evolution of the dan gene family in vertebratesgene, which is also the most frequently mutated gene...
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PEERCOMMUNITYINEVOLUTIONARYBIOLOGY
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RESEARCHARTICLE
Citeas:OpazoJC,HoffmannFG,ZavalaK,EdwardsSV(2020)EvolutionoftheDANgenefamilyinvertebrates.bioRxiv,794404,ver.2peer-reviewedandrecommendedbyPCIEvolutionaryBiology.doi:10.1101/794404 Posted:28June2020Recommender:KaterynaMakovaReviewers:2anonymousreviewersCorrespondence:[email protected]
EvolutionoftheDANgenefamilyinvertebratesJuanC.Opazo1,2,3,FedericoG.Hoffmann4,5,KattinaZavala1,ScottV.Edwards61InstitutodeCienciasAmbientalesyEvolutivas,FacultaddeCiencias,UniversidadAustraldeChile,Valdivia,Chile.2DavidRockefellerCenterforLatinAmericanStudies,HarvardUniversity,Cambridge,MA02138,USA.3MillenniumNucleusofIonChannels-AssociatedDiseases(MiNICAD).4DepartmentofBiochemistry,MolecularBiology,Entomology,andPlantPathology,MississippiStateUniversity,MississippiState,39762,USA.5InstituteforGenomics,Biocomputing,andBiotechnology,MississippiStateUniversity,MississippiState,39762,USA.6DepartmentofOrganismicandEvolutionaryBiology,HarvardUniversity,Cambridge,MA02138,USA.
Thisarticlehasbeenpeer-reviewedandrecommendedby
PeerCommunityinEvolutionaryBiologydoi:10.24072/pci.evolbiol.100104
ABSTRACTThe DAN gene family (DAN, Differential screening-selected gene Aberrant inNeuroblastoma) is a group of genes that is expressed during development and playsfundamental roles in limb bud formation and digitation, kidney formation andmorphogenesis and left-right axis specification. During adulthood the expression ofthese genes are associated with diseases, including cancer. Although most of theattention to this group of genes has been dedicated to understanding its role inphysiologyanddevelopment,itsevolutionaryhistoryremainspoorlyunderstood.Thus,thegoalofthisstudyistoinvestigatetheevolutionaryhistoryoftheDANgenefamilyinvertebrates,with the objective of complementing the already abundant physiologicalinformationwithanevolutionary context.Our results recovered themonophylyof allDAN gene familymembers and divide them into fivemain groups. In addition to thewell-knownDANgenes,ourphylogeneticresultsrevealedthepresenceoftwonewDANgene lineages; one is only retained in cephalochordates, whereas the other one(GREM3) was only identified in cartilaginous fish, holostean fish, and coelacanth.According to the phyletic distribution of the genes, the ancestor of gnathostomespossessed a repertoire of eight DAN genes, and during the radiation of the groupGREM1,GREM2,SOST,SOSTDC1,andNBL1wereretainedinallmajorgroups,whereas,GREM3,CER1,andDAND5weredifferentiallylost.Keywords:genefamilyevolution,Cerberus,differentialretention,evolutionarymedicine,evolutionaryslowdown,gremlin.
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Introduction
Geneticvariationharbored innon-model species representsapowerful resource togain insights inour
understandingofthegeneticbasesofbiologicaldiversity(Opazoetal.2005;Storzetal.2007;Faulkesetal.,
2015).Thecomparativeapproachhasgainedpopularity since thediscoveryofnon-model species thatare
resistant to diseases that affect human health, and also because some non-model species develop
pathologies in a similarway as ourselves (Protas et al. 2006; Gorbunova et al., 2012; Castro-Fuentes and
Socas-Pérez,2013;Manovetal.,2013;Henningetal.,2014;Braidyetal.,2015).
TheDANgenefamily(DANstandsforDifferentialscreening-selectedgeneAberrantinNeuroblastoma)is
a groupof genes characterized by the presenceof aDANdomain (http://pfam.xfam.org/family/PF03045),
lowinter-paralogconservationandexpressionduringearlydevelopment.DANgenesplayfundamentalroles
in limb bud formation and digitation, kidney formation, and morphogenesis as well as left-right axis
specification(Nolan&Thompson,2014).Inadultorganisms,theexpressionofDANgenesisassociatedwith
pathologiessuchascancerandnephropathies(Kolietal.2006;Walshetal.2010;Guetal.2012;Droguettet
al. 2014; Liangetal. 2015).As currently recognized, theDANgene family comprises sevenparalogs:NBL1
(neuroblastoma 1); CER1 (cerberus 1); DAND5 (DAN domain BMP antagonist family member 5); SOST
(sclerostin);SOSTDC1(sclerostindomaincontaining1);GREM1(gremlin1);andGREM2(gremlin2) (Avsian-
Kretchmer et al. 2004;Walsh et al. 2010; Nolan & Thompson, 2014). Historically, DAN genes have been
associatedwiththe inhibitionofthebonemorphogeneticprotein(BMP)signalingpathway,althoughmore
recentstudieshaveshownthattheyalsoactasantagoniststonodal,wingless-typeMMTVintegrationsite
(Wnt)andvascularendothelialgrowthfactor(VEGF)signalingcascades(Piccoloetal.1999;Elliesetal.2006;
Chiodelli et al. 2011). Most of the work on DAN genes has focused on understanding their role in
development (Nolan & Thompson, 2014, and references therein), and less effort has focused on their
evolutionaryhistory(Avsian-Kretchmeretal.2004;Walshetal;2010;Nolan&Thompson,2014;LePetillon
etal.2013;Opazoetal.2017).Understandingtheevolutionaryhistoryofgenefamiliespermitsinsightsinto
thewaysinwhichgenesoriginateanddiversify,aswellasratesofevolution,therelativerolesofdifferent
mutationalprocessessuchaspointmutationsandduplicationsand,most importantly, relatingallof these
phenomenatothephysiologicalandlife-historyidiosyncrasiesofdifferenttaxonomicgroups.
ThegoalofthisstudyistoinvestigatetheevolutionaryhistoryoftheDANgenefamilyinvertebrates,with
theaimofcomplementingthealreadyabundantphysiologicalinformationwithanevolutionaryperspective.
Therefore, we examined the diversity of DAN genes in representative species of all major groups of
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PEERCOMMUNITYINEVOLUTIONARYBIOLOGY 3
vertebrates, inferred homologous relationships by examining gene phylogenies and synteny conservation,
reconstructed ancestral gene repertoires, examined patterns of differential gene retention and quantified
rates ofmolecular evolution. Our results recovered themonophyly of all DAN gene familymembers and
groupedthemintofiveclades. Inadditiontothewell-knownDANgenes,ourphylogeneticresultsrevealed
the presence of two additional DAN gene lineages; one is only retained in cephalochordates (e.g.
amphioxus),whereasthesecondone(GREM3)hasonlybeenretainedbycartilaginousfish,holosteanfish,
and coelacanth. The phylogenetic distribution of genes suggests that the ancestor of gnathostomes
possesseda repertoireof eightDANgenes, and thatduring the radiationof vertebrates,GREM1,GREM2,
SOST, SOSTDC1, and NBL1 were retained in all major groups, whereas, GREM3, CER1, and DAND5 were
differentiallylost,creatingcomplexpatternsofretentionanddiversification.
Methods
DNAdataandphylogeneticanalyses
Tominimizepotentialerrorsintheannotationofthesequences,wemanuallyannotatedDANgenesin
representativespeciesofallmajorgroupsofchordates.Todoso,wefirstidentifiedgenomicfragments
containingDANgenesinEnsemblv86(Zerbinoetal.,2018)orNationalCenterforBiotechnologyInformation
(NCBI)databases(refseq_genomes,htgs,andwgs;Geeretal.,2010)basedonconservedsyntenys.Once
identified,genomicpieceswereextractedincludingthe5ʹand3ʹflankinggenes.Afterextraction,we
manuallycuratedtheexistingannotationorweannotatethenovobycomparingknownexonsequences
(querysequence)fromaspeciesthatshareacommonancestormostrecentlyintimetothespeciesofwhich
thegenomicpiece(subjectsequence)isbeinganalyzedusingtheprogramBlast2seqv2.5withdefault
parameters(Tatusova&Madden,1999).SequencesderivedfromshorterrecordsbasedongenomicDNAor
complementaryDNAwerealsoincludedtoattainabroadtaxonomiccoverage.Weincludedrepresentative
speciesfrommammals,birds,reptiles,amphibians,lobe-finnedfish,holosteanfish,teleostfish,cartilaginous
fish,cyclostomes,urochordatesandcephalochordates(SupplementaryTableS1).Aminoacidsequences
werealignedusingtheFFT-NS-istrategyfromMAFFTv.6(Katoh&Standley,2013).Ouralignmentcontained
424sequencesandalengthof797characters.Phylogeneticrelationshipswereestimatedusingmaximum
likelihoodandBayesianapproaches.Ourgoalwastoestimatethehistoryofagenefamily,notthephylogeny
oftheorganismallineagesthemselves,henceweusedtraditionalgene-treebasedphylogeneticmethods
(Edwards,2009;Yang&Rannala,2012).WeusedIQ-Tree(Kalyaanamoorthyetal.,2017)toselectthebest-
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fittingmodelofaminoacidsubstitution(JTT+I+G4)andtoobtainthemaximumlikelihoodtree
(Trifinopoulosetal.2016)andassessedsupportforthenodeswith1,000bootstrappseudoreplicatesusing
theultrafastroutine.BayesiansearcheswereconductedinMrBayesv.3.1.2(RonquistandHuelsenbeck,
2003),settingtwoindependentrunsofsixsimultaneouschainsfor5x106generations,samplingevery2,500
generations,andusingdefaultpriors.Oncetheanalysesweredone,weverifiedthattheestimatedsample
size(ESS)exceededtherecommendedvalueof200usingTracerver1.7.1(Rambautetal.2018).Therunwas
deemedconvergedoncethelikelihoodscoresreachedanasymptoticvalueandtheaveragestandard
deviationofsplitfrequenciesremained<0.01.Wediscardedalltreesthatweresampledbefore
convergence,andweevaluatedsupportforthenodesandparameterestimatesfromamajorityrule
consensusofthelast4,000trees.
AssessmentsofConservedSynteny
WeexaminedgenesfoundupstreamanddownstreamofeachmemberoftheDANgenefamilyonspecies
representativeofallthemajorgroupsofvertebrates.Syntenyanalyseswereperformedinhumans,chicken,
spottedgarandelephantshark.InitialorthologpredictionswerederivedfromtheEnsemblCompara
database(Vilellaetal.2009)andwerevisualizedusingtheprogramGenomicusv84.01(Muffatoetal.2010).
InthecaseoftheelephantsharkthegenomicsegmentscontainingDANgeneswereannotated,and
predictedgeneswerethencomparedwiththenon-redundantproteindatabaseusingBasicLocalAlignment
SearchTool(BLAST)(Altschuletal.1990).
Resultsanddiscussion
Genephylogeniesandsyntenyanalysesdefinehomology
OurphylogeneticanalysesrecoveredthemonophylyofeachmembersofthegenesintheDANgenefamily
withstrongsupportwiththeexceptionofDAND5(Fig.1).Inaddition,ourtreessuggestanarrangementin
whichthesegenesaredividedintofivemaingroups(Fig.1):1)acladecontainingtheSOSTDC1andSOST
genes;2)acladethatcontainstheCER1andDAND5genelineages;3)acladecorrespondingto2cerberus-
likesequencesofcephalochordates;4)acladecorrespondingtotheNBL1gene;and5)acladecontainingthe
GREMgenelineages(Fig.1).
Inthefirstclade,themonophylyofSOSTDC1andSOSTiswellsupported,asisthesistergroup
relationshipbetweenthem(showninshadesofred;Fig.1),suggestingthattheyshareacommonancestor
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morerecentlythanwithanyothermemberofthefamily(SupplementaryFig.S1A).Thesistergroup
relationshipbetweenthesetwogenelineageshasalsobeenrecoveredinotherstudies(Avsian-Kretchmeret
al.2004;Walshetal.2010;Nolan&Thompson,2014).Syntenyanalysesprovidefurthersupportforthe
identityofthesetwogenelineagesbecausegenesfoundup-anddownstreamareconservedinmost
surveyedspecies(Fig.2).ThetreetopologyinwhichtheSOSTDC1/SOSTcladeisrecoveredsistertoallother
membersofthegenefamilyhasalsobeenrecoveredinotherstudies(Avsian-Kretchmeretal.2004;Walshet
al.2010;Nolan&Thompson,2014).
Inthesecondgroup,showninshadesoforange(Fig.1),themonophylyofCER1genesiswell
supported,whereasthemonophylyofDAND5genesisnot(Fig.1).ThelackofsupportfortheDAND5clade
couldbeduetothepresenceofacoelacanthDAND5sequenceforwhichthephylogeneticpositionisnot
wellresolved(SupplementaryFig.S1B).However,genesfoundupstream(GADD45GIP1andRAD23A)and
downstream(NFIX,LYL1)ofthecoelacanthDAND5sequenceconfirmthatthegeneisintheexpected
positionforaDAND5ortholog,providingindependentsupportforthetopologyobtainedinourphylogenetic
analyses(Fig.1).Thus,thedatasuggeststhatthecoelacanthDAND5geneisatrueorthologoftheDAND5
geneofvertebratesandthatthelackofresolutionislikelyduetohighsequencedivergenceinthe
coelacanthgene.Inadditiontothecoelacanthsequence,syntenyanalysesprovidefurthersupportforour
phylogeny,becausegenesfoundup-anddownstreamofCER1andDAND5areconservedacrossspecies(Fig.
2).AlthoughwerecoveredasistergrouprelationshipbetweenCER1andDAND5(Fig.1),therelevantnode
wasnotsupported(Fig.1).Intheliterature,thereisnoclearpatternregardingthesistergrouprelationship
betweentheseparalogs(Avsian-Kretchmeretal.2004;Walshetal.2010;Nolan&Thompson,2014).
AlthoughthestudyofWalshetal.(2010)supportsthesistergrouprelationshipbetweenCER1andDAND5,
thestudyofAvsian-Kretchmeretal.(2004)recoveredatopologyinwhichCER1isthesistergroupofaclade
containingDAND5,NBL1andGREMgenelineages,whereas,Nolan&Thompson(2014)recoveredDAND5
sistertoacladecontainingCER1andGREMgenelineages.Insupportofourtopology,anaminoacid
alignmentthatincludesallhumanDANfamilymembersshowsthatCER1andDAND5aremoresimilarto
eachotherthantoanyothermemberofthegenefamily;moreover,theEnsemblandgenecardsplatforms
suggestthattheonlyparalogofCER1isDAND5andviceversa.Finally,accordingtoouranalyses,theclade
thatincludesCER1andDAND5wasrecoveredsistertothecladethatincludestheNBL1andGREMgene
lineagesandathirdcladeofDANgenesthatincludestwocerberus-likesequencesfromtwolanceletspecies
(Fig.1).
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OurphylogeneticanalysesrecoveredathirdcladeofDANgenesthatincludestwocerberus-like
sequencesfromtwolanceletspecies(BranchiostomafloridaeandB.belcheri;purplelineage;Fig.1).These
sequenceswererecoveredsistertotheGREM/NBL1clade(Fig.1).Thisphylogeneticarrangementwouldbe
compatiblewithtwopossibleevolutionaryscenarios.ThefirstsuggeststhattheDANgenerepertoireof
lanceletsrepresentsthegenecomplementoftheancestorofOlfactores,fromlatinolfactus,thegroupthat
includesvertebratesandurochordates,andduringtheradiationofthevertebratestheancestralgene
lineagegaverisetotheextantGREMandNBL1genes.Thesecondscenarioimpliesthatthelanceletgene
lineagecouldbeanancientmemberoftheDANgenefamilythathasonlybeenretainedin
cephalochordates.Insupportofthesecondscenario,wefoundcephalochordatesequencesintheNBL1and
GREMclades(SupplementaryFig.S1C),suggestingthatthegenomesofchordatespossessanalready-
differentiatedmemberoftheGREMandNBL1clades.
ThefourthcladecorrespondstotheNBL1genewhichwasrecoveredwithstrongsupport(pinkclade;
Fig.1)(SupplementaryFig.S1C).Theconservationofthegenesfoundup-anddownstreamprovidesfurther
supportfortheidentityoftheNBL1genelineage(Fig.2).ThephylogeneticpositionofNBL1isstillamatter
ofdebatebecausedifferentphylogenetichypotheseshavebeenproposedinearlierstudies.Nolanetal.
(2014)recoveredNBL1assistertothecladecontainingGREM1,GREM2,CER,andDAND5genelineages;
whereasinWalshetal.(2010)NBL1sequenceswererecoveredinatrichotomywiththeGREM1/GREM2and
CER1/DAND5clades.However,insupportofourstudyAvsian-Kretchmeretal.(2004)recoveredNBL1as
sistertotheGREMgenelineages.
Thefifthcladecorrespondstoawell-supportedgroupcontainingGREMgenelineages(shownin
shadesofgreen;Fig.1).Withinthisclade,themonophylyoftheGREM1andGREM2genesiswellsupported
(Fig.1).Asinothercases,syntenyanalysessupporttheidentityofbothgenelineages(Fig.3).Weidentifieda
cladethatcontainsasinglecopygeneinlanceletsandtunicates,whichinturnwasrecoveredassistertothe
GREM2clade(SupplementaryFig.S1D).Thepresenceofasinglecopygeneincephalochordatesand
urochordatesisexpectedgiventhatapreviousstudyreportedthatthevertebrateGREMgenesdiversifiedas
aproductofthetworoundsofwhole-genomeduplicationsearlyinvertebrateevolution(Singhetal.2015;
Sacerdotetal.2018;Simakovetal.2020),andcephalochordatesandurochordatesdivergedfrom
vertebratesbeforethesewhole-genomeduplications.Unexpectedly,ourphylogeneticanalysesidentifiedthe
presenceofathirdGREMgenelineage(GREM3)amonggnathostomevertebratesthathasnotbeen
describedbefore(Fig.1).Thisnewgenelineagewasrecoveredwithstrongsupport,andwasidentifiedin
threedistantlyrelatedspecies:elephantshark(Callorhinchusmilii),spottedgar(Lepisosteusoculatus)and
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coelacanth(Latimeriachalumnae).Syntenyanalysesprovidefurthersupportfortheidentityofthisnew
lineageasgenesfoundup-(RASGRP4,FAM98CandSPRED3)anddownstream(RYR1,MAP4K1,andEIF3K)
areconserved(Fig.3).Moreover,wefoundthathumanorthologsofthegenessyntenictoGREM3mapped
tochromosome19,suggestingthatthischromosomeastheputativegenomiclocationoftheGREM3genein
humans(Fig.3).Asimilardifferentialretentionofanancestralgeneinasmallgroupofdistantlyrelated
specieshasalsobeenobservedinothergenefamilies(Wichmannetal.2016).
Itisnecessarytohighlightthatalthoughwereportdisagreementintreetopologiesbetween
publishedstudiesandourresults,phylogenetictreesarenotalwaysdirectlycomparableastheyhave
differencesinthetaxonomicand/ormembershipsampling,twofactorsknowntoaffectphylogenetic
inference.
Definitionofancestralgenerepertoires
Theinterpretationofthephylogeneticdistributionofgenesincombinationwiththeevolutionary
relationshipsamongthem(i.e.genetree),inthelightoftheorganismalphylogeny(i.e.speciestree),
representsanappropriatewaytounderstandtheevolutionoftheDANgenefamily.Accordingtoourresults,
GREM1,GREM2,SOST,SOSTDC1,andNBL1arepresentinthemajorgroupsofgnathostomes(Fig.4;
SupplementaryFig.S1E),andwithineachparalogcladethespeciesarrangementdoesnotsignificantly
deviatefromtheorganismalphylogeny.Thisindicatesthatallthesegeneswerepresentinthecommon
ancestorofthegroup,andagreeswiththeexpectationsofthesimplestmodelofmultigenefamily
diversification,thedivergentevolutionmodel(Neietal.1997).InthecaseofGREM3,CER1,andDAND5,
althoughtheyarenotfoundinallmajorgroupsofgnathostomes,theirphyleticdistributionincombination
withthecorrespondingtreetopology(Fig.4)indicatethatthesegeneswerealsopresentinthecommon
ancestorofgnathostomes.Thus,ourresultssuggestthatthelastcommonancestorofgnathostomes,dated
between615to476mya(Hedgesetal.2015),possessedarepertoireofeightDANgenes:GREM1,GREM2,
SOST,SOSTDC1,NBL1,GREM3,CER1,andDAND5.Wenotethattheancestralconditionofeightgenesisonly
presenttodayincoelacanths(Fig.4)..Thisreconstructionholdsunderanyofthealternativerootings
previouslyproposedintheliterature.
Inferencesabouttherepertoireofthecommonancestorsofvertebrates,olfactoresandchordates
aresomewhatspeculativegiventhelimitedavailabilityofgenomesandthequalityofcurrentassemblies.
However,thephylogeneticpositionofkeytaxonomicgroups,suchascyclostomes,urochordates,and
cephalochordates,providessignificantinformationtoadvancehypothesesregardingthecomplementof
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PEERCOMMUNITYINEVOLUTIONARYBIOLOGY 8
genesinthesedifferentancestors.WeidentifiedGREM2andSOSTDC1sequencesincyclostomes
(SupplementaryFigs.S1AandS1D),indicatingthatatleastthesetwogeneswerelikelypresentinthe
commonancestorofvertebrates.However,giventhatcyclostomesequencesarenestedwithintheGREM2
andSOSTDC1cladesandnotsistertoacladewithmorethanoneDANgene,itispossiblethattheabsenceof
theotherparalogsincyclostomegenomesisduetogeneloss.
Inferencesaboutthecommonancestorofolfactoresarerestrictedgiventhelimitednumberof
urochordategenomesandthequalityoftheirassemblies.Tofurthercomplicatethissituation,urochordates
haveexperiencedanextraordinarynumberofgenelosses(Dehaletal.2002;Cañestroetal.2003;Cañestro
etal.2013;Albalat&Cañestro,2016),soitisdifficulttotellwhethertheabsenceofageneisduetothe
qualityofthedataorarealloss.Takingalltheseuncertaintiesintoaccount,wewereabletoidentifyone
urochordatesequence.ThissequencewasrecoveredassistertothreecephalochordateGREMgenes,which
inturnwasrecoveredassistertoGREM2(SupplementaryFig.S1D).GiventhatGREMgenesdiversifiedasa
productofthetworoundsofwholegenomeduplicationsinthelastcommonancestorofvertebrates(Singh
etal.2015;Sacerdotetal.,2018;Simakovetal.2020),asingleannotatedgenecopyinurochordatesis
expected.Thus,themostprobablescenarioisthatthecommonancestorofolfactorespossessedoneGREM
gene.Itisnotpossibletodrawinferencesfortheothermembersofthegenefamilyatthistime.However,
giventhattheancestorofolfactoresexistedbeforethetwovertebrate-specificwholegenomeduplications,
weexpectitsrepertoiretohavefewergenescomparedtotheancestorsofvertebratesorgnathostomes.
Finally,wefoundeightcephalochordatesequencesthathelpusdefinetherepertoireofgenesinthe
ancestorofchordates.OnesequencewasrecoveredsistertoalampreySOSTDC1gene,acladethatinturn
wasplacedsistertoallotherSOSTDC1sequences(SupplementaryFig.S1A).Agroupoftwosequenceswas
recoveredsistertothecladethatincludesNBL1andGREMgenes,andwethinkthiscladerepresentsan
ancientmemberofthegenefamilythatwasonlyretainedinthisgroup.Twoadditionalsequenceswere
recoveredassistertotheNBL1lineage;whereasthelastthreewereplacedsistertoaurochordateGREM
sequence,andthislatterclade,inturn,wasrecoveredsistertoGREM2(SupplementaryFig.S1C).Ourresults
agreewithLePetillonetal.(2013),whoalsofoundcopiesofNBL1andGREMorthologsincephalochordates.
However,incontrasttoLePetillonetal.(2013),wedidnotfindacephalochordateDAND5genelineagebut
insteadidentifiedacephalochordate-specificgenelineage(Fig.1,purpleclade).Bringingtheseresults
together,weproposethatthechordateancestorhadatleastfourDANgenes(SOSTDC1,NBL1,GREM,
DAND5andthecephalochordate-specificgenelineage).LePetillonetal.(2013)alsoidentifiedsequencesin
ambulacraria,thegroupthatincludesechinodermsandhemichordates,suggestingthatthedeuterostome
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PEERCOMMUNITYINEVOLUTIONARYBIOLOGY 9
ancestor,whichexistedbetween797and684mya(Hedgesetal.2015),hadatleastthreeDANsequences,
NBL1,GREM,andDAND5.
DifferentialretentionofDANgenesduringtheevolutionaryhistoryofgnathostomes
OurresultsshowthatcopiesofGREM1,GREM2,SOST,SOSTDC1,andNBL1havebeenmaintainedinthe
genomeofallmajorgroupsofgnathostomesduringthelast615mya(Hedgesetal.2015;Fig.4),whichcould
suggesttheyplaycriticalrolesduringdevelopment.Inlinewiththissuggestion,theinactivationordeletion
ofsomeofthesegenesproduceembryonicallylethaleffects(Khokhaetal.2003;Gazzeroetal.2006);in
othercases,thelossofageneisassociatedwithpathologicalconditions(Lietal.2008),althoughthereare
otherexampleswhereknockoutsdevelopminormorphologicalanomalieswithnosignificantconsequences
(Davisetal.2015;Vogueletal.2015).Althoughthesegenesarepresentinallmajorgroupsofgnathostomes,
someofthemcouldstillbeabsentinaparticulargroupwithinthesemoreinclusivetaxonomiccategories.
Forexample,ithasbeensuggestedthattheGREM2genewaslostinthecommonancestorofruminants,
hippopotamuses,andcetaceansbetween56.3and63.5millionyearsagoasaproductofachromosomal
rearrangement(Opazoetal.2017).
OurresultsalsorevealedthatGREM3,CER1andDAND5weredifferentiallyretainedduringthe
evolutionaryhistoryofgnathostomes.Thedifferentialretentionofgenescouldbeastochasticprocess,in
whichtheresultingdifferencesingenecomplementdonottranslateintofunctionalconsequences.This
phenomenoncouldbefacilitatedbyadegreeofredundancythatcouldfunctionasabackupwith
functionallyoverlappingparalogues,incasethatoneofthegenesislostorinactivated(Gitelman,2007;
Cañestroetal.,2009;Félix&Barkoulas,2015;Albalat&Cañestro,2016).However,itisalsopossiblethat
possessingmultiplecopiescouldhelpdirectthetrajectoryofphysiologicalevolutionbyproviding
opportunitiesfortheemergenceofbiologicalnovelty(Ohnoetal.,1968;Ohno,1970;Forceetal.,1999;
Hughes,1994;Zhang,2003).Forexample,thedifferentialretentionandduplicationoftheγ-globingenes
mighthaveplayedanimportantroleintheevolutionoflifehistoryinanthropoidprimatesbyfacilitatingan
extendedfetaldevelopment(Goodmanetal.1987;Opazoetal.2008).Similarly,thedifferentialretentionof
functionalcopiesoftheINSL4genemayhavebeenassociatedwithuniquereproductivecharacteristicsin
catarrhineprimates(Arroyoetal.2012a,b).
Fromanotherperspective,theabsenceofgenesinnaturalknockoutsrepresentsanopportunityto
understandtheirphysiologicalrole(Albertsoneta.2009).Thissuggestionisbasedontheorthology-function
conjecture:theexpectationthatorthologousgenesaremostlikelytohaveequivalentfunctionsindifferent
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PEERCOMMUNITYINEVOLUTIONARYBIOLOGY 10
organisms(Altenhoffetal.2012).Thisapproachhasbeenusefulforunderstandinghumandiseases,
especiallyforthoseexhibitingsimpleMendelianinherence,suchascysticfibrosis,albinismandother
diseases(Albertsonetal.2009).Forexample,ithasbeenshownthatintheblindcavefish(Astyanax
mexicanus),lossofpigmentationisproducedbymutationsinactivatingtheproteinencodedbytheOCA2
gene,whichisalsothemostfrequentlymutatedgeneincasesofhumanalbinism(Protasetal.2006).Thus,
bothretentionandgenelosscouldbeseenasevolutionaryeventsthathelpustounderstandthegenetic
basesofbiologicaldiversity.
OurresultspointtotheGREM3geneasthemostextremecaseofdifferentialretentionamongDAN
genes.Ithasbeenretainedincartilaginousfish,holosteanfishandcoelacanths(Fig.4),suggestingthatitwas
presentinthecommonancestorofgnathostomes,between615and473mya(Hedgesetal.2015),andwas
subsequentlylostindependentlyinthecommonancestorsoftetrapodsandteleostfish(Fig.5).Asimilar
caseofdifferentialretentionofanewlydiscoveredgenelineagewasshowninatumorsuppressorgene
family(Wichmannetal.2016).Inthiscase,inadditiontotheelephantshark,spottedgarandcoelacanth,the
newlydiscoveredgenelineagewasalsoretainedbyteleostfish(Wichmannetal.2016).Theabsenceof
GREM3inasignificantfractionofgnathostomespeciessuggeststhatGREM3couldbedispensable.However,
giventhatGREM3representsanewgenelineageofunknownbiologicalfunction,itisprematuretoevaluate
thephysiologicalandevolutionaryimpactsofretainingorlosingthisgene.Experimentalevidenceforthe
othermembersoftheclade(GREM1andGREM2)providesconflictingresults(Khokhaetal.2003;Davisetal.
2015;Vogueletal.2015),makingitdifficulttoanticipatethedegreeofdispensabilityoftherelatedGREM3
gene.
CER1wasalsopresentinthecommonancestorofgnathostomesandwasdifferentiallyretained
duringtheradiationofthegroup(Fig.4),beinglostinthecommonancestorofray-finnedfish
(Actinopterygii)between425and375mya(Betancur-Retal.2013)(Fig.4).Syntenyanalysesprovidefurther
supportforourscenariobecausethegenomiccontextofCER1isconservedinfish(Fig.5).CER1isaprotein
possessingnineconservedcysteinesandacysteineknotregionexpressedduringearlygastrulationinthe
primitiveendodermandsubsequentlyinsomitesandanteriorpresomiticmesoderm(Shawlotetal.1998;
Pearceetal.1999;Piccoloetal.1999;Beloetal.2000).CER1hasdevelopmentalrolesassociatedprimarily
withanterior-posterior,left-right,anddorso-ventralasymmetries(Beloetal.2009),accomplishingthemby
bindingtoBMPs,NodalandWntligands,whichinturnblocktheligand-receptorinteractionandactivation
(Bouwmeesteretal.1996;Piccoloetal.1999;Beloetal.2000;Silvaetal.2003;Avsian2004).The
physiologicalsignificanceofthisgeneseemstovarywiththetaxonomicgroup:forexample,CER1produces
.CC-BY-NC 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted June 27, 2020. . https://doi.org/10.1101/794404doi: bioRxiv preprint
PEERCOMMUNITYINEVOLUTIONARYBIOLOGY 11
strongerphenotypiceffectsinamphibiansthaninmammals(Simpsonetal.1999;Piccoloetal.1999;Beloet
al.2000).Infish,CER1developmentalfunctionsmaybeperformedbyitsparalogDAND5,whichis
functionallysimilar(Hashimotoetal.2004;Marquesetal.2004;Beloetal.2009;Lopesetal.2010;
Schweickertetal.2010;Hamadaetal.2012;Araujoetal.2014).Thus,fromanevolutionaryperspective,the
possessionofbothparalogs,CER1andDAND5,ingnathostomesotherthanfishwouldrepresentacaseof
functionalredundancy.Underthisscenario,thelossofoneofthesegenescouldbecompensatedbythe
retentionoftheother.
InthecaseofDAND5,wefoundthisgeneinallmainlineagesofgnathostomesotherthansauropsids
andcartilaginousfish(Fig.4).Inthecaseoftheelephantshark,webelievethattheabsenceofthegene
couldbeduetoaprobleminthecurrentgenomeassembly;accordingly,ourproposedscenariomainly
assumestheabsenceofthisgeneinsauropsids.OurresultsindicatethatDAND5waslostinthecommon
ancestorofsauropsids,thegroupincludingbirdsandnon-avianreptiles,between312and280mya(Hedges
etal.2015;Fig.5),eventhoughthecorrespondinggenomicregioniswellconserved(Fig.6).Ourproposed
lossofDAND5inbirdshasalsobeennoticedbyotherauthors(LePetillonetal.2013).DAND5hasbeen
describedasageneplayingfundamentalrolesdrivingasymmetriesintheearlystagesofdevelopmentby
inhibitingNodalactivity(Marquesetal.2004).Althoughinamphibians,fish,andmammalstheexpression
patternseemstobedifferent,themolecularmechanism,restrictingnodalsignalingintheleftsideofthe
embryoisconserved(Beloetal.2009).Inadditiontotherolesinearlydevelopment,duringadulthood
DAND5alsohelpspromotecancermetastasis,aswellasreactivationofmetastaticcellsinlungsbyreversing
theabilityofBMPtoinhibitcancerstemcellfunction(Guetal.2012).Experimentsinotherorgans,suchas
boneandbrain,showthatcancer-relatedfunctionsofDAND5arespecifictothelung(Guetal.2012).
Consequently,ithasbeenshownthatpatientsexpressinghighlevelsofDAND5haveoverallreducedsurvival
rates(Guetal.2012).Thelackofthisgeneinsauropsidsisdifficulttointerpret;wespeculatethat,here,the
lossofDAND5iscompensatedbyCER1,removinganyphysiologicalconsequencesandconsistentwiththe
ideathatgenefamiliespossesssomedegreeofredundancy(Gitelman,2007;Cañestroetal.,2009;Félix&
Barkoulas,2015;Albalat&Cañestro,2016).Thus,CER1andDAND5apparentlyperformsimilarphysiological
rolesduringdevelopment(Hashimotoetal.2004;Beloetal.2009).
Ratesofmolecularevolution:themammalianslowdown
.CC-BY-NC 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted June 27, 2020. . https://doi.org/10.1101/794404doi: bioRxiv preprint
PEERCOMMUNITYINEVOLUTIONARYBIOLOGY 12
Thephylogenetictreesindicatethattheratesofmolecularevolution,asmeasuredbybranchlengths,vary
withineachparalogclade(Fig.1).Themostnotoriouscaseistheobservedslowdowninbranchesleadingto
mammalsrelativetosauropsids(SupplementaryFig.S1AtoS1D).Thisisinterestingbecausesauropsidssuch
asbirds,crocodilians,lizards,snakesandturtlestendtohavegenome-wideratesofevolutionmuchlower
thaninmammals(Greenetal.2014).UsingtherelativeratetestinMEGA7(Kumaretal.2016)we
confirmedthatratesinaminoacidchangearesignificantlyslowerinmammalsrelativetosauropsids
(SupplementaryTableS2).Inmammals,thedivergencevaluesrangedfrom0.0112(SOSTDC1)to0.5143
(DAND5)substitutionspersite,whereasinsauropsidstheyrangedfrom0.0326(GREM2)to0.9248(DAND5).
Inallcases,exceptforGREM2,thesaurospiddivergenceswerehigherthaninmammals,andtheratioof
ratesinsauropsidsvs.mammalsvariedfrom1.79(DAND5)to14.66(GREM1).Weexpectgenesexpressedin
earlystagesoflifetobesubjecttomorestringentpurifyingselectionthanthoseexpressedinlater
development,andthereforetohavelowerevolutionaryrates(Goodman,1961,1963;Roux&Robinson-
Rechavi,2008).Thus,theslowerrateofevolutioninmammalscouldreflectanincreasedleveloffunctional
orstructuralconstraintsonthesegenes.Aslowdowninaspecificsetofgenesshouldbedistinguishedfroma
lineage-specificslowdown.Inthefirstcase,thereducedevolutionaryrateisprobablyduetodifferencesin
thestrengthofpurifyingselectionbetweenthedifferentlineages,whereaslineage-specificslowdownsare
moreplausiblybeexplainedbychangesinthesubstitutionrate,whichcouldbereducedbyloweringofthe
mutationratebyimprovedsystemsofDNArepairmechanisms,longergenerationtimes,orotherfactors
(Goodman,1985).
ConclusionsWeperformedanevolutionarystudyofthedifferentialscreening-selectedgeneaberrantinneuroblastoma
(DAN)genefamilyinvertebrates.Accordingtoourresults,thisgenefamilyhasevolvedbyacombinationof
modelsincludingdivergentevolution,birth-and-death,anddifferentialretention.Werecoveredthe
monophylyofallrecognizedgenefamilymembers,whichinturnwerearrangedintofivemainclades.
Importantly,inthisworkwedescribedthepresenceoftwonewDANgenelineages;onethatisonlypresent
incephalochordates(e.g.amphioxus),andother(GREM3)thatwasonlyidentifiedincartilaginousfish,
holosteanfishandcoelacanths.Theancestorofgnathostomevertebratespossessedarepertoireofeight
DANgenes.Duringtheradiationofthegroup,someofthem(GREM1,GREM2,SOST,SOSTDC1andNBL1)
wereretainedinthegenomeofallmajorgroups,whileothers(GREM3,CER1andDAND5)weredifferentially
.CC-BY-NC 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted June 27, 2020. . https://doi.org/10.1101/794404doi: bioRxiv preprint
PEERCOMMUNITYINEVOLUTIONARYBIOLOGY 13
retainedduringtheevolutionaryhistoryofthegroup.Therateofmolecularevolutionofmammalsisin
generallow,suggestinganincreasedevolutionaryconstraintregime.Finally,weexpectthatwithallthese
newinformationresearchersinthebiomedicalfieldcouldputtheirresultsinanevolutionaryperspective.
Dataaccessibility
Dataandsupplementarymaterialareavailableonline:https://zenodo.org/deposit/3911345
Acknowledgements
ThisworkwassupportedbytheFondoNacionaldeDesarrolloCientíficoyTecnológicofromChile(FONDECYT1160627) and Millennium Nucleus of Ion Channels Associated Diseases (MiNICAD), Iniciativa CientíficaMilenio,Ministry of Economy, Development and Tourism from Chile to JCO, National Science Foundation(EPS-0903787, DBI-1262901 and DEB-1354147) to F.G.H. S.V.E. acknowledges NSF grant DEB 1355343.Version 2 of this preprint has been peer-reviewed and recommended by Peer Community In Evol Biol(https://doi.org/10.24072/pci.evolbiol.100104)
Conflictofinterestdisclosure
Theauthorsofthispreprintdeclarethattheyhavenofinancialconflictof interestwiththecontentofthis
article.JuanC.OpazoisrecommenderforPCIEvolutionaryBiologyandPCIGenomics.
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