DraftgenomesequenceoftheAntarcticgreenspUWO241 - PDF document

iScience DraftgenomesequenceoftheAntarcticgreensp.UWO241 XiZhang,MarinaCvetkovska,RachaelMorgan-Kiss,NormanP.A.ner,DavidRoySmithnhuner@uwo.ca(N.P.A.H.)dsmit242@uwo.ca(D.R.S.) HIGHLIGHTS ChlamydomonasUWO241isagreenalgaoriginatingfromLakeBonney,AntarcticaWepresentadraftnucleargenomesequenceofUWO241(212Mb).TheUWOgenome OPENACCESS iScience ArticleDraftgenomesequenceoftheAntarcticgreenalgasp.UWO241XiZhang,MarinaCvetkovska,RachaelMorgan-Kiss,NormanP.A.HuandDavidRoySmithAntarcticaishometoanassortmentofpsychrophilicalgae,whichhaveevolvedvarioussurvivalstrategiesforcopingwiththeirfrigidenvironments.Here,weexploreAntarcticpsychrophilybyexaminingthe212MbdraftnucleargenomeofthegreenalgaChlamydomonassp.UWO241,whichresideswithinthewatercolumnofaperenniallyice-covered,hypersalinelake.LikecertainotherAntarcticalgae,UWO241encodesalargenumber(37)ofice-bindingproteins,putativelyoriginatingfromhorizontalgenetransfer.Evenmorestrik-ing,UWO241harborshundredsofhighlysimilarduplicatedgenesinvolvedin DepartmentofBiology,University

ofWesternOntario,London,ONN6A5B7,CanadaDepartmentofBiology,UniversityofOttawa,Ottawa,ONK1N6N5,CanadaDepartmentofMicrobiology,MiamiUniversity,Oxford,OH45056,Leadcontact*Correspondence:nhuner@uwo.cadsmit242@uwo.cahttps://doi.org/10.1016/j.isci. ,102084,February19,20212021TheAuthor(s). OPENACCESS RESULTSANDDISCUSSIONDraftnucleargenomesequenceofanalgafromanAntarcticlakeThehaploidnucleargenomeofUWO241wasassembleddenovousingacombinationoflong-readPacBio16.5Gb)andshort-readIllumina(40Gb)data,resultingin2,458scaffolds(N50=375.9kb)withanaccu-mulativelengthof211.6Mb(%GC=60.6)(Figures1Dand).Thislengthisconsistentwithowcytometry-merspectralanalysisofUWO241,whichpredictedanoverallgenomesizeof230Mb(FigureS1).Intotal,16,325protein-codinggeneswereannotated(allsupportedbytranscriptomicdata),capturingoftheChlorophyteBenchmarkingUniversalSingle-CopyOrthologsdatasets(FigureS1),indicatingahighlevelofgeneregioncompleteness.TheUWO241genomeisrichinfunctionalRNAs(630tRNAsand480rRNAs)aswellasnoncodingDNA(87%),havingthehighestaver

ageintrondensityyetobservedfromagreenalga(10introns/gene;avg.intronlength0.9kb).Theintergenicregionsaboundwithrepeats,ac-countingfor104Mb(49%)ofthetotalassemblylength,70Mbofwhicharerepresentedbytranspos-ableelements(TEs)(discussedbelow).Thepastdecadehasbroughtdraftnucleargenomesfor�25differentgreenalgalspecies,withespeciallystrongsamplingfromtheorderChlamydomonadales(Chlorophyceae)(Figure1D).TheUWO241genomeisthesecondtobesequencedfromtheMoewusiniacladeoftheChlamydomonadales(Nakadaetal.,2008),theothercomingfromtheacidophilicspeciesChlamydomonaseustigmaHirookaetal.,2017TheMoewusiniaiscloselyafliatedwiththeMonadinia,thecladetowhichtheAntarcticpsychrophilesICE-LandICE-MDVbelong(Figures1Aand1D)(Demchenkoetal.,2012).KeepinmindthattheChlamydomonasgenusispolyphyleticandthatUWO241,C.eustigma,andICE-LbranchclosertoDunaliellasalinaFigure1D),forexample,thantheydotoC.reinhardtii,whichhailsfromtheRein-hardtiniaclade(Possmayeretal.,2016Zhangetal.,2020).WhatimmediatelystandsoutfortheUWO241 DCB Figure1.Chlamydomon

assp.UWO241 (A)OriginsofisolationofUWO241andChlamydomonassp.ICE-MDV(LakeBonney),aswellasChlamydomonassp.ICE-L(seaiceoffofZhongshanStation);imagefromNASAEarthObservatory.(B)PhotographofLakeBonney(Wikimedia-Commons,2020(C)SimplieddiagramshowingenvironmentalconditionsinLakeBonney.(D)Treeofvariouschlamydomonadaleanalgaeandtheirnucleargenomestatistics;branchingorderbasedonpreviousphylogeneticanalyses(Nakadaetal.,2008Possmayeretal.,2016Zhangetal.,2020 OPENACCESS,102084,February19,2021 iScienc genomeascomparedtootheravailablegreenalgalnuclearDNAs(nucDNAs)isitsrelativelylargesize(approximatelytwicethatofC.reinhardtii),record-settingintrondensity,andhighrepeatcontent,outdoneonlybythatofICE-L(64%repeats)(Zhangetal.,2020).However,closeinspectionoftheUWO241codingregionsuncoveredsomethingmuchmoreunique:widespreadgeneduplicationtoadegreeunmatchedinanychlorophytestudiedtodate.HundredsofgeneduplicatesFunctionalannotationofthe16,325RNA-supportedgenemodelsrevealedthestandardcohortofproteinstypicallyencodedingreenalgalnucl

eargenomes(DataS1),aswellasmanyhypotheticalproteins(21.8%),parallelingthetrendsfromotheravailablechlamydomonadaleannucleargenesets,whicharegenerally20-30%hypothetical.Therewerenoobvioussignsofcontaminationintheassemblyorannotationsand,withoneconspicuousexception(discussedbelow),littleevidenceofhorizontalgenetransfer(HGT).Whenexaminingtheannotationsindetail,itbecameobviousthatmanygeneswererepresentedtwoormoretimeswithintheassembly.Toexplorethevalidityofthesemulti-copygenes,weperformedaseriesofBLAST(BasicLocalAlignmentSearchTool)-basedanalyseswithstrictdownstreamltering.AproteinBLASToftheUWO241genemodelsagainstthemselves(E-valuecut-off10)detected901pu-tativeduplicates(encompassing2,012genecopies)allwithpairwiseaminoacididentities80%.Welteredthisgenesettoonlythosewithnear-identicalproteinlengths(within10aminoacids)andpairwiseidentities,givingapared-downlistof336highlysimilarduplicates(HSDs),totaling1,339genecopies(Table1DataS2).Bysettingsuchastrictcutoff,wehaveundoubtedlyremovedsomegenuinedu-plicatesfromth

islist,butwewouldratherbeconservativeinourapproach,ensuringthatthegenepairsinquestionarebonadeduplicatesratherthanspuriousones.TheproteinsequencesoftheHSDsweresearchedagainsttheKEGG(KyotoEncyclopediaofGenesandGenomes)andPfamdatabases,providingafunctionalbreakdown(Table1DataS2).HSDsinUWO241areinvolvedinvariouscellularpathways,includinggeneexpression,cellgrowth,membranetransport,andenergymetabolism,butalsoincludehy-potheticalproteins(37%)andreversetranscriptases(11%)(Table1DataS2).HSDsforproteintranslation,DNApackaging,andphotosynthesiswereparticularlyprevalent,with19duplicationsofgenesforribo-somalproteins,10forhistones,andatleast4forproteinsofthechlorophylla/bbindinglight-harvestingcomplex(Table1Figures2A–2C).Aswiththepreviouslydescribedduplication(Cvetkovskaetal.,2018),manyoftheseHSDsarevirtuallyindistinguishablefromeachotherattheaminoacidleveland65areidenticalacrosstheirnucleotidecodingregions(DataS2ThearrangementsoftheHSDsareinformative.Approximately,20%containgenecopiesthataresituatedclosetooneanothe

r(ofteninahead-to-headorhead-to-tailorientation)andhaveverysimilarintron Table1.Summarystatisticsofhighlysimilarduplicategenes(HSDs)inUWO241. DatabaseExampleidentiÞersNumberofHSDs(%)Numberofgenecopies(%)ChlorophyllA-BbindingproteinPF005044(1%)25(2%)RibosomalproteinPF01015;PF01775;PF0082819(5%)42(3%)CorehistoneH2A/H2B/H3/H4PF001255(1%)99(7%)Ice-bindingprotein(DUF3494)PF119998(2%)21(2%)ReversetranscriptasesPF0007838(11%)151(11%)KEGG09,101CarbohydratemetabolismK13979(alcoholdehydrogenase)12(4%)89(7%)09,102EnergymetabolismK02639(ferredoxin);K08913(light-harvestingcomplexIIchlorophylla/bbindingprotein2)10(3%)51(4%)09,103LipidmetabolismK01054(acylglycerollipase)3(1%)15(1%)09,122TranslationK02868(largesubunitribosomalproteinL11e)27(8%)47(4%)HypotheticalproteinsNA125(37%)357(27%) Notallidentiersarelisted.Atotalof336HSDswereidentiedwithintheUWO241genome,encompassing1,339genecopies.HSDsshare90%pairwiseaminoacididentityandhavelengthswithin10aminoacidsofeachother. OPENACCESS,102084,February19,2021 iScienc numbersan

dintronicsequences(basedonpairwisealignments),implyingthattheyresultfromrecenttan-demduplicationevents(Figure2DataS2).Aclearexampleofthisistheduplicationofthelhcb2geneFigure2A).TheremainingHSDsaregenerallyfarapart(mostondistinctscaffolds)and,despitetheirmatchingcodingregions,many(50%)haveun-alignableintronicsequencesanddifferingnumbersofin-trons,suggestingthattheyderivefrommoreancientduplicationevents(Figures2Band2C;DataS2).ThisisthecaseforCvetkovskaetal.,2018),aswellasforhspa5(encodingheatshock70-kDaprotein5),thetwocopiesofwhicharefoundinthemiddleofdistinctscaffoldsandshare93%codingsequenceidentitybut25%nucleotidesimilarityacrosstheirintrons(Figures2Band2C).Whatevertheirarrangement,theexonicsequencesofmorethanhalfoftheHSDs(190)areunderstrongpurifyingselectionasevidencedbylow(1)nonsynonymoustosynonymoussubstitutionrates(dN/dS),rangingfrom0to0.5(avg.=0.2)FigureS2).ItispossiblethatthestrongcodingsequencepreservationoftheseduplicatescouldbeaidingthesurvivalofUWO241inLakeBonney,perhapsduetoincreasedgenedosage

(InnanandKondrashov,2010Kondrashov,2012),aspreviouslysuggested(Cvetkovskaetal.,2018).Butvariousnon-adaptiveexpla-nationsarealsoplausible.TheHSDs,however,representonlyafractionofduplicatedregionswithinthegenome.AgenomeinupheavalTheUWO241nucDNAcontainsthousandsofpartialduplicates,characterizedbygenefragmentsandpseudogenes,aswellasduplicatedsegmentsofintergenicandintronicDNA(FigureS3DataS3).Theseincompleteduplicates,whichrangeinsizefrom100-12,000bp,canexistinhighcopynumbers.70;(6)and,liketheHSDs,canbefoundintandemorondifferentscaffolds(FigureS3DataS3).ButunliketheHSDs,theyareinvariousstatesofdecay,possiblyreectinganongoingbirth-deathprocess,whichissupported C Figure2.ExamplesofduplicategenesinChlamydomonassp.UWO241 (A)Fourdistinctcopiesoflhcb2,alllocatedonscaffoldscf7180000014917.(B)Twodistinctcopiesof,locatedonscaffoldsscf7180000011611(hspa5-1)andscf7180000015050(hspa5(C)Pairwisealignmentofthededucedaminoacidsequencesofhspa5-1andhspa5 OPENACCESS,102084,February19,2021 iScienc bythefactthatmanyofthecomp

leteandpartialduplicatesaredirectlyassociatedwithoroccurneartoretrotransposons(RTs)(FigureS3DataS3),asoutlinedfortheduplicationoflhcb2Figure2RT-mediatedgeneduplicationisarecurringthemewithinnucleargenomes(QianandZhang,2014Panchyetal.,2016CasolaandBetran,2017KubiakandMakaowska,2017),includingthoseofgreenalgae(kalskietal.,2016),andtheUWO241genomecontainsthestandardhallmarksofsuchaphenomenon,suchaspoly-(A)tailinsertionsandtargetsiteduplications(FigureS3).Butthiscertainlydoesnotruleoutthepossibilitythatotherprocesses,suchasunequalcrossing-over(Zhang,2003),arecontributingtogeneduplicationwithinUWO241.Donotethat83%oftheHSDscontainintrons,acharacteristicnotgenerallyassociatedwithRT-medi-atedduplications,butnotunprecedented(CasolaandBetran,2017KubiakandMakaowska,2017).Retrocopiesofteninheritintronsfromparentalgenes,ankinggenomicDNA,orthefusionoftranscripts(CataniaandLynch,2008Zhuetal.,2009Szczesniaketal.,2011Kangetal.,2012Zhangetal.,2014).Altogether,weidentied401putativelyfunctionalRTsinthenucDNA,including77l

ongterminalrepeat(LTR)and324non-LTRRTs.Thesenumbersarelikelyunderestimatesofthetruetotalastheydonotincluderetropseudogenes,partialretroele-ments,oridentiedRTswithnoRNA-seqsupport,whichtogetheraccountfor�10%oftheassembly.What’smore,thereare�480duplicatedregionscontainingareverse-transcriptasedomain,includingonesinnoncod-ingDNA.UWO241hasmoreretroelementsthanallothersurveyedchlorophytes(4timesthatofC.reinhardtiiwiththeexceptionofICE-L,forwhichnon-LTRRTsaccountforastaggering23%ofthegenome(Zhangetal.,2020).InadditiontoRTs,theUWO241andICE-Lgenomesshareanotheratypicalfeature—genesforIBPs.Horizontallyacquiredandduplicatedice-bindingproteinsTheUWO241genomeencodesnofewerthan37proteinswithanice-bindingdomain(DUF3494)(Figure3whichisamongthelargestnumberofIBPseverrecordedinaphotosyntheticprotist.ThiswealthofIBPsappearstobetheconsequenceofHGTeventsincombinationwithgeneduplication.PhylogeneticanalysesoftheIBPgenes,whichrangeinsizefrom483-37,549bp,showtheirsimilaritytotypeIbacterialandarchaealIBPs(Fig-ure3

B),whichisconsistentwithpreviouswork(RaymondandMorgan-Kiss,2013).NucleargenesacquiredviarecentHGTeventsfrombacteriausuallylackintrons(KeelingandPalmer,2008),asdo14oftheIBPgenesfromUWO241;theremaininggenes,with4exceptions,allhaveasingle,shortintronattheir3’ends.ThelargestIBPgene,however,contains29introns.TheIBPgenesshowvaryingdegreesofsimilaritywitheachother(Fig-ure3C),including8groupingsofalmostidenticalgenes,suggestingacomplicatedhistoryofIBPgeneacqui-sitionandduplicationwithinUWO241.ThepresenceofpseudogenesandgenefragmentswithsimilaritytoIBPs(DataS3)indicatesthatsomepreviouslyfunctionalIBPcodingregionsmighthavebeenlost.ThesendingsaddtothegrowinglistofpsychrophilicandpsychrotolerantalgaeencodingIBPs(Blancetal.,2012RaymondandMorgan-Kiss,2013Mocketal.,2017),mirroringthepatternofice-associatedbac-teriaandfungi(Margesinetal.,2008).GenomesequencingofthepolardiatomFragilariopsiscylindrusiden-tied11IBPs(Mocketal.,2017),almostasmanyasfoundinICE-L(12)(Zhangetal.,2020).TheIBPsofUWO241andICE-Lshowasurprisingdegre

eofsimilaritywitheachotherasevidencedinthephylogeneticanalysis(Figure3B),especiallygiventhatthesetwoalgaewereisolatedfromlocationsthataremorethan2500kmapart(Figure1A).Chlamydomonassp.ICE-MDV,acloserelativeofICE-LandaresidentofLakeBonney(Figures1A,1C,and1D),currentlyholdstherecordforthegreatestnumberofIBPisoforms(50)inagreenalga(RaymondandMorgan-Kiss,2017).Inalltheseexamples,theIBPsarebelievedtohavebeenacquiredfrombacteriaviaHGT,andtheirexistenceisthoughttobeanadaptationtopolarenviron-ments(RaymondandKim,2012).ItmightseemobviouswhyaspeciesthatlivesintheAntarcticwouldacquireIBPs,whichcanhaveicerecrystallizationinhibitionactivitiesand,thus,protectcellsfromfreezingdamage(Davies,2014).However,thepotentialbenetsbestoweduponUWO241andICE-MDVbyhavingthesegenesisnotimmediatelyclear.UnlikeICE-L,UWO241doesnotliveoniceorsnow(Morgan-Kissetal.,2006)butdeepwithinlakewater,whichremainsatCyear-round(thisisalsotrueforICE-MDV).WedonotknowtheevolutionaryhistoryofUWO241orhowlongithasbeenisolatedinLakeBonney,meaningthepresence

ofIBPscouldbearemnantofanancestrallifestyleinvolvingcloseassociationwithiceandsnow.Genomeevolutioninapermanentlyice-coveredAntarcticlakeOnemustbemindfulnottoinstantlyinvokepositiveselectionwhentryingtoexplaintheevolutionofgenomicarchitecture(Lynch,2007BrunetandDoolittle,2018).ItistemptingtoproposethatpervasivegeneduplicationwithintheUWO241genomeisanadaptationtolifeinLakeBonney.Butonecouldalsoreasonthatthesefeaturesareneutral(orslightlydeleterious)outcomesofrandomgeneticevents,suchasthewhimsofselshelements.Aswithmanyaspectsofmolecularevolution,thetruthlikelyfallssomewherein-betweenthesetwoextremes. OPENACCESS,102084,February19,2021 iScienc ItisourbeliefthattheunderlyingmechanismsbehindtheduplicationswithintheUWO241nucDNA,beitretro-transpositionand/orotherprocesses,areneutralorevenmaladaptive.Likewise,wecontendthatmostoftheobservedduplicatesinthegenome,suchasthoseencodingreversetranscriptases,werexedthroughrandomgeneticdrift,perhapsexacerbatedbythehermeticenvironmentofLakeBonney.(Unfortunately,therearenod

ataontheeffectivepopulationsizeofUWO241andhowitcomparestothatofothergreenalgae,butchlor-ophytesappeartoberelativelyrareinAntarcticlakeautotrophiccommunities(Dolhietal.,2015)).Butifenoughduplicatesaregenerated,itstandstoreasonthateventuallyonewillariseresultinginanincreaseintness.Forinstance,ifanincreaseindosageofaparticulargeneisbenecial,thentheduplicationofthisgenecouldbexed(oratleastmaintainedafterthefact)bypositiveselection(InnanandKondrashov,2010Kondrashov,2012).Thisisarguablythebestexplanationfortheexistenceofthepetfduplicates(Cvetkovskaetal.,2018aswellassomeoftheotherHSDsinUWO241,includingtheIBPgenes.However,moreworkisneeded,includingadditionalgenomesequencesfromMoewusiniaalgae,especiallycloserelativesofUWO241,beforeonecan Figure3.Ice-bindingproteinsfromUWO241 (A)Maximumlikelihood(ML)phylogenetictreebasedontheaminoacidalignmentsof37IBPsinUWO241.(B)MLphylogeneticrelationshipsofIBPsinUWO241(red),ICE-L(green),Archaea(blue),andbacteria(black).(C)Aminoacidalignmentof37IBPsinUWO241viaClustalOmega,versi

on1.2.4,usingdefaultparameters. OPENACCESS,102084,February19,2021 iScienc denitivelysayifadaptationtoanextremeenvironmentiscontributingtotheretentionofHSDsinUWO241.ItisnoteworthyinthiscontextthatneithertheUWO241mitochondrialorchloroplastgenomes(Cvetkovskaetal.,2019)containduplicatedgenesorretroelement-likesequences.Geneduplicationisincreasinglybeingidentiedasameansofadaptationtoextremeenvironments(Kon-drashov,2012QianandZhang,2014).Moreover,duplicationeventsresultinginincreasedgenedosageareknowntoplayakeyroleintheinitialretentionofduplicategenes(InnanandKondrashov,2010).Thedatapresentedhereaddtothistheme.But,again,itisnotnecessarilytruethattheinfrastructurerespon-sibleforgeneratingputativelybenecialduplicationsisadaptive.Rather,somethingneutralcansome-timesgiverisetosomethinguseful,whichwethinkisthecaseforUWO241.Thequestionremains:whatprecisemolecularmechanism(s)arecausinggeneticduplicationsinthisalga?WefavoranRT-basedmodelbecauseofthecloseassociationbetweenRTsandduplicatesinthegenome,aswellastheprepon

deranceofreversetranscriptases.Butothermodelsarepossible.IfRTsarecontributingtogeneduplicationsinUWO241,thenthiscouldhelpexplainthegeneralupheavalweobservedthroughoutthegenomebutalsoraisesquestionsabouthowtheHSDsacquiredfunctionalregulatoryregions—retro-duplicationdoesnottypicallyincluderegulatoryelementsbuttheycanbeacquirebyRTsviaothermeans(KubiakandMakaowska,2017).Moreover,RTinsertionscanalsoalterthefunctionofnearbygenes,which,inturn,canhaveacascadeofeffects(Carellietal.,2016ConradandAntonarakis,2007Remarkably,similarevolutionaryprocessesappeartobeoperatingintheICE-Lgenome,inwhichgeneduplication,potentiallydrivenbyRTs,hasledtolargeexpansionsinvariousgenefamilies,includingIBPgenes(Zhangetal.,2020),aswellasHSDs(265duplicatescovering717genecopies)(Figure1Data).ManyoftheHSDsinICE-LhavesimilarfunctionstothoseinUWO241(DataS4C.eustigma,theclosestrelativeofUWO241forwhichadraftgenomesequenceisavailable,alsohasaconsiderablenumberofgeneduplicates(276),whichcouldbecontributingtoitssurvivalinanextremelyacidicenviro

nment(ookaetal.,2017).TheUWO241,ICE-L,andC.eustigmagenomesstandincontrasttootherexploredgreenalgalnucDNAs,whichdonothavelargenumbersofHSDs.Indeed,whenthesamebioinformaticsproced-uresusedtoidentifyandclassifyHSDsinUWO241werecarriedoutonavailablechlamydomonadaleange-nomes,smalltomoderatenumbersofgeneduplicationswereidentied(Figures1Dand),whichisconsistentwithpreviousanalysesofthesegenomes.ItwillbeespeciallyinterestingtoseeifICE-MDV—whichlikeUWO241livesdeepwithinthewatercolumnofLakeBonney—alsoharborsexpandedgenefam-iliesandHSDs.Whatevertheresult,Antarcticlakeshavealottoteachusaboutgenomeevolutionattheextremesoflife.LimitationsofthestudyAlargenumberofRTsandrampantgeneduplicationcancauseerrorsduringgenomeassembly(Ziminetal.,2017).WeperformedmultipleiterationsoftheUWO241assembly,usingdifferentprotocolsandal-gorithms,andarecondentthattheavailabledraftgenomesequenceinGenBankisofgoodquality.TheHSDs,inparticular,aresupportedbyRNA-seq,meaningthereexistsaspecictranscriptcorrespondingtoeachduplicategene.Butgiventhe

massiveextentofduplicationsintheUWO241genome,itislikelythatsomeregionsweremisassembled,especiallysegmentsofduplicatednoncodingDNA,andwillneedtoberesolvedthroughsubsequentsequencingprojects.Thatsaid,theoverallconclusionspresentedhereshouldnotbeaffected.ResourceavailabilityLeadcontactFurtherinformationandrequestsforresourcesshouldbedirectedtoandwillbefullledbytheLeadCon-tact,DavidR.Smith(MaterialsavailabilityThisstudydidnotgeneratenewreagentsorothermaterials.DataandcodeavailabilityTheassembledgenomesequencesandtherawsequencingdataofUWO241aredepositedattheUSNa-tionalCenterforBiotechnologyInformation(NCBI)databaseunderBioProjectaccessionPRJNA547753, OPENACCESS,102084,February19,2021 iScienc nucleotideaccessionVFSX00000000,andBioSampleaccessionsSAMN11975472andSAMN11975511.Thisstudydidnotgeneratenewcode.METHODSAllmethodscanbefoundintheaccompanyingTransparentmethodssupplementalleSUPPLEMENTALINFORMATIONSupplementalinformationcanbefoundonlineathttps://doi.org/10MC,NPAH,andDRSaresupportedbyDiscoveryGrantsfromtheN

aturalSciencesandEngineeringResearchCouncilofCanada(NSERC).WethankBojianZhongandJinlaiMiaoforsharingtheICE-Lgenomesequences,aswellasRoryCraigforusefuldiscussiononTEcuration.WealsothankYiningHuforherassis-tancewithcomputerprogramming.AUTHORCONTRIBUTIONSThestudywasconceptualizedbyMC,NPAH,andDRS.ThedatawereanalyzedbyMCandXZ.DRSandXZdraftedthemanuscriptandallauthorscommentedtoproducethemanuscriptforpeerreview.DECLARATIONOFINTERESTSTheauthorsdeclarenocompetinginterests.Received:November6,2020Revised:December8,2020Accepted:January14,2021Published:February19,2021Blanc,G.,Agarkova,I.,Grimwood,J.,Kuo,A.,Brueggeman,A.,Dunigan,D.D.,Gurnon,J.,Ladunga,I.,Lindquist,E.,andLucas,S.(2012).ThegenomeofthepolareukaryoticmicroalgaCoccomyxasubellipsoidearevealstraitsofcoldadaptation.GenomeBiol.,1–12Brunet,T.,andDoolittle,W.F.(2018).Thegeneralityofconstructiveneutralevolution.Biol.Philos.Carelli,F.N.,Hayakawa,T.,Go,Y.,Imai,H.,Warnefors,M.,andKaessmann,H.(2016).Thelifehistoryofretrocopiesilluminatestheevolutionofnewmammaliangene

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