bioRxiv preprint first posted online Jan. 12, 2016; doi: http://dx.doi.org/10.1101/036483. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
Aneo-sexchromosomeintheMonarchbutterfly,Danaus
plexippus
JamesR.Walters*andAndrewJ.Mongue
DepartmentofEcologyandEvolutionaryBiology,UniversityofKansas,Lawrence,KS,USA
*Authorforcorrespondence:JamesRWalters,DepartmentofEcologyandEvolutionary
Biology,UniversityofKansas,Lawrence,KS,USA
phone:785-864-6341
email:[email protected]
bioRxiv preprint first posted online Jan. 12, 2016; doi: http://dx.doi.org/10.1101/036483. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
Abstract
Wereportthediscoveryofaneo-sexchromosomeinMonarchbutterfly,Danaus
plexippus,andseveralofitscloserelatives.Z-linkedscaffoldsintheD.plexippusgenome
assemblywereidentifiedviasex-specificdifferencesinIlluminasequencingcoverage.
Additionally,amajorityoftheD.plexippusgenomeassemblywasassignedtochromosomes
basedoncountsof1-to-1orthologsrelativetothebutterflyMelitaeacinxia(withreplication
usingtwootherLepidopteranspecies),wheregenomescaffoldshavebeenrobustlymappedto
linkagegroups.Combiningsequencing-coveragebasedZ-linkagewithhomologybased
chromosomalassignmentsprovidedstrongevidenceforaZ-autosomefusionintheDanaus
lineage,involvingtheautosomehomologoustochromosome21inM.cinxia.Coverageanalysis
alsoidentifiedthreenotableassemblyerrorsresultinginchimericZ-autosomescaffolds.The
timingofthisZ-autosomefusioneventcurrentlyremainsambiguousduetoincomplete
samplingofkaryotypesintheDanainitribeofbutterflies.Thediscoveryofaneo-Zandthe
provisionalassignmentofchromosomelinkagefor>90%ofD.plexippusgeneslaysthe
foundationfornovelinsightsconcerningsexchromosomeevolutioninthisincreasingly
prominentfemale-heterogameticmodelspeciesforfunctionalandevolutionarygenomics.
Background
Majorrearrangementsofkaryotypeandchromosomestructureoftenhavesubstantial
evolutionaryimpactsonboththeorganismscarryingsuchmutationsandthegeneslinkedto
suchgenomicreorganization[1,2].Additionally,suchlarge-scalechromosomalmutations
oftenpresentnovelopportunitiestoinvestigatemolecularevolutionaryandfunctionalgenetic
processes.Oneprominentexampleofthisistheevolutionofneo-sexchromosomes,whichcan
arisefromthefusionofanautosomewithanexistingandwell-differentiatedallosome.This
effectivelyinstantaneoustransformationofaformerlyautosomalsetofgenesintosex-linked
lociisfertilegroundforcomparativeanalysesaimedatunderstandingthedistinctsetof
evolutionaryforcesactingonsexchromosomesrelativetoautosomes[3-6].Furthermore,
whentherelevanttaxaalsohappentobetractablegeneticmodelsystems,thereisopportunity
toexplorethefunctionalandmechanisticchangesassociatedwithsexchromosomeevolution.
Thecongruenceofneo-sexchromosomesexistinginamodelsystemisrelativelyrare,although
therearesomenotableexamples.
Numerousindependentoriginsofneo-sexchromosomesareknowninDrosophilafruit
flies,whererecentworkhasrevealedmuchabouttheevolutionaryandfunctionaldynamicsof
theseunusualsequences[3,7-11].Substantialinsightshavealsocomefromsticklebackfish,
whereneo-sexchromosomesappeartoplayanimportantroleinreproductiveisolation
betweenincipientspecies[12-14].Lookingbeyondtheseestablishedmodelsystems,therapid
expansionofgenomictechnologieshasallowedextensiveanalysesofgenecontent,sex-biased
geneexpression,dosagecompensation,andsequencedivergenceforrecentlyevolvedsex
chromosomesamongaverydiversesetoforganisms.Thisincludes,forexample,severalinsect
lineages[Teleopsidflies,agrasshopper,andStrepsiptera[15-17]],vertebrates[mammalsand
bioRxiv preprint first posted online Jan. 12, 2016; doi: http://dx.doi.org/10.1101/036483. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
birds[4,18,19],andplants[SileneandRumexgenera[20-22]].Aclearconsensusemerges
fromthisresearchthatthelackofrecombinationassociatedwithsexchromosomescatalyzesa
cascadeofevolutionarychangesinvolvingthedegenerationofoneallosome,theaccumulation
ofgeneswithsex-biasedexpression,increasedevolutionaryrates,and(often,butnotalways)
theacquisitionofdosagecompensation.Yetmanyofthedetailsinthisprocessremainelusive
andunresolved,includingtherateofallosomedivergence,theroleofpositiveselectionversus
drift,theimportancesex-specificselection,andthemechanismsunderlyingdosage
compensation(orthereasonsforitsabsence).Itisthereforeimportanttocontinuallyidentify
newopportunitiesfornovelinsightintotheevolutionofsexchromosomes.
Overwhelmingly,researchonsexchromosomesoccursinmale-heterogametic(XY)
species[5,23-25].Thisappearstobeparticularlytrueforneo-sexchromosomes,where
contemporarygenomicanalysesofneo-Zorneo-Wchromosomesarecurrentlylacking[with
onenotableexceptionforbirds[4]].Thisimbalanceisunfortunate,asZWsexdetermination
replacesmale-specificselectionwithfemale-specificselectionduringtheevolutionof
heterogamety,offeringanovelframeworkforelucidatingsexchromosomeevolution.What
prospectsarethereforimprovingthissituation?Birdsarethemostprominentvertebratetaxon
thatisfemale-heterogametic,butitappearsthatavianneo-sexchromosomesarequiterare,
andabsentfromprominentmodelspecies(e.g.,chicken,zebrafinch)[26,27].Fishesand
squamatesseemtobefarmorelabileinsex-chromosomeconstitution,withnumerous
independenttransitionsbetweenmaleandfemale-heterogametyandrelativelyfrequentsexautosomefusions[28],thustherearepotentiallygreatopportunitiesinthesetaxa.However,
noobviouslytractableZWmodelsystemwithneo-sexchromosomesisyetapparentforthese
lineages.
Formanyreasons,Lepidoptera(mothsandbutterflies)maybethemostpromising
female-heterogametictaxonforstudyingneo-sexchromosomes.SyntenyisunusuallywellconservedinLepidoptera[29-31],yettherearealsonumerousknownexamplesof
independentlyevolvedneo-Zandneo-Wchromosomes,severalofwhichhavebeenwellcharacterizedcytogenetically[6,32-34].Furthermore,comparativegenomicresourcesinthis
insectorderaresubstantialandgrowingquickly(www.lepbase.org).
Inthiscontext,wereportthefortuitousdiscoveryofaneo-Zchromosomeinthe
monarchbutterfly,Danausplexippus,andcloselyrelatedspecies.Monarchbutterflies,
renownedfortheirannualmigrationacrossNorthAmerica,alreadyhaveastrongprecedentas
amodelsysteminecology[35].Recentlymonarchshaveemergedasamodelsystemfor
genomebiology,withawell-assembledreferencegenome,extensivepopulationresequencing
data,andaprecedentforgenomeengineering[36-38].Thediscoveryofaneo-Zchromosome
substantiallyenrichesthevalueofthisspeciesasaresearchmodelingenomebiologyandlays
thefoundationforextensivefutureinsightsintotheevolutionandfunctionaldiversityofsex
chromosomes.
bioRxiv preprint first posted online Jan. 12, 2016; doi: http://dx.doi.org/10.1101/036483. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
Results
IdentifyingZ-linkedscaffoldsinD.plexippus
WeidentifiedZ-linkedscaffoldsintheD.plexippusgenomeassembly[36,39]by
comparingsequencingcoveragefrommaleandfemalesamples.ZchromosomeDNAcontentin
malesshouldbetwicethatinfemales,whileautosomesshouldhaveequalDNAcontent
betweensexes.Thusacorrespondingtwo-folddifferenceinsequencingcoverageisexpected
betweensexesfortheZchromosome,butnotautosomes,andcanbeusedtoidentifyZ-linked
scaffolds[16,40,41].Ahistogramofmale:femaleratiosofmediancoverageclearlyidentifies
twoclustersofscaffolds(Fig.1).Onelargeclusteriscenteredaroundequalcoveragebetween
sexes(Log2M:F=0)andasecond,smallerclusteriscenteredaroundtwo-foldgreatercoverage
inmales(Log2M:F=1).WecanthusclearlydistinguishtheZ-linkedscaffoldsasthosewith
Log2(M:F)>0.5,withtheremainderofthescaffoldspresumedtobeautosomal.
Onescaffold,DPSCF300028,appearedtohaveanintermediatecoverageratio,fallingat
Log2M:F≈0.7.Onelikelyexplanationforsuchintermediatevaluesisthatthescaffoldisa
chimeraofZ-linkedandautosomalsequencearisingfromanerroringenomeassembly[41].In
thisscenario,onlyaportionofthescaffoldisZ-linkedandgivesatwo-folddifferencein
coveragebetweensexes;theremainingautosomalfractionofthescaffoldyieldsequal
coverages.Theresultingestimateofaveragecoveragefortheentirescaffoldthenfallsatan
intermediatevaluebetweenexpectationsforZorautosomalscaffolds.Thisisclearlytruefor
DPSCF300028,asrevealedbyexaminingbasepair-levelsequencingcoverageacrossthescaffold
(Fig.2A).Whileaveragemalecoverageisconsistentacrosstheentirelengthofthescaffold,
femalecoverageexhibitsacleartransitionbetweencoverageequaltomales(theautosomal
portion)andcoverageonehalfthatofmales(theZ-linkedportion).Indeed,therearetwosuch
transitionsinscaffoldDPSCF300028,whichweestimatetooccurat0.76Mbpand1.805Mbp,
creatinga“sandwich”ofoneZsegmentflankedbyautosomalsegments.
Orthologcountslinkscaffoldstochromosomes.
Lepidopterashowaveryhighlevelofconservedsyntenyacrosssubstantialevolutionary
divergences[29-31].ThusitispossibletousecountsoforthologousgenestoassignD.
plexippusscaffoldstolinkagegroups(i.e.chromosomes)delineatedinothermothorbutterfly
species.WegeneratedpredictedorthologsbetweenD.plexippusandthreeotherreference
specieswheregeneticlinkagemappinghasbeenusedtoassigngenomicscaffoldsto
chromosomes:Melitaeacinxia(N=31),Heliconiusmelpomene(N=21),andBombyxmori(N=28)
[29,30,42].M.cinxiaandH.melpomenearebothnymphalidbutterfliesequallydivergedfrom
D.plexippus,whilethesilkmoth,B.mori,isdistinctlymoredivergent[43,44].
ToassignD.plexippusscaffoldstochromosome,wetabulatedperscaffoldthecountsof
one-to-onereferencespeciesorthologsperreferencespecieschromosome.D.plexippus
scaffoldswerethenassignedtothereferencechromosomewiththemaximumcountof
orthologs.Forafewscaffolds,atieoccurredinmaximumorthologcountperreference
chromosome,inwhichcasethescaffoldwasremovedfromfurtheranalysis;atmostthis
occurredforonly14scaffoldsperreferencespeciesandusuallyinvolvedsmallscaffolds
bioRxiv preprint first posted online Jan. 12, 2016; doi: http://dx.doi.org/10.1101/036483. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
harboringfewerthan5orthologs.Typicallythismethodyieldedaclear“best”reference
chromosomalassignmentforeachD.plexippusscaffold.
Thismethodofortholog-countchromosomal“lift-over”resultedinputative
chromosomalassignmentsfor>90%ofD.plexippusgenesrelativetoeachreferencespecies
(Table1,SupplementaryTableS1).Also,atleast4500orthologousgeneswereco-localizedto
chromosomebetweenD.plexippusandeachreferencespecies.Havingseveralthousand
orthologsmappedtochromosomeinD.plexippusandareferencespeciespresentsthe
opportunitytoexaminetheextentofchromosomalrearrangementsandgenemovement
betweenthetwospecies.HereweprimarilyreportthecomparisonwithM.cinxiabecausethis
speciesisbelievedtoretaintheancestrallepidopterankaryotypeof31chromosomes[29].
Furthermore,thiscountofchromosomesisclosesttothatreportedforDanausbutterflies
(N=30),indicatingitislikelythemostsimilarkaryotypetoD.plexippus[45].H.melpomeneand
B.moriareknowntohavemorederivedkarytoypesinvolvingseveralchromosomalfusions
relativetoM.cinxia;detailsofcomparisonstothesetwospeciesarereportedinthe
supplementarycontentandprovidecomparablesupportfortheprimaryfindingsreported
here.
Figure3summarizesthecross-tabulationofchromosomallinkagefor>4500orthologs
betweenM.cinxiaandD.plexippus.Theoverwhelmingmajorityoforthologsfallonthe
diagonal,indicatingsubstantialconservationofchromosomallinkageandrelativelylittlegene
shuffling,ashasbeenreportedelsewhereforLepidoptera[29-31].Thetwomostnotable
exceptionstothispatternbothinvolvetheZchromosome(Chr1).Inonecase[McChr9,
DpChr1]wecouldanticipatethisbecauseofthepreviouslyidentifiedchimericscaffold,
DPSCF300028.Thisscaffoldharbors34orthologsassignedtoMcChr1and23orthologsassigned
toMcChr9,consistentwiththechimericnatureofthescaffoldrevealedfrommale:female
coverageratios(Fig2A).
Thesecondcase[McChr1,DpChr21]appearedtoariseentirelyfromasinglescaffold,
DPSCF300001,thelargestscaffoldintheD.plexippusv3assembly.Thisscaffoldcarried107
orthologsassignedtoMcChr21,28orthologsassignedtoMcChr1,13orthologsassignedto
McChr23,andafewotherorthologsassignedtootherautosomes.Notably,despitethelarge
numberofapparentlyautosomalorthologs,theaveragemale:femalecoverageratiofor
DPSCF300001wasconsistentwithitbeingZ-linked[Log2(M:Fcoverage)=0.92].Nonetheless,
weplottedcoverageacrossthechromosomeanddetecteda~1Mbpportionatthe3’endof
thescaffoldwithcoveragepatternsconsistentwithbeinganautosome(Fig2C).TheM.cinxia
orthologsinthisautosomalportionwerelinkedexclusivelytoMcChr23.Therewasnotan
obviousshiftinsequencingcoveragebetweensexestoindicateamisassembledZ-autosome
chimerainvolvingMcChr21.Rather,itappearedthatnearlytheentiretyofscaffold
DPSCF300001hadtwicethecoverageinmalesthaninfemales,consistentwiththeentire
scaffoldbeingZ-linked,bothforregionsapparentlyhomologoustoMc1(Z)andMcChr21.
Aneo-ZchromosomeinD.plexippus
TheobservationthatasubstantialportionofscaffoldDPSCF300001wasZ-linkedand
homologoustoMcChr21,whileanotherlargesectionofthesamescaffoldwashomologousto
McChr1(i.e.,McChrZ),ledustohypothesizethatasingleZ-autosomefusioncouldexplainthe
bioRxiv preprint first posted online Jan. 12, 2016; doi: http://dx.doi.org/10.1101/036483. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
karyotypicdifferencesbetweenD.plexippus(N=30)andM.cinxia(N=31).Tofurtherinvestigate
thishypothesisofamajorevolutionarytransitioninsexchromosomecompositioninthe
Danauslineage,weexaminedthechromosomalassignmentsforallMonarchscaffolds
identifiedasZ-linkedviasequencingcoverageratios(Z-covscaffolds).Specifically,weidentified
theuniquesetofreferencechromosomestowhichZ-covscaffoldswereassigned,andthen
examinedthemale:femalecoverageratioforallscaffoldsassignedtothosereference
chromosomes.InthecaseofM.cinxiaasthereference,allZ-covscaffoldswereassignedeither
toMcChr1orMcChr21(Fig.4;comparableresultswereobtainedforH.melpomeneandB.
mori,SupplementaryFig.S2).ThisresultprovidesfurtherevidencethattheZinD.plexippusisa
neo-sexchromosomereflectingthefusionoftheancestralZchromosomewithanautosome
homologoustoMcChr21.
ThisanalysisintersectingZ-covscaffoldswithhomologytoM.cinxiarevealedtwo
scaffoldsthatdidnotfitwiththeexpectedpatternofsequencingcoverage(Fig.4).First,
scaffoldDPSCAF300044wasassignedtoMcChr1(Z)buthadLog2M:F≈0.25,muchmorelike
otherautosomesthanotherZ-linkedchromosomes.ThisscaffoldhadsevenZ-linkedorthologs
and4autosomal,suggestinganotherchimericscaffold.Indeed,examiningcoverageacrossthe
scaffoldrevealedacleartransitionincoverageaspreviouslyobservedforDPSCF300001and
DPSCF300028(Fig2B).Thusthelowmale:femalecoverageratioforthisscaffoldistheartifact
ofanassemblyerror.Againwewereabletopartitionthescaffoldintotwosections,one
autosomalandoneZ-linked,withabreakpointestimatedat0.29Mbp.
DPSCF300403wastheotherscaffoldwheretheM:Fratioofmediancoveragewas
inconsistentwiththehypothesisofaneo-Zchromosome.Thisscaffoldwasassignedto
McChr21buthadanautosomalcoverageratio.Coveragealongthechromosomewas
consistentwithitbeingentirelyautosomal(SupplementaryFigureS3).Inthiscasethescaffold
carriedonlyasingleone-to-oneorthologousgene(andonly5protein-codinggenestotal),so
theassignmenttoMcChr21istenuousandlikelyinaccurate.Thisscaffoldalsohadasingleoneto-oneorthologfoundinB.mori,andnoneidentifiedinH.melpomene.Wethereforeconsider
thisscaffoldlargelyuninformativeconcerningthepresenceofaneo-ZinD.plexippus.
Theneo-ZchromosomeexistsintheMonarch’scloserelatives
TheMonarchpopulationgenomicdatasetofZhaetal.(2014)alsocontainedmaleand
femaleresequencingsamplesforfourcloselyrelatedcongeners:D.gilippus,D.chrysippus,D.
erippus,andD.eresimus.Thispresentedtheopportunitytoassesswhetherthisneo-Zexistsin
thesespeciesinadditiontoMonarch.PublishedreportsofanN=30karyotypeinsomeofthese
speciesleadstothestrongpredictionthattheyallalsocarrythesameneo-Zchromosomal
arrangement[45].Asexpected,maleversusfemalesequencecoverageanalysisdoesclearly
showthesamescaffoldshomologoustobothMcChr1andMcChr21ashavingsequencing
coverageconsistentwithaneo-Z(Fig.5).Thusitappearsthattheoriginofthisneo-Zpredates
thediversificationofthegenusDanaus.
bioRxiv preprint first posted online Jan. 12, 2016; doi: http://dx.doi.org/10.1101/036483. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
Annotatingchromosomallinkage
Thecombinationofsequencingcoverageanalysisandcomparative“liftover”allowedus
toprovisionallyassignmostgenestochromosomesinD.plexippus.GenesfallingonZ-cov
scaffolds,orwithintheportionassessedasZ-linkedfornotedchimericscaffolds,havebeen
assignedtotheZchromosome.WefurtherpartitionedtheseZ-linkedgenesintobeingonthe
ancestral(anc-Z)orneo(neo-Z)portionoftheZ,basedonscaffoldhomologytoreference
chromosomes.InthecaseofDPSCF300001,welocalizedthefusionpointbetweenanc-Zand
neo-ZbyaligningM.cinxiaandH.melpomenescaffoldsfromtheZ(HmChr21,McChr1)or
relevantautosome(HmChr2,McChr21).Alignmentswithbothspecieswereconsistentin
placingthefusionpointatapproximately3.88Mbp(SupplementaryFig.S4).Otherwise,genes
andscaffoldswereassignedtochromosomesbaseddirectlyontheresultsofthe“lift-over”
relativetoM.cinxia.Table2givesatabulatedsummaryofresults,whileresultsforevery
proteincodinggeneareprovidedinSupplementaryTableS3.
Discussion
Thisdiscoveryofaneo-ZchromosomeinDanausbutterfliesandourdiscriminationof
genesfallingontheancestralversusrecentlyautosomalportionsarefundamentalobservations
thatprovidethefoundationforahostoffutureinferences.Theseresultscreatenovel
opportunitiestoaddressratesofmolecularevolution,theevolutionofdosagecompensation,
thepatternofallosomedivergence,andmanyotherimportantquestionsinsexchromosome
biology,allinanemerginggeneticandgenomicfemale-heterogameticmodelsystem.
Itseemsevidentfromtheresultspresentedherethatifthereremainsaneo-W
chromosome(i.e.,adegradedhomologoftheneo-Zsegment),itmustbesubstantiallydiverged
fromtheneo-Z.Weinferthisfromtheveryconsistent2:1coverageratioobservedonscaffold
regionscorrespondingtoMcChr21.Iftheneo-Wretainedsubstantialhomologytotheneo-Z,
wewouldexpectmanysequencingreadsemanatingfromtheneo-Wtoaligntotheneo-Z,and
shiftthisratiotowardsequality.Thisevidentlydoesnotoccur,stronglyindicatingsubstantial
divergencebetweentheneo-Zandanyneo-Wsequencethatisretained.Indeed,itisnoteven
clearatthispointwhetherthereisanyneo-Wchromosomeatall.Thisisanobviouspointfor
immediateinvestigation,perhapsbestapproachedusingcytogenetictechniques[6,32].
Brownetal.(2004)reportchromosomecountsfrommalebutterfliesofseveralspecies
fromthreegeneraintheDanainibutterflytribe:Danaus(N=30),Anetia(N=31),andLycorea
(N=30).ThemostrecentphylogeneticstudyofthesespeciesreportsAnetiawithinthemost
basallydiversifyinglineageinthisgroup[46].SoitistemptingtospeculatethattheZ-autosome
fusionreportedhereforDanausoccurredwithintheDanaini,afterthedivergencefromAnetia,
whichhas31chromosomes,presumablyreflectingasharedancestralkaryotypewithMelitaea.
However,thesamephylogeneticstudyreportsAnetiaassistertoLycorea(N=30),withinthe
samebasallysplittinglineage.Becausenootherchromosomecountsareknownforthe
numerousspeciesatintermediatedivergencesbetweenDanausandthe(Anetia,Lycorea)
lineage,weareleftwithtwoplausiblescenarios,assumingthereportedphylogenetic
relationshipsareaccurate.Inonecase,Anetiaindeedretainstheancestralkaryotypewhile
fusionsindependentlyoccurredinLycoreaandalsointhelineageleadingtoDanaus.The
alternativecaseisthataZ-autosomefusionpredatestheoriginsofallDanaini,witha
bioRxiv preprint first posted online Jan. 12, 2016; doi: http://dx.doi.org/10.1101/036483. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
subsequentchromosomalfissioninAnetia,producingoneextrachromosomeforN=31,a
chromosomecountthatisconvergentbutnothomologoustoMelitaea.Resolvingthesetwo
possibilitieswilllikelyrequireacomparativeanalysisofZ-chromosomehomologywithinthe
Danaini.
InanalyzingpatternsofchromosomalfusioninH.melpomeneandB.morirelativetoM.
cinxia,Aholaetal.(2014)reportasignificanttendencyforalimitedsetofancestral
chromosomes–particularlythesmallestones–tobeinvolvedinchromosomalfusionevents.
NeithertheancestralZnorMcChr21areamongthesesmall,repeatedlyfusedchromosomes.
Thusthechromosomalfusionreportedheredoesnotfitneatlywiththepatterndescribedby
Aholaetal.Nonetheless,HmChr2(homologoustoMcChr21)isthesecondsmallest
chromosomethatremainsunfusedbetweentheselineages[47].Soitisalsodifficulttoargue
stronglythatthisZ-autosomefusioninDanausisastrikingcontrasttothetrendof
chromosomalfusionsinvolvingsmallchromosomes.
Conclusion
Wehaveusedacombinationofgenomesequencingcoverageandcomparativegenomic
analysistodemonstratethatDanausbutterfliesharboraneo-Zchromosomeresultingfromthe
fusionoftheancestralZchromosomeandanautosomehomologoustoChr21inM.cinxia.Our
analysisalsoidentifiedandresolvedseveralZ-autosomechimericscaffoldsinthemostrecent
assemblyoftheD.plexippusgenome.Thisdiscoveryandprovisionalassignmentof
chromosomelinkagefor>90%ofD.plexippusgenespavesthewayformyriadanddiverse
investigationsintosexchromosomeevolution,whicharelikelytobeofdistinctimportance
giventheincreasingprominenceofmonarchbutterflyasafemale-heterogameticmodel
speciesforfunctionalandevolutionarygenomics.
Methods
Sequencingcoverageanalysis
IlluminashotgungenomicDNAsequencingdataforthreemaleandthreefemaleD.
plexippusindividualswereselectedforanalysisfromsamplessequencedbyZhanetal.(2014)
[38].Male-femalepairswereselectedonthebasisofapproximatelyequalsequencing
coverage.SampleswerealignedtotheD.plexippusversion3genomeassemblywithbowtie2
(v2.1.0),usingthe“verysensitivelocal”alignmentoption[39,48].Theresultingalignments
wereparsedwiththegenomecovandgroupbyutilitiesintheBedToolssoftwaresuite(v2.17.0)
toobtainaper-basemediancoveragedepthstatisticforeachscaffold[49].Genomic
sequencingdatafromotherDanausspecies,alsogeneratedbyZhanetal.2014,werealigned
tothesameassemblyusingStampy(v1.0.22)(defaultparameters,exceptfor
substitutionrate=0.1)[50].Detailsofallsampleidentity,includingGenBankSRAaccessions,are
giveninSupplementaryTableS2.
Coverageanalysescomparingmalesandfemaleswerelimitedtoscaffoldsoflengths
equaltoorgreaterthantheN90scaffold(160,499bp)[39].Also,incompletecaseswere
bioRxiv preprint first posted online Jan. 12, 2016; doi: http://dx.doi.org/10.1101/036483. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
excluded(i.e.,scaffoldswithnoreadsfromoneormoresamples).Intotal,140scaffoldswere
excluded,leaving5,257scaffoldsanalyzed.Foreachsample,eachscaffold’smediancoverage
wasdividedbythemeanacrossallscaffoldmediancoverages,therebynormalizingfor
differencesinoverallsequencingdepthbetweensamples.Samplesweregroupedbysexand
theper-scaffoldmeanofnormalizedcoveragedepthwascomparedbetweensexes,formulated
asthelog2ofthemale:femalecoverageratio.Autosomalscaffoldsareexpectedtoexhibit
equalcoveragebetweensexes,yieldingalog2ratioofzero.Zlinkedscaffoldsshouldhavea
ratioofone,duetothetwo-foldgreaterrepresentationinmales.Manipulation,analysis,and
visualizationofcoveragedatawasperformedusingcustomRscripts[51].
Forselectscaffoldswithintermediatemediancoverageratios,weusedBedtools
genomecovtocalculateper-basecoverage,inordertoidentifypotentialassemblyerrors
producingZ-Autosomalchimericscaffolds.Foreachsample,coverageperbasewasdividedby
themeanofallscaffoldmediancoverages,thusnormalizingforoverallsequencingdepth.The
normalizedcoverageperbasewasaveraged(mean)withinsexandvisualizedalongthelength
ofthescaffoldbyusingthemedianofa5kbpslidingwindow,shiftedby1kbpsteps.
PointestimatesforZ-autosomalbreakpointsinchimericscaffoldsweregeneratedusing
aslidingwindowanalysisofmale:femalecoverageratios.Putativebreakpointswereobtained
asthemaximumoftheabsolutedifferencebetweenadjacentnon-overlappingwindows.A
windowof150Kbpwith10kbpstepswasusedforDPSCF300001andthe5’breakpointof
DPSCF30028.Awindowof10kbpwith1kbpstepswasusedforDPSCF30044andinasecond,
localizedanalysisbetween1.5Mbandthe3’terminusofDPSCF30028tolocalizethesecond,3’
breakpoint.
Orthology-basedchromosomalassignmentsforD.plexippusscaffolds.
PutativechromosomallinkagewaspredictedforD.plexippusscaffoldsrelativetothe
genomeassembliesofthreereferencespecies,M.cinxia,B.mori,andH.melpomene,basedon
countsoforthologousgenes[29,30,42].OrthologousproteinswerepredictedbetweenD.
plexippusandeachreferencespeciesusingtheProteinorthopipeline[52].Usingonly1-to-1
orthologs,wetabulatedperD.plexipppusscaffoldthenumberofgenesmappedtoeach
chromosomeinthereferencespecies.EachD.plexippusscaffoldwastentativelyassignedto
thechromosomewiththehighestcountoforthologsinthereferencespecies.Scaffoldswere
excludedfromanalysiswhenmaximumorthologcountwastiedbetweentwoormore
scaffolds,thoughthissituationwasrareandalwaysinvolvedscaffoldswithlowcountsof
(orthologous)genes.
PointestimateoftheZ-autosomefusion
ThefusionpointinMonarchbetweenancestrallyZandautosomalsegmentswaslocalizedby
aligningthehomologousH.melpomeneorM.cinxiachromosomesagainstMonarchscaffold
DPSCF300001[29,47].Alignmentswerebasedonsix-frameaminoacidtranslationsusingthe
PROmeralgorithmandvisualizedwithmummerplot,bothfromtheMUMmersoftwarepackage
(v3.1)[53].WeinitiallyalignedthecompletesetofscaffoldsfromtheZ(HmChr21,McChr1)or
relevantautosome(HmChr2,McChr21),yieldingapreliminaryindicationthattheZ-Afusion
pointoccurredat~4MbponDPSCF300001.Torefineandbettervisualizethisphenomenon,
“pseudo-assemblies”werecreatedforeachchromosomeusingqueryscaffoldsproducing>500
bioRxiv preprint first posted online Jan. 12, 2016; doi: http://dx.doi.org/10.1101/036483. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
bpoftotalalignedcoverageonDPSCF300001.Selectedqueryscaffoldswereconcatenatedinto
asinglefastaentry,withorderingbasedontargetalignmentpositions.Foreachspecies,theZ
andautosomalpseudo-assemblieswereco-alignedtoDPSCF300001.Thetransitionpoint
betweencontiguousalignmentsofthetwopseudo-assemblieswasinterpretedasthe
approximatelocationoftheZ-AinMonarch.
Acknowledgements
ThismanuscriptisdedicatedtoChipTaylor,AnnRyan,andthemanyhard-workingmembersof
MonarchWatch.org.JimMalletandJohnDaveyprovidedhelpfulcommentsonthiswork.This
researchwassupportedbyNSF-DEB1457758(toJ.R.W.).Thecomputingforthisprojectwas
performedontheCommunityClusterattheCenterforResearchComputingattheUniversityof
Kansas.
Authorcontributions:JRWconceivedanddesignedresearch,performedanalyses,anddrafted
themanuscript.AJMperformedanalysesandhelpeddraftthemanuscript.Bothauthorsread
andapprovedthefinalmanuscript.
CompetingInterests:Theauthors(JRW&AJM)declarenocompetinginterests.
Additionaldatafiles:
Monarch_neoZ_Gene-Scaff.xlsxcontainspredictedchromosomallinkage,wheredetermined,
forallD.plexippusproteincodinggenes.
bioRxiv preprint first posted online Jan. 12, 2016; doi: http://dx.doi.org/10.1101/036483. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
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Table1.SummaryofassigningD.plexippusgenesandscaffoldstochromosomesviaorthology
“liftover”relativetoM.cinxia.
1:1orthologsidentified
6,740
1:1orthologsassignedtoM.cinxiachromosome
4,607(68.4%)
ProteincodinggenesinD.plexippus
15,130
D.plexippusproteincodinggenesassignedtochromosome
14,129(93.4%)
D.plexippusscaffoldsputativelyassignedtochromosomes
454
Table2.SummaryofprovisionalchromosomallinkageforD.plexippusproteincodinggenes,
withchromosomalidentityreflectinghomologytoM.cinxia
Chromosome
1
Anc-Z
Neo-Z
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
22
23
24
25
26
27
28
29
30
31
NotAssigned
NumberofGenes
1101
624
477
704
758
582
494
689
483
535
647
452
574
576
501
429
493
524
604
561
414
399
302
318
185
329
274
250
284
294
151
168
1055
bioRxiv preprint first posted online Jan. 12, 2016; doi: http://dx.doi.org/10.1101/036483. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
Figure1.Distributionofmediannormalizedmale:femalegenomicsequencingcoverage
ratiosforD.plexippusversion3assemblyscaffolds.Onlyscaffoldsoflengthequaltoor
greaterthantheN90scaffoldareshown.Thedottedlineat0.5representsthevalue
usedtopartitionscaffoldsasautosomal(grey)orZ-linked(red).
Figure2.Normalizedmaleandfemalecoveragealongthelengthofchimericscaffolds,
for(A)DPSCF300028,(B)DPSCF300044,and(C)DPSCF300001.Coveragesareplottedas
slidingwindows(width=5Kbp,step=1Kbp)ofmedianbasepairvalues.Theassociated
male:femaleratioofcoverageforeachwindowisplottedasaredlinebelowthepairof
sex-specificplots.AsterisksindicatetheestimatedbreakpointbetweenZlinkedand
autosomalsegmentsofeachscaffold,asdeterminedbythemaximumdifferencein
adjacent,non-overlappingwindowsofmale:femaleratio(seemethodsfordetails).
Figure3.Chromosomalco-linkagebetweenD.plexippusandM.cinxiaforpredicted
orthologousproteins.
Figure4.Ratiosofmale:femalemediannormalizedgenomicsequencingcoverage
plottedbyscaffoldlength.Scaffoldshomologousvia“liftover”proceduretoM.cinxia
chromosomes1/Z(blue)and21(green)areplottedindistinctcolors.Dottedlines
indicateexpectedvaluesforZ-linked(red)andautosomal(black)scaffolds.
Figure5.Ratiosofmale:femalemediannormalizedgenomicsequencingcoverage
plottedbyscaffoldlengthforfourspeciesofDanausbutterflies.
80
40
20
0
Frequency
60
Putative Autosomal scafffolds
Putative Z−linked scaffolds
−0.5
0.0
0.5
Log2( Male:Female coverage )
1.0
1.5
31
30
29
28
27
26
25
24
23
22
21
20
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
1
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
9
●
●
●
●
●
●
●
11
●
●
●
15
●
●
19
M. cinxia chromosome
●
●
●
17
●
●
●
●
13
●
●
●
●
●
●
●
●
●
10
●
●
●
●
●
●
●
100
●
●
7
●
●
●
●
●
●
●
●
●
500
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
5
●
●
●
●
Number
of genes
●
●
●
●
3
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
1:5
putative D. plexippus chromosome
Ortholog Chromosomal Co−localization: M. cinxia vs. D. plexippus
21
23
●
25
27
29
●
31
1
bioRxiv preprint first posted online Jan. 12, 2016; doi: http://dx.doi.org/10.1101/036483. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
A 2
1
2
1
steps
2
1
steps
0
Male:Female
coverage ratio
0
Female coverage
(Normalized)
0
Male coverage
(Normalized)
DPSCF300028
0
0.25
0.5
0.75
B 1
1.25
1.5
1.75
2
Scaffold Position (Mbp)
mcov.smooth[, 2]
2
1
2
1
steps
2
1
steps
0
Male:Female
coverage ratio
0
Female coverage
(Normalized)
0
Male coverage
(Normalized)
DPSCF300044
0
0.25
0.5
0.75
1
1.25
Scaffold Position (Mbp)
C mcov.smooth[, 2]
2
1
2
1
steps
2
1
steps
0
Male:Female
coverage ratio
0
Female coverage
(Normalized)
0
Male coverage
(Normalized)
DPSCF300001
0
0.5
1
1.5
2
2.5
3
3.5
Scaffold Position (Mbp)
mcov.smooth[, 2]
4
4.5
5
5.5
6
M. cinxia reference chromosome
●
1
●
21
●
Other
●
●
● ●
●
●
●
●●
0.0
0.5
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
● ● ●● ● ●
●
●
●
●●
●●
● ●
●● ●
● ● ● ● ●
●●
●● ●● ●● ●
●● ●●
●●●●
●
● ● ●●
●
●
●
●
●
●
●
●
●
●●● ●●● ●●● ●●
●
●
● ● ●●
●● ●
●
● ●● ●
●● ● ●●
●
●●●
●
● ●●
●
●●
●●●●
● ●●
● ●●●●
● ●●●
● ●● ●
●● ●
●
●●
●
● ●●
●●●
●● ●
●
●●
● ● ●
●●● ●●
●
●
● ●● ●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
● ●● ●
● ● ● ●●●
● ● ● ● ● ●● ●● ●
●
●
● ●●●
● ●
● ●●●
● ●●
●
● ●
●●●
●
●
●
●
● ●●
●●
● ●●● ●
●
●
●
●●
●
●
●
● ●
● ●●
●●●●
●●
●●
●● ●
●
●
● ●●
● ●●
●
●
● ● ● ●●
●●
●
●
● ●● ●●●●
●●
●
●
●●● ●●● ●
● ●
●●
●●● ●
●●●
● ●
●●● ● ●
●
●
●
●●
●
●●
●●
● ● ●●
● ●●
●
●
●
−0.5
Log2[ Male:Female coverage ]
1.0
●
●
5.5
6.0
Log10[ Scaffold Length (bp) ]
6.5
7.0
bioRxiv preprint first posted online Jan. 12, 2016; doi: http://dx.doi.org/10.1101/036483. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
Macrosynteny: B. mori vs D. plexippus
27
●
●
●
26
25
23
●
putative D. plexippus chromosome
●
●
●
●
●
●
●
●
●
20
●
●
18
●
17
●
●
●
●
●
●
●
●
●
●
●
●
●
11
●
●
●
●
Number
of genes
●
●
●
●
●
500
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
8
●
●
●
6
●
●
●
5
●
4
●
3
●
●
●
●
●
●
●
●
●
●
●
1
●
●
●
●
●
●
●
●
●
●
●
●
●
●
13
15
17
●
●
●
●
11
1
●
●
●
●
1 2 3 4 5 6 7 8 9
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
10
●
●
●
9
100
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
13
10
●
●
●
●
●
●
●
14
2
●
●
●
●
●
●
●
15
●
●
●
●
19
7
●
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●
●
●
●
●
●
●
●
●
●
●
●
21
12
●
●
●
●
22
16
●
●
24
●
●
●
●
1:5
28
19
21
●
23
25
27
B. mori chromosome
Macrosynteny: H. melpomene vs D. plexippus
20
●
●
19
putative D. plexippus chromosome
18
●
●
●
●
●
●
●
●
●
●
●
17
●
16
●
●
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●
15
14
13
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11
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9
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500
●
●
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●
Number
of genes
●
●
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●
●
●
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●
●
6
●
●
●
●
●
●
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5
●
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4
●
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1
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2
3
5
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4
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2
1
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3
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6
7
8
9
●
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●
●
●
●
10 11 12 13 14 15 16 17 18 19 20 21
H. melpomene chromosome
10
●
●
7
100
●
●
●
●
●
●
●
●
●
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●
●
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8
●
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12
10
●
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●
●
1:5
21
1
FigureS1.ChromosomalcolinkagebetweenD.plexippusand
B.mori(top)orH.melpomene
(bottom)forpredicted
orthologousproteins.
bioRxiv preprint first posted online Jan. 12, 2016; doi: http://dx.doi.org/10.1101/036483. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
B. mori reference chromosome
●
1
●
16
●
FigureS2.Ratiosofmale:female
mediannormalizedgenomic
sequencingcoverageplottedby
scaffoldlength.Scaffolds
assignedtochromosomes
putativehomologoustotheneoZchromosomeinD.plexippusare
plottedindistinctcolors.Top,
relativetoB.mori,to
chromosomes1(i.e.,Z;blue)and
16(green).Bottom,relativetoH.
melpomene,chromosomes1
(i.e.,Z;green)and2(blue).
Other
●
●
● ●
●
●
●
●●
0.0
0.5
●
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●
−0.5
Log2[ Male:Female coverage ]
1.0
●
●
5.5
6.0
6.5
7.0
Log10[ Scaffold Length (bp) ]
H. melpomene reference chromosome
●
2
●
Z
●
Other
●
●
● ●
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0.0
0.5
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●
−0.5
Log2[ Male:Female coverage ]
1.0
●
●
5.5
6.0
Log10[ Scaffold Length (bp) ]
6.5
7.0
bioRxiv preprint first posted online Jan. 12, 2016; doi: http://dx.doi.org/10.1101/036483. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
2
1
2
1
steps
2
1
steps
0
Male:Female
coverage ratio
0
Female coverage
(Normalized)
0
Male coverage
(Normalized)
DPSCF300403
0
0.025
0.05
0.075
0.1
0.125
Scaffold Position (Mbp)
0.15
0.175
0.2
FigureS3.NormalizedmaleandfemalecoveragealongthelengthDPSCF300403.Coveragesareplottedas
slidingwindows(width=5Kbp,step=1Kbp)ofmedianbasepairvalues.Theassociatedmale:femaleratioof
coverageforeachwindowisplottedasaredlinebelowthepairofsex-specificplots.
mcov.smooth[, 2]
H. melpomene Chr02 & Chr21(Z) vs DPSCF300001
H. melpomene Chromosome
A
Chr21(Z)
bioRxiv preprint first posted online Jan. 12, 2016; doi: http://dx.doi.org/10.1101/036483. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
Chr02
0
1000000
M. cinxia chromosome
B
4000000
3000000
2000000
DPSCF300001 position (bp)
5000000
6000000
5000000
6000000
M. cinxia Chr21 & Chr01(Z) vs DPSCF300001
Chr01(Z)
Chr21
0
1000000
4000000
3000000
2000000
DPSCF300001 position (bp)
FigureS4.PromeralignmentsofDPSCF300001againsttheZandhomologous
autosomefrom(A)H.melpomeneand(B)M.cinxia.Bestone-to-one
alignmentsweregeneratedusingdefaultparameters.ManualinspecEonof
alignmentcoordinatesrevealedthetransiEononDPSCF300001fromneo-Zto
anc-ZoccursinawindowbetweenposiEons3.878and3.886Mbp.
bioRxiv preprint first posted online Jan. 12, 2016; doi: http://dx.doi.org/10.1101/036483. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
TableS1.SummaryofassigningD.plexippusgenesandscaffoldstochromosomesviaorthology“liftover”
relativetothreedifferentreferenceassemblies.
M.cinxia
H.melpomene
B.mori
15130
15130
15130
totalnumberofproteincodinggenesintarget
14129
14427
14566
numberoftargetgenesassignedtochromosome
0.934
0.954
0.963
fractionoftargetgenesassignedtochromosome
454
514
508
6740
8190
7928
numberoftargetscaffoldsassignedto
chromosomes
numberof1:1orthologsidentified
4607
7150
7534
0.684
0.873
0.95
numberof1:1orthologsassignedtoreference
chromosome
fractionof1:1orthologsassignedtoreference
chromosome
SupplementaryTableS2.Sampleidentificationdetailsforsequencingdatausedincoverateanalyses.
Region Sample
North
Plex_MA_HI004_M
America
Plex_MA_HI035_F
Other
Danaus
Species
plexippus
Sex
male
plexippus
female
Plex_FLn_StM123_F plexippus
female
Plex_FLn_StM146_M plexippus
male
Plex_WSM_M36_M
plexippus
male
Plex_WSM_M38_F
plexippus
female
Erip_BRA_16005_F
erippus
female
Erip_BRA_16008_M erippus
male
Eres_CRC_92_F
eresimus
female
Eres_FL_27_M
eresimus
male
Gili_CRC_30_M
gilippus
male
Gili_TX_01_F
gilippus
female
Collectinglocation Date
Accession
Massachusetts,USA July32008 SRX679269
SRX679310
Massachusetts,USA August10
2009
SRX680105
St.Marks,Florida,
October
USA
2008
SRX681753
St.Marks,Florida,
October
USA
2009
SRX680118
Samoa
June2007
SRX681528
Samoa
June2007
Brazil
September SRX682069
2010
Brazil
September SRX682070
2010
SRX682071
CostaRica
July24
2010
SRX682072
Florida,USA
July20
2009
SRX682073
CostaRica
July24
2010
SRX998564
Texas,USA
October
2010