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 The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder.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.
Neo-sexchromosomesintheMonarchbutterfly,Danaus
plexippus
AndrewJ.Mongue1,PetrNguyen2,3,AnnaVolenikova2,3,andJamesR.Walters1*
1)DepartmentofEcologyandEvolutionaryBiology,UniversityofKansas,Lawrence,KS,USA
2)FacultyofScience,UniversityofSouthBohemia,37005ČeskéBudějovice,CzechRepublic
3)InstituteofEntomology,BiologyCentreCAS,37005ČeskéBudějovice,CzechRepublic
*Authorforcorrespondence:JamesRWalters,DepartmentofEcologyandEvolutionary
Biology,UniversityofKansas,Lawrence,KS,USA
phone:301-404-2743
email:[email protected]
Runningtitle:Neo-Zchromosomeinmonarchbutterfly
Keywords:sexchromosomes,evolution,Lepidoptera,genomics,chromosomalfusion
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 The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder.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.
Abstract
Wereportthediscoveryofaneo-sexchromosomeinMonarchbutterfly,Danaus
plexippus,andseveralofitscloserelatives.Z-linkedscaffoldsintheD.plexippusgenome
assemblywereidentifiedviasex-specificdifferencesinIlluminasequencingcoverage.
Additionally,amajorityoftheD.plexippusgenomeassemblywasassignedtochromosomes
basedoncountsof1-to-1orthologsrelativetothebutterflyMelitaeacinxia(withreplication
usingtwootherlepidopteranspecies),inwhichgenomescaffoldshavebeenmappedtolinkage
groups.Sequencing-coveragebasedassessmentsofZ-linkagecombinedwithhomologybased
chromosomalassignmentsprovidedstrongevidenceforaZ-autosomefusionintheDanaus
lineage,involvingtheautosomehomologoustochromosome21inM.cinxia.Coverageanalysis
alsoidentifiedthreenotableassemblyerrorsresultinginchimericZ-autosomescaffolds.
CytogeneticanalysisfurtherrevealedalargeW-chromosomethatispartiallyeuchromatic,
consistentwithbeinganeo-Wchromosome.Thediscoveryofaneo-Zandtheprovisional
assignmentofchromosomelinkagefor>90%ofD.plexippusgeneslaysthefoundationfor
novelinsightsconcerningsexchromosomeevolutioninthisfemale-heterogameticmodel
speciesforfunctionalandevolutionarygenomics.
Background
Majorrearrangementsofkaryotypeandchromosomestructureoftenhavesubstantial
evolutionaryimpactsonboththeorganismscarryingsuchmutationsandthegeneslinkedto
suchgenomicreorganization(LynchandWalsh2007;SoltisandSoltis2012).Additionally,such
large-scalechromosomalmutationsoftenpresentnovelopportunitiestoinvestigatemolecular
evolutionaryandfunctionalgeneticprocesses,forinstancetheevolutionofneo-sex
chromosomes,whichcanarisefromthefusionofanautosomewithanexistingandwelldifferentiatedallosome.Thiseffectivelyinstantaneoustransformationofaformerlyautosomal
setofgenesintosex-linkedlociisfertilegroundforadvancingourunderstandingofthedistinct
setofevolutionaryforcesactingonsexchromosomesrelativetoautosomes(Bachtrogetal.
2009;Pala,Hasselquist,etal.2012;Bachtrog2013;Šíchováetal.2013).Furthermore,when
suchaneventisobservedinatractablegeneticmodelsystem,thereisopportunitytoexplore
thefunctionalandmechanisticchangesassociatedwithsexchromosomeevolution.The
congruenceofneo-sexchromosomesexistinginamodelsystemisrelativelyrare,although
2
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peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
therearesomenotableexamples.Namely,independentoriginsofneo-sexchromosomesare
knowninDrosophilafruitflies(Countermanetal.2004;Floresetal.2008;Bachtrogetal.2009;
Zhouetal.2013;BrownandBachtrog2014;Nozawaetal.2014)andsticklebackfish,where
neo-sexchromosomesappeartoplayanimportantroleinreproductiveisolationbetween
incipientspecies(Kitanoetal.2009;Yoshidaetal.2014;Whiteetal.2015).
Lookingbeyondtheseestablishedmodelsystems,therapidexpansionofgenomic
technologieshasallowedextensiveanalysesofgenecontent,sex-biasedgeneexpression,
dosagecompensation,andsequencedivergenceforrecentlyevolvedsexchromosomesamong
averydiversesetoforganismsincludingseveralinsectlineages[Teleopsidflies,agrasshopper,
andStrepsiptera(BakerandWilkinson2010;MahajanandBachtrog2015;Palacios-Gimenezet
al.2015)],vertebrates[mammalsandbirds(Zhouetal.2008;Pala,Hasselquist,etal.2012;
Murataetal.2015)],andplants[SileneandRumexgenera(Houghetal.2014;Charlesworth
2015;Papadopulosetal.2015)].Aclearconsensusemergesfromthisresearchthatthelackof
recombinationassociatedwithsexchromosomescatalyzesacascadeofevolutionarychanges
involvingthedegenerationofoneallosome,theaccumulationofgeneswithsex-biased
expression,increasedevolutionaryrates,andoften,butnotalways,theacquisitionofdosage
compensation.Yetmanyofthedetailsinthisprocessremainelusiveandunresolved,including
therateofallosomedivergence,theroleofpositiveselectionversusdrift,theimportanceof
sex-specificselection,andthemechanismsunderlyingdosagecompensationorthereasonsfor
itsabsence.Itisthereforeimportanttocontinueidentifyingnewopportunitiesfornovelinsight
intotheevolutionofsexchromosomes.
Overwhelmingly,researchonsexchromosomesoccursinmale-heterogametic(XY)
species(VicosoandCharlesworth2006;Ellegren2011;Bachtrog2013;ParschandEllegren
2013).Thisappearstobeparticularlytrueforneo-sexchromosomes,wherecontemporary
genomicanalysesofneo-Zorneo-Wchromosomesarecurrentlylacking,withonenotable
exceptionforbirds(Pala,Hasselquist,etal.2012).Thisimbalanceisunfortunate,becauseZW
sexdeterminationoffersthenovelframeworkoffemale-specificselectionduringtheevolution
ofheterogametyandisacommonformofsexdeterminationinbothvertebratesand
invertebrates.Birdsarethemostprominentvertebratetaxonthatisfemale-heterogametic,but
3
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.
itappearsthatavianneo-sexchromosomesarequiterareandindeedabsentfromprominent
modelspecies,e.g.chicken,zebrafinch(Nandaetal.2008;Pala,Naurin,etal.2012).Fishesand
squamatesseemtobefarmorelabileinsex-chromosomeconstitution,withnumerous
independenttransitionsbetweenmaleandfemale-heterogametyandrelativelyfrequentsexautosomefusions(Pennelletal.2015),thustherearepotentiallygreatopportunitiesinthese
taxa.However,notractableZWmodelsystemwithneo-sexchromosomeshasbeenidentified
fortheselineages.
Formanyreasons,Lepidoptera,mothsandbutterflies,maybethemostpromising
female-heterogametictaxonforstudyingneo-sexchromosomes.Synteny,i.e.thechromosomal
placementoforthologousgenesbetweenspecies,isunusuallywell-conservedinLepidoptera
(Pringleetal.2007;TheHeliconiusGenomeConsortium2012;Aholaetal.2014;Kanostetal.
2016),yettherearealsonumerousknownexamplesofindependentlyevolvedneo-ZandneoWchromosomes,severalofwhichhavebeenwell-characterizedcytogenetically(Trautetal.
2008;Yoshidoetal.2011;Nguyenetal.2013;Šíchováetal.2013;Smithetal.2016).
Furthermore,comparativegenomicresourcesinthisinsectorderaresubstantialandgrowing
quickly(www.lepbase.org).
Inthiscontext,wereportthediscoveryofaneo-Zchromosomeinthemonarch
butterfly,Danausplexippus,andcloselyrelatedspecies.Monarchbutterflies,renownedfor
theirannualmigrationacrossNorthAmerica,alreadyhaveastrongprecedentasamodel
systeminecology(Urquhart1976;OberhauserandSolensky2004).Recently,monarchshave
emergedasamodelsystemforgenomebiology,withawell-assembledreferencegenome,
extensivepopulationresequencingdata,andaprecedentforgenomeengineering(Zhanetal.
2011,2014;Merlinetal.2013;Markertetal.2016).Thediscoveryofaneo-Zchromosome
furtherenrichesthevalueofthisspeciesasaresearchmodelingenomebiologyandlaysthe
foundationforextensivefutureinsightsintotheevolutionandfunctionaldiversityofsex
chromosomes.
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peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
Materialsandmethods
Sequencingcoverageanalysis
IlluminashotgungenomicDNAsequencingdataforthreemaleandthreefemaleD.
plexippusindividualswereselectedforanalysisfromsamplessequencedbyZhanetal.(2014).
Detailsofsampleidentities,includingGenBankSRAaccessions,aregiveninSupplementary
TableS1.Male-femalepairswereselectedonthebasisofapproximatelyequalsequencing
coverage.SampleswerealignedtotheD.plexippusversion3genomeassemblywithbowtie2
(v2.1.0),usingthe“verysensitivelocal”alignmentoption(LangmeadandSalzberg2012;Zhan
andReppert2013).Theresultingalignmentswereparsedwiththegenomecovandgroupby
utilitiesintheBedToolssoftwaresuite(v2.17.0)toobtainaper-basemediancoveragedepth
statisticforeachscaffold(QuinlanandHall2010).GenomicsequencingdatafromotherDanaus
species,alsogeneratedbyZhanetal.(2014b),werealignedtothesameassemblyusingStampy
(v1.0.22)(defaultparameters,exceptforsubstitutionrate=0.1)(LunterandGoodson2011).
Coverageanalysescomparingmalesandfemaleswerelimitedtoscaffoldsoflengths
equaltoorgreaterthantheN90scaffold(160,499bp)(ZhanandReppert2013).Also,
incompletecaseswereexcluded(i.e.,scaffoldswithnoreadsfromoneormoresamples).In
total,140scaffoldswereexcluded,leaving5,257scaffoldsanalyzed.Foreachsample,each
scaffold’smediancoveragewasdividedbythemeanacrossallscaffoldmediancoverages,
therebynormalizingfordifferencesinoverallsequencingdepthbetweensamples.Samples
weregroupedbysexandtheper-scaffoldmeanofnormalizedcoveragedepthwascompared
betweensexes,formulatedasthelog2ofthemale:femalecoverageratio.Autosomalscaffolds
areexpectedtoexhibitequalcoveragebetweensexes,yieldingalog2ratioofzero.Z-linked
scaffoldsshouldhavearatioofone,duetothetwo-foldgreaterrepresentationinmales.
Manipulation,analysis,andvisualizationofcoveragedatawasperformedinR(RDevelopement
CoreTeam2015).
Toselectscaffoldswithintermediatemediancoverageratios,weusedBedtools
genomecovtocalculateper-basecoverage,inordertoidentifypotentialassemblyerrors
producingZ-Autosomalchimericscaffolds.Foreachsample,coverageperbasewasdividedby
5
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peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
themeanofallscaffoldmediancoveragevalues,thusnormalizingforoverallsequencingdepth.
Thenormalizedcoverageperbasewasaveragedwithinsexandvisualizedalongthelengthof
thescaffoldbyusingthemedianofa5kbpslidingwindow,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(the
Glanvillefritillary,domesticsilkmoth,andpostmanbutterfly),basedoncountsoforthologous
genes(TheInternationalSilkwormGenomeConsortium2008;TheHeliconiusGenome
Consortium2012;Aholaetal.2014).OrthologousproteinswerepredictedbetweenD.
plexippusandeachreferencespeciesusingtheProteinorthopipeline(Lechneretal.2011).
Usingonly1-to-1orthologs,wetabulatedperD.plexippusscaffoldthenumberofgenes
mappedtoeachchromosomeinthereferencespecies.EachD.plexippusscaffoldwasassigned
tothechromosomewiththehighestcountoforthologsinthereferencespecies.Scaffoldswere
excludedfromanalysiswhenmaximumorthologcountwastiedbetweentwoormore
scaffolds,thoughthissituationwasrareandinvolvedscaffoldswithlowgenescounts.
PointestimateoftheZ-autosomefusion
ThefusionpointinMonarchbetweenancestrallyZandautosomalsegmentswas
localizedbyaligningthehomologousH.melpomeneorM.cinxiachromosomesagainst
MonarchscaffoldDPSCF300001(Aholaetal.2014;Daveyetal.2016).Alignmentswerebased
onsix-frameaminoacidtranslationsusingthePROmeralgorithmandvisualizedwith
mummerplot,bothfromtheMUMmersoftwarepackage(v3.1)(Kurtzetal.2004).Weinitially
6
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peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
alignedthecompletesetofscaffoldsfromtheZ(HmChr21,McChr1)orrelevantautosome
(HmChr2,McChr21),yieldingapreliminaryindicationthattheZ-autosomefusionpoint
occurredat~4MbponDPSCF300001.Torefineandbettervisualizethisphenomenon,pseudoassemblieswerecreatedforeachchromosomeusingqueryscaffoldsproducing>500bpoftotal
alignedcoverageonDPSCF300001.Selectedqueryscaffoldswereconcatenatedintoasingle
fastaentry,withorderingbasedontargetalignmentpositions.Foreachspecies,theZand
autosomalpseudo-assemblieswereco-alignedtoDPSCF300001.Thetransitionpointbetween
contiguousalignmentsofthetwopseudo-assembliesfromdistinctchromosomeswas
interpretedastheapproximatelocationoftheZ-autosomefusioninMonarch.
Cytogeneticanalysis
AllD.plexippustissuesusedforcytogeneticanalysiswerefromcaptive-bredbutterflies
rearedonanartificialdietprovidedbyMonarchWatch(MonarchWatch.org).Spread
chromosomepreparationsweremadefromgonadsofthirdtofifthinstarlarvaeofbothsexes
followingMediounietal.(2004).Inordertotestforthepresenceofsexchromatin,
preparationsofpolyploidsomaticnucleiweremadeaccordingtoTrautetal.(1986)from
Malpighiantubulesdissectedfromthesamematerial.
GenomicDNAwasisolatedseparatelyfrommalesandfemalesbystandardphenolchloroformextraction.Briefly,larvaltissueswerehomogenizedinliquidnitrogen,transferredin
lysisbuffer(100mMNaCl,10mMTris-HClpH8,0,50mMEDTA,100μg/mlProteinaseK,0,5%
Sarkosyl),andincubatedat37°Covernight.ThesampleswerethentreatedwithRNaseA(10
µg/ml)andpurifiedbythreephenol,onephenol-chloroform,andonechloroformextractions.
Male-andfemale-derivedhybridizationprobeswerelabeledbynicktranslationasdescribedin
Šíchováetal.(2015).
Genomicinsituhybridization(GISH)wasperformedasdescribedbyFukováetal.
(2005).Comparativegenomichybridization(CGH)wascarriedoutaccordingtotheprotocolin
Šíchováetal.(2013)withseveralmodifications,asfollows.Priortodenaturation,RNaseA
treatedslideswereincubatedin5xDenhardt’ssolution(0,1%Ficoll,0,1%polyvinylpyrrolidone,
0,1%bovineserumalbumin)at37°Cfor30min.A10µlhybridizationmixtureconsistingof
labeledfemaleandmaleprobes(350ngeach),sonicatedsalmonspermDNA(25µg),50%
7
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peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
deionizedformamide,and10%dextransulfatein2xSSCwasdenaturedandallowedto
reannealat37°Cfortwohours(c.f.Kallioniemietal.1992)beforeitwashybridizedtothe
denaturedfemalepreparation.
ResultsweredocumentedinaZeissAxioplan2microscope(CarlZeiss,Jena,Germany)
equippedwithappropriatefluorescencefilterset.ImageswerecapturedwithanOlympusCCD
monochromecameraXM10equippedwithcellSens1.9digitalimagingsoftware.Theimages
werepseudocolouredandsuperimposedwithAdobePhotoshopCS3.
DataAvailability
PutativechromosomalassignmentsforD.plexippusgenesareprovidedinSupplemental
TableS2.EstimatedbreakpointsreportedforchimericZ-autosomalassembliesareprovidedin
SupplementalTableS3.OtherintermediateRresultsfilesandcodeusedindescribedanalyses
areavailableuponrequest.
Results
IdentifyingZ-linkedscaffoldsinD.plexippus
WeidentifiedZ-linkedscaffoldsintheD.plexippusgenomeassembly(Zhanetal.2011;
ZhanandReppert2013)bycomparingsequencingcoveragefrommaleandfemalesamples.
MalesshouldhavetwicetheZchromosomeDNAcontentthanfemales,whileautosomes
shouldhaveequalDNAcontentbetweensexes.Thusacorrespondingtwo-folddifferencein
sequencingcoverageisexpectedbetweensexesfortheZchromosome,butnotautosomes,
andcanbeusedtoidentifyZ-linkedscaffolds(Martinetal.2013;Vicosoetal.2013;Mahajan
andBachtrog2015).Ahistogramofmale:femaleratiosofmediancoverageclearlyidentifies
twogroupsofscaffolds(Fig.1).Onelargeclusteriscenteredaroundequalcoveragebetween
sexes(Log2M:F=0)andasecond,smallerclusteriscenteredaroundtwo-foldgreatercoverage
inmales(Log2M:F=1).WecanthusclearlydistinguishtheZ-linkedscaffoldsasthosewith
Log2(M:F)>0.5,withtheremainderofthescaffoldspresumedtobeautosomal.
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Onescaffold,DPSCF300028,appearedtohaveanintermediatecoverageratio,fallingat
Log2M:F≈0.7.Onelikelyexplanationforsuchanintermediatevalueisthatthescaffoldisa
chimeraofZ-linkedandautosomalsequencearisingfromanerroringenomeassembly(Martin
etal.2013).Inthisscenario,onlyaportionofthescaffoldisZ-linkedandgivesatwo-fold
differenceincoveragebetweensexes;theremainingautosomalfractionofthescaffoldyields
equalcoverage.Theresultingestimateofaveragecoveragefortheentirescaffoldthenfallsata
valuebetweenexpectationsforZorautosomalscaffolds.ThisisclearlytrueforDPSCF300028,
asrevealedbyexaminingbasepair-levelsequencingcoverageacrossthescaffold(Fig.2A).
Whileaveragemalecoverageisconsistentacrosstheentirelengthofthescaffold,female
coverageexhibitsacleartransitionbetweencoverageequaltomales(theautosomalportion)
andcoverageonehalfthatofmales(theZ-linkedportion).Indeed,therearetwosuch
transitionsinscaffoldDPSCF300028,whichweestimatetooccurat0.76Mbpand1.805Mbp,
creatingaZsegmentflankedbyautosomalsegments.
Orthologcountslinkscaffoldstochromosomes.
Asmentionedabove,Lepidopterashowaveryhighlevelofsyntenyconservedacross
substantialevolutionarydistances(Pringleetal.2007;TheHeliconiusGenomeConsortium
2012;Aholaetal.2014;Kanostetal.2016).Thusitispossibletousecountsoforthologous
genestoassignD.plexippusscaffoldstolinkagegroups(i.e.chromosomes)delineatedinother
mothorbutterflyspecies.WegeneratedpredictedorthologsbetweenD.plexippusandthree
otherreferencespecieswheregeneticlinkagemappinghasbeenusedtoassigngenomic
scaffoldstochromosomes:Melitaeacinxia(N=31),Heliconiusmelpomene(N=21),andBombyx
mori(N=28)(TheInternationalSilkwormGenomeConsortium2008;TheHeliconiusGenome
Consortium2012;Aholaetal.2014).M.cinxiaandH.melpomenearebothbutterfliesequally
divergedfromD.plexippusandallthreearepartofthesamefamily,Nymphalidae;the
silkmoth,B.mori,isdistinctlymorediverged,locatedoutsideofthesubordercontainingall
butterflies(Wahlbergetal.2009;KawaharaandBreinholt2014).
ToassignD.plexippusscaffoldstochromosomes,wetabulatedperscaffoldthecounts
ofone-to-onereferencespeciesorthologsperreferencespecieschromosome.D.plexippus
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scaffoldswerethenassignedtothereferencechromosomewiththemaximumcountof
orthologs.Forafewscaffolds,atieoccurredinmaximumorthologcountperreference
chromosome,inwhichcasethescaffoldwasremovedfromfurtheranalysis;atmostthis
occurredforonly14scaffoldsperreferencespeciesandusuallyinvolvedsmallscaffolds
harboringfewerthan5orthologs.Typically,thismethodyieldedasingleobviousreference
chromosomalassignmentforeachD.plexippusscaffold.
Thismethodofortholog-countchromosomallift-overresultedinputativechromosomal
assignmentsfor>90%ofD.plexippusgenesrelativetoeachreferencespecies(Table1,
SupplementaryTableS2).Also,atleast4500orthologousgeneswereco-localizedto
chromosomebetweenD.plexippusandeachreferencespecies.Havingseveralthousand
orthologsmappedtochromosomeinD.plexippusandareferencespeciespresentsthe
opportunitytoexaminetheextentofchromosomalrearrangementsandgenemovement
betweenthetwospecies.HereweprimarilyreportthecomparisonwithM.cinxiabecausethis
speciesisbelievedtoretaintheancestrallepidopterankaryotypeof31chromosomes(Aholaet
al.2014).Furthermore,thiscountofchromosomesisclosesttothatreportedforseveral
Danausbutterflies,includingmonarch(N=30,seeFigure3),makingitthemostappropriate
comparisonavailable(Brownetal.2004).H.melpomeneandB.moriareknowntohaveless
similarkaryotypesinvolvingseveralchromosomalfusionsrelativetoM.cinxia;nonetheless,
detailsofcomparisonstothesetwospeciesarereportedinthesupplementarycontentand
providecomparablesupportfortheprimaryfindingsreportedhere.
Figure3summarizesthecross-tabulationofchromosomallinkagefor>4500orthologs
betweenM.cinxiaandD.plexippus.Theoverwhelmingmajorityoforthologsfallonthe
diagonal,indicatingsubstantialconservationofchromosomallinkageandrelativelylittlegene
shuffling,ashasbeenreportedelsewhereforLepidoptera(TheHeliconiusGenomeConsortium
2012;Aholaetal.2014;Kanostetal.2016).Thetwomostnotableexceptionstothispattern
bothinvolvetheZchromosome(Chr1).Inonecase[McChr9,DpChr1]wecouldanticipatethis
becauseofthepreviouslyidentifiedchimericscaffold,DPSCF300028.Thisscaffoldharbors34
orthologsassignedtoMcChr1and23orthologsassignedtoMcChr9,consistentwiththe
chimericnatureofthescaffoldrevealedfrommale:femalecoverageratios(Fig2A).
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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
orthologsinthisautosomalportion,withanestimatedbreakpointat5.82Mbp,werelinked
exclusivelytoMcChr23.Therewasnotanobviousshiftinsequencingcoveragebetweensexes
toindicateamisassembledZ-autosomechimerainvolvingMcChr21.Rather,itappearedthat
nearlytheentiretyofscaffoldDPSCF300001hadtwicethecoverageinmalesthaninfemales,
consistentwithZ-linkageforregionsapparentlyhomologousbothtoMc1(Z)andMcChr21.
Aneo-ZchromosomeinD.plexippus
TheobservationthatasubstantialportionofscaffoldDPSCF300001wasZ-linkedand
homologoustoMcChr21,whileanotherlargesectionofthesamescaffoldwashomologousto
McChr1,i.e.McChrZ,ledustohypothesizethataZ-autosomefusioncouldreadilyexplainthe
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.
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ThisanalysisintersectingZ-covscaffoldswithhomologytoM.cinxiarevealedtwo
scaffoldsthatdidnotfitwiththeexpectedpatternofsequencingcoverage(Fig.5).First,
scaffoldDPSCAF300044wasassignedtoMcChr1(Z)buthadLog2M:F≈0.25,muchmorelike
otherautosomesthanotherZ-linkedchromosomes.ThisscaffoldhadsevenZ-linkedorthologs
andfourautosomal,suggestinganotherchimericscaffold.Indeed,examiningcoverageacross
thescaffoldrevealedacleartransitionincoverageaspreviouslyobservedforDPSCF300001and
DPSCF300028(Fig2B).Thusthelowmale:femalecoverageratioforthisscaffoldislikelythe
artifactofanassemblyerror.Againwewereabletopartitionthescaffoldintotwosections,
oneautosomalandoneZ-linked,withabreakpointestimatedat0.29Mbpfromthe5’end.The
autosomalsectioncontainedapproximatelyequalcountsoforthologsassignedtotwodistinct
chromosomesinM.cinxiaandtheotherreferencespecies,solinkagetoaspecificautosome
couldnotbepredicted.SupplementaryTableS3summarizesbreakpointsandpredictedscaffold
assignmentsforthethreechimericZ-autosomescaffoldsidentifiedhere.
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
TheMonarchpopulationgenomicdatasetfromZhanetal.(2014b)alsocontainedmale
andfemaleresequencingsamplesfromthreecloselyrelatedcongeners:D.gilippus,D.erippus,
andD.eresimus.Thisallowedustoassesswhetherthisneo-Zexistsintheserelatedspecies
similarlytoD.plexippus.Analyzingmaleversusfemaleresequencinginthesespeciesdoes
indeedshowthesamescaffoldshomologoustobothMcChr1andMcChr21ashavingcoverage
differencesconsistentwithaneo-Z(Fig.5).Thusitappearsthattheoriginofthisneo-Z
predatesthediversificationofthegenusDanaus.
12
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
Thecombinationofsequencingcoverageanalysisandcomparativelift-overallowedus
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
placingthefusionpointatapproximately3.88Mbpfromthe5’endofthescaffold
(SupplementaryFig.S5).Otherwise,genesandscaffoldswereassignedtochromosomesbased
directlyontheresultsofthelift-overrelativetoM.cinxia.Table2givesatabulatedsummaryof
results,whileresultsforeveryproteincodinggeneareprovidedinSupplementaryTableS4.
Cytogeneticanalysis
PreparationsofhighlypolyploidnucleiofMalpighiantubulesfromD.plexippuslarvae
wereexaminedforthepresenceofaso-called“sexchromatin”,i.e.femalespecific
heterochromatinbodyconsistingofmultiplecopiesoftheWchromosome(TrautandMarec
1996).Largemulti-lobednucleiwereobservedonbothmaleandfemalepreparations(Fig.6),
whichsuggestsahighdegreeofpolyploidyintheexaminedcells,asexpectedinMalpighian
tubules(cf.Buntrocketal.2012).Allfemalenucleicontainedasingle,highlystained
heterochromatinbody(Fig.6a).Incontrast,nosuchheterochromatinwasdetectedinmale
somaticnuclei(Fig.6b).Thisdiscrepancyconfirmsthefemale-specificityofthe
heterochromatinobservedandindicatesaWchromosomeisacomponentoftheD.plexippus
genome.
Spreadmitoticcomplementsofmalescontained2N=60chromosomes.The
chromosomesweregenerallysmallanduniform,asistypicaloflepidopterankaryotypes
(Marecetal.2009),exceptfortwodistinctlylargerelements(Fig.7a).Femalemitotic
metaphaseconsistedof2N=60elementsaswell.Infemales,however,thetwolargest
chromosomesdifferinintensityoftheirDAPIstaining.Thedeeplystainedelementfound
13
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peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
exclusivelyinfemalespresumablyrepresentstheWsexchromosomeconsistingofA-Trich
heterochromatin(Fig.2b;Kapuscinski1979).
Inbothmitoticandpachytenenuclei,GISHclearlyidentifiedtheWchromosomeby
strongbindingoffemale-derivedprobe(Fig.7c,d).ItconfirmedthattheWchromosomeisone
ofthetwoexceptionallylargechromosomesintheD.plexippuskaryotype(Fig.7c).In
pachyteneoocytes,theWZbivalentwaseasilydiscerniblebytheheterochromaticW
chromosomethread.However,aboutonethirdofthechromosomewasnotstrongly
highlightedbyDAPIandtheintensityofitsstainingwascomparabletoautosomes,indicatinga
portionoftheWthatissubstantiallyeuchromatic(Fig.7e).Accordingly,thefemale-derived
probedidnothighlighttheWchromosomehomogeneouslyasthesignalwasweakeronboth
itsendsandtheeuchromaticsegment(Fig.7e-f).Female-derivedprobesinGISHalsostrongly
labeledoneinterstitialandafewterminalregionsofsomeautosomes,whichmostlikely
containclustersofrepetitivesequences(Fig.7d).
Comparativegenomichybridizationwithbothmale-andfemale-derivedprobeswasalso
usedtoassessthebroadmolecularcompositionoftheD.plexippusWchromosome(Fig.7h-i).
Hybridizationsignaloffemale-derivedprobelabeledbyfluoresceinwaslargelyconsistentwith
theresultsobtainedbyGISH.ThesignalhighlightednearlytheentireWchromosomethread,
withtheexceptionofitsterminiandeuchromaticsegment,inwhichtheprobedetectedonlya
smallinterstitialblock(Fig.7i,k,l).Themale-derivedprobelabeledbyCy3providedrelatively
weakhybridizationsignal,whichwasscatteredalongtheWchromosome.Thismaleprobe
highlightedonlytworegions:theWchromosomeendoppositetotheeuchromaticsegment
andtheregionhighlightedwithintheeuchromatinbyfemale-derivedprobe(Fig.7j,k,l).Both
probesdetectedthesameautosomalregionsasGISH(datanotshown).
Discussion
Usingacombinationofgenomicresequencing,comparativegenomics,andcytogenetic
analysis,wehavedocumentedthepresenceofaneo-ZchromosomeinDanausbutterflies,
alongwithwhatislikelyanaccompanyingneo-Wchromosome.Thisdiscoveryofneo-sex
chromosomesinDanausbutterfliesandourdiscriminationofgenesfallingontheancestral
14
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.
versusrecentlyautosomalportionsoftheZarefundamentalobservationsthatprovidethe
foundationforahostoffutureinferences.Theseresultscreatenovelopportunitiestoaddress
ratesofmolecularevolution,theevolutionofdosagecompensation,thepatternofallosome
divergence,andmanyotherimportantquestionsinsexchromosomebiology,allinafemaleheterogameticspeciesthatisalsoanemerginggenomicmodelsystem.
InanalyzingpatternsofchromosomalfusioninH.melpomeneandB.morirelativetoM.
cinxia,Aholaetal.(2014)reportasignificanttendencyforalimitedsetofancestral
chromosomes–particularlythesmallestones–tobeinvolvedinchromosomalfusionevents.
NeithertheancestralZnorMcChr21areamongthesesmall,repeatedlyfusedchromosomes;
thusthechromosomalfusionreportedheredoesnotfitneatlywiththispattern.Nonetheless,
HmChr2(homologoustoMcChr21)isthesecondsmallestchromosomethatremainsunfused
betweentheselineages(Daveyetal.2016).SoitisalsodifficulttoarguestronglythatthisZautosomefusioninDanausisastrikingcontrasttothetrendofchromosomalfusionsinvolving
smallchromosomes.
Motivatedbythebioinformaticdiscoveryofaneo-Zchromosome,weperformed
cytogeneticanalysisoftheD.plexippuskaryotypeinordertoprovidefurtherinsightinto
evolutionandmolecularcompositionofthemonarchsexchromosomes.Previously,an
observationofN=30chromosomeswasreportedonlyformales(Nageswara-RaoandMurty
1975).Ourcurrentanalysisconfirmsthesamechromosomenumbernotonlyinmalesbutalso
infemales(Fig.7a,b).Equalnumbersofchromosomesinmalesandfemales,alongwith
presenceofsexchromatininfemales,indicatesthatasingleWchromosomepersistsinthis
speciesalongsidetheneo-Z.Furthermore,detailedanalysisofmitoticcomplementsrevealeda
largechromosomepair(Fig.7a,b)andGISHclearlyidentifiedonechromosomeofthepairas
theWchromosome(Fig.7c).Asimilar,extraordinarilylargechromosomepairwasrecently
showntocorrespondtoneo-sexchromosomesinleafrollermothsofthefamilyTortricidae
(Nguyenetal.2013;Šíchováetal.2013).
GISHrepresentsasimplifiedversionofCGH,whichhasbeensuccessfullyusedfor
evaluatingthegrossmolecularcompositionoflepidopteranWchromosomes(e.g.Mediouniet
al.2004;Fukováetal.2005).Previousstudiesinseveralmothspeciescontrastedfluorescence
15
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peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
intensitiesofmaleversusfemalederivedprobesandidentifiedtwocommontypesofrepeats
onlepidopteranWchromosomes:(i)repetitivesequencescommontobothmalesandfemales,
i.e.presentinautosomesandZchromosome;and(ii)repetitivesequencesexclusivelyor
predominantlypresentinfemales(Saharaetal.2003).Inthesepreviouslystudiedspecies,the
Wprimarilycontainsthefirsttype,i.e.ubiquitousrepeats(e.g.Fukováetal.2005;Šíchováetal.
2013).Incontrast,themonarchWappearsdistinctfromtheWchromosomesoftheseother
speciesbecausethemajorityofthemonarchWchromosomeoverwhelminglycontainsrepeats
ofthesecondtype,i.e.female-limitedrepeats.ThemonarchWwaslabelledprimarilyby
female-derivedprobe(7i-k),indicatingthatitisprimarilycomprisedofrepetitivesequences
eitherspecifictoorgreatlyenrichedontheWchromosome.OnlytwosmallsegmentsoftheW
showednotablyhighdensitiesofubiquitousrepeatscommonlyenrichedontheentireWin
otherlepidopteranspecies.
ThisdiscrepancybetweenMonarchandotherspeciescouldberelatedtotherelatively
smallsizeoftheD.plexippusgenome.Themonarchbutterflyrepresentsthesmallest
lepidopterangenomeyetsequenced,withhaploidnuclearcontentofmale284Mbp(see
Dolezeletal.2003fortheconversionofpgofDNAtoMbp;GregoryandHebert2003)and
female273Mbp(Zhanetal.2011).Thissmallgenomeispresumablydepletedofrepetitive
sequencesfoundmoreubiquitouslyamongtheautosomesandZinotherlepidoptera.Indeed,
repeatcontentconstitutesonly13.1%oftheD.plexippusgenomeassembly(Zhanetal.2011).
TheresultsofCGHthuscouldreflectthefactthattheWchromosomerepresentsthelast
refugeformanysuchrepetitivesequencesinthemonarchgenomeafterotherwisebeing
purgedfromthegenome.
CytogeneticanalysesthusconfirmthattheD.plexippusWchromosomeiswell
differentiatedrelativetotheZ,whichisinagreementwithsequencingdatathatyieldavery
consistent2:1coverageratioonscaffoldregionscorrespondingtoMcChr21.Ifaneo-W
retainedsubstantiallyclosehomologytotheneo-Z,wewouldexpectmanysequencingreads
emanatingfromtheneo-Wtoaligntotheneo-Z,andshiftthiscoverageratiotowardsone.This
evidentlydoesnotoccur,indicatingsubstantialdivergencebetweentheneo-Zandanyneo-W
sequencethatisretained.
16
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.
CytogeneticanalysesfurtherindicatethatthemonarchWchromosomeexhibitsnotable
compositionalheterogeneity.BothGISHandCGHrevealedterminalgapsinfemale-derived
signal(Fig.2g,l).ThisissimilartoGISHresultsobtainedinacodlingmoth,Cydiapomonella
(Tortricidae),wherefemalederivedprobelabelledtheentireWchromosomeexceptforboth
subtelomericsegments(Fukováetal.2005).Van’tHofetal.(2012)proposedthatacopyofthe
Z-linkedlamininAgenewastransferredandmaintainedtoaWchromosomeofthepeppered
moth,Bistonbetularia(Geometridae),bygeneconversionresultingfromectopicrecombination
betweenrepeatslocalizedinterminalchromosomeregions.Thesamemechanismcouldbe
invokedtoexplainthelackoffemalespecificsignalsonthemonarchWchromosomeends.
AnotherregiondistinctlyidentifiedbyCGHcorrespondstoaninterstitialblocklocalized
withinaeuchromaticchromosomesegment.Theblockwasilluminatedbybothfemale-and
male-derivedprobes(Fig.2j,k,l),whichsuggestspresenceofrepetitivesequencescommonto
autosomesandZchromosome(Saharaetal.2003).Thisblock,togetherwiththeadjacent
terminalregion,formsachromosomesegmentwithdistinctmolecularcompositioncomprising
aboutonethirdoftheWchromosome.ThemonarchWthusshowsabipartiteorganization,
withonlytwo-thirdsofthechromosomebeinghighlyheterochromaticwhiletheremaining
thirdappearseuchromatic.AlsonoteworthyisthelargesizeofthemonarchWchromosome
relativetochromosomesotherthantheZ,whichisofcomparablylargesize.Thiscombination
ofsizeandbipartiteorganizationsuggeststhischromosomemaybeaneo-Wresultingfroma
W-autosomefusionoccurringinparallelwiththeneo-Zformation.However,cautionisadvised
inthisinterpretationbecauseWchromosomesizecanbemisleading(Schartletal.2016)and
cytogeneticexaminationofspeciesbearingneo-Zchromosomesrevealedconsiderable
differencesinthestructureoftheirWchromosomes(Šíchováetal.2013).
Finally,itshouldbenotedthatarelativelymodernW-autosomefusionwasrecently
reportedtobesegregatingintheAfricanQueenbutterfly,D.chrysippus,whereitcontrolscolor
patternandmale-killingandisdrivingpopulationdivergenceacrossahybridzone(Smithetal.
2016).GivenourresultsandthesamechromosomenumberN=30observedinthewild-typeD.
chrysippusdorippus,itseemsallDanausspeciesincludingD.chrysippussharetheneo-Zand
thuspresumablythisputativeneo-W(i.e.homologoustoMcChr21).Ifso,thismeanstheW-
17
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.
autosomefusioninD.chrysippuswouldbeacompoundneo-Winvolvingtwodistinctformer
autosomes.Thispatternofrelativelyfrequentkaryotypicchangeswithinthegenusfurther
recommendsDanausbutterfliesasanexcellentmodelsystemforstudyingsexchromosome
evolution.
Conclusion
Wehaveusedacombinationofgenomesequencingcoverage,comparativegenomic
analysis,andcytogeneticstodemonstratethatDanausbutterfliesharboraneo-Zchromosome
resultingfromthefusionoftheancestralZchromosomeandanautosomehomologousto
Chr21inM.cinxia.Also,atleastinthecaseofMonarchbutterflies,itappearsthatthisfusion
hasresultedinalargeneo-Wchromosomewithaprominenteuchromaticregion.Ouranalysis
alsoidentifiedandresolvedseveralZ-autosomechimericscaffoldsinthemostrecentassembly
oftheD.plexippusgenome.Thisdiscoveryandprovisionalassignmentofchromosomallinkage
for>90%ofD.plexippusgenespavesthewayformyriadanddiverseinvestigationsintosex
chromosomeevolution,whicharelikelytobeofdistinctimportancegiventheincreasing
prominenceofDanausbutterfliesasafemale-heterogameticmodelspeciesforfunctionaland
evolutionarygenomics.
Acknowledgements
ThankstoChipTaylor,AnnRyan,andtherestofMonarchWatch.orgfordonationoforganisms
usedinthisstudy.JimMalletandJohnDaveyprovidedhelpfulcommentsonthiswork.This
researchwassupportedbyNSF-DEB1457758toJ.R.W.Cytogeneticanalysiswassupportedby
theCzechScienceFoundationgrant14-35819PtoP.N.Thecomputingforthisprojectwas
performedontheCommunityClusterattheCenterforResearchComputingattheUniversityof
Kansas.
Authorcontributions:AJM,PN,AV,andJRWallperformedanalysesandcontributedtothe
manuscripttext.AJMadditionallyassembledanalysesandmanuscriptcomponentsandedited
thecompleteversion.Allauthorsreadandapprovedthefinalmanuscript.
18
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.
CompetingInterests:Theauthorsdeclarenocompetinginterests.
Additionaldatafiles:
Monarch_neoZ_Gene-Scaff.xlsxcontainsasummaryofZ-autosomechimericscaffold
breakpoints(TableS3)andthepredictedchromosomallinkage,wheredetermined,forallD.
plexippusproteincodinggenes(TableS4).
19
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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23
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.
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
24
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:femalegenomicsequencingcoverageratios
forD.plexippusversion3assemblyscaffolds.Onlyscaffoldsoflengthequaltoorgreaterthan
theN90scaffoldareshown.Thedottedlineat0.5representsthevalueusedtopartition
scaffoldsasautosomal(grey)orZ-linked(red).
Figure2.Normalizedmaleandfemalecoveragealongthelengthofchimericscaffolds,for(A)
DPSCF300028,(B)DPSCF300044,and(C)DPSCF300001.Coveragesareplottedassliding
windows(width=5Kbp,step=1Kbp)ofmedianbasepairvalues.Theassociatedmale:female
ratioofcoverageforeachwindowisplottedasaredlinebelowthepairofsex-specificplots.
AsterisksindicatetheestimatedbreakpointbetweenZlinkedandautosomalsegmentsofeach
scaffold,asdeterminedbythemaximumdifferenceinadjacent,non-overlappingwindowsof
male:femaleratio(seemethodsfordetails).
Figure3.Chromosomalco-linkagebetweenD.plexippusandM.cinxiaforpredicted
orthologousproteins.
Figure4.Ratiosofmale:femalemediannormalizedgenomicsequencingcoverageplotted
againstscaffoldlength.Scaffoldshomologousvia“liftover”proceduretoM.cinxia
chromosomes1/Z(blue)and21(green)areplottedindistinctcolors.Dottedlinesindicate
expectedvaluesforZ-linked(red)andautosomal(black)scaffolds.
Figure5.Ratiosofmale:femalemediannormalizedgenomicsequencingcoverageplottedby
scaffoldlengthforfourspeciesofDanausbutterflies.Thedottedlineatlog2(M/F)=0.5
representsthethresholdusedtodiscernautosomal(<0.5)fromZ-linked(>0.5)scaffolds.
Figure6.Multi-lobed,highlypolyploidnucleioftheMalpighiantubulesfromDanausplexippus
larvaestainedbyorcein.(a)Afemalenucleuswithasingledeeplystainedsexchromatinbody
(arrow).(b)Amalepolyploidnucleuswithnoheterochromatin.Bars=20µm.
Figure7.CytogeneticanalysisofsexchromosomesinD.plexippus.Chromosomeswere
counterstainedbyDAPI(blue).(a)Malemitoticmetaphasecomplementconsistingof2N=60
elements.Notethelargechromosomepair(arrowheads).(b)Femalemitoticmetaphase
nucleuscomprising2N=60chromosomes.Thelargestelementsdifferinintensityoftheir
staining(arrowheadandarrow,thelattermarkinghighlystainedchromosome).(c-g)Female
complementsexaminedbygenomicinsituhybridization(GISH).Femalederivedprobewas
labeledbyCy3(red).(c)Thesamemitoticcomplementasin(b).TheprobeidentifiedtheDAPI
positivechromosomeasaWchromosomeandthusindicatedtheotherlargechromosometo
betheZchromosome.(d)Femalepachytenecomplementconsistingof30bivalents.TheWZ
bivalentwasclearlyrecognizedbyfemale-derivedprobe,whichhighlightedwholeW
chromosomeexceptitsterminalsegments.Theprobealsomarkedthreeterminalandone
interstitialregionsofseveralautosomes(asterisks).(e)TheWZbivalentstainedbyDAPI.Note
thatonlyabouttwothirdsoftheWchromosomethreadisdeeplystainedandapparently
25
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.
heterochromatic.Themorelightlystainedeuchromaticsegmentismarkedbyanarrow.(f)
Hybridizationsignaloffemale-derivedprobehighlightingtheWchromosomethread.Notethat
thesignalisweakerintheeuchromaticregion(arrow).(g)CompositeimageofDAPIandthe
probe.(h-l)ApachyteneWZbivalentprobedbycomparativegenomichybridization(CGH).
Male-derivedprobewaslabeledbyCy3(red),female-derivedprobebyfluorescein(green).(h)
TheWZbivalentstainedbyDAPI.TheweaklystainedZiswrappedaroundthestronglystained
W,whichisfoldedinhalf.(i)Hybridizationsignaloffemale-derivedprobe.Notethattheprobe
labeledonlyasmallregionoftheeuchromaticsegment(arrow).(j)HybridizationsignalofmalederivedprobestronglyhighlightstwoWsegments(arrows).(k)Boththefemale-andmalederivedsignalsmerged.(l)CompositeimageofDAPIandbothprobes.Bars=5µmin(a-g)and
2.5µmin(h);(e-g)and(h-l)havethesamescale.
26
80
40
0
20
Frequency
60
Putative Autosomal scafffolds
Putative Z−linked scaffolds
−0.5
Figure 1
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
●
●
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9
●
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●
●
●
●
●
●
●
●
●
●
●
●
●
11
●
●
●
15
17
19
M. cinxia chromosome
●
●
●
●
●
●
●
●
13
●
●
●
●
10
●
●
●
●
●
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●
100
●
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7
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500
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5
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Number
of genes
●
●
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●
3
●
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1
Figure 2
●
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]
Figure 3
4
4.5
5
5.5
6
M. cinxia reference chromosome
●
1
●
21
●
Other
●
●
● ●
●
●
●
●●
0.0
0.5
●
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● ● ● ●●●
● ● ● ● ● ●● ●● ●
●
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● ●
● ●●●
● ●●
●
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● ● ●●
● ●●
●
●
●
−0.5
Log2[ Male:Female coverage ]
1.0
●
●
5.5
Figure 4
6.0
Log10[ Scaffold Length (bp) ]
6.5
7.0
1.0
0.5
plexippus
erippus
eresimus
gilippus
−0.5
0.0
●
0
1
2
3
4
5
6
Scaffold length (Mb)
D. plexippus
D. erippus
D. gilippus
D. eresimus
●●
●
●
1.0
1.0
1.0
●●
● ●
1.0
●
●
●
0
1
2
3
4
Scaffold length (Mb)
5
6
0
1
2
3
4
Scaffold length (Mb)
5
6
0.5
−0.5
0.0
Log2(M/F)
0.5
−0.5
0.0
Log2(M/F)
0.5
0.0
●
−0.5
0.0
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Log2(M/F)
0.5
●
−0.5
Log2(M/F)
Log2(M/F)
Danaus spp. median depth of coverage by scaffold
Figure 5
0
1
2
3
4
Scaffold length (Mb)
5
6
0
1
2
3
4
Scaffold length (Mb)
5
6
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.
Figure 6
Figure 7
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.
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
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
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
●
●
●
●
●
●
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17
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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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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
●
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7
100
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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
●
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
6.5
7.0
Log10[ Scaffold Length (bp) ]
H. melpomene reference chromosome
●
2
●
Z
●
Other
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Log2[ Male:Female coverage ]
1.0
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5.5
6.0
Log10[ Scaffold Length (bp) ]
6.5
7.0
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).
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
0.15
0.175
0.2
Scaffold Position (Mbp)
mcov.smooth[, 2]
FigureS3.NormalizedmaleandfemalecoveragealongthelengthDPSCF300403.Coveragesareplottedas
slidingwindows(width=5Kbp,step=1Kbp)ofmedianbasepairvalues.Theassociatedmale:femaleratioof
coverageforeachwindowisplottedasaredlinebelowthepairofsex-specificplots.
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