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Science, Walther-Rathenau-Str. 47,17487Greifswald, Germany. Greifswald, Institutefor Mathematics andComputer University of Morith Arndt Companys, 2308010Barcelona,Catalonia, Spain. Barcelona, Catalonia, Spain. 618 5 1 within themetazoanshaschanged.Traditionally (inprecladistic of acoelomorphshasbeencomplexandtheirassumedaffinities detail inthefollowingparagraphs.Thehistoryofclassification withinthisphylumandforthatreasonwillbediscussed in Xenacoelomorpha.Acoelomorphsarethebest-characterized simple bodyplan.With thexenoturbellids theyconstitutethe taxon :agroupofmostlymarinewormswith a Acoel flatwormstogetherwiththenemertodermatidsconstitute the Neural system,Evolution,bHLH,GPCR, KEY WORDS:Acoelomorpha,, the subjectofthispaper. the genomesortranscriptomesofthesethreegroupsanimalsis acoels. Howthisprocessof‘progressive’ cephalizationisreflectedin simple sub-epithelialnetof systems withdifferent degreesofcephalization;fromtherelatively work becausethemembersofthreecladeshavenervous Xenacoelomorpha models.Theserepresentanidealsystemforthis at least100years.We arenowtacklingthisissueusing from simplerneuronalarrayshasbeenamatterofintensedebatefor (centralized) nervoussystems.Howcomplexbrainsareassembled time. A majorfocusofourresearchistheorigin‘cephalized’ their expressionpatternsduringdevelopment,inbothspaceand used toanalysetheevolutionaryhistoryofgenefamiliesandstudy different membersofthethreeclades.Thedataobtainedhasbeen the sequencingofcompletegenomesandtranscriptomes molecular/developmental tools,themostimportantofwhichhasbeen nervous system.Thisresearchplanhasbeenaidedbytheuseof main aimistounderstandthestructureanddevelopmentof systematic studyofthemicroscopicanatomytheseworms;our the pastfewyears,ourgroup,alongwithothers,hasundertakena would includeitasafourthphylumwithintheDeuterostomia.Over authors placeitasthesistergroupofremainingbilaterians,some The groupstillhascontentiousphylogeneticaffinities; thoughmost includes threeclades:Acoela,NemertodermatidaandXenoturbellida. Xenacoelomorpha is,mostprobably, amonophyleticgroupthat and PedroMartinez Elena Perea-Atienza perspective The nervoussystemofXenacoelomorpha:agenomic RESEARCH ARTICLE © 2015.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2015)218,618-628doi:10.1242/jeb.110379 Barcelona (IBUB), Av. Diagonal, 643,08028Barcelona,Catalonia, Spain. Environmental GenomicsGroup, Fabeckstraße Germany. 60-62,14195Berlin, for GenomeResearch andMedical SystemsBiology, and Evolutionary Phylogenetic affinities of the members of Xenacoelomorpha of themembers of Phylogenetic affinities INTRODUCTION ABSTRACT * Institució Catalana deRecerca iEstudisAvançats (ICREA),Passeig Lluís deGenètica,Universitat deBarcelona,Av.Departament Diagonal, 643,08028- Author ([email protected]) for correspondence 2 Institut deBiomedicinalaUniversitat de 1,5, 1 , BrendaGavilán * Symsagittifera to thecompactbrainof 1 , MartaChiodin 4 , Xenoturbella Dahlem Centre 3 Ernst 1 , JosepF. Abril its metazoanaffinities. within thegroupremainsimmunetospecificproblemsposedby clearly tobeamonophyletictaxon,theinferenceofspecifictrends provoke debate.However, andsinceXenacoelomorphaseems and theotherplacingthemwithinDeuterostomia,continueto alternative views,oneplacingtheAcoelomorphaasbasalbilaterians Lundin, 1997;2000;LundinandSterrer, 2001).Now, these Ehlers, 1985;Smithetal.,1986;LundinandHendelberg, 1996; morphological data(Tyler andRieger, 1975;Tyler andRieger, 1977; Xenacoelomorpha, aclassificationalsosupportedbysome Xenoturbellida, andthuserectedanewmonophyleticgroup,the within theAmbulacraria,groupedwithanothertaxon, ). ThoseauthorssuggestedthatAcoelomorphasit with affinities totheAmbulacraria(Echinodermsand et al.,2011) thatAcoelacouldinsteadbeadeuterostomiangroup, Philippe andcollaboratorsproposed(Philippeetal.,2007; et al.,2009).However, usingalternativemethodologiesanddatasets, was confirmedinsomeexhaustivephylogenomicanalyses(Hejnol ancestral bilateralanimal(BaguñàandRiutort,2004).Thisposition be clearlyestablished,turningthemintogoodproxiesforan (Ruiz-Trillo etal.,1999).Theposition ofacoelsseemedto showed thattheAcoelaweresistergroupofremaining agreed upon(Deuterostomia,EcdysozoaandLophotrochozoa) evolving, recoveredthethreemajorbilateriansuperphylacurrently 1997). A deeperanalysisusingscoresofacoels,someslow- Rhabdithophora nestingwithintheprotostomes(Carranzaetal., Catenulida branchingasthefirsttwocladesofbilateriansand Platyhelminthes werepolyphyletic,withtheAcoelaand rDNA) tobuildphylogenetictreesshowed,forthefirsttime,that taxon (Hyman,1951).Theintroductionofmolecularcharacters(18S Platyhelminthes, whichwasconsideredthemostbasalbilaterian analysis) theAcoelomorphawereincludedwithinphylum brain’ (Raikova,2004c).However, since itiswellknownthatthese some authorstoproposethat acoels haveasimple‘commissural immunochemical markers,inparticular antibodiesagainst5-HT, led Achatz andMartinez,2012). Thesystematicuseofthese the nervoussysteminthisgroup possible(Raikovaetal.,2004a; such as5HT, GYIRF-amideandFMRF-amide hasmadeanalysisof neuroanatomy. Theuseofasinglesetimmunochemicalmarkers, immunocytochemistry haveaddedtoourknowledgeof acoel years, severalstudiesusingelectronmicroscopy and 2004a; HejnolandMartindale,2008;Raikova,2008).Inthepast 10 degree ofcentralizationintheanteriordomain(Raikovaet al., group. Itischaracterized,accordingtosomeauthors,bya low case herebecauseitshowsahighdegreeofvariabilitywithin the (Haszprunar, 1996).TheAcoelomorphnervous systemisapertinent considered tobeimportantforascertainingphylogeneticinformation For manyyears,thestructureofnervoussystemhas been The acoel nervous systemincontext acoelnervous The 1,2 , KatharinaJ.Hoff 3 AlbertJ.Poustka , 4

The Journal of Experimental Biology level, whichissuggestiveofwhat couldbetheinitial subepidermal nervousplexus, aringofprocesseslocatedatthe nervoussystem comprises,inadditiontoa Xenoturbella al., 2000b).Noareasofobvious condensationhavebeendetectedin 1)(Raikovaet net withoutanysubmuscularnervous structures(Fig. simplest, consistingofawell-developed basalintraepidermalnerve most basalcladeofXenacoelomorpha.Itsneuroanatomyis the relative taxonomicpositionswithinthegroup.Xenoturbellida is the to infercorrelations(trends)betweentheirneuroanatomyand their characteristics ofthenervoussystemindifferent taxaallowus 2001; Reuteretal.,2001a;2001b).Thestructural plexus (Raikovaetal.,1998,Raikova2000a; et al., stomatogastric systemandthepresenceofapervasiveperipheral Nemertodermatids andXenoturbellids;forinstance,thelack ofa distributed andspacedaroundtheanterior–posteriorbodyaxis. three tofivepairsofsimilarneuritebundlesthatareregularly a generalpattern:thepossessionofanintraepidermalplexus and acoels hasahighdegreeofstructuralvariability, althoughallshare by allthesedifferent studieshasshownthatthenervoussystemof to characterizethedetailedultrastructure.Theinformationprovided Cebrià, 2008),itisnecessarytousecomplementarymethodologies the nervoussystemunstained(Grimmelikhuijzenetal.,2002; antibodies onlylabelparticulargroupsofcells,leavingtherest Fig. RESEARCH ARTICLE miamia (Raikova etal.,2000b;Israelsson,2007); from: the anteriorring-commissurebybilobedbrain,withadenseneuropil,plusonetothreecommissures.Nervoussystemsinformation basiepidermal locationofthenervoussystemandtendencytopossessasecond,anterior,statolith. (6)Replacementof ringcommisure.Thestatocysthasone lossofthe along theanterior–posteriorbodyaxis.(4)Presenceofastatocystwithtwostatoliths,typicalNemertodermatida.(5)Progressive Xenoturbella et al.,2010)(currentstudy).AdaptedfromadiagraminAchatzandMartinez(AchatzMartinez,2012). There areafewmorphologicalfeaturessharedbyacoels,

.Schematicorganizationofthenervous systemindifferentmembersoftheXenacoelomorpha. 1. (Correa, 1960); Xenacoelomorpha nerve netlocatedbasiepidermally. extending Presenceofastatocyst.(3)ring-commisurepluslownumberneuritebundles,

, yettheypossesastatocyst(Israelsson, 2007).The Xenoturbella 2 westbladi S. funilis Acoelomorpha

Nemertoderma (Crezée, 1975); 1 4 westbladi N. westbladi Acoela

I. pulchra Diopisthoporus 3 gymnopharynngeus (Raikova etal.,2004b); (Achatz andMartinez,2012); Bitesticulata

Paratomella rubra 5 Bursalia Prosopharungida D. gymnopharyngeus Isodiametra pulchra clade, whichincludessome of themoststudiedacoels(e.g. 2011), acladethatincludesallthenamed ‘higheracoels’.This sister groupofProsopharyngida isCrucimusculata(Jondeliusetal., 1)(Crezée,1975).The commissures pluseightneurite bundles (Fig. group, thenervoussystemis organized inonetothree ring groups, theCrucimusculataand theProsopharyngida.Inlatter 1)(Crezée,1978).ThecladeBursaliaisdividedintwomajor (Fig. bundles thatextendfromtheposteriortoendofanimal connect, dorsolaterally, tworingsandapairofdorsolateralneurite a denseplexusaroundthestatocystwithtwoneuralextensions that sister groupofBursalia,indicatethatthenervoussystemconsists of Paratomellidae, agroupthatbranchedlaterandisconsidered the data obtainedfor 1)(Tyler, 2001;AchatzandMartinez,2012).The ventral tracks(Fig. a smallerringlocatedmoreanteriorly. Thesetworingsarelinkedby commissure withtwoganglioniclobesposteriortothestatocyst, and Diopisthoporidae, thenervoussystemisorganized asaring-shaped most recentgroups.Inthebasalfamilyofacoels, the increment inthecomplexityofneuralstructures,particularly Achatz andMartinez(AchatzMartinez,2012)–revealsan members ofAcoela–wefollowthedetaileddescriptiongivenby comparison ofthegeneralneuroanatomywithindifferent 2)(Raikovaetal.,2004b).A quick concentration ofneurons(Fig. stages oftheevolutionaryprocessleadingtoananterior Hofstenia miamia S. roscoffensis The JournalofExperimentalBiology(2015)doi:10.1242/jeb.110379

Solenofilomorpha (Smith andTyler, 1985); funilis hc samemberofthe Paratomella rubra,whichis (Bery etal.,2010;BeryandMartínez,2011; Semmler or (1) Putativexenacoelomorphancestor. (2) Symsagittifera roscoffensis Crucimusculata

Isodiametra pulchra P. rubra 6

(Crezée, 1978); Symsagittifera roscoffensis ), showsamore- X. westbladi H. 619

The Journal of Experimental Biology 620 RESEARCH ARTICLE on indevelopment, theHoxpatterningsystem playsaclearly cnidarians (Magieetal.,2005; Hejnol andMartindale,2009).Later in agreatvarietyoftaxa,including non-bilateriantaxasuchasthe SoxB internalized cellsisreadilyaccounted forbytheexpressionofa differentiate intoneurons.Thedifferentiation ofneuronsfromthese internalized atlaterstages of embryonicdevelopmentand et al.,2000).Someoftheprogenythesemicromeres are lineages, specificallytheepidermisandallneuralcells(Henry located attheanimalpole,whichgiverisetoectodermal The nervoussystemoftheacoelsoriginatesfrommicromeres the acoelnervoussystemanditsmolecularregulatorymechanisms. paragraphs wesummarizewhatisknownabouttheembryology of of thenervoussysteminXenacoelomorpha.Infollowing Unfortunately, verylittleisknownabouttheembryologicalorigin towards theanteriorconcentrationofnervoussystem. visualize theevolutionofxenacoelomorphsasfollowingatrend (mostly thosebelongingtoCrucimusculata).Thuswecould complexity, betweenthosefoundinxenoturbellidsandtheacoels that couldbeinterpretedasintermediate,intermsofstructural neurons andprocessesintheformofaringcommissure;condition The nemertodermatids,instead,haveananteriorconcentrationof – itisalsofoundinthesistergroupofBilateria:. net ofneurones(Raikovaetal.,2000b)isnowregardedasancestral phylum Xenacoelomorpha,thepresenceofanintraepithelialnerve 2011). within theXenacoelomorpha(Hejnoletal.,2009;Philippe the Crucimusculata,isclearlyanevolutionaryderivedcharacter condensation seeninsomeacoels,particularlythosebelongingto (Achatz andMartinez,2012;Achatzetal.,2013).Thus,the 1) brain withacellularcortexanddenseinternalneuropile(Fig. complex nervoussystem,withanteriorconcentrations,abilobed processes emergefromthe‘brain’ andextendtotheperiphery. Scalebars:75 all alongtheanterior–posteriorbodyaxis.A lateralpair(ln1)islightlystained.A intheanteriordomain.Somenervous highconcentration ofneuritesisevident . (B) tothemostfrontalregionof stained, commissureslinktheneuritebundlesinvicinityofbrain(doublearrowheads).Neuralprocessesextendclearly net (arrowheads).Airregular pattern,thincommissuresconnecttheneuritebundlesallalonganterior–posteriorbodyaxis,formingacellular few, intensely stained withsynaptotagminantibody.lateral pairs(ln2).Inan Threepairsofneuritebundlesareshown:themedian(mn)medio-lateral(ln1)and Fig. control systemdevelopment acoelnervous anditsmolecular The Since thexenoturbellidsrepresentmostbasalcladewithin

.Synaptotagmin-andElav-positivestructuresintheanteriorpartofadult 2. orthologue, agenethatisearly expressedinpro-neuralcells S. roscoffensis AB In2 In1 stained withElavpolyclonalantibody. Two stronglystainedmedio-lateralneuritebundles(mn)areshowninthemostcentralregion, mn In1 In2 μ m. and thefunctionality ofthenervoussystem: theso-calledbasic was twogenefamiliesclosely relatedwithboththedevelopment relationship withtheformation ofneuraltissue.Ourstartingpoint of themembersgenefamilies thatarewellknownfortheir members oftheXenacoelomorpha, weundertookathoroughstudy In ordertounderstandtheprocess ofneurogenesisinthedifferent 2010). (Hejnol andMartindale,2009;Morenoetal.,SikesBely, posterior) havebeenidentifiedintheacoelspeciesstudiedtodate neurons. OnlythreeHox important roleinprovidingpositionalcuestothedifferentiating genes position as,orinclosevicinityto,thecellsexpressingneural longifissura expressed inanteriorneuralstructurestheacoel orthologue oftheposterior ‘classical’ neural geneshavebeenstudiedintheacoelspecies.The encompassing thethreegermlayers.Besidesthesegenes,fewother needed, buttheposterior ectodermalneural expression,althoughfurthercorroborationis morphogenetic proteins) and theNemertodermatids. known aboutthecontrolofneurogenesisinbothXenoturbellids showing nopreferencefordorsalorventrallocations.Nothing is have nervecordsequallydistributedalongtheirdorso–ventral axis, GPCRs) (BHLHsand geneproducts andeffector regulatory Neuronal proteins) andtheirantagonists,ADMPs( differential dorsal–ventralexpression ofBMPs(bonemorphogenetic should leadtoexcitingdiscoveriesbecause,despitehaving dorsal–ventral patterning.Theidentificationofsuchanetwork whereas noinformationisavailableregardingtheregulation of its involved inantero–posteriorpatterningtheacoelnervoussystem, The twomostanterior Although scarce,mostofthemoleculardatarelatestogenes Nk2.1 Symsagittifera roscoffensis The JournalofExperimentalBiology(2015)doi:10.1242/jeb.110379 (Hejnol andMartindale,2009),probablyinthesame and Otp In1 (Hejnol andMartindale,2008). genes (oneanterior, onecentraland (Srivastava etal.,2014),allacoelspecies Hox mn ParaHox Hox nervous system In1 genes seemtoberestricted gene isexpressedincells gene caudal( . (A)S.roscoffensis anti-dorsalizing Cdx) isalso

The Journal of Experimental Biology in fourgroups(α of thetransmembranedomain.Thisfamilycanbefurthersubdivided lipids, peptidesorglycoproteins)throughtheextracellularportion interact withagreatvarietyofligands(suchasamines,purines, as classA)isthelargest. Rhodopsins haveshortN-terminibut Schiöth andFredriksson,2005).Therhodopsinfamily(alsoknown rhodopsin, adhesion,frizzledandsecretin(Fredrikssonetal., 2003; main familiesfollowingtheso-calledGRAFSsystem:glutamate, highly variableN-terminithatallowustoclassifytheminto five intracellular signallingmolecules.Thisspecificityisdueto their this ,GPCRsbindtoaverydiversearrayofligands and and identifythissuperfamilyofgenes.Despitetheconservation of (7TM) thatcanbeusedasamotifingenomicanalysistosearch for known toshareahighlyconservedseventransmembranedomain sensation andalsointheregulationofhomeostasis.GPCRsare involved insensoryfunctionssuchasphotosensitivityorolfactory between speciesandsubtypes.Within neuraltissuetheyare eukaryotic species.Nevertheless,theirtotalnumberdiffers widely transmembrane receptors,andtheyareveryabundantinmost domain butlackingabasicformthelastgroup,F. two different domains:‘orange’ andWRPW. ProteinswithaCOE HER (HairyandenhancerofsplitbHLH)proteinsalsoincludes proteins. GroupEcontainstheproteinsrelatedto are unabletobindDNA,butactasnegativeregulatorsofgroupA to theACGTGorGCGTGcoresequences.MembersofGroupD C includesaPAS domaininadditiontotheHLHdomain,binding B bindtocoreDNA sequencesnamed ‘Eboxes’ (CANNTG).Group characteristics (AtchleyandFitch,1997;Jones,2004).GroupsA and are relatedthroughevolutionandhavesimilarstructural included inhigher-order groupsnamedA,B,C,D,EandF, which families ofbHLHsinmetazoans(Simionatoetal.,2007),all PAS (Per–Arnt–Sim)or‘orange’. involved inprotein–proteininteractionssuchasthe‘leuzinezipper’, dimeric complexes.SomebHLHsalsoincludeadditionaldomains variable loopregion.Theynormallyactivatetheirtarget genesas a basicregionforDNA bindingandtwo DNA-binding domainwhich,asthenamesuggests,iscomposedof These neuronalprecursorproteinspossessahighlyconservedbHLH precursors requiredforCNSregeneration(Cowlesetal.,2013). studies havedetectedbHLHsexpressedinstemcellsandneuronal guidance (Bertrandetal.,2002;Guillemot,2007).Moreover, recent cellular subtypespecification,migratorybehaviourandaxonal development; theycontrolaspectssuchasneuralfatecommitment, (Jones, 2004).bHLHsareregulatorsofnervoussystem in awiderangeofeukaryotes,includingfungi,plantsandmetazoans differentiation isthebHLHgenesuperfamily. Thisclassispresent developmental processessuchascellproliferationand A summarydescriptionofthesefamiliesfollows. the processofprogressive‘cephalization’ thatoccursinthisclade. within Xenacoelomorpha,shouldprovideuswithkeyinsightsinto eventually thestudyofexpressionpatternsforallthesegenesand (GPCR) families.Changesinnumbersandsubfamilies, helix–loop–helix (bHLH)andtheG-protein-coupledreceptor RESEARCH ARTICLE extracellular hormone-binding domain.Theglutamate family(also class B,togetherwithAdhesion, canberecognizedbyan that ofadhesion,fromwhichit evolved.Thisfamily, belonging to growth factordomains.Thesecretin 7TMdomainisverysimilarto domains, suchascadherin,laminin, andcalcium-bindingepidermal family ischaracterizedbylong N-terminiwithdifferent functional GPCRs formthelargest andmostdiversesuperfamilyof Molecular phylogeneticanalysishasrevealed45orthologous A key, well-knowngroupoftranscriptionfactorsinvolvedin , β , γ and δ ) and13subfamilies.Theadhesion α -helices, interruptedbya Drosophila the patternbecomes progressivelylessintense andtheneurite 2B). Throughtheposteriorpartof thebodyaxis undetectable (Fig. clearly visiblecomparedwith the othertwopairsthatarealmost anterior partofadultanimals(the brain);withtwoneuritebundles previously reported(Fig.2A). anterior stainingappearstobemoredetailedthanhas been because ofthespecificityanti-synaptotagminantibody, the the threecommissuresimmediatelyposteriortobrain.Perhaps commissures arelessprominent.Commontoallknownpatterns are and themedio-ventralones,whereasinourexperiments, two morecommissuresclearlyconnectthedorsalneuritebundles studies (Semmleretal.,2010).However, inthosepatternsatleast terminal differentiation regulators. interpretation ofgenepatterns,suchasthoseregulatoryand example ofhowwecangeneratemarkersthatwillaidinthe provide asummarydescriptionoftheseexpressionpatterns,asan protein synaptotagminandtheRNA-bindingfactorElav. Here,we focused ourattentionontwowell-knownproteins:thesynaptic epitopes usinginformationderivedfromtheGenomeProject.We 1891, wegeneratedspecificantibodiesagainst‘pan-neuronal’ nervous systemoftheacoel In ordertobetterunderstandthestructureanddevelopmentof phylum. regulatory anddownstreameffector geneshaveevolvedwithinthis Xenacoelomorpha opensawindowonknowledgeofhowupstream Understanding theevolutionaryhistoryofGPCRsandbHLHsin signalling (Strotmannetal.,2011; Krishnanetal.,2013). roles inearlyembryonicdevelopment,tissuepolarityandcell receptors oftheWntpathway, andarewellknownfortheircrucial module’. Themembersofthelastfamily, frizzled(classF),actas long N-terminalligand-bindingdomainknownasthe‘Venus flytrap known asclassC)comprisesneuronalmodulatorswithatypical The and theanti-tyrosinated This patternisreminiscentofthatobtainedwithboththeanti-5-HT rostral domain,theneuritebundlesbecomelessintenselystained. 2A,doublearrows).Aswemovefurtherposteriorfromthe (Fig. commissures canbedetectedthatdirectlyconnectallthebundles in thebrainarea.Immediatelyposteriortobrain,five (Semmler etal.,2010).Theonlyprominentcommissuresarelocated contrary totheobservationsmadewithanti-5-HT antibody 2A,arrows) net notcompletelyvisibleattheposteriorend(Fig. through verythincommissures,formingasmall,mostlyirregular, most ofthelengthorganism. Theyareclearlyinterconnected pair, amedio-ventralpairandventro-lateralpair)runningalong synaptotagmin, consistsofsixlongitudinalneuritebundles(adorsal The adultnervoussystem,asrevealedbytheantibodyagainst 2012). ancient featuresharedbyCnidaria andBilateria(Nakanishietal., vectensis Bilateria. Ashasbeenshowedforthecnidarian involved inneuraldevelopmentanddifferentiation processesacross highly conservedRNA-bindingproteinsthatare,mostprobably, revealed species-specificantibodies through Structure of the of Structure RESULTS ANDDISCUSSION Adult Elav-positivestructures Adult synaptotagmin-positivestructures In elav S. roscoffensis, Elavimmunoreactivityisobserved inthemost , post-transcriptionalregulationbytheseproteinsisan genes, whicharealsousedasneuronalmarkers,encode The JournalofExperimentalBiology(2015)doi:10.1242/jeb.110379 Symsagittifera roscoffensis Symsagittifera α -tubulin antibodiesdescribedinprevious Symsagittifera roscoffensis vonGraff nervous system nervous Nematostella 621

The Journal of Experimental Biology affinity in families hadnomembers.OnlyonegenefromfamilyA hadunclear more thanonebHLHmemberperfamily;wealsofoundthatsome 1997). Inbothgenomes,S.roscoffensis six high-ordergroups:A,B,C,D,EandF(AtchleyFitch, supplementary materialFig. the PFAM 27.0 databank(hostedattheEMBL-EBI;see known proteinsequencesfromseveralmetazoans: X. bockii. 1949. Thegenomescomprise18genesin one, andaligned themwiththetotallength of themostclosely weak statisticalsupport.Thus, we appliedBLAST tothem, oneby relatives ofNeuroD,ASCa,ASCb andARNT –weresorted, with similarly unclear of theacoelsequence.Infact, thesequencealignedwith occurred forHES/HEY: wewerenotabletodeterminetheaffinities families becauseofitslowbranch support.A similarsituation relative thatwecouldnotallocatetooneofthesetworelated 1).InthecaseofgroupA,wefoundone putativeOligo/Beta3 (Table E (HES/HEY).NorepresentativesofgroupDorFwereidentified (Max, MTFandSRBP);oneingroupC(ARNT); Beta3, PTFa,PTFb,twist,andaputativeOligo);threeingroup B families: ASCa,ASCb,Beta3,E12/E47,MyoD,Net,NeuroD, members ofthefollowinggroups:14ingroupA (membersofthe 2007; Gyojaetal.,2012). topology tothoseobtainedinpreviousstudies(Simionatoet al., supplementary materialFigsS1,S2,bHLHtrees)presentasimilar – althoughstilllimitedoftheMetazoa.Ourtrees (see andcnidarians,provideduswithgoodrepresentation bilaterians. Thespecieschosen,includingprotostomes, of genesnotbelongingtothefamiliesalreadycharacterizedinother 622 RESEARCH ARTICLE Saccoglossus kowalevskii 1).To thealignments,weaddedsequencesfrom (Table Hydra magnipapillata Drosophila melanogaster, of We identifiedtheputativebHLHgenesinsequencedgenomes antibodies. accurately definetherelativedomainsidentifiedbyallthese antibodies (Semmleretal.,2010).Doubleandtriplestainingwill unlike thedatapublishedonuseofRFamideandserotonin contrast tothefourventralbundles,whicharealmostimperceptible, dorsal neuritebundlesarenotablylabelledwithElav, inclear seems tobemorelimitedtheanteriorregionofadult.The body oftheanimal,whereasElavimmunoreactivitypattern study showanextensivesetofneuralstructurescoveringthewhole 5HT andtheRFamideimmunoreactivitypatternsdescribedinthat the specificanti-Elavantibody, somecleardifferences arise.The al., 2010)intheadult immunochemical patternsobtainedbySemmleretal.(Semmler dorsally. From thisstructure,neuralprocessesextendfrontally, laterallyand 2B). ventral side,theneuritebundlesconverge intoanarea(Fig. presence oflesswell-packedfibres.Anteriortothestatocyst,on thickness ofthedorsalneuritebundlesdecreases,reflecting bundles becomethinnerandinappearance.We notethatthe bHLHs gene families involved inneurogenesis –characterizing Xenacoelomorpha Exploring thegenomeof The 18bHLHscharacterizedin It isinterestingtonoteherethatwhenwecomparethe S. roscoffensis The phylogeneticanalysisperformedtoclassifythemused S. roscoffensis. Interestingly, wefoundnospecificcases and thexenoturbellid H. sapiens S. roscoffensis , courtesyofDrGyoja(Gyojaetal.,2012) and N. vectensis

S1). Thegeneswerecategorizedinto Capitella teleta HES/HEY. Some genes–putative S. roscoffensis with thoseobtainedhere Xenoturbella bocki and , Acropora digitifera S. roscoffensis X. bocki,wefoundno , alldownloadedfrom were classifiedas Homo sapiens and 33in Westblad and , (supplementary materialFig. regions. Thisprocedureprovidedsupportforourfamilyassignments related sequences,specificallycheckingbyhandforconserved alignments. We thinkthisreflectsthe factthatAcoelaappearsto although usingthesamemetazoan referencesequencesforthe X. bocki important tonotethatthebootstrap supportisalwayshigherinthe As mentionedabove,in genomes ofNemertodermatidaareneededtoclarifytheissue. within AcoelomorphaandperhapsarespecifictoAcoela;complete these families.Therefore,wesuggestthattheduplicationstookplace case forX.bocki orthologues inthePTFaandPTFbfamilies.Eventhiswasnot do notseemtobethenormin have duplicatesinsomefamilies(Simionatoetal.,2007).Duplicates to theHES/HEY families(supplementarymaterialFig. families. A similarthinghappened withtheacoelsequencerelated were checkedmanuallybeforebeingassignedtotherespective aligned withlowbootstrapsupport(ARNT, NeuroDandAtonal) with unclearaffinities inthephylogenetictree.Again,sequences Xb_Orphan-HLH3. A similarcaseoccurswithXb_Orphan-HLH4, diverged orthologueofNeuroD;weprovisionallynamedit it totheNetfamily. Blastanalysis suggeststhatitcouldbealsoa However, carefulanalysisofthesequencedidnotallowustoassign ,seemstobeaputativememberoftheNetfamily. the high-ordergroupB,withweakbranchsupportin the 48bHLHsincludedinouranalysis.A componentclusteredin HLH1 andXb_Orphan-HLH2,didnotmatchwithcertaintyanyof E (HESandHEY).Two sequences,whichwenamedXb_Orphan- Myc, SRBP, TF4andUSF);oneingroupC(ARNT);4 NSCL, PTFa,PTFbandtwist);7ingroupB(Max,MITF, Mlx, Beta3, E12/E47,hand,mist,MyoRa,MyoRb,neurogenin,NeuroD, different groups:16ingroupA (relativesof:ASCa,ASCb,Atonal, in putative ‘Oligo’)thathasarepresentative in is onefamily, andonlyone,MyoD(plustheproblematiccaseof more accurateastowhentheselossesoccurred.Interestingly, there of comprehensivedatafromNemertodermatida,itisdifficult tobe were producedbeforethediversificationofAcoela.Inabsence Group C:HESandHEY. Thispatternclearlyshowsthatsomelosses and neurogenin;fromGroupB:Mlx,Myc,TF4USF; from missing inacoelsare:fromGroupA:Atonal,hand,mist,MyoRa Specifically, thefamiliesthatarepresentinxenoturbellidsbut of 1).Thegenome same familiesarenotpresentinbothofthem(Table bocki Xenoturbella bocki more in-depthanalysesinthefuture. We havenotdeterminedthebHLHsubfamiliesyet:thiswillrequire may atsomepointimprovetheclassificationofthesefourgenes. lineage. Theintroductionofanitemizedrepresentationspecies enormously fromknownrelativeswithintheXenacoelomorpha that hasbeenlostinallthebilateriansanalysed;ortheyhavedrifted this: thesequencesarehomologoustoanancestralbilateriangene of theknownbilaterianfamilies.Two hypotheseswouldexplain HLH3 andXb_Orphan-HLH4,whichdonotseemtobelongany sequences: Xb_Orphan-HLH1,Xb_Orphan-HLH2,Xb_Orphan- Some species,suchasH.sapiens The 33bHLHsfoundinX.bocki When welookgloballyatallthe bHLHgenesinbothtaxa,itis Remarkably, thehigh-ordergroupswithrepresentationin Xenoturbella S. roscoffensis genome alsohavemembersin trees thaninthoseincluding The JournalofExperimentalBiology(2015)doi:10.1242/jeb.110379 . , whichhadonlyonememberbelongingtoeachof ‘orphan’ bHLHs has fewerfamiliesthanthatof X. bocki

S1, SR_tree). S. roscoffensis. and thecnidarian , werealsoclassifiedintothe we identifiedfour‘orphan’ S. roscoffensis S. roscoffensis S. roscoffensis, butnot We foundonlytwo ; however, the

S2, Xb_tree). N. vectensis sequences, X. bocki X. , .

The Journal of Experimental Biology RESEARCH ARTICLE

Table 1. Number of bHLH family members

Group A

Species Total ASCa ASCb Amber Atonal Beta3 Delilah E12/E47 Hand Mesp Mist MyoD MyoRb MyoRa Net NeuroD Neurogenin NSCL Oligo Paraxis Peridot PTFa PTFb SCL Twist Xenoturbella bocki 3321011011010110(1?) 1 1 1 0 0 0 1 1 0 1 Symsagittifera roscoffensis 18 1(1?) 10010100010011010(1?) 0 0 2 2 0 1 Homo sapiens* 118230220423142214323201 1 32 Nematostella vectensis* 82 3 1 1 0 0(2?) 0(1?) 1 2(3?) 1 0(2?) 0 1 0 1 0(1?) 0(1?) 0 0(2?) 1 1 0 1 3 1 Drosophila melanogaster* 59400311111110110110101 2 11 Acropora digitifera* 70311000121001010000110 1 31 Hydra magnipapillata* 33 3 1 N 0 0(1?) 0(1?) 1 0 0 0(1?) 0 0 0 1 0(1?) 0(1?) 0 0(1?) 1 N 0 1 0 0 Group B Group C D D? E F

Species AP-4 Fig-alpha MAD Max MITF Mlx Mnt Myc SRBP Tf4 USF Ahr ARNT Bmal Clock Hif Sim SRC Trh Emc Pearl HES HEY COE Orphans The JournalofExperimentalBiology(2015)doi:10.1242/jeb.110379 Xenoturbella bocki 0001110111101000000003(4?) 1(2?) 0 2(3?) Symsagittifera roscoffensis 0001100010001000000000(1?) 0 (1?) 0 0 Homo sapiens* 1151521421322223(4?)2(3?)31(2?) 4 0 7(10?) 3(6?) 4 4 Nematostella vectensis* 2 0 1 1 1 1 1 4(6?) 2 1 2 2 1(2?) 1(2?) 1 0(2?) 0(2?) 0 0(2?) 1(2?) 1 11(12?) 1(2?) 1 8–16 Drosophila melanogaster* 100111111112113111110111(2?) 1 0 Acropora digitifera* 101111152122113(4?) 0 0 0 1 1 1 7(9?) 1(3?) 1 14–17 Hydra magnipapillata* 1011100400111000(1?)0(1?)00(1?) 0(1?) ND 1 0 1 6–11 The number of orthologous family members and the total number of bHLHs are indicated. The data for X. bocki and S. roscoffensis are derived from our analysis. The information from the other representative species (*), H. sapiens, N. vectensis, D. melanogaster and H. magnipapillata, was obtained from Simionato et al. (Simionato et al., 2007). The data from A. digitifera (plus the latest identifications in N. vectensis) was obtained from Gyoja et al. (Gyoja et al., 2012). 623

The Journal of Experimental Biology NCBI proteindatabase(http://blast.ncbi.nlm.nih.gov/Blast.cgi). sequences byrunningBLAST withallthecandidatesagainst D. melanogaster. The clusteringwascheckedforalltheacoel regulatory genes. specific changesingenomecomposition,particularfor evolution ofthenervoussystem,anditscentralization,islinkedto , weshouldstarttogetabetterpictureofhowthe complements andabetterdescriptionoftheneuroanatomythese family oftranscriptionalregulators.Moreover, withaccesstothe understanding oftheevolutionarydynamicsthisimportant different membersofthisphylumshouldprovideuswithabetter A moredetailedcharacterization ofbHLHcomplementsin members ofNemertodermatidaandalsosomemorebasalacoels. to exploreafullrangeofxenacoelomorphs,incorporating genome ofonlyoneacoelspeciesandxenoturbellid.We need conclusions becausewehavegatheredcompletedatafromthe Simionato etal.,2007). there havebeenseveralfamilylosses,particularlyinAcoela(see 23. To us,ithasbecomeclearthatintheXenacoelomorphalineage (Simionato etal.,2007;Gyoja2012),rangesbetween18and 624 RESEARCH ARTICLE Dicty_CAR relative, makingatotalof245 different GPCRs and 225rhodopsinreceptors; however, wedidnotfindany following complements:9glutamate, 6frizzled,5adhesion/secretin In thegenomeof queenslandica evolutionary perspective.Thespeciesusedinouranalysiswere: Mendoza etal.,2014)andwerechosentooffer anextensive sequences usedasreferenceswereprovidedbyAlexdeMendoza (de analysed eachfamilytoclassifyallthegenesfound.TheGPCR Mendoza etal.,2014).ForS.roscoffensis, wefurtherphylogenetically have representativesinthelasteukaryoticcommonancestor (de mammals, especiallytherhodopsinfamily, althoughmostofthem for moredetails).GRAFSGPCRsaretypicallyexpanded in plus themembersofDicty_CAR(seeMaterialsandmethods families (Fredrikssonetal.,2003;SchiöthandFredriksson,2005), of genescontainingthetypicalGPCR7TMdomainGRAFS We minedthegenomesof shared between there weremore;atotalof29.Nevertheless,thenumberfamilies dataset for 2007). Surprisingly, thenumber offamiliesrepresentedinour (Ledent andVervoort, 2001;Ledentetal.,2002;Simionato apparently presentintheancestorofcnidariansandbilaterians ancestor).A totalof29–33thesefamiliesare deuterostomes (andmaythereforehavebeenpresentinthe these, 44arethefamiliessharedbyprotostomesand phylogenetic analyses. Xenoturbella more derivedacoels,theso-called‘highacoels’.Incontrast, roscoffensis general, verydivergent orthologoussequences.Inaddition, one shouldexpectthatthegenomeof Therefore, ifthebHLHsequencesfollowsamegeneralpattern, with ahigherrateofnucleotidesubstitutionsinmostgenesanalysed. have longbranchesinmostphylogenetic(phylogenomic)analyses; roscoffensis Analysis ofGPCRfamilynumbers GPCRs However, weshouldbecautiousaboutdrawingverygeneral Of the48bHLHfamiliesknown,45arepresentinBilateria. S. roscoffensis belongs tothecladeCrucimusculata,whichincludes we detectedonly16families.Inthegenomeof , appear tohaveshortbranchesinallmolecular N. vectensis X. bocki S. roscoffensis S. roscoffensis is notinthisnumericrange.Infact, , n h ndrasaaye Tbe1) and thecnidariansanalysed(Table S. kovalevskii we foundforeachfamilythe and S. roscoffensis , H. sapiens, X. bocki for thepresence C. teleta encodes, in X. bocki and A. S. S. (Strotmann etal.,2011). we didnotobserveaspecies-specific expansioninthissubfamily (Krishnan etal.,2012). consequently lostorrarelypresentinmostmetazoanspecies cAMP receptormighthavebecomesomehowredundantand . Afterthediversificationofthoseinmetazoan, , andrhodopsininthecommonancestorof before thesplitofunikontsfromcommonancestor evolved fromsuchamoreprimitivereceptortype;theformertwo Frizzled, adhesionandrhodopsinclassesarethoughttohave sequence informationandtheAUGUSTUSgenepredictions. given asaveragesofboththepredictionsbasedongenomic that allthe Rhodopsin and1Dicty_CAR;atotalof304GPCRs(Table glutamate receptors,7Frizzled,21Adhesion/Secretin,258 (Table with thereference species,evenwiththexenoturbellids, inwhose acoel genome,whichrepresents averylownumberwhencompared We detectedfivemembersoftheadhesion/secretinfamily inthe humans, 6intheacoelsstudiedand7xenoturbellids(Table receptors (LagerströmandSchiöth,2008).With 11 membersin role incellsignallingandtissuepolarity, wheretheyfunctionasWnt one ofthemostevolutionarilyconservedbecauseitsfundamental any Smoothened-likereceptor. Interestingly, thefrizzledfamilyis Frizzled 9/10(supplementarymaterialFig. with Frizzled1/2/7,one5/8andtwomore with We alsofoundsixFrizzled-like receptors:threeclusteredtogether centralized nervoussystemand However, although GABA (AronowiczandLowe, 2006; Krishnanetal.,2013). have acentralizednervoussystemandlacksneuronsthatrelease eight GABA receptorsisobserved inS.kowalevski Conn, 2010).Itisremarkablethatasimilarspecificexpansionwith neuronal excitabilityintheCNSofvertebrates(Niswenderand that GABA receptorsplayarole insynaptictransmissionand greater numberthanthethreetypesfoundinhumans.Itisknown roscoffensis duplication ofgenomesspecifictovertebrates.Intriguingly, humans, althoughthismaybeadirectconsequenceofthe represents asimplificationcomparedwiththeeightthatfoundin detected threeglutamatemetabotropicreceptors(GMR);which In theglutamatefamily(seesupplementarymaterialFig. H. sapiens, to species-specificdiversification,whichisespeciallyremarkablein except forD.melanogaster representatives inallthefamiliesthanourreferencespecies, Adhesion andsecretinfamilies Frizzled family Glutamate family systems arestructurallysimplerthatvertebrates. than of Acoela;ithasanervoussystemmorphologicallymorecomplex is wherewefind this trendwithintheXenacoelomorphamonophyleticgroup,which from eachGPCRfamily(althoughnotinallcases).We alsoobserve vectensis, wenoticedthattheformerhas,ingeneral,fewermembers of genesinX.bocki Table The Dicty_CARdomaincorrespondstoacyclicAMP receptor. X. bocki

2). InthecaseofX.bocki

2 showsthatthetwospeciesstudiedhavefewer C. teleta The JournalofExperimentalBiology(2015)doi:10.1242/jeb.110379 X. bocki , butwithfewerGPCRsinallthefamilies. has sixγ S. roscoffensis and S. kowalevski and theS.roscoffensis rhodopsinvaluesare , comparedwith -aminobutyric acid(GABA)receptors,a N. vectensis. . ItisknownthatGPCRsaresubjected , despiteitbeingadivergent member S. roscoffensis , thecomplementswere:17 does nothaveanobviously Despite thehighercomplexity A. queenslandica

S4). We didnotdetect has aclearbrain,both , whichdoesnot

2). Note

S3) we and

2), N. S.

The Journal of Experimental Biology genomic sequenceandtheAUGUSTUSgenepredictions. and therearemany putativedefinitionsforsuch aword,especially phylum. We understandthattheterm‘complexity’ isaloadedone, structural complexityofthedifferent nervoussystemswithin the correlate themolecularcomplexity ofthesefamilieswiththe functionality ofnervetissue. The mainaimofthisstudywasto the bHLHsandGPCRs,involved inthespecificationand group, wehaveundertakenasystematic studyoftwosuperfamilies, for thefirsttimeusinggenomesequenceofmembers this al., 2004a;Raikovaet2004b;Achatz2013).Here, and systems (Raikovaetal.,2000a;Raikova2000b; et relatively simplemorphology, includingthatoftheirnervous The phylumXenacoelomorphaisformedofanimalswith a of performed onthedifferent families ofGPCRsfoundinthegenome the future,samedetailedanalysisof human orthologue;theytendtoformindependentgroupsinstead. In sequences weidentifiedin genome. Whatweobservedinthisstudyisthatmostofthe reference sequences,whichisthemostreliableandstudied issues, wealignedallmembersofthissubfamilywithhuman acoel sequencesareextremelydivergent. Inordertoresolvethese reference speciesgenomesarenotyetfullyannotated,whereasour the mostdifficult toanalyse.To complicateitfurther, someofthe The rhodopsinfamilyisthelargest andmostdiversified,thus analyses arenecessarytoconfirmthisfinding. family members,althoughinabasalposition.However, further clusters withsomemembersofthe arrow) wouldbeagoodcandidatesecretinrelativebecauseit Sr_orphan1_aug3sy.g27699.t1 (supplementarymaterialFig. whether thereisanysecretinhomologue.Infact,thegenewecall which ofthegenesdetectedbelongtoadhesionfamilyand Metazoa (Strotmannetal.,2011). Itwouldbeinterestingtoascertain are presentinmostbilaterians,butnotoldermembersofthe detected andanalysedthemembersofbothfamiliestogether. They they shareasimilar7TMcoresequence.Forthisreason,we (Bjarnadóttir etal.,2007). Fig. search, weassignedthisgene,Sr_GPR125(supplementarymaterial homologues GPR123,GPR124andGPR125.AfteraBLAST material Fig. genome wedetected21members.Ofthosefive(supplementary Note thatthevaluescorrespondingto al., 2012). numberrisesto683(Krishnanet ** Thenumberofhumanrhodopsinsvariesaccordingtothelevelstringencyutilizedinsearch.Inotherpublications * FromdeMendozaetal.(deal.,2014). Symsagittifera roscoffensis Xenoturbella bocki Nematostella vectensis* Amphimedon queenslandica* Species Table RESEARCH ARTICLE Homo sapiens* Saccoglossus kowalevskii* Conclusions Rhodopsin family Drosophila melanogaster* Capitella teleta* Secretin receptorsevolvedfromadhesionandtherefore X. bocki S5, fourtharrow),asaputativeorthologuetohumanGPR125 2. NumberofGPCRseachfamilyintheindicatedspecies .

S5), onlyoneclearlyclusteredwiththehuman S. roscoffensis X. bocki hdpisAhso/ertnGlutamate Adhesion/Secretin Rhodopsins 225 258 1049 147 297** 246 71 980 C. teleta and S.roscoffensis S. roscoffensis and do nothaveadirect H. sapienssecretin 5 21 58 64 48 22 18 39 should be rhodopsin numbersaretheobtainedasaveragesofpredictionsderivedfromraw

S5, first primordia. case, pavingthewaytodecipherevolutionof brain (Moroz,2012).Knowledgeofthegenomesisnow, inany putative ‘cephalizing’ trendsthatweobserveinthisandother these nervoussystems;andperhapsitwillprovideinsightintothe should giveusabetter, richerunderstandingoftheevolution activities andanalysisoftheirevolvingregulatoryinteractions circuits inthedifferent nervoussystems.Thestudyofgene have toreflectthefunctionalcomplexityorof Moreover, structuralcomplexity, perhapsjustsuperficial,doesnot that theviewwehaveofbothgenomesisstillincomplete. paradox requiresanddeservesfurtheranalysis.We arewellaware patterning anddownstreamsensoryfunctions.Thisputative nervous systemandthenumberofgenesinvolvedinboth an inversecorrelationbetweenthestructuralcomplexityof Xenoturbella the complementofgenes(anditsdiversity)ishigherin molecular datashowthatinthecaseoftwofamiliesanalysed, anterior endoftheanimal(closetoseveralsensorystructures).Our have acondensedbilobedbrainwithdenseneuropileinthemost Acoela, particularlythosebelongingtothecladeCrucimusculata, comprise anintraepidermalnetofnervecells,somemembers (mentioned above)haveshownthatwhereas members oftheCnidaria).Severalmorphologicalstudies complex than‘simpler’ neuronalnets(asseen,forinstancein ‘cephalized’ (centralized) nervoussystemsarestructurallymore external inputs.Thisisadefinitionthatwouldassume high degreeofinterconnectivityandacapacityforprocessing neuronal concentrationinonepoleoftheanimal,whichimpliesa narrow conceptofcomplexity:onethatreflectsthedegree in thecontextofbiologicalsystems.However, hereweuseavery 7.0. with AdobePhotoshopCS.Diagrams wereproducedusingAdobeIllustrator Images wereprocessedusingImageJ (Abramoff etal.,2004) and finished or aLeicaSPIImicroscope(Leica Microsystems,Weztlar, Germany). Confocal laser-scanning microscopywasperformed withaLeicaTCSSPE and Martinez(AchatzMartinez,2012). Immunostaining wasperformedfollowingtheprotocolsoutlinedinAchatz OR)]. antibody [AlexaFluorgoatanti-rabbit532(MolecularProbes,Eugene, antibodies (bothpreviouslypre-absorbed)andreactedwiththesecondary primary anti-synaptotagmin(dilution1:200)oranti-Elav1:800) Image acquisition, processing andpreparation acquisition,processing Image Immunohistochemistry MATERIALS ANDMETHODS 9 17 37 43 23 23 10 51 The JournalofExperimentalBiology(2015)doi:10.1242/jeb.110379 than inSymsagittifera rzldDcyCRTotal Dicty_CAR Frizzled 7 6 4 6 11 3 5 5 . Thiswouldmeanthatthereis S. roscoffensis 1 2 0 0 0 0 0 1 Xenoturbella was incubatedin seems to 304 1152 258 245 379 294 104 1076 625

The Journal of Experimental Biology also consideredand thetreeswereinferredbyNeighbour-Joining Tree bootstrap replicates (N100)waschosen.A neighbour-joining approach was of invariablesites(‘mPROTGAMMAILG’ option).Thebest treeoutof100 discrete gammadistributionofamong-site variationratesandaproportion (Stamatakis, 2014),withtheamino acidevolutionarymodelLGusinga Maximum-likelihood analysiswas carriedoutusingRAxML (v8.0.12) using Geneioussoftware(v5.3.6, Biomatters, http://www.geneious.com). options. Conservedregionsfromthose alignmentsweremanuallyselected 2008) withthe‘max’ iterate 1000and‘localpair’ (equivalenttoL-INS-i) the Pfamdatabasepreservingoriginalaccesscodes. sequences for and Satoh(GyojaSatoh,2013)referencewasusedforbHLHs.bHLH described bydeMendozaetal.(deal.,2014)whereasthe Gyoja also considered.GPCRreferencesequenceswereclassifiedinfamilies as bHLHs family, sequencesfrom case ofGPCRs,sequencesfromA.queenslandica 626 RESEARCH ARTICLE sapiens, against adatabaseofproteinsequencesfromdifferent species,including predictions foreachfamily, orsubfamilyinthecaseofGPCRs,werealigned alignment inordertoproducethepresentedphylogenetictrees.Then, final 2013).We havemanuallyfilteredthemoreinformativecolumnsof curated andalignedwithClustalW2(Larkinetal.,2007;McWilliam etal., The bestpredictionsforeachdomainsearchanddatasetweremanually annotation overgenomesequences(AUGs),forboth sequences assembledfromRNA-seqreads(ESTs), aswellgene Genome assembliesincludingscaffolds andcontigs(SCFs), Genome sequences,annotation andselection Phylogenetic analysis predicted (AUGs)proteinsforfurtheranalyses. filtered outfromthesetofgenomic(SCFs),transcriptomic(ESTs) and significant hitforanyofthepreviouslydescribedHMMmodelswerethen ‘domE 1e more power),‘E1e− parameters: ‘max’ (todisableallheuristics,whichentailslessspeedbut binding domain)fortheHLHfamily. hmmsearchwasrunwiththefollowing PF05462 (Dicty_CAR)fortheGPCRfamily;andPF00010(HLHDNA- Adhesion/Secretin), PF00003(7tm_3,Glutamate),PF01534(Frizzled)and 2014) hiddenMarkovmodels(HMM):PF00001(7tm_1),PF00002(7tm_2, used toscanthesequencesusingfollowingPfam(v27.0)(Finnetal., proteins fromAUGUSTUSpredictions.HMMER(v3.0)(Eddy, 2011) was from thosethreesets–ORFsgenomeandtranscriptomesequences specific proteindomainswasperformedonalltheaminoacidsequences transcript andproteinsequencesthatwereusedinthisstudy. A searchfor transcripts. Thedescribedannotationprotocolproducedthesetsofputative predictions alreadyincludedputativeproteinstranslatedfromthepredicted larger than20aminoacidsusingcustomPerlscripts;whereasAUGUSTUS sequences weretranslatedintoallpossibleopenreadingframes(ORFs) parameter setandallavailablehints.Genomictranscriptomicnucleotide hints. AUGUSTUSwasrunontheunmaskedgenomeusingoptimized results aswelltheRepeatMaskeroutputtablewerealsoconvertedto alignments wereconvertedtohintsforAUGUSTUS.PASA andCEGMA al., 2008).RNA-Seqreadsweremappedagainsttherepeatmaskedgenome, redundantly) andusedforoptimizingAUGUSTUSparameters(Stanke,et unmasked genomefrom which ranPASA (Haasetal.,2003)onassembledRNA-Seqdataandthe initial genesetwasgeneratedbyWebAUGUSTUS (Hoff andStanke,2013), of ultra-conservedcoreproteingenesfromtheunmaskedgenome.A second coordinates. CEGMA (v2.4)(Parraetal.,2007)wasusedtoproduceaset above mentionedcustomlibrary, andtoproduceatablethatlistsrepeat al., 2014)wasusedtoproducearepeat-maskedgenomeversionwiththe RepeatScout (v1.0.5)(Priceetal.,2005).RepeatMasker(v3.3.30)(Smit transcriptome assemblies,respectively. A repeatlibrarywasgeneratedusing (Xie etal.,2014)wereusedintheprotocolforgenomeand genome). SOAPdenovo(v2)(Luoetal.,2012)andSOAPdenovo-Trans (v1) (sequences willbepubliclyavailablealongwiththepublicationof bocki Full alignmentswereperformedusingMAFFT (v6.864b)(KatohandToh, , weredownloadedfromtheserverofUniversityGreifswald D. melanogaster, − 5’ (maximumdomainE-valuetoreportahit).Sequenceswith S. kowalevski 5’ (maximumtotalE-valuetoreportsequences)and Xenoturbella. Bothgenesetsweremerged (non- S. kowalevski and A. digitifera C. teleta , N. vectensis were directlydownloadedfrom and H. magnipapillatawere were considered;forthe S. roscoffensis and C. teleta. and In the H. X. jraótr .K,Giaddti,K,Igmnsn . iz,M . Fredriksson, M.A., Mirza, M., Ingemansson, K., Geirardsdóttir, T. K., Bjarnadóttir, ey . adn,A,Mrie,P n atnti,V. Hartenstein, and P. Martinez, A., Cardona, A., Bery, ey .adMrie,P. Martinez, F. and Guillemot, A. Bery, and D.S. Castro, N., Bertrand, the,W .adFth W. M. Fitch, and W. R. Atchley, auà .adRuot M. Riutort, and J. Baguñà, The authorsdeclarenocompetingorfinancialinterests. members oftheXenacoelomorphagenomeconsortium. Finally, weacknowledgethecontinuous,insightful,discussionswithall comments thatwereinstrumentalintheimprovementofsubmittedmanuscript. de Barcelona.We wouldliketothanktherefereesforsuggestionsand by ManelBoschandtheaccesstoCLSMMicroscopyfacilityofUniversitat process onanalysingandclassifyingtheGPCRs.We appreciatethesupportgiven databases. Moreover, A.deMendozawasespeciallyhelpfulthroughthewhole (Okinawa InstituteofScienceandTechnology) foraccesstotheirGPCRandbHLH thank specificallyAlexdeMendoza(IBECSIC-UPF, Barcelona)andFukiGyoja held attheWhitneyMarineLaboratory, St.Augustine,Florida.We wouldliketo which wassupportedbytheNationalScienceFoundation(NSF).TheMeeting This workwaspresentedatthe‘EvolutionofFirstNervousSystemsII’ meeting, Genome Projectconsortiumpaper. available alongwiththeforthcomingpublicationofXenacoelomorpha whole genome/transcriptomeoriginalsequenceswillbemadepublicly number assignedinthepreliminarygenome/transcriptomeanalyses.The S10. Allthesequencesincludedinourstudykeeporiginalreference columns canbefoundinFastaformatassupplementarymaterialFigsS6to further refinementsanddetailsaddedusingAdobeIllustrator7.0. and processedintoimageswithFigTree (v1.4.1)(Rambaud,2007),with Builder (njtreev1.9.1)(Li,2006).Finally, thecomputedtreeswereedited http://jeb.biologists.org/lookup/suppl/doi:10.1242/jeb.110379/-/DC1 Supplementary materialavailableonlineat PhD (APIF-UB)fellowshipfromtheUniversitatdeBarcelona. de Catalunya(2009-SGR-001018)werealsousedinthisproject.E.P.-A. hasa 32806) fundstheworkinlaboratoryofP. Martinez.FundsfromtheGeneralitat acoel collectiontrips.A grantfromtheSpanishMinistryofEconomy(BFU2012- Programme ‘ASSEMBLE’,whichcoveredsomeofthecostsassociatedtoour We wouldliketoacknowledge thegeneroussupportobtainedfromEU the manuscript.P.M. directedthewholeproject. and testingoftheanti-peptideantibodies.Allauthorscontributedtowriting immunochemical analysisappearinginthispaper. M.C.contributedtothedesign the genomicandphylogeneticanalysis.E.P.-A. andB.G.performedallthe over thegenomicandtranscriptomicsequences.E.P.-A. andB.G.performedall K.H. providedallgenemodels.J.F.A. didtheinitialscreeningoffunctionaldomains manuscript. A.P. generatedraw-sequencingdataandprovidedtheassemblies. All authorscontributedtothedesignofexperimentsandwriting baof .D,Mglas .J n a,S.J. Ram, and P. J. Magalhaes, M.D., Abramoff, caz .G n atnz P. Martinez, and J.G. Achatz, rnwc,J n oe C.J. Lowe, and J. Aronowicz, Competing interests Acknowledgements References Supplementary material Funding Author contributions caz .G,Cidn . avnoe,W,Tlr .adMrie,P. Martinez, and S. Tyler, W., Salvenmoser, M., Chiodin, J.G., Achatz, Evol. central nervoussystemofajuvenileacoel, protein-coupled receptors. H. B. Schiöth, and R. Zoologica regenerating nervoussystemofthe acoel specification ofneuralcelltypes. helix classoftranscriptionfactors. acoelomorph . Integr. Comp.Biol. 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