurseals)andPhocidae(trueorearlessseals).OdobenidaeandOtariidaeconsti-
tutetheOtaroidea,thesistergroupofPhocidae.ThepioneeringkaryologicalstudiesofPinni-
pediaandlatercomparisonsbasedondifferentialstainingtechniquesshowedthatpinnipeds
havenarrowrangeofdiploidnumbers2n=32to36andarecharacterizedbyapronounced
karyologicalconservatism[8–12].AllOtariidaehavestrikinglysimilarkaryotypeswiththe
chromosomenumber2n=36.Thechromosomenumberofthewalrusis2n=32.ThePhoci-
daehavetwochromosomenumbers,2n=34and2n=32,separatedbyasinglefusion[8].
GenomesofCarnivorahavebeeninvestigatedindetailbothbyclassicandbymolecular
cytogeneticmethodsthatshowedtrendsofkaryotypeevolutionintwobranchesofthisorder
—CaniformiaandFeliformia.Todate50speciesofCarnivorahavebeenstudiedbyfluorescent
insituhybridization(FISH)withflow-sortedwhole-chromosomeprobesfromnineCarnivora
speciesandhuman[13,14].Theconservedsyntenicsegmentsandrearrangementsofthese
blockswereestablishedformostfamiliesdepictingfascinatingkaryotypicchangesduringCar-
nivoraradiation.Twomajorpatternsofgenomeevolutionwererevealedforthisorder:fast
genomereshufflingduringspeciationinCanidae,UrsidaeandMephitidaeandsignificantlevel
ofconservationamongfelids,viverrids,andmusteloids(Mustelidae,Procyonidae)[13,15–25].
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ChromosomeEvolutioninPinnipeds
ComparativecytogeneticsofCarnivoraprovidedthebasisforreconstructionofthekaryo-
typeofthesupposedcommonancestor(AncestralCarnivoraKaryotype,ACK)[19,26–29].In
theoriginalattempttoreconstructACKbasedoncomparativeG-bandinganalysispinniped
karyotypesplayedanimportantrole[26].ThefirsthypotheticalstructureofACKusingchro-
mosomepaintingwasbasedonPinnipeddata[28].Howevercurrentversionsofreconstructed
ACKcontainsomeunsettledquestionsconcerningthestateofseveralancestralsegmentsthat
requireadditionalsupport.
TodatesequencingdatafortenCarnivoraspecies:cat,tiger,lion,giantpanda,domestic
dog,polarandbrownbears,ferretandtwopinnipeds—walrusandWeddel’sseal,havebeen
published(http://www.ensembl.org,http://www.ncbi.nlm.nih.gov/genome).Sequencingofthe
Baikalsealgenomeisinprogress(unpublished).Comparativechromosomepaintingmaps
provideprimarybackbonefordenovowholegenomeassembly.Yethigh-resolutionG-banded
chromosomesandmolecularassignmentofhomologiesthroughchromosomepaintingisonly
availableforonepinnipedspecies—theharbourseal(Phocavitulina)thatbelongstofamily
Phocidae[28,30].Howevertwootherfamiliesofpinnipeds(OtariidaeandOdobenidae)are
lackingthechromosomepaintingdata[13].
Here,wereportcomparativechromosomemapsforrepresentativesfromallthreepinniped
families:thewalrus(Odobenusrosmarus,Odobenidae),Stellersealion(Eumetopiasjubatus,
Otariidae)andtheBaikalseal(Pusasibirica,Phocidae)establishedbylocalizationofhuman,
domesticdogandselectedsetofstonemartenchromosome-specificpaintingprobes,compiled
withpreviouslypublishedchromosomepaintingdata.Weconductanalysisinthecontextof
chromosomeevolutionintheorderCarnivoraandineutherianmammalsandrefinetheACK
basedonthenewevidencefromPinnipediachromosomepainting.
MaterialsandMethods
Speciessampled
Weusedsamplesfromwildanimals.Tissuesfromthewalruswerecollectedduringaboriginal
quotasealinginthecoastalwatersofBeringSea(Mechigmenbay,ChukotkaAutonomous
Okrug,RussianFederation).FlapperandearskinbiopsysamplesfromStellersealionswere
collectedontheTyuleniyIsland(theSeaofOkhotsk,Russia)whenpup’swerebranded.Iso-
fluranewasusedforinhalationalanaesthesia.TissuesamplesoftheBaikalseal(LakeBaikal,
Barguzinbay,RussianFederation)weretakenfromthemalekilledbyfishnetsandreportedfor
thescientificresearch.Allsampleswerecollectedaccordingtoproceduresapprovedbythe
CommitteeontheEthicsofAnimalExperimentsoftheInstituteofMolecularandCellular
BiologySBRAS.TheinformationaboutspeciesusedinthisstudyislistedinTable1.
Storageandtransportationoftissuesamples
Thelackofcytogeneticdataforwildmarinemammalsandthelimitednumberofavailablepri-
marycelllinesmaybeexplainedinpartbytheremotenessoftheirhabitatsandsoby
Table1.
No Latinnames Codes 2N Sex Commonnames
1 Odobenusrosmarus OROS 32 M Walrus
2 Eumetopiasjubatus EJUB 36 M Northernsealion
3 Pusasibirica PSIB 32 M Baikalseal
4 Canisfamiliaris CFA 78 M Domesticdog
5 Martesfoina MFO 38 M Stonemarten
6 Homosapiens HSA 46 M Human
doi:10.1371/journal.pone.0147647.t001
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ChromosomeEvolutioninPinnipeds
difficultiesofcollectinganddeliveringasepticviabletissuesamplestothelaboratories.Whileit
took10–14daystotransportspecimenstothelaboratoryfromremoteareasofcollectionaddi-
tionaleffortstopreventbacterialandmycoticcontaminationwereundertaken.Uponcollec-
tionalltissuepieceswerethoroughlywashedandscrubbedincontinuouscoolwaterflowto
removedirtandsand.Thensampleswereincubatedfor20–30miningrowthmediumwith5
timesexcessofantibioticmixture(gentamicinsulphate+ampicillin+amphotericinb),followed
bytheincubationinmediumwith3timesexcessofantibiotics.Aftertheantibiotic/antimycotic
treatmentallsampleswereplacedintoindividual5mlplastictubescontainingsteriletransport-
ingmedium(growthmediumαMEM(Gibco)with15%bovinecalfserum(HyClone),genta-
micinsulphate50mg/L,ampicillin100mg/Landamphotericinb2.5mg/L).Forcepsand
scissorswerewashedinsoapywater,rinsedinwater,thensoakedin96%ethanolandflame-
sterilisedoverthealcoholburnerbeforemanipulatingeachsample.Tubeswithsuchprocessed
sampleswerekeptintherefrigeratoratatemperature+4–10°C.Everytime(approximately
onceintwoorthreeday)whenthemediumintubeswasacidifyingandchangingthecolour
samplesweremovedintonewsteriletubescontainingfreshtransportingmediumusingaseptic
precautionsandflame-sterilizedtools.
Chromosomepreparationandbandingtechniques,chromosome
nomenclature
Theprimaryfibroblastcelllineswerederivedfrombiopsiesofear,lung,sineworflappertis-
suesofO.rosmarus(OROS),E.jubatus(EJUB)andP.sibirica(PSIB)usingconventionaltech-
niques[31].Metaphasepreparationsweremadeasdescribedearlier[22,32,33].StandardG-
bandingstainingwasperformedaccordingto[34]method.ChromosomesofOROS,EJUBand
PSIBwerearrangedbylength.
Chromosome-specificpaintingprobespreparationandcharacterization
Setsofhuman,domesticdogandstonemartenchromosome-specificpaintingprobeswere
describedpreviously[22,35,36].Inthisinvestigationweusedthedogchromosomalnomencla-
turespublishedby[22].Whole-chromosomedoglibrarywasusedforFISHonO.rosmarus
(OROS),E.jubatus(EJUB)andP.sibirica(PSIB)genomes.Paintingwithhumanprobeswas
doneonEJUBandPSIBchromosomes.
Imagecaptureanddataprocessing
Digitalimagesofthehybridizationsignalswerecapturedasdescribedby[22,33,36]usingthe
VideoTestsystem(St.-Petersburg)withaCCDcamera(Jenoptic)mountedonaZeissmicro-
scopeAxioscope2.MetaphasespreadsimageswereeditedinCorelPaintShopProPhotoX2.
Results
Hybridizationofdogprobesontochromosomesofthreepinniped
species
HybridizationexamplesofCFAprobesareshowninFig1A–1C.EachCFAprobehighlighted
1to5fragmentsinthekaryotypesofwalrus,StellersealionandtheBaikalseal.TheCFAauto-
somalprobesdetected68homologouschromosomalsegmentsinthegenomesofinvestigated
pinnipedspecies.TwelveautosomesandX-chromosomeareconservedintotointhestudied
karyotypes.ChromosomemapsofOROS,EJUBandPSIBarepresentedinFigs2,3and4
respectively.ThehomologytochromosomalsegmentsofMFOisbasedonapublished
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ChromosomeEvolutioninPinnipeds
Fig1.Examplesoflocalizationofdog(A-C)andhuman(D-F)paintingprobeswithGTG-bandingofthesamemetaphasetotheright.(A)CFA1/26
onEJUBchromosomes;(B)CFA21+23/18onOROSchromosomes;(C)CFA16/21+28onPSIBchromosomes;(D)HSA19/3onOROSchromosomes;(E)
HSA12/16onPSIBchromosomes;(F)HSA7/1onOROSchromosomes.
doi:10.1371/journal.pone.0147647.g001
comparativechromosomedatabetweenMFOandCFA[13]andshowntotherightofpinni-
pedchromosomes.
Mappingofhumanprobesontochromosomesofthewalrus,Stellersea
lionandtheBaikalseal
The22HSAwholeautosomalprobesrevealed32homologouschromosomalsegmentsinthe
genomesofEJUB,OROSandPSIB.Patternsofchromosomepaintingwithhumanchromo-
some-specificpainting-probesaredemonstratedinFig1D–1F.EachHSAprobepainted1or2
fragmentsinthekaryotypesofstudiedspecies.HumanchromosomeprobesHSA6,11andX
eachdelineatedacompletechromosomeinthewalrus,StellersealionandtheBaikalsealkar-
yotypes.ProbescontainingHSA5and16eachdetectedanentirechromosomeintheEJUB
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ChromosomeEvolutioninPinnipeds
Fig2.GTG-bandedkaryotypeofwalrus(Odobenusrosmarus,2n=32)withtheassignmentof
homologytohuman(HSA),dog(CFA)andstonemarten(MFO)chromosomes.Thesquaredenotesthe
centromerepositiononcorrespondingchromosome.*—segmentswerenotpaintedbyanydogprobe.
doi:10.1371/journal.pone.0147647.g002
chromosomalset,HSA9paintedawholechromosomeinEJUBandPSIB.10chromosome
breaks,oneinversionand15(forEJUB)or16(forPSIBandOROS)fusionsarerequiredto
convertthehumankaryotypeintothepinnipedkaryotypes.
Mappingofselectedstonemartenpaintingprobesontochromosomesof
pinnipedspecies
Stonemartenchromosomes15and5weremappedontometaphasesoftheBaikalseal,probe
combinationsofMFO14/15and12/5weremappedontothewalrusmetaphasestoclarify
arrangementofancestralcarnivoresyntenicelementsambiguousafteranalysesofhumanand
dogpaintingprobelocalizations.
Fig3.GTG-bandedkaryotypeoftheStellersealion(Eumetopiasjubatus,2n=36)withthe
assignmentofhomologytohuman(HSA),dog(CFA)andstonemarten(MFO)chromosomes.The
squaredenotesthecentromerepositiononcorrespondingchromosome.*—segmentswerenotpaintedby
anydogprobe.
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ChromosomeEvolutioninPinnipeds
Fig4.GTG-bandedkaryotypeoftheBaikalseal(Pusasibirica,2n=32)withtheassignmentof
homologytohuman(HSA),dog(CFA)andstonemarten(MFO)chromosomes.Thesquaredenotesthe
centromerepositiononcorrespondingchromosome.*—segmentswerenotpaintedbyanydogprobe.
doi:10.1371/journal.pone.0147647.g004
Discussion
Thereweremanyattemptstoshiftintoaquaticorsemi-aquaticlifestyleindifferentmamma-
lianorders—Cetartiodactyla(whales,dolphins,porpoises),Afrotheria(manatee,dugong),Car-
nivora(pinnipeds,otters,polarbear),Rodentia(beavers,watervoleandothers).Pinnipeds
hunt,sleepandspendmostoftheirlifeinthewaterbutcomeouttobeartheirpups.Theques-
tionaboutgeneticimpactoftheuniquepositionofpinnipedsasmarinesemi-aquaticanimals
amongmostlyterrestrialCarnivoraisveryintriguing.Surprisinglysuchdrasticchangeinthe
modeoflifeisnotaccompaniedbycriticalchangesatthegeneticlevel.Adaptationstoaquatic
environmentarereflectedinamino-acidsubstitutionsofpositivelyselectedgenomicregions
foundinpinnipedsandconvergentwithothermarinemammals(whaleandmanatee)andyet
accompaniedbyremarkablylowsubstitutionrateinmutationproneCpGislands[37].There
isalsoindicationoflowlevelofvariationinskullmorphologybetweenpinnipedsandfissipeds
[38].Thesemodernfindingsreflectontheoldhypothesisofchromosomalconservatismin
marinemammals[39]thatstates“speciationcausedbychromosomalrearrangementsisrare
withinmammalianorderscharacterizedbylowreproduction,goodmobilityandcontinuous
rangeofdistribution”.
ChromosomeevolutioninPinnipedia
Theconceptofhighdegreeofchromosomeconservationinpinnipedsisbasedontheclassical
cytogeneticdatathathaverevealedsimilardiploidnumbers(32–36)acrossthisgroupofsemi-
aquaticmammalsandshowedhighsimilarityofG-bandingpatternwithothermammals.Itis
supportedbypaintingfindingsofthelownumbersofconservativeancestralelementsinhar-
boursealcomparedtoothermammals[8–11,28].Ourdataprovidefurthersupportdocument-
ingremarkablechromosomeconservationacrossallfamiliesofpinnipeds.22human
autosomepaintingprobesdelineated32conservativeautosomesegmentsinthegenomesof
threestudiedspecies.Thiscorrespondstoassessmentoftheirgenomesaslow-rearrangedrela-
tivelytotheancestraleutheriangenome.Similarlylownumberofautosomesegmentswas
detectedbyhumanpaintingprobesintheconservativekaryotypesofmusteloids,felids,hyenas
andsomeviverrids[13,40,41]andconservedkaryotypesofothermammalslikeaardvarkand
goldenmole(30).Muchhighernumbersarefoundinothermammals(47forcloselyrelated
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ChromosomeEvolutioninPinnipeds
pangolins,40–50incetartiodactylsandperissodactyls,42inbats)orevenhighlyrearranged
Carnivorakaryotypes(67–73incanids,44inursids,40inskunks)indicatinghigherdivergence
fromancestraleutheriankaryotype.Chromosomepaintinginpinnipedshereconfirmslong-
standinghypothesisaboutkaryotypeconservatisminmarineanimals.Theunderlyingreasons
forthisphenomenonwerediscussedandincludelowdegreeofinbreeding,lowreproductivity
rateandabsenceofphysicalgeographicbarriers[39].Othermarineanimalswithexceptionof
afrotherianmanatee(41)alsohavelownumberofconservativeancestralunits:otter(32),pilot
whale(34),finlessporpoise(34)andbottlenosedolphin(30).Latterthreespeciesbelongtothe
orderCetartiodactylaandatthesametimeothermembersoftheorderhaveover45conserva-
tivechromosomesegments[13,33,42–44].Bothgroups—CetaceaandPinnipediaarerather
specious(withmorethan80and32species)demonstratingthatconservativekaryotypedoes
notimpedethespeciationandthatthespeciesradiationisnotalwaysaccompaniedbyappar-
entchromosomerearrangements.
Werevealedinthegenomesofthreestudiedpinnipedsconservedsyntenicsegmentassocia-
tionscommonforthewholecladeEutheria(HSA3/21,4/8,7/16,10/12/22,14/15and16/19;
[24,29](Table2).IntotalpinnipedkaryotypesdifferfromancestralEutheriankaryotype
[45,46]by5fusions(HSA1/8,2/20,2/13,19+3/21,12/22+18))andonefission(HSA1).Four
syntenicsegmentassociations(HSA1/8,2/13,2/20,19+3/21)arecharacteristicforthewhole
orderCarnivorawithfewexceptions[14]andrepresentelementsofancestralCarnivorakaryo-
typewith2n=38[47].SyntenyHSA12/22/18wassuggestedtobeacytogeneticcharacter
Table2. Correspondenceofpinniped(theStellersealion,thewalrus,theBaikalsealandthecommonseal1)chromosomeswithdog,human,
ancestralcarnivorekaryotype(ACK2),stonemarten3andcat3chromosomes.
EJUB OROS PSIB PVIT CFA HSA ACK MFO FCA
1 2 2 sm1 20/23/35/30/27 19p/3/21 1 2 A2p/C2
2 4 4 m1 22/28/19/36/33/28 13/2q 3 3 A1p/C1q
3 3 3 sm2 16/28/15/19/32/13/3 4q/8p/4p+q 2 1 B1
4 6 5 m2 13/34/17/6/5/15/2/5 8q/1p+q 4 4 F2/C1p
5 1q 1q sq 11/2/3/4/35 5 5 5 A1q
6 8 6pprx m3pprx 2/29/10/15/10 10p/12/22 8 9 B4
7 10 10 m5p 28/24/10/17 20/2p 9 8 A3
8 7 9 sm4 3/25/8 15/14 7 7 B3
9 9 8 sm3 37/12/1 6 6 6 B2
10p 11p 6pdist m3pdist 6 7/16p 18 18 E3
10q 11q 7p m4p 37/8 1q 16 16 F1
11 1p 7q m4q 4/31 10q 12 12 D2
12 13 11 m6 5/21/18 11 11 10 D1
13 12 12 sm5 18/14/16 7 10 11 A2q
14 14 13 m7 26/7/1 12/22/18 13 13 D3
15 5q 14 m8 1/11/9 9 14 14 D4
16 5p 1p sp 9/5 17 15 15 E1
17 15 15 m9 1/2/5 19q/16q 17 17 E2
X X X X X X X X X
CorrespondencewithhumanchromosomesisbasedonourFISHresults,humanchromosomesegmentsaredesignatedaccordingtopublisheddata:
1Frönickeetal.[28],
2Perelmanetal.[14],
3Nieetal.[13]).
HereweusedogchromosomenomenclatureaccordingtoYangetal.,[22].dist—distalpartofchromosome,prx—proximalpartofchromosome.
doi:10.1371/journal.pone.0147647.t002
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ChromosomeEvolutioninPinnipeds
linkingCarnivoraandCetartiodactyla[13,42,44].Itwasalsosuggestedthatthereisanassocia-
tionHSA1q/10q(withHSA1qbeingreallysmallsize—HSA1q42.1–q43)thatiscommonfor
allBoreutherianmammals[48].Howeverwedidnotseethesegmentof1qonthecorrespond-
ingpinnipedchromosomes(OROS1,EJUB11,PSIB7)anditislikelythatthesizeofthis
HSA1qsegmentisatthethresholdofchromosomepaintingresolution.Thereisnocommon
cytogenetictraittodistinguishallpinnipedsfromtherestofCarnivoraandtoprovidecyto-
geneticlinkformonophyleticoriginofthisgroupversusoncesuggesteddiphyly[5].
BaikalsealisendemicspeciesfoundonlyinLakeBaikalwithitsoriginstillbeingunderdis-
cussion.MitogenomicdataindicatethatithasseparatedfromotherPusaspeciesatleast4
MYA[49].Previously,onlyconventionallystainedkaryotypeofPusasibiricawasreported
[50].HerewepresentG-bandedkaryotypeofPusasibiricawithestablishedhomologiesto
humananddoggenomes.Zoo-FISHofharbourseal(Phocavitulina,PVIT)withhuman
whole-chromosomeprobesdisclosed30autosomalconservedsyntenicsegments[28]com-
paredto32revealedinPusasibirica.HighsimilarityofGTG-bandingofP.vitulina[30]andP.
sibiricaindicatesthattwoconservativefragmentsweremissedinthefirstanalysisofpinniped
bycomparativechromosomepainting:thedistalpartofp-armofPVITm3ishomologousto
HSA12andthedistalpartofp-armofPVITm7ishomologoustoHSA7.Itmeansthatthe
genomeofharborsealcontainssyntenieshomologoustoHSA7/16and12/22+18andconse-
quentlythereare32autosomalsegmentshomologoustohumanconservativefragmentsinthe
genomeofPVITlikewiseinthegenomeofPSIB.OtherwisecomparisonofG-bandingand
humanprobespaintingpatternsinPhocavitulinaandPusasibiricakaryotypesheredidnot
revealanydifferencesshowinghighlevelofkaryotypeconservationduringspeciationinPhoci-
nae.Thatprovesthatwhole-chromosomeprobeswouldnotbehelpfultouncoverthehistory
ofspeciationinPhocinaetosolveunsettledquestionaboutPhoca/Pusadivisionandindicates
thenecessityforfurtherphylogeneticstudies[51].
Karyotypeofthewalrus,theonlylivingrepresentativeofOdobenidae,wasdescribedprevi-
ously[10].Attemptstorevealrearrangementsbetweenwalrusandotherpinnipedsdidnot
yieldconclusiveresults[10,52].Usingcombinationofhumananddomesticdogpainting
probesofhighpowerofresolutionwerevealedtwofusionscharacteristicforOdobenidae:HSA
5/10and9/17(ACK5/12,14/15)thathadremainedunresolvedinstudiesofG-bandedkaryo-
typesofOtariidaeandOdobenidae.Nowwithlinksofthewalruschromosomestowell-anno-
tatedhumananddoggenomesthechromosomeassignmentofexistingwalrusgenome
scaffoldsmaybedeductedtoprovidevaluablelinkageinformationandtotrackevolutionary
changesinchromosomesatthefinesequencelevel(http://www.ncbi.nlm.nih.gov/genome/
14031).
OnthebaseofFISHdatawithdogpaintingprobeswewereabletodetectfusionsofconser-
vativeelementshomologoustostonemartenautosomes(Martesfoina,MFO)[13].Soweused
somewholechromosomeprobesofMFOtochecktheseassumptionsandtoreconstructances-
tralcarnivorekaryotypereorganizationduringformationofseal’sgenomes.Hereweapplythe
ACKchromosomenumberscorrespondingto[47]nomenclature.Fusionshomologousto
HSA5/17,7/16+10+12/22and10/1(ACK5/15,8/18and12/16)foundintheBaikalseal
genomearecommonwithPhocavitulinaandarelikelymarkersforthewholePhoca/Pusa
branch.FusionHSA7/16+1(ACK16/18)iscommonforStellersealionandthewalrus.This
rearrangementcommonformonospecificOdobenidaeandrepresentativeofOtariidaepro-
videsstrongsupportforthegrowingmolecularevidenceinsolvingthelong-standingpuzzleof
affinityofOdobenidaewitheitherOtariidaeorPhocidae[4].Incidentallythisfusionisalso
foundinbearswithexceptionofgiantpanda[19,23,24].Howevertheorderofdogchromo-
someprobesisdifferentonthissyntenicgroup(CFA7/38/6inbearsandCFA6/7/38inwalrus
andsealion)andmostlikelyindicateshemiplasicnatureofthisfusionratherthancommon
PLOSONE|DOI:10.1371/journal.pone.0147647 January28,2016 9/15
ChromosomeEvolutioninPinnipeds
chromosomalsignatureforArctoidea(Ursidae+Pinnipedia+Musteloidea)oraffinityofUrsi-
daeandPinnipedia[2,6,53,54](Fig5).ThegenomeofStellersealionretainedancestralstate
anddiffersfromAncestralMusteloidKaryotype[33]byonlyonefusionACK16/18(HSA
7/16+1).ThisagreeswithconclusionofthecomparativeG-bandinganalysesthatotariidkar-
yotyperetainedthemostprimitivefeaturesandstrikinglyresemblesconservativeprocyonid
karyotypes[10].Chromosomepaintingherepreciselyshowedrearrangementsseparatingpin-
nipedkaryotypeserasinguncertaintiesofsoleG-bandingcomparisonsandrevealedcharacter-
isticchromosomalmarkersforbranchingorderinPinnipedia.
RefinementofAncestralCarnivoreKaryotypebypinnipedpaintingdata
FirstancestralcarnivorekaryotypereconstructionwasdonebyWurster-HillandGraybased
oncomparativechromosomeanalysesofG-bandedkaryotypesoflargenumberofcarnivore
speciesincludingavailabledataforpinnipeds[9,26].Itwascalled“hypotheticalprimitivekar-
yotype”andsuggestedtocontainatleast16autosomeswithminimumfundamentalnumberof
58.Itwasnotableattempttoreconstructforthefirsttimeancestralkaryotypeforthewhole
mammalianorderthatturnedsuccessfulowingtoremarkablyconservativechromosomesets
inbothcarnivorebranchesoffeloids(Felidae,Viverridae)andcanoids(Procyonidae,Pinnipe-
dia).Appearanceofthegenemappingdataforhumanandcatledtothenextvariantofances-
tralkaryotypecalledCARthatcontained21–22autosomepairswithFN=60–68[27,55]and
wasimportantlyshowntobeveryclosetotheprimateancestralkaryotype.ExtensiveFISH
dataprovidedreliablebasefornewreconstructionsofACK.Yetlackofpaintingdataforsev-
eralkeybranchesofCarnivora(Otariidae,Odobenidae,Prionodontidae,Nanndiniidae)con-
tributestosomeuncertaintiesinACK.
Pinnipediaoccupyuniquepositionamongcanoids:iffamilieswithdrasticallyrearranged
karyotypes(Canidae,Ursidae,Mephitidae)arenottakenintoaccountthenpinnipedsrepre-
sentthemostbasalcanoidbranchandbecomeimportantlineageforancestralkaryotype
reconstruction.BasedonchromosomepaintingdatainCarnivoratwoversionsofACKdiffer-
ingbydiploidnumbershavebeenproposed:i.e.2n=42[27,29]and2n=38[28,47].Thedif-
ferencesareprovidedbyfusedorsplitstatusofautosomeelementshomologoustoFCAA1p/
C1qandC1p/F2.Ourdatademonstratethatinkaryotypesofthreestudiedpinnipedspecies
thesefragmentsarepresentasajointelementandthisisanargumentfortheACKdiploid
number2n=38asitwasfirstsuggestedinZ-CARversionofancestralkaryotypebasedon
Phocidaepainting.AlsoallpinnipedshavefusedstateforFCAA2p/C2,which,incontrast,is
presentastwoseparatepiecesinallfeloidspeciesstudiedbychromosomepaintingsofar.This
fusionisancestralforthewholeorderCarnivora.
RefinedversionofACKwith2n=38thatincludespaintingdataofpinnipedkaryotypesis
presentedonFig6.Themappingofdogpaintingprobesinthreepinnipedspeciesprovides
Fig5.KaryotypeevolutionpathwaysinPinnipedia.
doi:10.1371/journal.pone.0147647.g005
PLOSONE|DOI:10.1371/journal.pone.0147647 January28,2016 10/15