in the

www.elsevier.com/locate/gene

PhylogeneticrelationshipsoftheFox(Forkhead)genefamily

intheBilateria

FrancßoiseMazeta,Jr-KaiYub,DavidA.Liberlesc,LindaZ.Hollandb,SebastianM.Shimelda,*

aSchoolofAnimalandMicrobialSciences,TheUniversityofReading,P.O.Box228Whiteknights,ReadingRG66AJ,UK

bScrippsInstitutionofOceanography,UniversityofCaliforniaatSanDiego,LaJolla,CA,USA

cComputationalBiologyUnit,CentreforComputationalScience,UniversityofBergen,5020Bergen,Norway

Received15April2003;receivedinrevisedform28May2003;accepted29May2003

ReceivedbyR.DiLauro

Abstract

TheForkheadorFoxgenefamilyencodesputativetranscriptionfactors.ThereareatleastfourFoxgenesinyeast,16inDrosophilamelanogaster(Dm)and42inhumans.Recently,vertebrateFoxgeneshavebeenclassifiedinto17groupsnamedFoxAtoFoxQ[GenesDev.14(2000)142].Here,weextendthisanalysistoinvertebrates,usingavailablesequencesfromD.melanogaster,Anophelesgambiae(Ag),Caenorhabditiselegans(Ce),theseasquirtCionaintestinalis(Ci)andamphioxusBranchiostomafloridae(Bf),fromwhichwealsoclonedseveralFoxgenes.Phylogeneticanalyseslendsupporttothepreviousoverallsubclassificationofvertebrategenes,butsuggestthatfoursubclasses(FoxJ,L,NandQ)couldbefurthersubdividedtoreflecttheirrelationshipstoinvertebrategenes.WewereunabletoidentifyorthologsofFoxsubclassesE,H,I,J,MandQ1inD.melanogaster,A.gambiaeorC.elegans,suggestingeitherconsiderablelossinecdysozoansortheevolutionofthesesubclassesinthedeuterostomelineage.Ouranalysessuggestthatthecommonancestorofprotostomesanddeuterostomeshadaminimumcomplementof14Foxgenes.D2003ElsevierB.V.Allrightsreserved.

Keywords:Phylogeny;Evolution;Amphioxus;Ciona

1.Introduction

TheFoxgenefamilyencodestranscriptionfactorscon-taininganapproximately100aminoacidDNA-bindingdomainknownastheforkheaddomain(WeigalandJa¨ckle,1990;reviewedbyGranadinoetal.,2000).Thisdomainformsastructureknownasawingedhelix,suggestingtheFoxgenesarerelatedtothelargeassemblageofgenescontainingwingedhelixdomainsthatoccurinbotheukary-

Abbreviations:Ag,Anophelesgambiae;Bf,Branchiostomafloridae;BLAST,BasicLocalAlignmentSearchTool;Ce,Caenorhabditiselegans;Ci,Cionaintestinalis;Dm,Drosophilamelanogaster;Dr,Daniorerio;FKHRP1,ForkHeadRelatedPseudogene1;Fr,Fugurubripes;FREAC,ForkHeadRelatedActivator;Hs,Homosapiens;slp,sloppypaired;SMART,SimpleModularArchitectureResearchTool;Xl,Xenopuslaevis.

*Correspondingauthor.Tel.:+44-118-9875123x7084;fax:+44-118-9310180.

E-mailaddress:s.m.shimeld@reading.ac.uk(S.M.Shimeld).0378-1119/$-seefrontmatterD2003ElsevierB.V.Allrightsreserved.doi:10.1016/S0378-1119(03)00741-8

oteandprokaryotegenomes(Clarketal.,1993).Amongstthisassemblage,however,theFoxgenesformadiscretegrouprecognisableonthebasisofthehighdegreeofaminoacidsequenceidentityoftheirforkheaddomains(GajiwalaandBurley,2000).Foxgeneshavebeenidentifiedinmanyanimalsaswellasinyeastandotherfungi,butnotinplants.ThissuggestsanevolutionaryoriginoftheFoxgenefamilyinacladeofunicellularorganismsthatgaverisetoboththefungalandanimallineages(Baldauf,1999).Infungi,fourFoxgeneshavebeenidentified,whilecomparisonofmammaliangenomesshowsatleast34distinctorthologs,suggestingamajorexpansioninthecomplexityofthisfamilyduringanimalevolution.

AnearlyphylogeneticanalysisshowedthattheFoxgenescanbesubgroupedinseveralsubclasses(KaufmannandKnochel,1996).Subsequently,ananalysisincludingover130vertebrategenesand3invertebrategenesextendedthisclassification,defining15subclassesofFoxgenesnamedfromAtoO(Kaestneretal.,2000).Eachsubclasswaswellsupportedbybootstrapanalysis,butmostofthe

80F.Mazetetal./Gene316(2003)79–89

deeperrelationshipsbetweensubclasseswereunclear.Morerecently,twomoresubclasses,PandQ,weredefinedforatotalof17(Hongetal.,2001;Shuetal.,2001;Schubertetal.,2001).

Foxgeneshavealsobeenfoundinseveralinvertebratetaxa;however,asystematicapproachtotheirclassificationisnotyetavailable.Correspondingly,wehaveundertakenamolecularphylogeneticanalysisofMetazoanFoxgenes,includingFoxsequencesfromthebasalchordatesCionaintestinalis(Ci)(anascidian)andBranchiostomafloridae(Bf)(amphioxus)aswellasfromvertebratesandfromthreeprotostometaxawithsequencedgenomes(Drosophilamel-anogaster[Dm],Anophelesgambiae[Ag]andCaenorhab-ditiselegans[Ce]).OurresultsshowthatmostinvertebrateFoxgenescanbeascribedasorthologoustospecificcladesofvertebrategenes,andthatthesecladestypicallycorre-spondtothepreviouslydefinedvertebratesubclasses.AfewinvertebrateFoxgenesdonotshowarobustrelationshipwithanyspecificcladeofvertebrategenes,andsomecladesofchordategenesdonothaveprotostomeorthologs.Over-all,weidentify14cladesofFoxgeneswithmembersinbothchordatesandecdysozoans,settingthisasthemini-mumFoxgenecomplementofthecommonancestorofprotostomesanddeuterostomes.Wealsoexaminetheevi-dencefortheroleofselectioninFoxsubclassdiversificationandidentifycandidatesitesunderlyingtheadaptiveradia-tionofFoxgenesubclasses.

2.Materialsandmethods2.1.Acquisitionofsequences

Sequenceswereacquiredfromthefollowingdatabases:D.melanogaster:http://flybase.bio.indiana.edu/(17sequen-ces);C.intestinalis:http://ghost.zool.kyoto-u.ac.jp/(forcDNAsequences)andhttp://www.jgi.doe.gov/(forgenomicsequences)(11sequencesintotal).Wenote,though,thatourlistofC.intestinalissequencesisunlikelytobeexhaustive,andthatmoreFoxgenesarelikelytobepresentinthistaxonasrevealedbytheongoinggenomeassemblyprocess(N.Satoh,personalcommunication).Fugurubripes:http://www.jgi.doe.gov/andhttp://www.hgmp.mrc.ac.uk(release1)(24sequences).C.elegans(14sequences),Daniorerio(Dr),Xenopuslaevis(Xl)(16sequences),Homosapiens(Hs)(36sequences)andyeast(4sequences):http://www.ncbi.nlm.nih.gov/.A.gambiae:http://www.ensembl.org/Anopheles_gambiae/(20sequences).InitialsearchesofsequenceannotationbykeywordweresupplementedwithBLASTXandBLASTPsearchestoidentifyincorrectlyannotatedorunannotatedsequences.WealsoconsultedtheSimpleModularArchitectureResearchTool(SMART)database(Schultzetal.,2000)toensurethemethodsusedhereforproteindomainidentificationhadnotidentifiedadditionalsequencesthatoursearcheshadmissed.Com-parisonofF.rubripesandD.reriogenes(notshown)

revealedthatmostD.reriogeneshadapparentorthologsinF.rubripes.Similarly,allthemurineFoxgeneswefoundwereorthologoustoH.sapiensgenes.ThereforeweomittedD.rerioandmouseFoxgenesfromouranalysis,astheywouldnotaddfurtherinsightintorelationshipsbetweeninvertebrateandvertebrategenes.TheexceptiontothisistheD.reriogeneDrFoxH,whichisincludedintheanalyses,aswecouldnotfindaF.rubripes(Fr)FoxHortholog.SeveralamphioxusFoxgeneshavebeenpreviouslyidenti-fied(Shimeld,1997a;Toressonetal.,1998;Schlakeetal.,2000;Yasuietal.,2001;MazetandShimeld,2002;Yuetal.,2002a,b,2003).Tothesesequences,weaddedseveralthathavebeenrecentlygeneratedinourrespectivelaboratories,giving11intotal.Fullcharacterisationoftheseadditionalgeneswillbepublishedelsewhere.2.2.Phylogeneticanalyses

FoxgenesfromalltaxawerealignedwithCLUSTALX(Thompsonetal.,1997)toidentifytheforkheaddomainandanyadditionalconserveddomainswhichmightbepresent.Theforkheaddomainprovedtobetheonlyconsistentlyalignablesectionofthesequencesinthedataset;hence,thesewerecollatedandrealignedforphylogeneticanalysis.Besidestheforkheaddomain,someFoxproteinsalsohaveanotherconservedpeptidemotifsimilartotheeh-1domainencodedbyseveralhomeoboxgenes(SmithandJaynes,1996;Shimeld,1997b).However,therelativepositionofthiseh-1domaintotheforkheaddomainisvariableandtheconsensusweak(datanotshown).Withoutfunctionalanal-ysis,itisdifficulttodetermineifthisrepresentsdivergenceofaprimitivefunctionaldomain,orconvergentevolutionofsimilarsequencewithorwithoutafunctionalrole.Conse-quently,wedonotbelievethatthiseh-1-likedomainisareliablephylogeneticcharacter,andwehavenotuseditinouranalyses.

PreliminaryphylogeneticanalysisoflargedatasetswasconductedusingNeighborJoiningimplementedbyCLUS-TALX.Incompletesequences,probablepseudogenes(e.g.,ForkHeadRelatedPseudogene1[FKHRP1]fromH.sapiens)andhighlydivergentsequenceswithanincompleteforkheaddomain(e.g.,crg-1fromD.melanogaster)werenotincludedinthephylogeny,sincepreliminarysequencecomparisonssuggestedthattheseresultedfromrelativelyrecentlineage-specificevolutionaryeventsandarethereforenotrelevanttodeeperphylogeneticrelationships.PairwiseKa/KsratioswerecalculatedusingboththeNG(NeiandGojobori,1986)andtheYN(YangandNielsen,2000)methods.Covarion-basedrelativeratetestswereperformedonproteinsequencestodetectchangesintheselectivepressuresactingonthesegenes(Gu,1999;GuandVanderVelden,2002).Siteswereidentifiedforsubclass-specificevolutioniftheyshowedposteriorprobabilitiesgreaterthan0.5formorethanhalfofthesignificantdivergencesfortherespectiveclass.TreeswereviewedandmanipulatedwithTreeViewPPC(Page,1996).

F.Mazetetal./Gene316(2003)79–8981

3.Resultsanddiscussion

3.1.MolecularphylogeneticanalysisoftheFoxgenesToexaminetherelationshipsbetweeninvertebrateFoxgenesandthevertebrateFoxgenesubclasses,weunder-tookamolecularphylogeneticanalysisoftheFoxfamily.Initially,weincludedalltheavailableFoxsequencesfromC.intestinalis,B.floridae,C.elegans,D.mela-nogaster,A.gambiae,H.sapiens,F.rubripesandX.laevis,exceptforasmallnumberofhighlyderivedgenes(seeMaterialsandmethods).WealsoincludedthefourFoxgenesfromyeast,whichformtwopairsofrelatedgenes.WefoundnoevidencethateitherpairismorecloselyrelatedtoonesubsetofanimalFoxgenesthananyother(notshown).Consequently,theyeastgenesarelikelytobeequallyrelatedtoalloftheanimalgenes.Infurtheranalyses,weexcludedthefungalFoxgenes,duetotheirhighlevelofdivergencefromtheanimalFoxgenes.

ToexaminetherelationshipofbasalchordateFoxgeneswithputativevertebrateorthologs,weanalysedvertebrate,C.intestinalisandB.floridaesequences.WealsoincludedD.melanogastersequences,togiveapreliminaryindicationofwhethercladesofchordategenescouldbeextendedtoincludeecdysozoanorthologs.TheresultsofthisanalysisareshowninFig.1.Thisanalysis,likepreviousones(Kaestneretal.,2000),showedthatmostvertebrategenesfellintowell-definedcladessupportedbyhighbootstrapvalues.ThisincludedallF.rubripes,D.rerioandX.laevisgenes,whichwereallcloselyrelatedtoatleastoneH.sapiensgene(Fig.1anddatanotshown).ThissuggestsadditionalFoxsub-classesdonotremaintobediscoveredinhumans.MostFoxgenesofbothbasalchordatesandD.melanogasterwereassociatedwithspecificvertebratecladeswithreasonablebootstrapsupport(discussedindetailinSec-tions3.3–3.13).

ToexaminetherelationshipsofallprotostomeFoxgeneswithchordateFoxgenes,weconstructedastrippeddatasetofhumangenes,includingonehumanmemberofeachofthecladesidentifiedinFig.1.Wheremultiplehumangeneswerepresentinaclade,thegeneusedwasselectedonthebasisofitssubclassnumber(thatis,weusedthegenenamedas1,HsFoxD1,HsFoxE1,etc.)asthephylogenyindicatedallmembersofthecladewereequallyvalidchoicesforsuchananalysis.Wealsoincludedall‘orphan’chordategeneswhichcouldnotbeascribedtoaspecificclade.WealsoincludedallC.elegans,A.gambiaeandD.melanogasterFoxgenes(withtheexceptionthatweincludedonlyoneofthegenepairsslp1/slp2andfd4/fd5,asthesehavebeenpreviouslyshowntoresultfromrelativelyrecenttandemduplications).Thisdatasetallowsrelationshipsbetweeninvertebrateandvertebrategenestobeclearlyvisualised.TheresultsoftheanalysisareshowninFig.2.

3.2.ClassificationofvertebrateandinvertebrateFoxgenesVertebrategeneshavebeenpreviouslycategorisedintosubclassesonthebasisofamolecularphylogeneticanalysis.Essentially,eachsubclasswasamonophyleticgroupofchordategenesseparatedfromotherFoxgenesbyhighbootstrapvalues.Mostofthesesubclassescontainedmul-tipleFoxgenesfromseveraldifferentspecies,representingamixtureoforthologousandparalagousrelationships.Weexaminedourtreestoseeifecdysozoanandbasalchordategenescouldbeascribedtoindividualvertebratesubclasses.Ifbasalchordategenescanbeascribedasorthologoustoavertebratesubclass,wecanconcludethesubclassevolvedasanindependentcladeofgenespriortotheradiationofcrownvertebratesandwasprimitivelypresentinallverte-brates.Ifanecdysozoangenecanbeascribedasortholo-goustoavertebratesubclass,wecanconcludethesubclassevolvedasanindependentcladeofgenespriortotheradiationofcrownbilateriansandwasprimitivelypresentinallbilateriananimals.

Formostofvertebratesubclasses,suchrelationshipswerediscernable.Insomeinstances,multipleinvertebrategeneswerefoundtobeorthologoustospecificcladesofvertebrategeneswithinasubclass.Intheseinstances,wesuggestsubdividingthesubclasstoindicatetheserelation-ships.Finally,severalvertebratesubclassesdidnothaveidentifiableorthologsinsomeorallofthetaxaexamined,andonerobustcladeofinvertebrategenesdidnothaveanorthologinvertebrates.Intotal,weidentified14cladesofgeneswithrepresentativesinchordatesandecdysozoans,definingthisastheminimumcomplementofFoxgenesinthecommonancestoroftheBilateria.Adetailedclade-by-cladedescriptionofourfindingsfollowsinSections3.3–3.13.Thenumbersinbracketsrepresentpercentageboot-strapsupportfromFigs.1and2,respectively.Ifonlyasinglevalueisgiven,thisisfromFig.1,unlessotherwisestated.TheseresultsaresummarisedinFig.3.3.3.FoxAtoFoxG

Thegenesinthesesevensubclasseshavebeencompar-ativelywellstudied,andmostoftherelationshipswedetectedinphylogeneticanalyseshavebeenpreviouslydeterminedonthebasisofdirectsequencecomparisonsorsmallerphylogeneticanalyses.WenotedmonophyleticgroupingsofFoxA(99%/100%),FoxB(87%/97%),FoxC(100%/100%),FoxD(99%/100%),FoxF(99%/98%)andFoxG(90%/91%),eachincludingatleastonebasalchordateandecdysozoanortholog.VertebrateFoxEgeneswerealsomonophyleticinFig.2(91%).NoecdysozoanFoxEortho-logswereidentified.3.4.FoxHandFoxI

PutativechordateFoxHgenesgroupedwithstrongsupport(97%).However,noecdysozoanFoxHcandidates

82F.Mazetetal./Gene316(2003)79–89

F.Mazetetal./Gene316(2003)79–8983

Fig.2.UnrootedneighborjoiningtreecontainingsequencesfromC.elegans(Ce),D.melanogaster(Dm),A.gambiae(Ag)andH.sapiens(Hs).Onlyonegeneofeachsubclasshasbeenselectedforhumangenes.Subclasseswithgenespresentinvertebratesplusatleastoneecdysozoanareshaded,togetherwiththeassociatedbootstrapsupportvalue.Numbersrepresentpercentagebootstrapsupportfortheadjacentnode,andvalueslowerthan60%arenotshown.H.sapiens,C.elegansandD.melanogastergenesarereferredtobygenename;foralistofaccessionnumbers,pleaseseeTable1.A.gambiaegenesarereferredtobyanabbreviatedgenename.Foralistoffullgenereferencenumbersforthesegenesanddetailsonhowtoaccessthesequences,pleaserefertoTable1.

wereidentified.Similarly,chordateFoxIgenesgroupedstrongly(100%),butecdysozoanFoxIgeneswerenotidentified.3.5.FoxJ

ouramphioxussequencegroupedwithFOXJ1(100%),whiletheC.intestinalissequencegroupedwithFOXJ2(99%).Thesetwocladesgroupconsistentlyassistercladesinourphylogenies,butwithlimitedsupport.3.6.FoxK

NoputativeecdysozoanFoxJgeneswereidentified.Interestingly,ourphylogeniessuggestedtheFoxJcladeincludedtwogenesbeforetheradiationofchordates,since

OurphylogeneticanalysisgavestrongsupporttothesubclassificationofD.melanogasterLD13167andA.gam-

Fig.1.NeighborjoiningtreecontainingsequencesfromB.floridae(amphi),C.intestinalis(CiorGCi),D.melanosgaster(Dm),F.rubripes(Fr),H.sapiens(Hs),X.laevis(Xl).ThesingleD.reriosequenceisindicatedbytheprefixDr.Numbersrepresentpercentagebootstrapsupportfortheadjacentnode.Onlybootstrapsupportvaluesgreaterthan60%areshown.Shadedboxesindicateindividualsubclassesandthebootstrapvaluessupportingtheirdefinition.Thiswasanunrootedanalysis;however,forclarity,thedepictedtreehasbeendrawnasrootedwiththeFoxPsubclass.

84F.Mazetetal./Gene316(2003)79–89

Fig.3.Foxgenedistributionbysubclassinthetaxaanalysedaspartofthisstudy.Filledboxesindicatethatamemberofthatsubclasshasbeendetectedintheindicatedspecies.Forsimplicity,onlyhumangenesareshownasrepresentativeofvertebrates.NotetheabsenceinallthreeecdysozoanlineagesofFoxE,H,I,J,MandQ1orthologs.

biaeAg16550asFoxKorthologs(100%/96%).Ag3502isalsolikelytobeamemberofthissubclass(82%inFig.2),However,wedidnotidentifyaFoxKgeneinanyotherinvertebratetaxon.3.7.FoxL

VertebrateFoxL2groupswithA.gambiaeAg59(99%inFig.2)andtheC.intestinalislocusWno743d17(100%).Amongstthechordates,FoxL1orthologswereidentifiedonlyinvertebrates(99%).TheA.gambiaesequenceAg446groupswithFoxL1inFig.2withmodestsupport.ExaminationofsequencealignmentsshowsthatAg446isorthologoustoD.melanogasterfd2,asbothgenesshareauniquederivedindelintheforkheaddomain(notshown).WethereforeconcludeAg446andfd2areprobablyorthologoustoFoxL1.Consequently,wesuggestsplittingtheFoxLsubclasstoreflecttheobser-vationthatbothFoxL1andFoxL2haveorthologsinecdysozoans.3.8.FoxM

OuranalysesonlyidentifiedaFOXMsequenceinthehumangenome.However,wenoteFoxMgeneshavebeenisolatedfromothervertebrates(forexample,mouseandrat,accessionnumbersO08696andP97691,respectively,andthechickenEST603956051F1availablefromhttp://www.chick.umist.ac.uk/).3.9.FoxN

TheFoxNgenesresolvedintotwoclades,onewithFOXN1andFOXN4relatedtoA.gambiaeAg2273andAg2274,D.melanogasterjumuandamphioxusBfFoxN1(100%/100%),andasecondwithFOXN2andFOXN3

relatedtoD.melanogasterches-1andA.gambiaeAg8820(100%/100%).Consequently,wesuggestsplittingtheFoxNsubclasstoreflectthisseparation.NoC.elegansgeneswereassociatedwiththeseclades.AsingleX.laevisandasingleF.rubripesFoxNgenefelloutsidetheseclades(Fig.1);however,withoutmoresequencesinthissubclass,itisdifficulttointerpretthisresult.ThetwoFoxNcladesconsistentlyappearedassistercladesinourphylogenies(87%inFig.2).3.10.FoxO

TheC.elegansgenedaf-16(whichincludestwoseparateforkheaddomains;Oggetal.,1997)andthreeverysimilarA.gambiaegenes(Ag11033,Ag11036,Ag6490)andD.melanogasterQ95V55(cg3143)groupedrobustlywithvertebrateFoxOgenes(100%/99%).3.11.FoxP

TheD.melanogastersequencecg16899,theA.gambiaesequenceAg14855andtheC.eleganssequenceF26D12.1groupedrobustlywiththevertebrateFoxPgenes(100%/100%);however,wedidnotidentifyaFoxPorthologinotherinvertebrates.3.12.FoxQ

TheprototypevertebrateFoxQsubclassgenewasthehumangeneHFH11.Inadditiontothis,wehaveidentifieddefinitiveFoxQgenesinF.rubripesandC.intestinalis(100%).Noecdysozoangenesgrouprobustlywiththisclade.However,asecondcladeofgenesappearsinourtreesthatincludesD.melanogaster,A.gambiaeandC.elegansgenes(100%inFig.2)andC.intestinalisGCiWno740j23(66%)andB.floridaeAmphiFoxQ2

F.Mazetetal./Gene316(2003)79–89

Table1

SubclassmembershipforinvertebrateandH.sapiensFoxgenesSubclassFoxA

SpeciesH.sapiensH.sapiensH.sapiens

D.melanogasterA.gambiaeaC.elegansC.intestinalisB.floridaeB.floridaeH.sapiens

D.melanogasterD.melanogasterA.gambiaeA.gambiaeC.elegansC.intestinalisB.floridaeH.sapiensH.sapiens

D.melanogasterA.gambiaeB.floridaeH.sapiensH.sapiensH.sapiensH.sapiens

D.melanogasterA.gambiaeC.elegansC.intestinalisB.floridaeH.sapiensH.sapiensH.sapiensC.intestinalisB.floridaeB.floridaeH.sapiensH.sapiens

D.melanogasterA.gambiaeC.elegansC.intestinalisB.floridaeH.sapiens

D.melanogasterD.melanogasterD.melanogasterC.elegansA.gambiaeB.floridaeH.sapiensC.intestinalisH.sapiensC.intestinalisH.sapiensB.floridaeH.sapiensC.intestinalisH.sapiens

D.melanogaster

Currentname(s)

FOXA1(HNF-3a)FOXA2(HNF-3h)FOXA3(HNF-3g)forkhead,fkhAg19661Ce-fkh1CiFoxA5AmHNF-3-1AmHNF-3-2FOXB1fd4fd5Ag394Ag2511lin-31

GciWno407-j18AmphiFoxB

FOXC1(MF1,FKHL7)FOXC2(MFH1)fd1

Ag11032AmphiFoxC

FOXD1(FREAC4)FOXD2(FREAC9)FOXD3

FOXD4(FREAK5)fd3Ag8042unc-130

CiFoxD,GciWno586o10AmphiFoxDFOXE1(TITF2)FOXE2FOXE3

GciWno830m18AmphiFoxE4AmphiFoxE5FOXF1FOXF2

biniou,DMFoxFAg11090F26B1.7CiFoxFAmphiFoxF

FOXG1(BF1,HBF2)slp1slp2cg9571

CeT14G12.4Ag351

AmphiBF-1

FOXH1(FAST1)GciWno152a23

FOXI1(FREAC6,HFH3)GciWno743d17FOXJ1AmphiFoxJFOXJ2Cieg45o16

FOXK1a-c(ILF1-3)LD16137

85

AccessionnumberorsourceofsequenceaU39840NM_021784L12141J03177

ENSANGG00000019661U51163AF002988X96519Y09236AF071554P32028P32029

ENSANGG00000020221ENSANGG00000002511L11148

www.jgi.doe.govAJ506162AF048693Y08223P32027

ENSANGG00000011032FMandSMS,unpublishedU59831AF042832L12142U13223Q02360

ENSANGG00000008042NM_064010

www.ghost.zool.kyoto-u.jpAAN03853U89995X94553U42990

www.ghost.zool.kyoto-u.jpAAK85731

FMandSMS,unpublishedU13219U13220AAK97051

ENSANGG00000011090AAB37792

FMandSMS,unpublishedFMandSMS,unpublishedX74143,X74142,X74144P32030P32031

www.flybase.bio.indiana.eduAAA82436

ENSANGT00000024640AF067203AF076292

www.ghost.zool.kyoto-u.jpU13224

www.jgi.doe.govU69537

JK-YandLZH,unpublishedAF155132

www.ghost.zool.kyoto-u.jpU58196

www.flybase.bio.indiana.edu

(continuedonnextpage)

FoxB

FoxC

FoxD

FoxE

FoxF

FoxG

FoxHFoxIFoxJ1FoxJ2FoxK

86

Table1(continued)SubclassFoxKFoxL1

SpeciesA.gambiaeA.gambiaeH.sapiens

D.melanogasterA.gambiaeH.sapiensA.gambiaeC.intestinalisH.sapiensH.sapiensH.sapiens

D.melanogasterA.gambiaeA.gambiaeB.floridaeH.sapiensH.sapiens

D.melanogasterA.gambiaeH.sapiensH.sapiensA.gambiaeA.gambiaeA.gambiaeC.elegansH.sapiensH.sapiensH.sapiens

D.melanogasterA.gambiaeC.elegansH.sapiensC.intestinalisD.melanogasterA.gambiaeC.elegansC.intestinalisB.floridaeH.sapiens

D.melanogasterA.gambiaeA.gambiaeC.elegansC.elegansC.elegansC.elegansC.elegans

F.Mazetetal./Gene316(2003)79–89

Currentname(s)

Ag3502Ag16550FOXL1fd2

Ag17877FOXL2Ag10771

GciWno608e22FOXM1

FOXN1(WHN)FOXN4jumuAg2273Ag2274BfFoxN1

FOXN2(HTLF)FOXN3(CHES1)ches-1Ag8820

FOXO1a,b(FKHR,FKHRP1)FOXO3a,b(FKHRL1,FKHRL1P)Ag11033Ag11036Ag6490daf-16FOXP1FOXP2FOXP3cg16899Ag14855F26D12.1

FOXQ(HFH11)Ciad036o10cg11152Ag410

C25A1.2(FKH-10)CiGCiWno740j23AmphiFoxQ2FREAC10cg32006Ag16120Ag14807PES-1

B0286.5(FKH-6)F40H3.4

C29F7.4(FKH-3)K03C7.2

AccessionnumberorsourceofsequenceaENSANGG00000003502ENSANGG00000016550U13225Q02360

ENSANGG00000017877AF301906

ENSANGG00000010711www.jgi.doe.govU74612Y11746AF425596NM_079578

ENSANGG00000002273ENSANGG00000002274JK-YandLZH,unpublishedU57029U68723AJ252199

ENSANGG00000008820U02310

NM_001455,AF032887ENSANGG00000011033ENSANGG00000011036ENSANGG00000006490AF020342AF250920AAL10762Q9BZS1AAF54432

ENSANGG00000014855NP_500833AF153341

www.ghost.zool.kyoto-u.jpAAM18014

AAAB01008846_410CAB02761

www.jgi.doe.govAY163864AF042831

www.flybase.bio.indiana.eduENSANGG00000016120ENSANGG00000014807Q27253AAA80692AAC67430CAB07324NP_508664

FoxL2

FoxMFoxN1/4

FoxN2/3

FoxO

FoxP

FoxQ1FoxQ2

Orphans

A.gambiaegenesarerepresentedbyENSEMBLreferencenumbers,excepttheFoxQ2genewhichisnotannotatedandrepresentedbyitsscaffoldcoordinates.Bothcanbeaccessedusingthesearchfacilityatwww.ensembl.org/Anopheles_gambiae.

a(92%).Sincegenesfromoneorganism(C.intestinalis)arepresentinbothclades,thesimplestinterpretationistosplittheFoxQgenesintotwosubclasses.Wepropose(assuggestedbyYuetal.,2003)thatthesebenamedFoxQ1andFoxQ2respectively,asthesenamesareconsistentwithnomenclatureinpublishedliterature.Insummary,FoxQ1geneshavebeenidentifiedinchordatesbutnotothertaxa,whileFoxQ2geneshavebeenidentifiedinecdysozoansandbasalchordates,butnotvertebrates.

3.13.Orphangenes

Inadditiontotheseclades,therewereseveralgeneswhoserelationshipstovertebratesubclasseswecouldnotresolve.ThesearetheC.eleganssequencesPES-1,B0286.5,F40H3.4,C29F7.4andK03C7.2,thehumangeneFREAC10andtheA.gambiaesequenceAg14807.Further-more,theD.melanogastersequencecg320006andA.gambiaesequenceAg16120areorthologs(99%inFig.2),

F.Mazetetal./Gene316(2003)79–8987

butwedidnotdetectsequencesorthologoustotheseinanyothertaxon.Intotal,however,theseorphangenesmakeupasmallproportionoftheFoxgenecomplementofeachtaxon.

Insummary,ourphylogeneticanalysisallowedustoascribemostinvertebrateFoxgenestoaspecificvertebratesubclasswithreasonablebootstrapsupport.Thesesub-classesrepresent‘orthologygroupings’suchthatweinferallthegenesinasubclassaredescendedfromasinglegeneinthecommonancestorofthetaxainwhichthatsubclasshasbeenidentified.AschematicrepresentationofsubclassdistributioninthetaxaexaminedisshowninFig.3.Table1liststhehumanandinvertebrategeneswehaveassignedtoeachsubclass,coupledwithrelevantaccessionnumbers.

3.14.Subclassespresentinchordatesbutmissinginothertaxa

Asdiscussedabove,wehavenotfoundmembersinothertaxaofseveralofthesubclassesdefinedinvertebrates.Currently,therearenoecdysozoangenesinthesubclassesE,H,I,J,MandQ1.Severalpossibilitiesmightexplainthis:

1.Thedatamaybemissing.Weconsiderthisunlikelyintaxaforwhichcompleteornear-completegenomesequencesandadvancedassembliesareavailable.

2.Thesubclassesmayhavebeenlostbyindividuallineages.3.Thegeneswehavenotbeenabletoassigntospecificsubclassesmaybemissingorthologsnowunrecognisableduetotheirlevelofdivergence.

4.Somesubclassesmayhaveevolvedspecificallyinthechordatelineage.Newmembersofgenefamiliescanevolvebyduplication,andunderthisscenario,duplica-tionwouldhavebeenfollowedbyrapiddivergenceofonegene,obscuringtheoriginalrelationshipandpullingthatgene(andthesubclassthatevolvedfromit)basallyinphylogeneticanalyses.Toinvestigatethispossibilitymoreclosely,weexaminedourdatasetsforevidenceofadaptivesequencechangeinspecificlineages.3.15.EvidencefortheactionofselectioninFoxgeneevolution

First,wetestedforevidenceofadaptiveevolutioninoursequencedatasets.UsingthepairwiseNei–Gojobori(NeiandGojobori,1986)methodtocalculatetheratioofnonsynonymoustosynonymousnucleotidesubstitutionrates(Ka/Ks)betweengenesinthedataset,wefoundthatmanyofthepairwisecomparisonsnotsurprisinglyshowedsaturationofKs.Ofthosecloserrelationshipsthatdidnot,therewasevidenceforasmallnumberofeventsofpositiveselectionorreducedfunctionalconstraintsanalysisofthefoursuchinstancesinthefourhumangenes(D2/A2;E1/F2;N4/E1;N4/E2)didnotindicate

phylogeneticsupportforrecentgeneduplicationsandmayindicateevidenceforKshavingbeenunderestimatedratherthantruepositiveselection.KsunderestimationcanbecausedbynonrandomGCusage,codonbias,orothersimilargenome-levelevents(Liberles,2001).RepeatingtheanalysiswiththemaximumlikelihoodapproachofYangandNielsen(2000)didnotsupportpositiveselectivepressuresinthesecases,butdidforothercomparisonsinvolvingsubclassP,alsoatveryhighKs.

WhileDNA-basedmethodstodetectpositiveselectivepressuresaresubjecttosaturationandgenomicphenomena,protein-basedmethodscanbetterbeusedtodetectselectivepressuresleadingtowardsalteredproteinfunctionatlongevolutionarydistances.Thesemethodshavepreviouslybeenimplementedtostudyfunctionaldivergenceingenefamiliesofelongationfactorsandofleptin(Gaucheretal.,2001;SiltbergandLiberles,2002).

Onemethodforthisanalysis,Diverge,looksforsite-specificshiftsinsubstitutionratesbetweencladeswheredifferentfunctionsareidentified,usingarelativeratetest(Gu,1999;GuandVanderVelden,2002).GroupswithatleastfoursequencesfromsubclassesP,O,N1/4,N2/3,F,Q1,I,D,E,A,B,CandGwereselectedfromthetreeinFig.1.EvidenceforsignificantshiftsinsubstitutionpatternsisseenbetweenmanysubclassesinTable2a.Aminoacidpositionswithposteriorprobabilitiesgreaterthan0.5inmorethanhalfofthesignificantcomparisonsforthatsubclassareindicatedinTable2b.Thosewithposteriorprobabilitiesgreaterthan0.5inallsignificantcomparisonsareindicatedinbold.Suchresiduesarecandidatesforhavingfunctionalrolesintheselectivedivergenceofsubclassesandmayrepresenttheresiduesresponsibleforsubclass-specificactivity.InTable2b,residuesmarkedinitalicsappeartobefacingtheDNAbindingsurfaceinthecrystalstructureofGajiwalaetal.(2000)(PDBentry1DP7)andarecandidatesforselectivedivergenceinDNAbindingspecificity.Position30insubclassEismarkedinbothboldanditalicsandwouldbeaparticularlystrongcandidateforfunctionalshifts.ThispositionisalsoshiftinginsubclassesP,O,N1/4,N2/3,A,andC.ThepositionisvariablebetweenSerandAlainsubclassE,anddifferentsubclassesuseSer,Ala,Thr,Asn,Cys,orarevariablebetweentheseaminoacids,mostlysmallpolaraminoacidswithdifferenthydrogenbondingfunctionality.Covarionprocessesmaybeinoperation,asotheraminoacidpositionsshowsimilarpatternsofvari-ation.UnravelingtheroleofindividualandcoordinatedsubstitutionsafterasdeterminantsoffunctionaftergeneduplicationintheFox(Forkhead)genefamilyremainsanimportantchallenge.

4.Conclusions

4.1ThemajorityofbilaterianFoxgenesshowclear,

orthologousrelationshipswithFoxgenesinothertaxa.

88F.Mazetetal./Gene316(2003)79–8952.0F90C.09812..00FF7932B..00398311...000FFF377481A...00068241202....0000FFFF77085604E....00001351533331.....00000FFFFF8928627521D.....00000623166203221......000000FFFFFF804534300351......I00000032268723022240.......0000000FFFFFFF008492018016200Q.......0000000se10714855ti22132122s........00000000taFFFFFFFFse86104056t24323542a........rF00000000noituti409146026t322222202s.........b000000000usFFFF.dg3FFFFFl/844858603on2226742402bitfN.........000000000niihsdefto2714358864ae5232131122cic..........n0000000000dnedi4FFFFFFFFFFiv/2670095124ere17759106635arN..........0000000000oroefzsess57858925802moa24233222132r...........flc00000000000tbuFFFFFFFFFFFnes12254086047r67037971062enfO...........fe00000000100iedwytletbn732452480171ad230233212222ce............i000000000000friaFFFFFFFFFFFFnpgmi350506557350so050713231517c............sP000000000000nsoaswiraÃpM43//121mQoONNFQIDEABCGCTable2b

Residueswithposteriorprobabilitiesgreaterthan0.5ingreaterthanhalfofthesubclassspecificcomparisonsareindicatedSubclassResiduesP14,23,25,30,31,34,39,40,45,53,60,61,74,76,77,79,81

O

12,13,14,19,20,21,22,23,24,25,26,27,28,30,31,34,36,37,38,39,40,41,42,44,45,46,47,48,49,50,51,52,53,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,102,103

N1/4

12,13,14,19,20,22,23,24,25,26,27,28,30,31,34,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,

74,

75,

76,

77,

78,

79,

80,

81,

82,102,103

N2/314,21,22,25,28,30,34,36,39,43,44,45,46,47,48,49,50,51,53,59,60,61,62,76,81,82,102

F27,36,46,51Q125,34,48,77I14,21,24,25,26,27,31,34,36,40,44,45,46,47,48,51,53,59,76,77,80,81,103

D21,24,31

E14,21,26,28,30,31,36,44,45,46,49,52,53,60,61,62,82,103A

12,13,14,19,20,21,22,23,24,25,26,27,28,30,36,37,38,39,40,41,42,44,45,47,49,51,52,53,59,61,63,64,65,66,67,68,69,70,71,72,73,74,

75,76,77,78,80,81,102,103B31,39,40,46,51,53,59,61,76C26,27,28,30,31,34,

36,39,44,

46,47,51,53,61,

79,82

G

28,

31,40,60

Thosewithposteriorprobabilitiesgreaterthan0.5inallcomparisonsareindicatedinbold,whilethosefacingtheDNAbindinginterfaceinthecrystalstructureofGajiwalaetal.(2000)areindicatedinitalics.

4.2FourteencladesofFoxgeneshavemembersinboth

chordatesandecdysozoansandcanbeconsideredprimitiveforbilateriananimals.

4.3TheFoxE,H,I,J,MandQ1subclassesareabsentfrom

allthreeecdysozoantaxa.Thesemayhavebeenlostontheecdysozoanlineageorhaveresultedfromduplica-tionsspecifictothedeuterostomelineage.

4.4TheFoxQ2subclassisapparentlymissinginverte-bratesandhaspresumablybeenlost.

4.5Thereisevidenceforsite-specificchangesinsub-stitutionratesduringFoxgenesubclassdiversification.

Acknowledgements

Wethankananonymousrefereeforhelpingtoimprovetheaccessibilityofthemanuscript.F.M.andS.M.S.acknowledgethesupportoftheBBSRC.

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