Uncovering the Ancestry of B Chromosomes in Moenkhausia sanctaefilomenae

20 Pages·2016·4.9 MB·English

RESEARCHARTICLE
Uncovering the Ancestry of B Chromosomes
Moenkhausia sanctaefilomenae
in (Teleostei,
Characidae)
RicardoUtsunomia1*,DuílioMazzoniZerbinatodeAndradeSilva1,FranciscoJ.Ruiz-
Ruano2,CristianAraya-Jaime1,JoséCarlosPansonato-Alves1,Priscilla
CardimScacchetti1,DiogoTeruoHashimoto3,ClaudioOliveira1,VladmirA.Trifonov4,
FábioPorto-Foresti5,JuanPedroM.Camacho2,FaustoForesti1
1 DepartamentodeMorfologia,InstitutodeBiociênciasdeBotucatu,UniversidadeEstadualPaulista,
Botucatu,SãoPaulo,Brazil,2 DepartamentodeGenética,UniversidaddeGranada,Granada,Spain,
3 CAUNESP,UniversidadeEstadualPaulista,Jaboticabal,SãoPaulo,Brazil,4 InstituteofMolecularand
CellularBiologySBRAS,Nobosibirsk,Russia,5 DepartamentodeCiênciasBiológicas,Faculdadede
Ciências,UniversidadeEstadualPaulista,Bauru,SãoPaulo,Brazil
*[email protected]
Abstract
OPENACCESS
Citation:UtsunomiaR,SilvaDMZdA,Ruiz-Ruano
Bchromosomesconstituteaheterogeneousmixtureofgenomicparasitesthataresome-
FJ,Araya-JaimeC,Pansonato-AlvesJC,Scacchetti
PC,etal.(2016)UncoveringtheAncestryofB timesderivedintraspecificallyfromthestandardgenomeofthehostspecies,butresultfrom
ChromosomesinMoenkhausiasanctaefilomenae interspecifichybridizationinothercases.ThemodeoforigindeterminestheDNAcontent,
(Teleostei,Characidae).PLoSONE11(3):e0150573.
withtheBchromosomesshowinghighsimilaritywiththeAgenomeinthefirstcase,butpre-
doi:10.1371/journal.pone.0150573
sentinghighersimilaritywithadifferentspeciesinthesecond.ThecharacidfishMoenkhau-
Editor:AndreasHouben,Leibniz-InstituteofPlant
siasanctaefilomenaeharbourshighlyinvasiveBchromosomes,whicharepresentinall
GeneticsandCropPlantResearch(IPK),GERMANY
populationsanalyzedtodateintheParanaandTietêrivers.Toinvestigatetheoriginof
Received:November30,2015
theseBchromosomes,weanalyzedtwonaturalpopulations:onecarryingBchromosomes
Accepted:February17,2016 andtheotherlackingthem,usingacombinationofmolecularcytogenetictechniques,
Published:March2,2016 nucleotidesequenceanalysisandhigh-throughputsequencing(IlluminaHiSeq2000).Our
resultsshowedthati)BchromosomeshavenotyetreachedtheParanapanemaRiver
Copyright:©2016Utsunomiaetal.Thisisanopen
accessarticledistributedunderthetermsofthe basin;ii)Bchromosomesaremitoticallyunstable;iii)therearetwotypesofBchromo-
CreativeCommonsAttributionLicense,whichpermits somes,themostfrequentofwhichislightlyC-banded(similartoeuchromatininAchromo-
unrestricteduse,distribution,andreproductioninany
somes)(B ),whiletheotherisdarklyC-banded(heterochromatin-like)(B );iv)thetwoB
1 2
medium,providedtheoriginalauthorandsourceare
typescontainthesametandemrepeatDNAsequences(18SribosomalDNA,H3histone
credited.
genes,MS3andMS7satelliteDNA),withahighercontentof18SrDNAintheheterochro-
DataAvailabilityStatement:Allsequencingdata
maticvariant;v)alloftheserepetitiveDNAsarepresenttogetheronlyintheparacentro-
filesareavailablefromtheNCBIdatabase(accession
number(s)KU184525-KU184571,KU129073- mericregionofautosomepairno.6,suggestingthattheBchromosomesarederivedfrom
KU129201,KU129202-KU129595,KU129596- thisAchromosome;vi)thetwoBchromosomevariantsshowMS3sequencesthatare
KU130117).
highlydivergentfromeachotherandfromthe0Bgenome,althoughtheB -derived
2
Funding:ThisworkwassupportedbyFundaçãode sequencesexhibithighersimilaritywiththe0Bgenome(thissuggestsanindependentori-
AmparoàPesquisadoEstadodeSãoPaulo-13/
ginofthetwoBvariants,withthelessfrequent,B typepresumablybeingyounger);andvii)
00953-8granttoRicardoUtsunomia,Conselho 2
thedN/dSratiofortheH3.2histonegeneisalmost4–6timeshigherforBchromosomes
NacionaldeDesenvolvimentoCientíficoe
Tecnológico,andCoordenaçãodeAperfeiçoamento thanforAchromosomesequences,suggestingthatpurifyingselectionisrelaxedforthe
dePessoaldeNívelSuperior.Thefundershadno
PLOSONE|DOI:10.1371/journal.pone.0150573 March2,2016 1/20
UncoveringtheAncestryofBChromosomesinMoenkhausiasanctaefilomenae(Teleostei,Characidae)
roleinstudydesign,datacollectionandanalysis, DNAsequenceslocatedontheBchromosomes,presumablybecausetheyaremostly
decisiontopublish,orpreparationofthemanuscript. inactive.
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist
Introduction
Bchromosomesaredispensablegenomicelementsthatarepresentinapproximately15%of
eukaryotes.Thesechromosomesexhibitaparasiticnature,andtheirinteractionwiththehost
genomedeterminestheirpopulationfrequency,whichishighlydynamicasaconsequenceof
“thearmsrace”betweenAandBchromosomes[1–3].BecauseBchromosomesdonotalways
occurinpairs,theirsegregationdoesnotconformtoaMendeliansystem,whichmayfacilitate
transmissionrateshigherthan0.5amongthesechromosomes,resultingintransmission
advantagescollectivelyreferredtoas“drive”[3,4].
TheoriginofBchromosomeshasbeeninvestigatedinvariousorganisms.Basically,they
mayarisefromtheAchromosomesofthecurrenthostspecies(intraspecificorigin)orbe
derivedinterspecificallythroughhybridization[3].AnintraspecificoriginofBchromosomes
hasbeendemonstrated,forinstance,inmaize[5,6],themigratorylocust[7],rye[8],andthe
fishAstyanaxparanae[9].However,examplesofBchromosomesarisingthroughinterspecific
hybridizationhavebeenreportedintheplantgenusCoix[10],thefishPoeciliaformosa[11],
andthewaspNasoniavitripennis[12,13].
AlthoughthesequencecompositionofBchromosomesisunknowninmostcases,several
studiesusingfluorescentinsituhybridization(FISH)andnext-generationsequencing(NGS)
haveallowedbettercharacterizationofrepetitiveDNAsequencesandsingle-copygenes
locatedonBchromosomes[14,15].Notably,thesedataprovidednewinsightsabouttheorigin
ofBchromosomes[8,16–18]andhavesuggestedthatsomeoftheDNAcontainedinBchro-
mosomesispotentiallyfunctional,asribosomalDNAwithintheBchromosomeofthegrass-
hopperEyprepocnemisploransisabletoyieldthecorrespondingphenotype(i.e.,anucleolus)
[19].
Moenkhausia(Teleostei,Characidae)isasmallfreshwatercharacidfishgenusthatiswidely
distributedinSouthAmericanriverbasinsandcomprisesapproximately86species[20].M.
sanctaefilomenaeshowsaconservedkaryotypewithrespecttothestandardgenome(2n=50
biarmedchromosomes)butalsocarriesmicro-Bchromosomesinallpopulationshitherto
analyzed[21–25].Specifically,severalpopulationscollectedintheTietêRiverbasinshow
euchromatic,orpartiallyortotallyheterochromaticBchromosomes[21,23–25],whereas
onepopulationcollectedintheParanáRivershowsonlytheeuchromaticvariant,whichis
restrictedtomales[22].
Recently,afterkaryotypingthreepopulationsfromtheTietêRiverbasinandperforming
chromosomepaintingusingaB-specificprobe,Scudeleretal.[25]concludedthatthehetero-
chromaticBtypehadanintraspecificorigin,duetosharingDNAsequenceswithseveralA
chromosomes,andthatitaroseindependentlyfromtheeuchromaticBchromosome,asa
paintingprobeproducedfromtheformerBtypedidnotpaintthelattervariant.Inanattempt
toextendtheseconclusionsfurther,inthepresentstudy,weanalyzedanaturalpopulationcar-
ryingtheeuchromaticandheterochromaticvariantsbymeansofacombinationofcytogenetic
(C-banding,microdissection,chromosomepainting,FISHmapping,silverstaining),molecular
(PCRamplification,cloning,SangerDNAsequencingandIlluminasequencing),phylogenetic,
andbioinformaticstechniques,additionallyprovidingthefirstreportforaB-lackingpopula-
tioninthisspecies.
PLOSONE|DOI:10.1371/journal.pone.0150573 March2,2016 2/20
UncoveringtheAncestryofBChromosomesinMoenkhausiasanctaefilomenae(Teleostei,Characidae)
MaterialsandMethods
EthicsStatement
Samplingwascarriedoutonprivatelands,andtheownersgavepermissiontoconductthis
study.Theanimalswerecapturedusingnets,transportedtothelaboratoryandkeptinafish
tankfor2days,andtheywereanesthetizedbeforetheanalyses.Theanimalswerecollectedin
accordancewithBrazilianenvironmentalprotectionlegislation(CollectionPermissionMMA/
IBAMA/SISBIO—number3245)andtheproceduresforthesampling,maintenanceandanaly-
sisofthefisheswereperformedincompliancewiththeBrazilianCollegeofAnimalExperi-
mentation(COBEA)andapproved(protocol504)bytheBIOSCIENCEINSTITUTE/UNESP
ETHICSCOMMITTEEONTHEUSEOFANIMALS(CEUA).
Sampling,chromosomebandingandDNAextraction
IndividualsofM.sanctaefilomenaeweresampledin2riversoftheParanáRiversystem,with
23specimens(11femalesand12males)beingcollectedfromtheBatalhaRiver(BR),belonging
totheTietêRiverbasin(Bauru,SP;22°24’23.65’S,49°05’51.38”W),and16individuals(11
femalesand5males)fromtheNovoRiver(NR),intheParanapanemaRiverbasin(Ocauçu,
SP;22°28’13.32”S,49°55’26.17”W).Theseriversareseparatedfromeachotherbyapproxi-
mately170Kmasthecrowfliesandbyseveralhundredkilometresthroughtheriversconnect-
ingthem.Afteranalysis,allspecimensweredepositedinthefishcollectionoftheLaboratório
deBiologiaeGenéticadePeixes(LBP)atUNESP,Botucatu,SãoPaulo,Brazil,undervoucher
numbersLBP19830(BatalhaRiver)andLBP19831(NovoRiver).Aftercapture,theanimals
weretakentotheLaboratory,transferredtoanaerated40Lglassaquarium(60x30x30cm)at
25°Candkeptthereuntilsacrificeforthreedaysatmost.Foodadministrationwasnotcarried
out.
Beforeanalysis,theanimalsweresacrificedbyoverdoseofanaestheticin1%benzocainein
water.Mitoticchromosomeswereobtainedfromcellsuspensionsfromtheanteriorkidney
accordingtoForestietal.[26].C-bandingwascarriedoutaccordingtoSumner[27],andactive
nucleolarorganizerregions(NORs)wererevealedaccordingtoHowellandBlack[28].The
chromosomeswereclassifiedasmetacentric(m),submetacentric(sm),subtelocentric(st)and
acrocentric(a)accordingtoLevanetal.[29].GenomicDNA(gDNA)wasobtainedfromliver
cellsusingthePromegaWizardGenomicDNAPurificationKitaccordingtothemanufactur-
er’sinstructions.
Chromosomemicrodissection
Cellsuspensionsweredroppedontocleancoverslips.Thecoverslipswerethenwashedina1x
PBSsolutionfor1minute,incubatedinatrypsinsolution(1%Trypsin,1xPBS)for20seconds
andwashedagainin1xPBS.Finally,thepreparationswerestainedwith5%GiemsainPBSfor
5minutes.ForthemicrodissectionofB ,weusedcellsuspensionsfromindividual69693,
1
whichpresentedonlythisvariant(Table1).TomicrodissecttheB variant,C-bandingwas
2
performedtoallowbetteridentificationoftheheterochromaticBchromosomeinthemeta-
phasespread.Inaddition,oneautosomefromaB-carryingindividualwasalsomicrodissected
(pairNo.1,henceforthreferredtoasA ).Itmustbenotedthatallmicrodissectionexperiments
1
werecarriedoutusingsingle-copychromosomes.
Eachmicrodissectedchromosomewastransferredtoamicropipettecontainingacollection
solution(1.5μg/μlproteinaseK,0.1%SDS,0.1%TritonX-100,1mMEDTA,10mMTris-
HCl,pH8.0,10mMNaCl)andplacedfor1hat60°Cinamoistchamber.Then,thepipette
tipswerebrokenintoa0.2mlmicrotubecontaining5μlofsterileMilliQwater,followedby
PLOSONE|DOI:10.1371/journal.pone.0150573 March2,2016 3/20
UncoveringtheAncestryofBChromosomesinMoenkhausiasanctaefilomenae(Teleostei,Characidae)
Table1. Intra-andinterindividualvariationinthenumberofB andB chromosomesintheBRpopulation.
1 2
Idno. Sex Cellswith0–6B chromosomes Cellswith0–2B chromosomes Total
1 2
0 1 2 3 4 5 6 Mean Median MI 0 1 2 Mean Median MI
69616 M 2 2 12 3 2 2.05 2 0.31 21 0 0 21
69618 M 0 1 1 5 7 3.29 3.5 0.20 14 0 0 14
69626 F 3 13 13 2 1.45 1 0.65 31 0 0 31
69628 F 3 14 8 2 3 1.60 1 0.80 0 12 18 1.60 2 0.20 30
69676 M 0 4 21 13 2.24 2 0.22 38 0 0 38
69678 F 8 15 6 0.93 1 0.48 2 16 11 1.31 1 0.45 29
69679 M 4 0 8 18 2 2.44 3 0.23 3 22 7 1.13 1 0.31 32
69680 F 2 4 14 4 1.83 2 0.25 24 0 0 24
69682 M 4 14 4 1.00 1 0.36 22 0 0 22
69637 M 2 3 10 10 2.12 2 0.34 25 0 0 25
69693 F 0 15 14 3 1.63 2 0.28 32 0 0 32
69695 M 3 20 5 1.07 1 0.29 28 0 0 28
69696 F 0 4 24 5 2.03 2 0.14 33 0 0 33
69710 F 0 2 8 18 2 2.67 3 0.16 30 0 0 30
69713 M 1 5 13 9 2.07 2 0.29 28 0 0 28
69714 F 3 26 4 1.03 1 0.21 33 0 0 33
69729 M 0 6 9 8 6 2 1 2.75 3 0.35 32 0 0 32
69730 F 0 0 4 11 11 3.27 3 0.19 26 0 0 26
69731 M 11 18 10 0.97 1 0.54 39 0 0 39
69739 M 0 13 16 2 1.65 2 0.24 0 29 2 1.06 1 0.06 31
69740 M 0 5 4 9 2.22 2.5 0.31 18 0 0 18
69741 F 1 5 10 12 2.18 2 0.34 28 0 0 28
69743 F 0 1 10 20 3 2.74 3 0.15 34 0 0 34
Mean 1.97 2 0.32 0.22 0.22 0.26
SE 0.15 0.17 0.03 0.25 0.26 0.08
MI=mitoticinstabilityindex,F=female,M=male,SE=standarderror.
doi:10.1371/journal.pone.0150573.t001
amplificationusingtheGenomePlexSingleCellWholeGenomeAmplificationKit(WGA-
Sigma).
Whole-genomesequencing(WGS)andsatelliteDNAidentification
ToperformadeepersearchforsatDNAsintheM.sanctaefilomenaegenome,wesequenced
gDNAfromtwoindividualscollectedfromtheBatalhaRiver(carryingupto6B)andtheNovo
River(0B)ontheIlluminaHiSeq2000platform,yielding2x101bppaired-endreads.Aftera
qualitytrimmingstep(filteringoutreadswithlessthan90%ofbasesshowingaqualitylower
thanQ20)withTrimmomatic[30],wesampled200,000pairsofreads(100,000readsfrom
eachpopulation)forclusteringusingRepeatExplorer[31]consideringpaired-endreads,with
clusteringandassemblyoverlaplengthsequalto55and40bp,respectively.Forthisanalysis,
wealsobuiltacustomdatabaseofrepeatedsequencesbyrunningRepeatModeler[32]onthe
assembledA.mexicanusgenome(GenBankaccessionnumberAPWO00000000.1)asacom-
plementtoRepBaseforclusterannotation.Thiscustomdatabaseresultedin1,243sequences,
consistingof589,736bpandN50=613bp.WesubsequentlysearchedforclustersofsatDNA
families(i.e.,unannotated)withsphereorringshapesandagraphdensityhigherthan0.1.
Next,wemanuallyprocessedtheassembledcontigswithGeneiousProv8.04usingtheHigh
PLOSONE|DOI:10.1371/journal.pone.0150573 March2,2016 4/20
UncoveringtheAncestryofBChromosomesinMoenkhausiasanctaefilomenae(Teleostei,Characidae)
Sensitivity/Slowoptiontovisualizedotplotgraphicstodetecttandemrepetitions.Wethen
splitthesesequencesintomonomers,alignedthemandobtainedaconsensussequenceofthe
monomericunits.
RepetitiveDNAprobes
5SrDNA,U2snDNAandH3histonegeneprobeswereobtainedviaPCRdirectlyfromthe
genomeofM.sanctaefilomenaeusingbothprimersdescribedpreviously[6,33–35]andothers
describedhere,asfollows.Toyieldanoptimum-sized18SrDNAprobe((cid:1)600bp),anewsetof
primerswasdesignedbasedonsequencesoftheorganismsavailableinGenBank:18S6F(5’-
CTCTTTCGAGGCCCTGTAAT-3’)and18S6R(5’-CAGCTTTGCAACCATACTCC-3’).
ProbesoffortwosatDNAswereobtainedusingthefollowingdivergentprimers:MS3F(5’-
TGGTTCCCAATTTGCAATCAAG-3’)andMS3R(5’-ATCGGACCTTTCTTCGCTTTACA-
3’);MS7F(5’-CACAAGCCTTATGTTCACCATGA-3’)andMS7R(5’-GTACAGTAAA
GTTGTAAGTGGT-3’).
FISH
PriortotheFISHexperiments,allprobeswerelabelledwithdigoxigenin-11-dUTPorbiotin-
16-dUTP.ThepaintingprobeswerelabelledusingtheGenomePlex(WGA3Reamplification
Kit-Sigma)followingthemanufacturer’sprotocol,andtherepetitiveDNAprobeswerelabelled
viaPCR.
FISHwasperformedunderhighstringencyconditionsusingthemethoddescribedbyPin-
keletal.[36].Thepre-hybridizationconditionsweredifferentaccordingtotheprobesused.
Thus,slidesprobedwithrepetitivesequenceswereincubatedwithRNAse(50μg/ml)for1hat
37°C,andthechromosomalDNAwasdenaturedin70%formamide/2xSSCfor5minat70°C.
Foreachslide,30μlofhybridizationsolution(containing200ngofeachlabelledprobe,50%
formamide,2xSSCand10%dextransulphate)wasdenaturedfor10minutesat95°C,then
droppedontotheslidesandallowedtohybridizeovernightat37°Cinamoistchambercon-
taining2xSSC.Slidesprobedwithwhole-chromosomepaintswereincubatedwith0,005%
pepsin/10mMHClfor10min,andthechromosomalDNAwasdenaturedin70%formamide/
2xSSCfor3minat70°C.Foreachslide,30μlofhybridizationsolution(containing200ngof
labelledprobe,50%formamide,2xSSC,10%dextransulphateand3μgofsalmonsperm
DNA)wasdenaturedfor10minutesat85°Candallowedtopre-hybridizefor30minat37°C,
thendroppedontotheslides,followedbysealingwithrubbercementandhybridizationat
37°Cinamoistchambercontaining2xSSCfor36h.Post-hybridization,allslideswerewashed
in0.2xSSC/15%formamidefor20minat42°C,followedbyasecondwashin0.1xSSCfor15
minat60°Candafinalwashatroomtemperaturein4xSSC,0.5%Tweenfor10min.Probe
detectionwascarriedoutwithavidin-FITC(Sigma)oranti-digoxigenin-rhodamine(Roche),
andthechromosomeswerecounterstainedwithDAPI(4’,6-diamidino-2-phenylindole,Vector
Laboratories)andanalyzedusinganopticalphotomicroscope(OlympusBX61).Imageswere
capturedusingImageProplus6.0software(MediaCybernetics).Fromeachindividual,amini-
mumof10cellswasanalyzedtoconfirmtheFISHresultsandestimatethenumberofBchro-
mosomespercell.
DNAamplification,cloningandsequencing
Weamplified,clonedandsequencedpartialH3histonegenesandbothsatDNAsfromvarious
samples,includingamicrodissectedeuchromaticBchromosome(B ),amicrodissectedhetero-
1
chromaticBchromosome(B ),gDNA0BfromM.sanctaefilomenaefromtheNovoRiver(0B
2
gDNA)and0BgDNAfromA.fasciatus.Inaddition,MS3andMS7satDNAsequenceswere
PLOSONE|DOI:10.1371/journal.pone.0150573 March2,2016 5/20
UncoveringtheAncestryofBChromosomesinMoenkhausiasanctaefilomenae(Teleostei,Characidae)
obtainedfromthelongestautosomepair(A ).Thereactionswereperformedin1xPCRbuffer,
1
1.5mMMgCl ,200μMeachdNTP,0.5UofTaqpolymerase(Invitrogen),0.1μMeachprimer
2
and50ngofDNA.Thecyclingprogramforamplificationoftheseregionsconsistedofanini-
tialdenaturationat95°Cfor5min,followedby32cyclesat95°Cfor45s,56°Cfor30sand
72°Cfor1minandafinalextensionof72°Cfor15min.ThePCRproductswerevisualizedin
2%agarosegels,andthefragmentobtainedfromeachsamplewasextractedfromthegeland
clonedintothepGEM-TEasyVector(Promega,Madison,Wisconsin,USA).DNAsequencing
wasperformedwiththeBigDyeTMTerminatorv3.1CycleSequencingReadyReactionKit
(AppliedBiosystems)followingthemanufacturer’sinstructions.Althoughdifferentstrategies
wereadopted(i.e.,testingdifferentprimerpairsandaddingDMSOtothePCR),wefailedto
amplifyanyregionofthe45SrDNAsequencefromBchromosomes,probablybecauseGC-
richsequencesareusuallyunder-amplifiedinWGAsteps[7].
ExtractionofMS3andMS7fromIlluminareads
ToobtainadetailedandreliablescoreofhaplotypeabundancefortheMS3andMS7satDNAs
sequencesfromgenomiclibraries,weextractedthemonomersdirectlyfromtheIllumina
reads.Becausethereadsaresmallerthanthemonomersizeofbothsatellites,wejoinedthe
paired-readsusingfastq-join(https://code.google.com/p/ea-utils/wiki/FastqJoin)withamini-
mumoverlappingsizeof6bp.
ForMS7,wealignedthejoinedreadsagainstadimersequenceofsatDNAwithRepeatMas-
kersoftware[37]andusingacustomPythonscript(https://github.com/fjruizruano/ngs-
protocols/blob/master/rm_getseq.py);weemployedthealignmentinformationfromtheout-
putfile(withtheextension.out)toextractonlythealignedregion.Then,wemappedthese
sequenceswithGeneiousProv8.04againstthedimerandextractedthecentralregioncorre-
spondingtoonemonomerbymanuallydeletingthosesequencesthatdidnotcoveranentire
monomer.ForMS3,afterGeneiousmapping,wecuttheendsmappedoutofthereference
monomerandaddedthemtotheotherendtoobtainsequencesstartingandendingatthe
samepositions,whichwasachievedwithanothercustomPythonscript(https://github.com/
fjruizruano/ngs-protocols/blob/master/sat_cutter.py).
Sequenceanalysis
ConsensussequencesfromforwardandreversestrandswereobtainedusingGeneiousPro
v8.04,andalignmentsweregeneratedusingtheMusclealgorithm[38]underthedefault
parameters.DNAdiversityanalyses,consideringindels,wereperformedwithDnaSPv5.05
[39].MinimumspanningtreeswerebuiltonthebasisofpairwisedifferencesusingARLE-
QUINv3.5.1.3[40]andwerevisualizedwithHAPSTARv0.7[41].
Comparisonsofsynonymoussubstitutionspersynonymoussite(dS)andnon-synonymous
substitutionspernon-synonymoussite(dN)ofH3.2fromeachlibrary(B ,B ,A and0B
1 2 1
gDNA)werecarriedoutusingthenonparametricKruskal-Wallistest,followedbyDunn’s
multiplepost-hoctest,consideringα=0,05.
Results
Individualsfromthetwoanalyzedpopulationsshowedasimilarstandardkaryotype,allexhib-
iting2n=50biarmedchromosomes(6m+16sm+28st),withnosex-relatedchromosomal
dimorphism.Additionally,mitoticallyunstableBchromosomeswereobservedinthegenomes
ofallstudiedspecimenscollectedfromtheBRpopulation,asmanifestedintheintraindividual
variationinBnumbers(0–6).However,allindividualsfromtheNRpopulationlackedB
chromosomes.
PLOSONE|DOI:10.1371/journal.pone.0150573 March2,2016 6/20
UncoveringtheAncestryofBChromosomesinMoenkhausiasanctaefilomenae(Teleostei,Characidae)
IntheBRpopulation,thereweretwotypesofBchromosomesobservedonthebasisofthe
C-bandingpatternandpopulationfrequency.Themorefrequentvariant(designatedB )
1
showedalightC-bandingpattern(similartoeuchromatinintheAchromosomes),whereas
thelessfrequentvariant(B )showedadarkC-bandingpattern(similartoheterochromatinin
2
theAchromosomes)(Fig1).WhileB wasfoundinall23individualsanalyzedfromtheBR
1
population(100%prevalence),B waspresentonlyinfourofthem(17%prevalence)(Table1).
2
BothBtypeswerefoundinmalesandfemales.
SimilarDNAcontentinthetwoBchromosomevariants
Whole-chromosomepainting(WCP)withtheB andB probesshowedsimilarhybridization
1 2
signalsforthetwoprobesonbothBvariants,inadditiontothepericentromericregionsof
approximatelyone-thirdoftheAchromosomes(Fig1a–1d).Thiswasalsoapparentwhendou-
bleWCPwithbothB-derivedprobeswasperformedonmetaphasecellsfromB-lackingNR
individuals,withbothhybridizationsignalsco-locatinginmostcases(Fig1eand1f).
FISHmappingrevealedremarkabledifferencesbetweenthetwopopulationsregardingthe
numberofH3histone,5Sand18SrDNAclusters,whiletheU2snDNAshowedexactlythe
samepatternofchromosomaldistribution(Fig2).
Themostextremedifferencebetweenthetwopopulationswasfoundfor18SrDNAwhich,
waslocatedonlyontheshortarmofchromosomepairno.6inNR,whereasinBR,distalclus-
tersappearedonmanychromosomesinahomozygous(13,14,15,17)orheterozygous(2,12,
20,22,24,25)state,inadditiontotheclusteronchromosome6.SequentialFISHandsilver
impregnation(withthelatterindicatingactiverDNAclusters)showedthattherDNAcluster
onchromosome6wasalwaysactive,whilemostotherclustersonotherchromosomes(includ-
ingBchromosomes)intheBRpopulationwereinactive(S1Table,S1Fig).ConsideringtheB
chromosomes,NORactivitywasobservedonlyforB inapproximately10%ofthecellsana-
1
lyzedinasingleindividual,indicatingthatthecontributionoftheBchromosomestorRNA
synthesisdoesnotseemtoplayanimportantroleinthecellularphysiologyofB-carryingM.
sanctaefilomenaespecimens.
Amongthetandemrepeatgenefamiliesassayed,bothtypesofBchromosomescarriedonly
18SrDNAandH3histonegenesites,buttheheterochromaticvariant(B )carriedalarger18S
2
rDNAclusterthantheeuchromaticone(B )(Figs2and3).
1
ToimproveourknowledgeoftheDNAcontentoftheBchromosomes,wesearchedforsat-
elliteDNA(satDNA)tandemrepeatsintwosetsofIlluminaHiseq2000Paired-Endreads
obtainedfromwhole-M.sanctaefilomenaegenomesequencingrunsfromaB-lackingindivid-
ualfromtheNRpopulationandaB-carryingindividualfromtheBRpopulation.Becauseall
individualsanalyzedfromtheBRpopulationcarriedBchromosomes,itwasnecessarytoana-
lyseaB-lackinggenomefromtheNRpopulation.However,itwasunfortunatethatthetwo
repeatfamiliespresentontheBchromosomes(18SrDNAandH3histonegenes)showed
extensivespreadingacrossAchromosomesintheB-carryingpopulation,thusimpedingthe
detectionofchangesinDNArepeatcoveragebetweentheB-carryingandB-lackinggenomes.
Forthisreason,weusedtheIlluminareadstosearchforsatelliteDNAsthatmightbeusefulas
additionalB-specificFISHmarkers.
Sequenceclusteringanalysisresultedin11,149clusters,constitutinggenomicproportionsof
25.6%and24.4%for+Band0Bindividuals,respectively.Wedesignedprimerpairsforseven
putativesatelliteDNAs,thenPCRamplifiedtheseDNAsandselectedthoseshowingaladder-
likepatterninagarosegels.Next,wegeneratedDNAprobesforFISHandmappedthemtoA
andBchromosomes(datanotshown),andweselectedthetwosatDNAfamiliesshowingaclus-
tereddistributionthatwerepresentonbothAandBchromosomes(Fig3eand3f),henceforth
PLOSONE|DOI:10.1371/journal.pone.0150573 March2,2016 7/20
UncoveringtheAncestryofBChromosomesinMoenkhausiasanctaefilomenae(Teleostei,Characidae)
Fig1.MetaphaseplatesofM.sanctaefilomenaeafterwhole-chromosomepaintingwithB andB
1 2
probesandtheirrespectivesequentialC-bandingpatterns.(a-d)representM.sanctaefilomenaefromthe
BatalhaRiver.(e-f)representM.sanctaefilomenaefromtheNovoRiver.
doi:10.1371/journal.pone.0150573.g001
referredtoasMS3(CL27)andMS7(CL96).TheMS3satDNAshowedaconsensussequenceof
186bpwithaclusterdensityof0.16(S2Fig)anddidnotshowanysimilaritytothecustomdata-
base.Notably,thisclusterwas2xmoreabundantintheBRlibrary(0.236%)thanintheNR
library(0.117%).TheMS7satDNAexhibitedaconsensussequenceof100bpwithaclusterden-
sityof0.55(S2Fig)andyieldedsimilarityhitswithDNA/TcMar-Tc1(54%ofhits)intheA.
mexicanusdatabase.Intermsofabundanceindifferentlibraries,therewasalmostnodifference
observedforthisclusterbetweenthetwopopulationsanalyzed(0.0285%forBRand0.0295%
forNR).FISHanalysiscorroboratedtheabundancedataandrevealedthatMS3waslocatedin
thepericentromericregionof13chromosomepairsintheBRpopulation,butonly6pairsin
theNRpopulation.Therefore,thehigherabundanceofMS3intheB-carryingpopulationwas
notduetoBchromosomes(eventhoughtheyactuallycarrythissatellite)buttoitspresenceon
twiceasmanyAchromosomes.Conversely,MS7waslocatedinthetelomericregionsof15
pairsinbothanalyzedpopulations.
Takentogether,theseresultsindicatethatbothBvariantscontainessentiallythesameDNA
repeats(H3histonegenes,18SrDNAandMS3andMS7satDNAs).Thefactthatautosome
PLOSONE|DOI:10.1371/journal.pone.0150573 March2,2016 8/20
UncoveringtheAncestryofBChromosomesinMoenkhausiasanctaefilomenae(Teleostei,Characidae)
Fig2.M.sanctaefilomenaekaryotypesconstructedfrommitoticmetaphasecellssubjectedtoFISH
withdifferentrepetitiveDNAprobes.a)NovoRiverpopulation.b)BatalhaRiverpopulation.Notethe
differentialdistributionofH3histonegenesand5Sand18SrDNAclustersontheAchromosomesinboth
populations.
doi:10.1371/journal.pone.0150573.g002
pairno.6istheonlyAchromosomecarryingalloftheserepeatfamiliesstronglypointstothe
possibilitythatbothBchromosomeswerederivedfromthepericentromericregionofthis
chromosome.
ThetwoBchromosomevariantsshowasimilardegreeofmitotic
instability
BecausethenumberofBchromosomesvariedamongcellswithinthesameindividual,weper-
formedananalysisofthedegreeofmitoticinstabilitycausingthisvariation.Forthispurpose,
weusedamitoticinstabilityindexpreviouslydevelopedinamigratorylocust[42]thatisbased
ontheassumptionthatthemediannumberofBchromosomesintheadultrepresentsthe
numberofBchromosomesinthezygotestage.Thismitoticinstabilityindex(MI)measures
thesumofdeviationsinBnumbersinasampleofcellswithrespecttothemedian,normalized
perBchromosome.
ThefactthatbothBchromosometypescontainedH3histonegenesand18SrDNAhelped
ustoidentifythetwoBtypesin657mitoticmetaphasecellssubjectedtodoubleFISHandsub-
sequentC-bandingin23individualsfromtheBRpopulation(mean=29cellsperindividual,
SD=6)toaccuratelyscorethenumberandtypeofBchromosomes(Fig4).Ineachindividual,
PLOSONE|DOI:10.1371/journal.pone.0150573 March2,2016 9/20
UncoveringtheAncestryofBChromosomesinMoenkhausiasanctaefilomenae(Teleostei,Characidae)
Fig3.MetaphaseplatesofM.sanctaefilomenaefromtheBatalhaRiverafterFISHwithdifferent
repetitiveprobesandsequentialC-bandingtoshowtheclusteringof18SrDNA,H3histonesand
satDNAsondifferentBchromosomes.NotethatB iseuchromaticshowingsmallblocksof18SrDNA,
1
andB isheterochromaticshowinglargeramountsof18SrDNA.
2
doi:10.1371/journal.pone.0150573.g003
wecalculatedthemeannumberofBchromosomespercell,themediannumberofBsandthe
mitoticinstabilityindex(MI).TheresultsrevealedthatB andB showedasimilarMI,butB
1 2 1
wasalmostnine-foldmorefrequentthanB (Table1).
2
AcomparisonofthemeannumberofBchromosomesandMIperindividualbetweenthe
presentdatawiththosepreviouslyreportedfortheTieteRiverin[21]and[23],bymeansof
theKruskal-Wallistest,revealedsignificantdifferencesforboththemeanvalues(H=21.16,
df=2,N=46,p<0.0001)andMI(H=15.4,df=2,N=46,P=0.0005).Giventhatthethree
sampleswerecollectedfromthesameriver,butatdifferenttimes,theseresultssuggestaten-
dencyofMItoincreaseacrossyears.However,weobservedvariationinthemeannumber
ofBchromosomesperindividualthatpresentedaninvertedV-shape(Fig4).Thismightsug-
gestthatthemitoticinstabilityofBchromosomeshasincreasedacrosstheyears,whilethe
numberofBsperindividualhasreachedamaximum,inaccordwiththeexistenceofatoler-
ancethreshold.
PLOSONE|DOI:10.1371/journal.pone.0150573 March2,2016 10/20