Origin of Pax and Six gene families in sponges: Single ... · queenslandica (Larroux et al., 2008)....

Developmental Biology 343 (2010) 106–123

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Developmental Biology

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Evolution of Developmental Control Mechanisms

Origin of Pax and Six gene families in sponges: Single PaxB and Six1/2 orthologs inChalinula loosanoffi

April Hill a,⁎,1, Werner Boll b,1, Carolin Ries b, Lisa Warner a, Marisa Osswalt c,2, Malcolm Hill a, Markus Noll b,⁎a Department of Biology, University of Richmond, Richmond, VA 23113, USAb Institute of Molecular Life Sciences, University of Zürich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerlandc Biology Department, Boston College, Boston, MA 02467, USA

⁎ Corresponding authors. A. Hill is to be contacteUniversity of Richmond, Richmond, VA 23113, USA. FaInstitute of Molecular Life Sciences, University of Zürich8057 Zurich, Switzerland. Fax: +41 44 635 6829.

E-mail addresses:[email protected] (A. Hill), mar1 Equal contribution.2 Present address: Infinity Pharmaceuticals, Cambridg

0012-1606/$ – see front matter © 2010 Elsevier Inc. Adoi:10.1016/j.ydbio.2010.03.010

a b s t r a c t
a r t i c l e i n f o
Article history:Received for publication 14 October 2009Revised 11 February 2010Accepted 16 March 2010Available online 25 March 2010

Keywords:Pax genesSix genesPax–Six gene regulatory networkEvolutionPoriferaHaliclonaChalinula loosanoffi

Pax genes play an important role in networks of transcription factors that determine organogenesis, notablythe development of sensory organs. Other members of this regulatory network include transcription factorsencoded by the Six gene family. Sponges lack organs and a nervous system, possibly because they have notevolved a Pax/Six network. Here we show that the demosponge Chalinula loosanoffi encodes only one Paxand one Six gene, representatives of the PaxB and Six1/2 subfamilies. Analysis of their temporal transcriptionpatterns during development shows no correlation of their mRNA levels while their spatial patterns showsome overlap of expression in adult tissue, although cellular resolution was not achieved. These results donot suggest that these genes form a major network in this basal phylum, although its existence in a minorfraction of cells is not excluded. We further show that sponge PaxB can substitute for some of the Pax2, butnot of the Pax6 functions in Drosophila. Finally, we have analyzed the phylogeny of Pax and Six genes andhave derived a model of the evolution of the Pax gene subfamilies in metazoans. It illustrates a diversificationof Pax genes into subfamilies mostly in triploblasts before the protostome–deuterostome split, whereas fewsubfamilies were lost in various phyla after the Cambrian explosion.

d at Department of Biology,x: +1 804 289 8233. M. Noll,, Winterthurerstrasse 190, CH-

[email protected] (M. Noll).

e, MA 02139, USA.

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© 2010 Elsevier Inc. All rights reserved.

Introduction

Pax genes encode a small family of transcription factors, includingthe DNA-binding paired domain and, in some of its subfamilies, also aprd-type homeodomain (Noll, 1993; Hanson and van Heyningen, 1995;Chi and Epstein, 2002). Paired domains and Pax genes were discoveredin a test of the gene network hypothesis (Bopp et al., 1986, 1989). It wasbased on our concept of the evolution of gene networks and theirintegrated functions by the deployment of network-specific domains,regardless of whether these are of a protein-coding or cis-regulatorynature (Frigerio et al., 1986). Thus, the evolution of Pax genes and theirnetworks regulating integrated functions (Noll, 1993) was an eminentquestion ever since their discovery and probable restriction tometazoans (Burri et al., 1989). An early analysis, based on the limitednumber of Pax genes isolated from Drosophila (Bopp et al., 1986, 1989),

mouse (Gruss and Walther, 1992), and humans (Burri et al., 1989),predicted the existence of four subfamilies, Pax2/5/8, Pax3/7, Pax1, andPax4/6, before the protostome–deuterostome split, while consideringDrosophila Pox neuro (Poxn) to be a member of the Pax2/5/8 subfamily(Noll, 1993). This pedigree was extended to include more primitivemetazoans, primarily through the isolation and analysis of Pax genesfrom cnidarians (Sun et al., 1997; Gröger et al., 2000; Miller et al., 2000;Sun et al., 2001; Kozmik et al., 2003;Hoshiyama et al., 2007;Matus et al.,2007), a placozoan (Hadrys et al., 2005), and sponges (Hoshiyama et al.,1998; Larroux et al., 2006). The chief conclusion from these studies wasthat two of the subfamilies, Pax2/5/8 and Pax3/7, also exist inanthozoan cnidarians as PaxA/B/C and PaxD genes (Miller et al., 2000;Matus et al., 2007),whereas inmedusozoan cnidarians (Sun et al., 1997;Gröger et al., 2000; Kozmik et al., 2003) and sponges (Hoshiyama et al.,1998; Larroux et al., 2006) only a single subfamily, represented by PaxBand PaxA, was found. It was also proposed that the PaxA gene wasmostclosely related to Poxn and that the ancestral PaxCmight have been theprecursor of the Pax6 subfamily (Miller et al., 2000). It remainedunclear,however, whether additional paired-domain subfamilies existed in thebasal metazoan phylum of sponges (Hoshiyama et al., 1998; Larroux etal., 2006).

Additional interest in the evolution of Pax genes and theirnetworks was raised by the discovery of a conserved role of Pax6 ineye development of bilaterians (Quiring et al., 1994; for reviews, see

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107A. Hill et al. / Developmental Biology 343 (2010) 106–123

Hanson and van Heyningen, 1995; Gehring and Ikeo, 1999; Kozmik,2005), in agreement with one of the predictions of the gene networkhypothesis that gene networks are conserved (Noll, 1993). Ectopicexpression in leg, wing, and antennal discs of Drosophila Eyeless (Ey)or its mouse homolog Pax6 is able to induce small ectopic eyes on thecorresponding adult structures (Halder et al., 1995). This property iseven conserved in PaxB of Tripedalia cystophora, a jellyfish withcomplex eyes, and in Pax2 of Drosophila, although the ectopic eyes aresmaller and induced only on legs (Kozmik et al., 2003). However,ectopic eyes were as big as those induced by Ey when the bindingspecificity of PaxB was changed to that of Pax6 (Kozmik et al., 2003)by the substitution of only three amino acids in the paired domain(Czerny and Busslinger, 1995). Since PaxB also regulates transcriptionof the eye crystallin in the jellyfish (Kozmik et al., 2003), these resultsdemonstrate a close relationship of paired domains between thePaxB/2/5/8 and Pax6 subfamilies and suggest that a PaxB-like proteinwas the primordial Pax protein in eye evolution (Kozmik et al., 2003).In addition, jellyfish PaxB retained Pax2 functions, as shown by theextensive, although incomplete, ability to substitute for the eye-specific function of Pax2 in Drosophila spapol mutants (Kozmik et al.,2003).

Normal or ectopic eye development in Drosophila is induced by anetwork of transcription factors (Chen et al., 1997; Pignoni et al., 1997;Shen and Mardon, 1997; Halder et al., 1998; Treisman, 1999) that isactivated by a combination of signaling pathways (Kumar and Moses,2001) and appears to be conserved in bilaterians (Treisman, 1999;Kawakami et al., 2000). A prominentmember of this Pax–Six–Eya–Dacgene network, in addition to the Pax6 homologs ey and twin of eyeless(toy) (Czerny et al., 1999; Kronhamn et al., 2002), is the sine oculis (so)gene of Drosophila (Cheyette et al., 1994) or its Six1/2 orthologs inother bilaterians (Seo et al., 1999). Six genes encode a family oftranscription factors containing a conserved Six domain, required forinteractionwith the transcription factor Eya (Pignoni et al., 1997), andan adjacent Six-type homeodomain. Six genes can be grouped intothree subfamilies, Six1/2, Six3/6, and Six4/5, that are characterized bydiagnostic tetrapeptides close to the N-terminus of their homeo-domains (Seo et al., 1999). The presence of Six gene clusters inmammals that include one member of each subfamily led to theproposal of an ancestral Six gene cluster in the progenitor ofprotostomes and deuterostomes (Boucher et al., 2000). Indeed, sucha cluster of Six genes probably already existed before the cnidariansdiverged from bilaterians, as Six genes of all three subfamilies wereisolated from Cladonema radiatum, a jellyfishwith lens eyes (Stierwaldet al., 2004). Even in themore basal phylum of sponges Six genes exist,although only an incomplete sequence of a Six domain, isolated fromthe demosponge Haliclona sp., has been reported (Bebenek et al.,2004). However, it was neither clear to which subfamily this Six genebelonged nor whether additional subfamilies exist in Haliclona. Asingle Six gene belonging to the Six1/2 subfamilywas also found in thedraft sequence of the genome of the demosponge Amphimedonqueenslandica (Larroux et al., 2008). Like Pax genes, Six genes appearto be restricted to metazoans (Kawakami et al., 2000). No Pax or Sixgenes were found by BLAST searches in the genomes of the twochoanoflagellates, Monosiga brevicollis (draft genomic sequence ofDOE Joint Genome Institute) and Proterospongia sp. (draft genomicsequence of The Broad Institute), a member of the closest extantrelatives of metazoans (King et al., 2008), nor are they present in thegenomeof thefilose amoeboid symbiont, Capsaspora owczarzaki (draftgenomic sequence of The Broad Institute), another unicellularopisthokont closely related to metazoans (Ruiz-Trillo et al., 2004,2007). Members of both families participate in homologous Pax–Six–Eya–Dac gene networks that are conserved from insects to mammalsand not restricted to eye development, but deployed in many otherprograms of organogenesis (Kawakami et al., 2000), such as inmyogenesis (Heanue et al., 1999), nephrogenesis (Xu et al., 2003) orinner ear development (Ozaki et al., 2004).

Given metazoan monophyly, one might surmise that spongespossess a core set of genes that supports all characteristics of being ananimal. Recent work strongly suggests that sponges utilize animal-specific genetic pathways to create the sponge body (Adell et al., 2003;Perovic et al., 2003; Hill et al., 2004; Funayama et al., 2005; Larroux etal., 2006). Analysis of the draft sequence of the first sponge genome ofA. queenslandica (previously named Reniera sp.) and of cDNAsequences of the demosponge Oscarella carmela illustrates thatsponges possess at least one member of many of the transcriptionfactor and signal transduction families known to be crucial indevelopment of complex metazoans (Larroux et al., 2006, 2008;Nichols et al., 2006). Here we isolate a PaxB and a Six1/2 gene from themarine demosponge Chalinula loosanoffi (also known as Haliclonaloosanoffi) and show that they are the only members of their genefamilies in this species. Thus, these genes are probably the represen-tatives of the primordial Pax and Six genes. Analysis of their temporalexpression patterns during development and tissue aggregation doesnot suggest that they are part of a Pax–Six gene network in a majorfraction of cells in which they are expressed. However, such a networkis not excluded to exist in a small portion of developing or adult spongecells. Chalinula PaxB can substitute to a large extent for Pax2 in thedeveloping Drosophila eye but is unable to induce even small ectopiceyes in Drosophila legs, wings, or antennae and hence has no Pax6function. Finally, we derive a model of Pax gene evolution by anextensive phylogenetic analysis of Pax genes, including Pax genes fromseveral genome projects that we specifically annotated to obtain abetter coverage of lower phyla.

Materials and methods

Collection of sponges

Chalinula loosanoffi (this species was recently reclassified from H.loosanoffi (Hartman, 1958) to C. loosanoffi) was collected from eitherLong Island Sound at Anchor Beach,Milford, CT, or the Chesapeake Bayat Virginia Institute of Marine Science, Gloucester Point, VA. Whennecessary, sponges were reared in recirculating seawater tanks, buttissue was usually processed immediately and stored at −80 °C.

Culturing of larvae and primmorphs

Larvaewere collected from individual sponges placed in beakers ofsterile, filtered seawater. Reproductive sponges released larvae intothe water from where newly released larvae were collected with apipet. Larvae were cultured in 24-well plates in sterile, filteredseawater, which was replaced daily, and were staged as described(Hill et al., 2004). All tissues were stored in RNA stabilization solutionRNAlater (Ambion) overnight and stored at −80 °C for subsequentisolation of RNA.

For culturing of sponge cell aggregates, adult Chalinula wasdissociated into single cells in Ca2+,Mg2+-free seawater and filteredthrough nylon mesh cups as described (Leith, 1979). Aliquots ofdissociated cells were stored in RNAlater (Ambion). Sponge cellaggregates (primmorphs)were obtained from single cells as described(Custodio et al., 1998; Müller et al., 1999). Primmorphs of approx-imately 5 mm in diameterwere stored in RNAlater for subsequent RNAisolation. Smaller primmorphs were cultured at 16–22 °C in suspen-sion by constant circulation in natural seawater, supplemented with0.2% RPMI-1640 medium, 0.060 mM silicate, as described (Krasko etal., 2002). After transfer of the primmorphs to a Petri dish in the samemedium supplemented with 0.030 mM Fe3+, circulation was omittedto allow the primmorphs to attach to the substrate, proliferate, andspread across the dish (Krasko et al., 2002). Spreading and differen-tiating aggregateswere cultured for 5 days before theywere harvestedfor RNA isolation.

108 A. Hill et al. / Developmental Biology 343 (2010) 106–123

Isolation of genomic DNA, isolation of RNA, and RT–PCR analysis

Chalinula genomic DNA was isolated by a modified CTAB method(Doyle and Doyle, 1987; Cullings, 1992). After grinding 100 mg ofsponge tissue in 0.35 ml of CTAB buffer (50 mMTris–HCl, pH 8.0, 0.7 MNaCl, 10 mM EDTA, 1% hexadecyl-trimethylammonium bromide, and0.1% β-mercaptoethanol), 0.35 ml of CTAB buffer was added, andproteins were digested with 0.020 ml of Proteinase K (20 mg/ml) at65 °C for 1 h. The solution was extracted with 0.60 ml of chloroform–

isoamyl alcohol (24:1) and theDNAprecipitatedwith an equal volumeof isopropanol at−20 °C overnight. After centrifugation, the pelletwaswashedwith 70% ethanol, allowed to air dry, and the DNA dissolved inTE, pH 8.

Chalinula RNA was isolated from free-swimming larvae orreaggregated adult tissue by use of the Nucleospin RNA II kit(Clontech) and treated with DNAse I to degrade any contaminatinggenomic DNA. For RT–PCR, 0.50 µg of RNA was reverse-transcribedwith the ThermoScript Reverse Transcriptase kit (Invitrogen) andoligo(dT) primers. To detect PaxB transcripts, the cDNA was amplifiedby PCR with Platinum Taq DNA polymerase (Invitrogen) and theprimers 5′-CCAACTAGGCGGACTCTTCG-3′ (forward) and 5′-CTTTGTCAGGGAGGTCAAGC-3′ (reverse), spanning the first to thefourth coding exon of PaxB (positions 1182 to 3069 of the genomic, orpositions 149 to 1323 of the cDNA sequence). To detect Six1/2mRNA,the cDNAwas amplified by PCRwith the primers 5′-CGAAAGCGTGCT-GAAAGCAAAGG-3′ and 5′-CACGGGGGGATGGGTATGGATTC-3′ thatspan positions 224 to 543 of the cDNA sequence. Since this sequencedid not include an intron, a “cDNA synthesis” reaction without reversetranscriptase was amplified by PCR as a control to exclude artifactscaused by amplification of contaminating genomic DNA. RT–PCRcontrol reactions were performed with Chalinula-specific actin geneprimers as described (Hill et al., 2004).

In situ hybridization to tissues

Sponge tissues were fixed overnight in sterile seawater, 4%paraformaldehyde, 0.02% glutaraldehyde, transferred into ascendingconcentrations of methanol, and stored in 100% methanol at −80 °C.Fixed tissues were rehydrated through a methanol and PTw (PBS, 0.1%Tween-20) series and subsequently fixed again in PBS, 4% paraformal-dehyde. Tissues were washed in detergent solution (50 mM Tris–HCl,pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% SDS, and 0.5% Tween-20) for30 min, followed by six washes in PTw, processed to a 1:1 solution ofPTw and hybridization buffer (5× SSC, pH 5, 50% formamide, 0.050 mg/ml heparin, 0.25% Tween-20, 1% SDS, and 0.100 mg/ml sonicated anddenatured salmon sperm DNA), prehybridized in 0.500 ml of hybrid-ization buffer at 60 °C for at least 3 hours, and hybridized with 10 ng ofriboprobe at 60 °C overnight. After hybridization, tissueswerewashed 7times in hybridization buffer at 60 °C and gradually processed to roomtemperature throughwashes in TBST (25 mMTris–HCl, pH 7.5, 140 mMNaCl, 2.5 mM KCl, 0.1% Tween-20) and hybridization buffer (1:1). Aftera fewwashes in TBST, tissueswere incubated in0.500 mlof TBST, 1%BSAto block nonspecific binding of antibodies, and stained histochemicallywith alkaline phosphatase according to standard protocols. For thepreparation of sections, the tissue was embedded in paraffin and cutwith a Leica Microsystems RM2245 rotary microtome.

All probes were labeled with the Dig RNA labeling kit (Roche). ForPaxB, either the entire cDNA or a 371-bp region spanning the paireddomain, amplified by the primers 5′-GGCGTGAACCAACTAGGCGGACT-3′and 5′-CGACTGCGAACGATGCGATTTAT-3′, was used to generate ariboprobe. Both probes produced the same in situ staining pattern. ForSix1/2, a 320-bp region spanning two-thirds of the Six domain andhalf ofthe adjacent homeodomain, amplified by the primers 5′-CGAAAGCGTGCTGAAAGCGAAGG-3′ and 5′-CACGGGGGGATGGGTATG-GATTC-3′, was used to generate a riboprobe. All DNA fragments werecloned in the TOPO TA Cloning Dual Promoter Vector (Invitrogen), from

which both sense and antisense riboprobes were produced that werepartially hydrolized to an average length of 100–200 nucleotides suitablefor hybridization.

Isolation and sequencing of PaxB gene

Paired box DNA was isolated from genomic DNA by PCR withdegenerate primers of conserved paired box sequences, encoding thepeptides HGCVSKI and M(I)FA(T)WEIR and biased toward thecorresponding Cnidaria and Porifera Pax gene sequences: 5′-CAYGGIT-GYGTIWSYAARAT-3′ and5′-CKDATYTCCCAIGYRAAIAT-3′. The resultingDNA fragment of about 450 bpwas cloned and verified to encodepart ofthepaired domain byDNAsequencing. This fragment provided the basisfor an inverse PCR strategy (Ochman et al., 1988) to walk along thegenomicDNAandobtain the complete PaxBgene locus. All PCRproductswere separated by agarose gel electrophoresis; fragments of expectedsizes were cut out of the gel, eluted, and cloned by use of the TOPO TACloning kit (Invitrogen); and clones were sequenced with the Big DyeTerminator kit (Applied Biosystems) on a 3730 DNA Analyzer (AppliedBiosystems).

To determine the exon/intron structure of the PaxB gene, PaxB cDNAwas amplified by PCR from 3′RACE-ready PaxB cDNA, synthesizedaccording to the manufacturer's protocol by use of a 3′RACE kit(Clontech) from 1 µg of total RNA isolated from adult Chalinula tissue.PCR of the 3′RACEwas performedwith 5′-CCAACTAGGCGGACTCTTCG-3′as forward primer (positions 1182–1201 of genomic PaxB DNA) and theUPM reverse primer (Clontech). An aliquot of the 3′RACE reaction wasused for nested PCR with 5′-GGACTCTTCGTGACCGGTCG-3′ as forwardprimer (positions 1192–1211of genomicPaxBDNA)and theNUP reverseprimer (Clontech) under the same PCR conditions. The remaining 5′portion of PaxB cDNA was isolated by PCR by trying various forwardprimers, the sequence of which had been derived from genomic PaxBDNA sequence. It generated a PaxB cDNA spanning the genomic PaxBsequence from positions 819 to 5339.

Isolation and sequencing of Six1/2 gene

For Six gene isolation fromgenomic and cDNA, PCRswere performedwith the degenerate primers used previously for the isolation of Six1/2and Six4/5 genes of the jellyfish C. radiatum (Stierwald et al., 2004).Single and identical genomic and cDNA fragments of 269 bp, spanningthe region encoding amino acids 107 to 196 of Cl-Six1/2, were obtainedand extended by 120 bp and 24 bp at their 5′ and 3′ ends, respectively,by use of nested primers whose sequenceswere obtained from a partialsequence of a Six1/2 gene isolated from a Haliclona sp. (Bebenek et al.,2004). Finally, the cDNA was extended by 5′ and 3′RACE with theGeneRacer RLM-RACE kit (Invitrogen). The Six1/2 cDNA generatedconsisted of several fragments covering a total length of 1713 bp.Multiple sets of primers derived from the cDNA sequence were used foramplification by PCR of the corresponding genomic DNA, revealing asingle intron of 418 bp interrupting the coding region after the secondnucleotide of the codon for Ser207. PCR products were purified andcloned as described above. Cloneswere sequencedwith the SequiThermEXCEL II kit (Epicenter) on a LiCor Systemor sequencedwith theBigDyeTerminator kit (Applied Biosystems) on a 3730 DNA Analyzer (AppliedBiosystems).

DNA constructs and generation of transgenic flies

Chalinula PaxB was expressed in Drosophila by the Gal4/UASsystem (Brand and Perrimon, 1993). Target constructs were gener-ated by cloning the complete Cl-PaxB cDNA into the pP{UAST} vector.The Pax6-specific amino acids I, Q, and N were introduced into thepaired domain of Cl-PaxB at positions 42, 44, and 47 of the paireddomain by PCR-based mutagenesis of the PaxB cDNA by use of thefollowing primers: 5′-GCTATTTAGGTGACACTATAG-3′ (sp6pri), 5′-


CTACTTATGTCGCAAGGACGTA-3′ (splink), 5′-GCGACATAAGTA-GAATCCTACAAGTGTCTAACGGTTGCGTG-3′ (IQNmut), and 5′-GTAA-TACGACTCACTATAGGG-3′ (T7pri).

Fig. 1. Structure of the C. loosanoffi PaxB gene and its product. (A) Map of the Cl-PaxB gene. Torigin is at the 5′end of the tentative first exon, which is not coding. Regions encoding the ppart of the protein (yellow) are colored, while untranslated 113 bp 5′-leader and 344 bp 3′-tPax genes are marked with asterisks. The brown box downstream of the PaxB gene represen(arrows). The GenBank accession number for the genomic DNA and cDNA of the Cl-PaxB gepaired domain is shown in red, the octapeptide in blue, the homeodomain in green, while thefilled triangles. (C) Conserved intron positions in the paired domain and homeodomain of Cl-features in secondary structure, the β-hairpin (β1 and β2) and the six α-helices in the PDdifferent Pax genes by gray arrowheads, for Cl-PaxB by red arrowheads. For the two conservPax genes whose intron positions are close to those conserved strictly in other Pax genes a

P-element constructs were coinjected with the transposase donorplasmid pUChsΔ2-3 (designed by D. Rio; FlyBase FBmc0000938) intotheposterior pole of stage 2 yw embryos. P-elementmediatedgerm line

he eight exons of PaxB are mapped with respect to a scale (in kb) shown below whoseaired domain (red), octapeptide (blue), the homeodomain (green), and the remainingrailer regions are shown in gray. Introns whose positions have been conserved in otherts a conserved ORF, FAM40A, which is transcribed in a direction opposite to that of PaxBne is GQ985310. (B) Sequence of the ORF of the 589-amino-acid Cl-PaxB protein. Thepeptide conserved in different sponges is underlined. Positions of introns aremarked byPaxB. Paired domain (PD) and homeodomain (HD) are depicted with their characteristicand the three α-helices in the HD. Intron positions in these domains are indicated fored introns, the sequence flanking the splice donor and acceptor sites are shown below.re in parentheses. For abbreviations of species and Pax genes, see Fig. S2.


transformation led to several stable transgenic lines, which wereestablished in a y w background. To express Cl-PaxB, several of thetransgenic lineswere crossed toflies carryingGal4drivers, yw; spa-Gal4(Jiao et al., 2001) or y w; Sp/CyO; dpp-Gal4-40C.6/TM6B (Staehling-Hampton et al., 1994).

Phylogenetic analysis of Pax and Six protein families

Metazoan Pax and Six protein sequences were obtained from thepublished literature or BLAST searches of the NCBI GenBank, exceptfor sequences from A. queenslandica and Saccoglossus kowalevskii,obtained from the respective Trace Archive draft genomes at NCBI,and some sequences from Trichoplax adhaerens (Ta PaxB2) that wereretrieved by BLAST search of the T. adhaerens Grell-BS-1999 v1.0genome database at the DOE Joint Genome Institute. Pax genesequence information of Schmidteamediterranea and Capitella capitatawas retrieved by BLAST search from the SmedGD v1.3.14 (Robb et al.,2008) and the Capca1 database of the DOE Joint Genome Institute,respectively. Paired domain sequences of selected Pax proteins andSix domain and Six-type homeodomain sequences of selected Sixproteins were aligned with the multiple sequence alignment programClustalX version 2.0 with default parameters (Larkin et al., 2007), andalignments were manually refined where necessary.

Maximum likelihood (ML) phylogenetic inferences were derivedby use of the PhyML program version 3.0 (Guindon and Gascuel,2003). For the analyses of Pax and Six proteins, we evaluated the bestamino acid substitution model by ProtTest v2.2 (Abascal et al., 2005)and applied the LG model of sequence evolution accordingly (Le andGascuel, 2008). Two classes of sites were assumed, one class beinginvariable and the other free to change and follow a gamma shapedistribution, which was calculated by use of a discrete approximationwith four categories of sites. As starting tree, either the BioNJ tree orfive random trees were chosen, and the best resulting tree wasregarded as output tree. Tree topologies were estimated with the NNIand SPR methods (Hordijk and Gascuel, 2005). Statistical branchsupport for the ML analysis was assessed with the approximatelikelihood ratio test (aLRT; Anisimova and Gascuel, 2006). Phyloge-netic trees are displayed with the Tree Explorer which is included inthe Mega4 software (Tamura et al., 2007). Only branches with aLRTvalues larger than 0.50 are resolved and trees are rooted at midpoint.

Results

C. loosanoffi contains a single Pax gene

To isolate Pax gene DNA from C. loosanoffi, genomic DNA wasamplified by PCRwith degenerate primers specific for highly conservedpairedbox sequences,whichgenerated a singleDNA fragment encodinga partial paired domain of a PaxB-type gene. A second amplification byPCR produced additional DNA fragments encoding partial paireddomains of other Pax gene subfamilies: Pax6 [100% identity to zebrafish(NP_571379)] and Pax3/7 [(87% identity to Drosophila paired(AAB59221)]. However, only the PCR product of the first amplificationdetected a hybridization signal on Southern blots of genomic ChalinulaDNA, apparent as a single band (Fig. S1), which implies that thesequences amplified by the second PCR did not originate from Chalinula

Fig. 2. Alignments of paired domains, octapeptides, and homeodomains of Pax proteins. (A)and subfamilies of Pax genes. Extent of β-hairpins and α-helices of paired domains are bsequence shown at the top. Gaps in the alignment are marked by dashes and not included inare colored as defined in Fig. 5. (B) Homeodomains of Pax proteins labeled as in (A) but inclthe Cl-PaxB sequence shown at the top, while amino acids similar to themost frequent aminoincluded in numbering of amino acids. Homeodomain sites diagnostic for Pax3/7 (yellow)partial homeodomains of Pax2 in arthropods and chordates are labeled in pink and blue coloPax2a, also extends only over the N-terminal half with about 50% similarity to cnidariancomplete paired domains and two PAI subdomains with associated octapeptides and home

but rather from contaminating tissues, probably of other marinemetazoans and, more importantly, that the C. loosanoffi genomeincludes only a single Pax gene. This notion of a single Pax gene insponges is supported by BLAST searches in the NCBI trace archive of theA. queenslandica (Reniera) genome that yielded only one paired domain,that of PaxB, on 23 independent sequences.

Structural features of Chalinula PaxB

Extending the genomic DNA from the PaxB-type gene fragment byrepeateduse of inverse PCR (Ochman et al., 1988) and combining itwiththe corresponding cDNA, we isolated and sequenced most of the PaxBgene as overlapping fragments comprising 5349 bp, at least 4521nucleotides of which are transcribed. The gene consists of at least eightexons, the last seven of which comprise the entire open reading frame,encoding a protein of 589 amino acids that includes a paired domain,homeodomain, and octapeptide (Figs. 1A and B). The N-terminal paireddomain is encoded by exons 2 and 3 that are separated by an intronwhose location is conserved amongmanymembers of the Pax2/5/8 andPax6 subfamilies in other metazoans (Fig. 1C). A single intron also splitsthe coding region of the homeodomain at a position conserved in mosthomeodomains of those Pax subfamilies that include a homeodomain(Fig. 1C).

Immediately downstream of the C. loosanoffi PaxB gene, a largeopen reading frame was detected (Fig. 1A) that encodes a protein of758 amino acids well conserved in other metazoans, includingmammals, nematodes, and insects (NCBI Blast: 42% identity withmouse FAM40A (Q8C079) and 38% identity with CG11526-PA ofDrosophila melanogaster). The polypeptide contains several putativetransmembrane domains, and its N-terminal portion resembles theacidic N1221 domain (PFAM PF07923) in yeast. The function of thisputative protein is presently unknown.

The paired domain of Chalinula PaxB ismost closely related to that ofPaxB fromother sponges,A. queenslandica (91% identity; 96% similarity)and Ephydatia fluviatilis (83%; 95%), and very similar to that of cnidarianPaxB or members of the Pax2/5/8 subfamily (N73%; N88%) (Fig. 2A).The octapeptide (Burri et al., 1989), amotif essential for the recruitmentof the Groucho corepressor by interaction with the SP domain andpossibly the Q/TLE N-terminal domain of Groucho (Eberhard et al.,2000) and conserved in Pax2/5/8 and cnidarian PaxB proteins even asnonapeptide YSINGILGI (Kozmik et al., 2003), is weakly conserved,sharing four identical amino acids with these Pax subfamilies (Fig. 2A).The only other octapeptide identified in a sponge, namely in PaxB ofA. queenslandica, shares the last 5 amino acidswith that of Chalinula, butonly 2 with octapeptides of cnidarian PaxB or Pax2/5/8 proteins(Fig. 2A). That the octapeptidewe identified in PaxB of sponges (Fig. 2A)may be functional is suggested by our search in the A. queenslandicagenome for a Groucho protein, the sequence of which is strikinglyconserved. Each of its two highly conserved portions, consisting of a115-amino acid Q/TLE N-terminal domain and a 300-amino acidC-terminal moiety of seven WD-40 repeats, display about 70% identityand 80% similarity with respect to Drosophila Groucho.

The homeodomain of Cl-PaxB is closest to the homeodomain of thePax6 subfamily (about 38% identity) and to the conserved N-terminalhalf of the homeodomain of the Pax2/5/8 subfamily in chordates andechinoderms (Fig. 2B). Strikingly, in arthropods, this partial

Paired domains and octapeptides labeled in the left margin by abbreviations of speciesoxed. Dots indicate identities, gray color similarities of amino acids with the Cl-PaxBthe numbering of amino acids. Paired domain sites diagnostic for different subfamilies

uding phyla. The three α-helices are boxed. Dots indicate identities in amino acids withacid at a given position are in gray. Gaps in the alignment aremarked by dashes and notand Pax6 (red) protein subfamilies are colored. The homologies extending beyond thers, respectively. Note that the homeodomain of the Pax2-like protein of S. mansoni, SmpPaxB homeodomains. Fig. S2 shows an expanded and more representative list of 134odomains and includes sequence accession information.

Fig. 3. Six1/2 protein of C. loosanoffi and alignment of its Six domain (SD) and adjacent Six-type homeodomain (HD) with other SDs and Six-type HDs. (A) Sequence of the ORF of the446-amino-acid Cl-Six1/2 protein. The SD (purple) and abutting Six-type HD (green) are colored, and the position of the single intron, which is conserved in human Six genes(Gallardo et al., 1999; Boucher et al., 2000), is marked by a filled triangle. The GenBank accession number for the genomic DNA and cDNA of the Cl-Six1/2 gene is GQ985311.(B) Alignment of SDs and Six-type HDs. SDs and Six-type HDs are labeled in the left margins by abbreviations of species, subfamilies of Six genes, and, in the upper part, by phyla ofspecies. The extents of SDs and HDs are marked by purple and green lines and those of α-helices in HDs are boxed. Dots indicate identities and gray color similarities of amino acidswith the Cl-Six1/2 sequence shown at the top. Gaps in the alignment are marked by dashes and not included in numbering of amino acids. SD and Six-type HD sites diagnostic fordifferent Six subfamilies are colored in green (Six1/2), orange (Six3/6), and blue (Six4/5). An expanded and more representative list of 62 SDs, used for construction of thephylogenetic tree in Fig. 6, is shown in Fig. S3, which includes NCBI accession numbers and full names of species.


homeodomain is conserved only over the 12 N-terminal amino acidswith regard to the Pax6-type homeodomain, but extends amongarthropods over the length of a typical homeodomain (about 67%

similarity between Drosophila and Tribolium or Aphid; marked pink inFig. 2B). This extension of homology of the partial homeodomain overthe length of a true homeodomain is also observed among vertebrate


Pax2/58proteins (markedblue in Fig. 2B). It remains to be seenwhethera similar extension of homology of the partial homeodomain we foundin Pax2a of Schistosoma mansoni (Fig. 2B) also exists for otherPlatyhelminthes. Like the homeodomains of other sponge PaxB proteinsbut in contrast to those well conserved among PaxB proteins in otherlower phyla (Cnidaria and Placozoa), the homeodomain of Cl-PaxBdisplays virtually no sequence conservation in the 5 N-terminal and7 C-terminal amino acids (Fig. 2B). The distal part of the third α-helix isdegenerated, and the amino acids I/V47 and N51, in ‘standard’homeodomains essential for the recognition of the (A/T)AAT corebinding site (Laughon, 1991; Pomerantz and Sharp, 1994), are notconserved either. Also, other amino acids of the Pax6-type home-odomain proposed to be important for its interaction with DNA as wellas the Pax6-type paired domain (Bruun et al., 2005) show only a lowdegree of conservation. Curiously, in all three sponge genes, the twoturns between the three alpha-helices are best conserved in relation tohomeodomains of the Pax6 subfamily (Fig. 2B). In addition to the paireddomain and homeodomain, we found a ‘sponge-specific’ triskaideka-peptide SPNMPSSNSDEAA (Fig. 1B), which is the only additional‘domain’ that is conserved in PaxB proteins of demosponges (85%identity) but not of other lower metazoans, or in Pax2/5/8 proteins ofhigher metazoans.

A single Six gene in C. loosanoffi

A Six gene was isolated from C. loosanoffi as genomic and cDNA byPCR with degenerate primers (Stierwald et al., 2004), and subsequent5′- and 3′-extension by RACE of the cDNA, which was used to clone thecorresponding genomic DNA (cf., Materials and methods). Since PCRgenerated only a single DNA fragment, despite the use of degenerateprimers able to amplify Six genes of all subfamilies, only one subfamilyof Six genes exists in C. loosanoffi. This subfamily was most likelyrepresented by a single gene, as evident from Southern blot analysis(Fig. S1). This conclusion is consistent with BLAST searches for the Sixdomain and adjacent homeodomain in the genome of A. queenslandicathat detected only a single Six gene, Six1/2, on 10 independentsequences in the NCBI trace archive. The gene belongs to the Six1/2subfamily because it includes in the N-terminal portion of its homeo-domain the tetrapeptide ETSY (Seo et al., 1999; Kawakami et al., 2000),aswell as in its Six domain andhomeodomain other aminoacids that arediagnostic for the So/Six1/2 subfamily of Six proteins (Fig. 3). As ischaracteristic of Six-type homeodomains (Kawakami et al., 2000), theCl-Six1/2homeodomaindeviates at twopositions conserved in all otherhomeodomains, R5 and Q12, that are changed to serines (Fig. 3B). TheCl-Six1/2 gene encodes a highly conserved Six domain (77% identity or92% similarity with that of mouse, Mm Six2; Fig. 3B) and adjacent Six-type homeodomain (88% identity with that of Mm Six2; Fig. 3B) on apresumptivefirst exon, separated by a 418-bp intron from its other exonencoding the C-terminal moiety of the Cl-Six1/2 protein (Fig. 3A). Thepresence of a single intron at this position has been highly conserved inhuman SIX2 and SIX6 genes (Gallardo et al., 1999; Boucher et al., 2000).The combined domains are best conserved among Six1/2 proteins ofdemosponges (97% similarity with those of E. fluviatilis or A. queen-slandica). Surprisingly, thesedomains are considerably less conserved inSixC of the calcareous sponge Sycon calcaravis (73% similarity;Hoshiyama et al., 2007) than in Six1/2 of the mouse or So of Drosophila(N88% similarity).

Phylogenetic analysis of Cl-PaxB and Cl-Six1/2

Phylogenetic analysis of Pax genes, based on their paired domains,shows the Cl-PaxB gene to fall within a well-supported sponge-specific clade that includes Pax genes from marine and fresh waterdemosponges (Fig. 4). The poriferan Pax genes cluster within thePaxB/2/5/8 subfamily, but relationships within this clade are poorlyresolved. This subfamily forms a clade with the paired domains

encoded by the Poxn/PaxA/PaxC and Pax6 subfamilies (Fig. 4). Moreinformative is an approach that considers within paired domains the41 sites of subfamily-specific amino acids (Fig. 5A). According to thiscriterion, the sponge PaxB sequences resemble the PaxB subfamily atleast at 36 sites, while they match the Pax6 subfamily at most at 22positions.

Phylogenetic analysis of Six genes, based on the amino acidsequences of their Six domains and Six-type homeodomains, unambig-uously grouped the Cl-Six1/2 gene with the Six genes of the marinedemosponge A. queenslandica, Aq-Six1/2, and of the fresh waterdemosponge E. fluviatilis, Ef-SixC (Hoshiyama et al., 2007). Thecalcareous sponge S. calcaravis is not included in the same branch, butall poriferan Six genes fall within the Six1/2 subfamily in theML analysiswhich produced a phylogenetic tree that is consistent within the threesubfamilies (Fig. 6).

Interestingly, T. adhaerens, the only known species of the phylumPlacozoa, has two Six genes that clearly belong to two subfamilies,Six1/2 and Six3/6 (Figs. 3B and 6). This suggests that the Six3/6subfamily was lost in sponges or, what seems more probable, Poriferaare basal to Placozoa and the Six3/6 subfamily originated after theseparation of Placozoa from Porifera.

PaxB and Six1/2 expression analysis

Transcript levels of Cl-PaxB are very low during all postreleaselarval stages, from free-swimming larvae to larvae attached to thesubstrate, which may imply expression in only a small fraction of alllarval cells, whereas overall transcript levels are substantiallyincreased in adult tissue (R in Fig. 7). In contrast, transcript levels ofCl-Six1/2 are consistently much higher than those of PaxB throughoutall larval stages and in adults (Fig. 7). Real-time RT–PCR indicates thatSix1/2 transcript levels also increased in adult tissue compared tolarval stages when normalized to actin transcript levels (data notshown). Similarly, in dissociated and reaggregated adult cells, astriking difference is observed between transcript levels of PaxB andSix1/2 (Fig. 7). PaxB transcript levels are highest during primmorphcellular attachment and spreading, whereas levels in disaggregatedtissue and reaggregating primmorphs are low. Conversely, Six1/2 ismost highly expressed in disaggregated cells and reaggregatedproliferating primmorphs, while considerably lower levels areapparent during primmorph cellular attachment and spreading(Fig. 7).

Analysis of transcript patterns by in situ hybridization showed thatPaxB transcripts are abundant in reproductive Chalinula adults(Fig. 8A) but not detectable in embryos and prerelease larvae thatare part of that tissue (inset of Fig. 8A). Six1/2 is also expressed inadult tissue undergoing reproduction. However, in contrast to PaxB,transcripts of Six1/2 are observed in larvae, mainly in their outerepithelial layer but not in their inner cell mass, as evident fromsections of reproductive adult tissue (Fig. 8B). Although RT–PCRanalysis demonstrated very low levels of PaxB transcripts but veryhigh levels of Six1/2 transcripts during postrelease larval develop-ment (Fig. 7), we were only able to assess spatial expression in earlierstages, i.e., in embryos and prerelease larvae (insets of Figs. 8A and B).After release from the mother sponge, free-swimming larvae of thisspecies are especially fragile, and no fixation methods were able topreserve intact larvae suitable for in situ hybridization analysis.

It was possible, however, to compare PaxB and Six1/2 transcriptpatterns in adult tissue since both genes are transcribed to reachrelatively high levels in adult sponges (R in Fig. 7). Preservation oftissue and cell structure in sections of adults is particularly challengingbecause of their high spicule content (red arrows in Figs. 8C and D);nevertheless, global expression patterns of PaxB and Six1/2 wereanalyzed in adult tissue. The leuconoid morphology of the adultChalinula sponge includes four major ‘regions’ (Figs. 8B–D). The first isthe atrial opening of the oscular chimney, the osculum'smajor conduit

Fig. 5. Diagnostic sites of paired domains. (A) Amino acids at 41 diagnostic positions, numbered at the top, of paired domain subfamilies are shown. At each of these positions, theamino acid of at least one subfamily differs characteristically from that in other subfamilies. Amino acids with a single incidence or a frequency below 20% at a certain position withina subfamily are not listed. One or several amino acids diagnostic for a subfamily are marked by the color of the subfamily indicated in the left margin. If more than one subfamilyshares the same amino acid at a position, the amino acid is considered to be diagnostic for the subfamily that displays the highest incidence of this amino acid within the subfamily,weighed by the number of paired domains of that subfamily among the 126 paired domains analyzed (Fig. S2). The amino acids are shown in four different letter sizes, withdecreasing size indicating frequencies ≥90%, ≥66%, ≥33%, and b33%. (B) Table listing numbers of differences at diagnostic positions between paired domain subfamilies. Onlydifferences equal to or larger than a factor of 5 in frequency of the amino acid are considered to be significantly different. (C) Frequencies of amino acid changes at diagnostic sites ofpaired domains. The number of amino acid changes taken from (A) is plotted as a function of their position within the paired domain. Below the histogram, contacts of amino acidswith the DNA backbone (P) and/or base in the major (M) or minor (m) groove are indicated (Xu et al., 1999).


through which the water is extruded. A pinacodermal layer of cellsseparates the atrial opening of the oscular chimney from the spongebody, the largest portion of which consists of the choanosome ormesohyl. This internal region comprises the choanocyte chambersconsisting of choanocytes, their associated canals, and a variety ofother cell types (e.g., archeocytes and schlerocytes). Finally, theoutermost region is the external pinacodermal layer lining the surface

Fig. 4. Phylogenetic tree of Pax genes. The cladogram, inferred by maximum likelihood, wastatistical support is indicated as approximate likelihood ratio test (aLRT) values. For branchpaired domain sequences, see Fig. S2. Eight paired domains from Ctenophora, Platyhelminthebe grouped unambiguously with any of the paired domain subfamilies and hence were labetheir cognate phyla, Pax protein subfamilies are schematically indicated with their character

of the sponge facing the environment. PaxB and Six1/2 are transcribedin the pinacodermal lining of the atrial opening of the oscular chimney(black arrows in Figs. 8B–D) as well as in the immediately adjacentchoanosome (Figs. 8C and D). However, it is unclear whether they aretranscribed in the same cells. Transcripts of PaxB appear moreabundant in the pinacodermal cell layer lining the oscular chimneythan those of Six1/2. Although transcripts of both PaxB and Six1/2were

s obtained by the analysis of 133 paired domains (Fig. S2). Next to the branches, theires with aLRT values below 0.50 forking is not resolved. For NCBI accession numbers ofs, Annelida, Nematoda, Echinodermata, and Hemichordata formed a clade but could notled ‘Pax?’ (see also legend to Fig. S2). To the right of the names of the Pax proteins andistic domains, the paired domain (red), octapeptide (blue), and homeodomain (green).

Fig. 6. Phylogenetic tree of Six genes. The cladogram, inferred by maximum likelihood, was obtained by the analysis of the 62 Six domains and Six-type homeodomains shown inFig. S3. Next to the branches, their statistical support is indicated as approximate likelihood ratio test (aLRT) values. For branches with aLRT values below 0.50 forking is not resolved.For accession numbers of Six domains and Six-type homeodomains, see Fig. S3. The Six proteins, whose names and phyla are indicated to the right of the cladogram, are grouped intothree subfamilies (Seo et al., 1999) labeled at the right.


detected within the choanosome, transcripts of neither gene weredetectable in a narrowband of the outer choanosome and the adjacentexternal pinacodermal layer (inset in Fig. 8C). It is not probable thateither gene was expressed at high levels in choanocytes becausechoanocyte chambers have a defined, circular shape. Since it was notpossible to determinewhether PaxB and Six1/2were transcribed in thesame nor in which cell type because of tissue damage caused byspicules during sectioning, this level of resolution will require another

spongemodel whose tissue can be better preserved for in situ analysis.We are currently pursuing these questions in the freshwater spongeEphydatia muelleri, where the global expression pattern we observe issimilar to that of Chalinula (data not shown). Although globalcoexpression is observed for PaxB and Six1/2 in some regions ofadult tissue (Figs. 8B–D), given the nonoverlapping expression profilesduring embryonic development (Figs. 8A and B), larval development(Fig. 7), and cell reaggregation (Fig. 7), it is difficult to reconcile these

Fig. 7. Transcript levels of Cl-PaxB and Cl-Six1/2 in developmentally staged Chalinulatissues. Chalinula PaxB (Cl-PaxB) and Six1/2 (Cl-Six1/2) transcript levels were analyzedby RT–PCR in free swimming larvae (0–8 h, 8–16 h, 16–24 h, and more than 24 h old),attached larvae (A), metamorphosing larvae (M), rhagon/juvenile adults (R),dissociated adult cells from disaggregated tissue (DC), primmorph aggregates (P),and metamorphosing primmorph tissue (MP). Transcript levels of actin (Cl-Actin),which are constant throughout development, are shown as control below.


results with exclusive roles of PaxB and Six1/2 in a Pax–Six network ofChalinula. Our results rather suggest that many cells express Six1/2 inwhich PaxB is inactive or transcribed at much lower levels duringdevelopment and in adults.

Cl-PaxB partially rescues the spapol eye phenotype

Previous experiments have shown that a cnidarian PaxB cansubstitute for Pax2 functions in the Drosophila eye (Kozmik et al.,2003). Similarly, we tested the ability of a sponge PaxB to substitutefor the D-Pax2 function in spapol mutants of Drosophila, in which theeye-specific enhancer spa of D-Pax2 is deleted (Fu and Noll, 1997).

Fig. 8. Patterns of Cl-PaxB and Cl-Six1/2 transcripts in prerelease larvae and in reproductivevisualized by immunohistochemical staining with alkaline phosphatase after in situ hybridiz(A, B) and nonreproductive (C, D) adult C. loosanoffi tissues. Reproductive adult tissues inchybridization with PaxB transcripts was hybridized with an antisense probe, while the right pview in the inset demonstrates the absence of PaxB transcripts in an embryo/prerelease lantisense probe, reveals Six1/2 transcripts in embryos and prerelease larvae. The inset shepithelial layer (el) but not the inner cell mass (icm). (C, D) Hybridization with antisense prlower magnification of the sponge body to highlight the absence of PaxB transcripts in the exto pinacodermal cells that line the atrial opening of the oscular chimney (ao) and to some

These mutants exhibit severe eye defects resulting from the missingD-Pax2 expression in cone and primary pigment cells of developingeye discs (Fu and Noll, 1997). Thus, Cl-PaxB protein was expressed inspapol mutants under the indirect control of the spa enhancer by spa-Gal4 driving the expression of UAS-Cl-PaxB. Whereas wild-type eyeshave regular hexagonal facets with mechanosensory bristles project-ing from alternate facet vertices (Fig. 9F), spapol eyes are reduced insize, their ommatidia form irregular arrays of variable size withbroken or missing bristles, and their corneal lenses are irregular, oftenfused, and contain necrotic pits (Fig. 9A). Each of eight independenttransgenic UAS-Cl-PaxB lines was able to partially rescue the spapol

phenotype when combined with spa-Gal4 (Figs. 9B–D). The rescuedeyes seemed to be normal in size, showed more regularly arrangedommatidia, and bristles were present over the entire surface of theeye. However, necrotic pits were still observed, mainly in the anteriorhalf of the eyes, affecting in different transgenic lines between 1% and10% of the ommatidia (Figs. 9B–D). By contrast, rescue with D-Pax2,also driven by spa-Gal4, was nearly complete, and no necrotic pitswere apparent (Fig. 9E).

Sponge PaxB protein is unable to induce ectopic eyes in Drosophila

A cnidarian PaxB is also able to substitute to some extent for eye-specific Pax6 functions by inducing ectopic eyes in Drosophila (Kozmiket al., 2003), which are, however, smaller than those induced by thePax6 homologs, Ey or Toy (Halder et al., 1995; Czerny et al., 1999).Accordingly,we testedwhether sponge PaxB can similarly substitute for

and nonreproductive adults of C. loosanoffi. PaxB (A, C) and Six1/2 (B, D) transcripts areation of DIG-labeled riboprobes to whole mount (A) and sections (B–D) of reproductivelude many embryos and prerelease larvae (A, B). (A) The left half showing abundantart, hybridized with a sense probe, exhibits no staining above background. The enlargedarva hybridized with antisense probe. (B) Reproductive adult tissue, hybridized withows an enlarged view of an embryo/prerelease larva with transcripts evident in theobes of PaxB (C) or Six1/2 (D) to nonreproductive adult tissues. The inset in C shows aternal pinacoderm (ep) and adjacent choanosome (c). Black and red arrows (B–D) pointof the abundant spicules, respectively.

Fig. 9. Rescue of the spapol eye phenotype by expression of Cl-PaxB under control of the spa enhancer. Scanning electronmicrographs of left eyes (anterior to the left) of females withthe following genotypes: (A) y w; UAS-Cl-PaxB (line CR6)/+; spapol, (B–D) y w spa-Gal4; UAS-Cl-PaxB (lines CR3, CR5, and CR6)/+; spapol, (E) y w spa-Gal4; UAS-D-Pax2/+; spapol,and (F) y w; UAS-Cl-PaxB (line CR6)/+.


these Pax6 functions by expressing UAS-Cl-PaxB under the control of adpp-Gal4 driver in the leg, antenna, and wing disc. In control flies,expression of UAS-ey (Halder et al., 1995) under the same controlgenerated many ectopic eye structures on legs, wings, and antennae.Ectopic expression of jellyfish PaxB led to substantially smaller eyestructures or single ommatidia on legs (Kozmik et al., 2003). By contrast,none of the UAS-Cl-PaxB transgenic lines was able to induce ectopic eyemorphogenesis under the same conditions. Neither did ectopicexpression of UAS-Cl-PaxB in imaginal discs lead to deformedappendages, as observed with UAS-ey (Punzo et al., 2001) or UAS-Tc-PaxB transgenes (Kozmik et al., 2003; data not shown), an effect weattribute to developmental pathway interference (Jiao et al., 2001). Topotentially increase the binding specificity of Cl-PaxB for enhancers ofEy target genes, the three aminoacidsQ, R, andHat positions 42, 44, and47 of the Cl-PaxB paired domain, which are conserved in the PaxB/2/5/8 subfamily, were replaced by the Pax6-specific amino acids I, Q, and N(Czerny and Busslinger, 1995), amodification that strongly enhanced inTc-PaxB its efficiency to induce ectopic eyes (Kozmik et al., 2003). In Cl-PaxB, however, none of eight independentUAS-Cl-PaxB(IQN) transgeniclines produced offspring with ectopic eyes or appendage defects whencombined with the dpp-Gal4 driver.

Discussion

Gene networks have been highly conserved in metazoans, aspredicted by the gene network hypothesis (Frigerio et al., 1986; Noll,1993). A striking example is the conservation of the Pax–Six–Eya–Dacnetwork that determines eye development (for a review see Treisman,1999), but which is also instrumental in myogenesis, nephrogenesis,and in the development of other organs (Kawakami et al., 2000). Herewe investigated the origin of two gene families that participate in thisnetwork by examining their presence in the basal phylum of sponges.We have shown that the demosponge C. loosanoffi has only one

member each of these gene families, PaxB and Six1/2, which thus arerepresentatives of the founders of these gene families. The Six3/6 andSix4/5 subfamilies and the ‘archetypal’ Six gene cluster (Boucher et al.,2000), therefore, evolved inmetazoans only after the poriferan lineagesplit but were both already present when bilaterians diverged fromcnidarians (Stierwald et al., 2004). The Pax gene subfamilies, on theother hand, have emerged only by the time of the protostome–deuterostome split, as predicted (Noll, 1993).

In addition, we have shown that, like PaxB of the cubomedusanjellyfish Tripedalia (Kozmik et al., 2003), Chalinula PaxB protein is ableto substitute for Pax2 functions in Drosophila eye development but,unlike the cnidarian PaxB, cannot perform Pax6 functions inDrosophila. As Tripedalia has complex eyes with lenses, this mightreflect that the Pax–Six–Eya–Dac network has evolved in medusozo-ans, but not in sponges. Indeed, the temporal expression patterns ofPaxB and Six1/2 in developing embryonic and larval tissue andreaggregating adult cells of Chalinula donot suggest the existence of anexclusive primordial Pax–Six network in the ancestor of sponges andeumetazoans. However, our results do not exclude that such a networkcould exist given overlapping expression patterns in adult sponges.Furthermore, it is possible that a network could play a role in a smallfraction of cells of developing sponges, such as in the photoresponsivecells of parenchymella larvae (Leys and Degnan, 2001; Maldonado etal., 2003). If the generation of these photoresponsive larval cellsdepends on a Pax–Six network, the hypothesis that this network is aprecursor to metazoan sensory systems (Maldonado et al., 2003) and/or gave rise to eyes of Cnidaria or Chordatawould receive considerablesupport. The Chalinula larvae used in this study are small, fragile, anddifficult to collect, which made it impossible to determine whetherPaxB and Six1/2 are expressed at the site of the future or existingpigmented ring at the posterior pole where the photoresponsiveciliated epithelial cells are located (Maldonado et al., 2003). Shouldhowever a Pax–Six network exist in sponges, it seemsmore likely to be


active in pinacodermal cells and their associated choanosome thatmayplay a role in the contractile activity of the adult rather than inphotoresponsive larval cells.

An alternative possibility, consistent with the absence of atemporally correlated expression of PaxB and Six1/2 in Chalinula, isthat the Pax–Six gene network evolved in metazoans only after theirdivergence from sponges. An attractive hypothesis is that homologousgene networks evolved and diversified in parallel with the diversifica-tion of the gene families of a particular network, here those of Pax andSix genes. For example, Pax2 and Pax6 evolved by duplication of PaxB, asdiscussed below, both taking over different tasks in eye development ofvertebrates and flies, illustrating how gene duplication and functionaldiversification accelerated the diversification of eyes (Kozmik, 2005).Such a hypothesis is consistent with the many homologous Pax–Sixgene networks, inwhich the replacement of amember of one subfamilyby a member of a different subfamily leads to a different integratedfunction of the network (Noll, 1993). Among these diverse functions ofhomologous Pax–Six gene networks are crucial roles in the develop-ment of the eye, inner ear, skeletal muscles, kidney, limb, and brain(Kawakami et al., 2000).

Origin and evolution of Pax genes

Based on their paired domains, modern Pax genes can be dividedinto four or five subfamilies, PaxB/2/5/8, PaxD/3/7, Pax1/9, Pax6, andPoxn, depending onwhether or not Poxn-like genes are included in thePaxB/2/5/8 subfamily (Noll, 1993). Pax genes have been found in

Fig. 10. Evolution of Pax genes in metazoans. The scheme was derived from the 136 pairedgene whose modern descendant, PaxB, has been maintained in Porifera. Subsequent duplicaPax gene subfamilies Pax3/7, Pax1/9, Poxn, Pax2/5/8, and Pax6. In several lines of Diploblasubfamilies indicated behind the phyla at the right. The phylum of Cnidaria is characterizeMedusozoa. Note that the four duplications giving rise to all Pax subfamilies have occurred byoccurred later. Duplication of Pax genes within subfamilies continued, as exemplified by thesubfamilies. Although paired domains that formed a single cluster in Fig. 4 but could not benot belong to the Pax3/7 or Pax6 subfamily because they do not include a homeodomainsubfamily since, unlike Pax1/9 that have no introns in their paired domains, they are interrincluded in the scheme are Pax genes that have lost one of the two subdomains of the pairewhere the RED domain was lost in the Pax3/7 subfamily of Platyhelminthes.

metazoans but not in plants, lower eukaryotes, or bacteria, aspredicted (Burri et al., 1989). While in anthozoans clearly more thanone subfamily of Pax genes exists (Miller et al., 2000), medusozoansmay include only a single type, the PaxB subfamily (Sun et al., 1997;Kozmik et al., 2003). This raises the question as to the origin of Paxgenes, i.e., whether more primitive metazoans, such as Porifera orPlacozoa, possess only a single subfamily of Pax genes. While a PaxBgene was found in the only characterized placozoan species T.adhaerens (Hadrys et al., 2005) and in the demosponges E. fluviatilisand Reniera sp. (Hoshiyama et al., 1998; Larroux et al., 2006), itremained unclear whether additional subfamilies of Pax genes werepresent in these animals. We have shown here that only one Pax geneis present in the genome of the demosponge C. loosanoffi and that itbelongs to the PaxB subfamily.

In an attempt to trace the evolution of Pax genes back to their origin,we have analyzed 136 paired domain sequences, covering a wide rangeof phyla, i.e., parazoans and eumetazoans, diploblasts and triploblasts, aswell as protostomes and deuterostomes (Fig. S2). To increase thecoverage of lowerphyla,wehave annotated a number of Paxgenes fromthe genome sequencing projects of the placozoan T. adhaerens, theplanarian S. mediterranea, the annelid C. capitata, and the hemichordateS. kowalevskii. A dendrogram including 133 of these paired domains(Fig. 4) is consistentwith the grouping of Pax genes intofive subfamiliesamong which the Poxn/PaxA/PaxC clade is assumed as separatesubfamily, in addition to the previously established subfamilies ofPax1/9, PaxD/3/7, PaxB/2/5/8, and Pax4/6 (Noll, 1993; Sun et al., 1997;Galliot et al., 1999;Miller et al., 2000). Eightpaireddomains could not be

domains shown in Fig. S2. Pax genes evolved from a single PaxB-type ancestral Ur-Paxtions (filled circles) of Pax genes before the radiation of Bilateria generated the modernsts and Triploblasts Pax genes were lost (open squares), resulting in their spectrum ofd by different subfamily spectra in the class of Anthozoa and the remaining classes ofthe time of the Cambrian explosion, about 500 Mya, whereas most losses of subfamiliesmembers of the Drosophila prd/gsb/gsbn subfamily or the vertebrate Pax2/5/8 and Pax6assigned unambiguously to a particular subfamily, their proteins, indicated as ‘Pax?’, do. The unassigned Platyhelminthean proteins do not belong to the Poxn or the Pax1/9upted by an intron in α3 but not by one close to α4, which is diagnostic for Poxn. Notd domain, PAI or RED (Hobert and Ruvkun, 1999), except in the newly discovered case


assigned to any of these subfamilies and formed a separate group(Fig. 4). However, an assignment of these paired domains to a newsubfamily is presently not sufficiently supported.

In a more perspicuous approach to assess to which subfamily acertain paired domain belongs, we have examined which amino acidpositions in paired domains are diagnostic for a paired domainsubfamily. An extension of this analysis, previously used for the PaxB/2/5/8 and Pax6 subfamilies (Kozmik et al., 2003), is shown for allpaired domain subfamilies in Fig. 5A. It has been derived from the 125complete paired domains of Fig. S2 that have been assignedunambiguously to a specific subfamily in the phylogenetic tree ofFig. 4. According to these 41 sites diagnostic for paired domainsubfamilies (Fig. 5A), the paired domain of Poxn is clearly closer tothat of PaxB than those of PaxA and PaxC. Thus, Poxn differs from PaxBat only 9 but from PaxA and PaxC at 14 and 13 of these positions(Fig. 5B). In addition, with as few as 4 differences at the 41 diagnosticsites, the paired domains of the PaxB subfamily are closest to those ofthe Pax2/5/8 subfamily, while the paired domains of PaxD are mostsimilar to those of the Pax3/7 subfamily (Fig. 5B), in agreement withthe dendrogram shown in Fig. 4.

Based on these considerations, we have derived a pedigree ofpaired domain subfamilies that originates with the single Pax genefound in Porifera (Fig. 10). For this pedigree, we have adopted aclassical phylogeny in which Porifera are basal to Placozoa (Philippe etal., 2009), although this has been questioned recently by the proposalthat Placozoa are basal to all Metazoa (Dellaporta et al., 2006;Schierwater et al., 2009). Clearly, our analysis of Pax and Six genefamilies supports the classical pedigree with sponges at its root betterbecause only one subfamily of each gene family is found in sponges,whereas Placozoa have two subfamilies of Six genes, Six1/2 and Six3/6(Figs. 3B and 6). However, as we shall see, these alternative proposalsof an urmetazoon, regardless of whether a sponge or placozoon, willnot seriously affect the pedigree of Pax genes in Metazoa.

The demosponge C. loosanoffi has only a single PaxB gene, includingan octapeptide and a homeodomain. While the dendrogram of paireddomains indicates that T. adhaerens PaxB2 belongs to the Poxnsubfamily (Fig. 4), analysis of its paired domain (Fig. S2) by use ofdiagnostic paired domain sites (Fig. 5A) shows that it is closer to thePaxB (11 differences) than the Poxn subfamily (15 differences). Thisview is supported by the fact that the placozoan PaxB2 includes ahomeodomain, like PaxB but unlike Poxn. Thus, the placozoan T.adhaerensgenomecontains two PaxBgenes (Hadrys et al., 2005; Fig. S2),both encoding a homeodomain and one of them an octapeptide, but noPax genes of a subfamily different from PaxB. Therefore, theUr-Pax genethat appeared in Porifera and Placozoa was a PaxB gene, including anoctapeptide and a homeodomain, as previously proposed (Noll, 1993).This gene duplicated in the placozoan lineage where one of theduplicated genes lost its octapeptide. As the PaxD/3/7 subfamily isfound in triploblasts and anthozoan diploblasts (Figs. 4, S2, and 9), itsprecursor was generated by duplication of PaxB between the parazoan–eumetazoanand thediploblast–triploblast splits. This argument rests onthe conclusion that it is improbable that the PaxD and Pax3/7subfamilies arose independently by convergent evolution, as they differin a consistent manner at 16 diagnostic positions from the PaxBsubfamily (Fig. 5A). Since at least some cnidarian Medusozoa have noPax genes of the PaxD subfamily, PaxDwas lost again in these cnidarians(Fig. 10). Another duplication of PaxB in which the homeodomain waslost produced the precursor of the modern PaxA gene as observed incnidarians (Fig. 10). Since bilaterian Poxn is clearly closer to PaxB thanPaxA (Fig. 5B) and also shares the octapeptidewith PaxB (Fig. S2),whichis absent from PaxA, we propose that both PaxA and Poxnwere derivedfrom PaxB by independent duplications rather than Poxn from PaxA, aspreviously proposed (Miller et al., 2000). Therefore, PaxAwas generatedin diploblasts before the cnidarian radiation (Fig. 10). By contrast, PaxCoriginated by a later duplication from PaxB in anthozoans. Thus, theorigin of anthozoan PaxD preceded that of PaxA, which in turn occurred

before that of PaxC, in agreement with the increasing number ofdeviations from PaxB at the diagnostic paired domain sites (Fig. 5B).

In the triploblast lineage, a similar argument can be made for theorigin of the Pax1/9 subfamily. Since these genes are found inprotostomes and deuterostomes, a duplication of one of the ancestralPax genes occurred before the protostome–deuterostome split intriploblasts to generate the precursor of the Pax1/9 subfamily (Fig. 10).At this time, the ancestral PaxB and PaxD genes existed, either ofwhichcould have produced the Pax1/9 subfamily by duplication and loss ofthe homeodomain in the precursor of Pax1/9 (Fig. 10) because bothshow a similar number of deviations from the Pax1/9 subfamily at thediagnostic sites of their paired domains (Fig. 5B). In the same timeinterval before the protostome–deuterostome split, PaxB rather thanPaxD generated the ancestral Pax6 gene because Pax6 is much closer toPaxB (Fig. 5B). Since during this very period Pax2/5/8 emerged fromPaxB, whichwas losing part of its homeodomain, we propose that Pax6and Pax2/5/8 were generated by the same duplication event of theancestral PaxB (Fig. 10). Thus, one of the duplicated genes lost theoctapeptide and evolved into Pax6, the other evolved into Pax2/5/8,losing the C-terminal half of its homeodomain in vertebrates andPlatyhelminthes and retaining only the N-terminal fifth of its homeo-domain in arthropods (Fig. 2B). Interestingly, the amino acid sequencefollowing the conserved part of the homeodomain has been highlyconserved in arthropods although with no recognizable similarity tohomeodomains (Fig. 2B). A similar conservation of the sequencefollowing the N-terminal half of the homeodomain was observed inchordates (Fig. 2B).

Remarkably, the genomes of the two species of the phylumPlatyhelminthes that have been sequenced have Pax genes with acomplete paired box that can be assigned unambiguously to only twosubfamilies, Pax2/5/8 and Pax6. The two Pax genes Pax2b,c of thePlatyhelminthean species S. mediterranea that cannot be assigned to aparticular subfamily have an intron in α3, unlike Pax1/9 that have nointrons interrupting their paired domain, but no intron close to α4(Fig. 1C), which is diagnostic for Poxn (s. below). Hence, in thisphylum Pax1/9 and Poxn were lost, while the Pax3/7 subfamily wasretained but lost the RED domain (Fig. 10). Similarly, Poxn appears tohave been lost in nematodes and chordates, whereas Pax3/7 was lostin echinoderms and hemichordates before their divergence fromchordates, as their unassigned Pax genes do not encode a homeo-domain (Fig. 10). Pax genes that could not be assigned to a specificsubfamily but grouped in a single clade (‘Pax?’ in Fig. 4) were found inPlatyhelminthes, Annelida, Nematoda, Echinodermata, and Hemi-chordata (Fig. 10). It is conceivable that these are members of anadditional subfamily, although at present, such a hypothesis is notsufficiently supported. If this was the case, this subfamily originatedby an additional duplication of PaxB and concomitant loss of itshomeodomain before the split of deuterostomes from protostomeswith its subsequent loss in Chordata and Ecdysozoa.

Origin and conservation of intron positions in Pax genes

A discussion of the evolution of Pax genes would be incompletewithout a consideration of the positions of introns interrupting theirconserved domains. These may be regarded as fingerprints of thehistory of a gene family and permit an estimate of the time at whichintronsmust have been present during evolution. Consequently, someof them might be diagnostic for Pax gene subfamilies. All Pax geneshave an intron in the first codon, or only a few nucleotides upstream,of the paired domain. Since this position is always close to theN-terminus of the coding region, its precise location might havebeen under less stringent selection, provided the paired domainremained intact, and could have varied its position by a sliding duringevolution (Stoltzfus et al., 1997; Rogozin et al., 2000). Another intronthe position of which is highly conserved, from PaxB in sponges toPax2/5/8 in Platyhelminthes, arthropods, sea urchins, and chordates


(Fig. 1C), splits the third α-helix of the paired domain. This intron isalso present in two Platyhelminthean Pax genes, Sm Pax2b,c (Fig. 1C),that could not be assigned to a particular subfamily but lacka homeodomain and hence are probably diverged members of thePax2/5/8 subfamily. The position of this intron is also conserved inPax6 of arthropods and the urochordate Ciona intestinalis, but is absentin Pax6 of sea urchins and vertebrates, which instead have an intronshifted to a position between the second and third α-helix (Fig. 1C).Similarly, in Caenorhabditis elegans this intron that splits α3 is absentfrom both Pax2 and Pax6. In Pax2, it is replaced by an intron locatedbetween α2 and α3 at the same position as in Pax6 of sea urchins andvertebrates; and in Pax6, by an intron at the N-terminus of α3, aposition shifted with regard to an intron of Drosophila Poxn by onenucleotide (Fig. 1C). The positions of all these introns may beexplained by rare events of intron sliding over very short distancesduring evolution (Stoltzfus et al., 1997; Rogozin et al., 2000) as well asby nearby hot spots to which introns may slide (Fig. 1C).

It is striking that the position of the first intron interruptingα6 of thepaireddomain is conserved in Pax6while that of the second intron inα6is conserved and restricted to Pax2 (Fig. 1C). This suggests that theseintrons were present in the ancestral Pax6 and Pax2 gene, respectively.Aswe propose that these genes originated in a duplication event of theirancestralPaxBgene, the simplestmodelwould predict that thepositionsof intronswere also fixed during this event, whichwould imply that theancestral PaxB gene had an intron at only one of these two positions.Most Pax3/7 genes have lost this intron, but it was retained in Pax3/7 ofvertebrates, although its position was shifted by one nucleotide ascompared to its position in Pax6 (Fig. 1C). The intron found inα5 of thepaired domains of Drosophila gsbn and C. elegans Pax2 may again haveresulted by intron sliding to a hot spot from the respective positions inα6 of Pax3/7 and Pax2/5/8.

Similar arguments can be made for the presence at earlier times inevolution of the two introns that split α1 of the homeodomain in theprecursor of Pax6 genes andα3 of the homeodomain in the precursorsof PaxB/C/D, Pax3/7, and Pax6 (Fig. 1C). In general, many introns havebeen lost during evolution that split the paired domain and homeo-domain of Pax genes. Thus, all introns interrupting the paired domainof the Pax1/9 gene subfamily were lost. By contrast, evidencesupporting the gain of introns by Pax genes is scarce. An example,however,may be the intron interruptingα1 of the Pax6 homeodomain(Fig. 1C), which appears to be present in Pax6 genes of most, if not all,bilaterian phyla and hence may have been acquired during theduplication of PaxB giving rise to the ancestral Pax6 and Pax2/5/8 genes (Fig. 10). Another example of an intron gainmay be the introninterrupting the linker between the PAI andRED subdomains in Pax2ofC. elegans (Fig. 1C).

An interesting intron position is conserved between Pax3/7 ofvertebrates, Pax6 of C. elegans, and Poxn of all phyla examined, whichis located between the coding regions of the PAI and RED subdomainsof the paired domain (Fig. 1C). At this position, small triplet shifts ofthe intron–exon boundary are apparently tolerated or may be evenadvantageous, as long as they result in the insertion of only few aminoacids in the linker between the PAI and RED domains (Fig. S2). Hence,it is possible that this intronwas present in PaxBwhen its duplicationsgenerated the ancestral PaxD/3/7, Poxn, and Pax6 genes, i.e., beforethe separation of triploblasts from diploblasts (Fig. 10). If the paireddomain was generated by fusion of the two subdomains, PAI and RED,a homeodomain and a homeodomain-like fold (Xu et al., 1999), it ispossible that this intron is a relic of this event and hence was presentalready in the first PaxB gene. Alternatively, this intron may havearisen independently at hot spots in various Pax gene subfamilies andphyla. In Pax2 of C. elegans, this intron may again have shifted to aposition closer to the third α-helix of the paired domain (Fig. 1C).

In summary, these considerations suggest that the first Pax gene inmetazoans was of the PaxB-type, as previously proposed (Gröger etal., 2000; Kozmik et al., 2003), and included, in addition to its paired

domain, an octapeptide and homeodomain (Noll, 1993; Balczarek etal., 1997). In addition, it included probably at least five introns, if theintron separating the PAI and RED subdomains was present, whichwere located at the N-terminus of the paired domain, in α3 and α6 ofthe paired domain, between the PAI and RED domain, and inα3 of thehomeodomain. These introns were lost in some lineages, whereasother introns were probably added later (for a review, see Jeffares etal., 2006). It appears that after the Cambrian explosion of triploblastsintron loss of Pax genes was much more frequent than intron gain.Finally, new Pax genes not only originated by gene duplication butwere also lost, such as Pax3/7 in Echinodermata and Hemichordata orPoxn in Chordata. Moreover, no new subfamilies of Pax genes weregenerated after the Cambrian explosion, although duplication of Paxgenes continued to occur.

Relation to earlier models of Pax gene evolution

While our model of the evolution of Pax genes is largely consistentwith previous models (Noll, 1993; Breitling and Gerber, 2000; Galliotand Miller, 2000; Miller et al., 2000; Hadrys et al., 2005; Matus et al.,2007), it is much more specific and deviates in a number of salientfeatures. In addition to the paired domain, the first Pax gene ofPorifera or Placozoa encoded a homeodomain and an octapeptide andthus was not a PaxA but a PaxB gene. Rather PaxA has been derivedfrom the ancestral PaxB gene by duplication and loss of the homeoboxand octapeptide (Fig. 10). Moreover, in contrast to the proposal ofothers (Galliot and Miller, 2000; Miller et al., 2000), we have arguedthat Poxn was derived by duplication from PaxB rather than PaxA.Therefore, contrary to a recent report (Hoshiyama et al., 2007), thediversification of the Pax gene family began after the divergence ofEumetazoa from Parazoa (Fig. 10).

The PaxD subfamily of Pax genes originated by duplication of theancestral PaxB gene, including its octapeptide and homeodomain,before the diploblast–triploblast split and gave rise to the modernPax3/7 subfamily (Miller et al., 2000; Fig. 10). It lost its octapeptide inthe anthozoan lineage but retained it in many modern Pax3/7 genes(Hadrys et al., 2005) where it was discovered (Burri et al., 1989).Surprisingly, this subfamily of Pax genes was lost at least twice, first inMedusozoa and again in Echinodermata and Hemichordata (Fig. 10).It appears that loss of Pax gene subfamilies occurred repeatedlyduring evolution. Thus, Poxn and Pax1/9were lost in Platyhelmintheswhile Poxn was lost in nematodes and chordates (Fig. 10).

The remaining Pax gene subfamilies originated by gene duplicationbefore the protostome–deuterostome split, Pax1/9 from the ancestralPaxB or PaxD gene, where it lost the homeobox (Fig. 10), and Pax2 andPax6 from the ancestral PaxB gene, during which Pax2 lost most of thehomeobox, and Pax6, the octapeptide (Matus et al., 2007; Fig. 10).

The enigma of diagnostic paired domain sites

From the expanded list of paired domains (Fig. S2), a table wasderived for those sites that are diagnostic for the various paireddomain subfamilies (Fig. 5A). Astonishingly, most of the 41 aminoacids at these positions do not contact the DNA of an optimal Pax6-binding site, and of those that do, about half are located in the regionthat links the two paired subdomains (residues 61–76) and interactwith the minor groove while only N47 interacts with a base in themajor groove (Xu et al., 1999; Fig. 5C). Moreover, of the three aminoacids, 42, 44, and 47, known to distinguish between DNA binding sites(Czerny and Busslinger, 1995), the first two do not contact the DNAeither (Xu et al., 1999). Conversely, of the 38 amino acids that contactthe DNA binding site (Xu et al., 1999), only 9 are at diagnostic sites(Fig. 5C). Indeed, a plot of the number of amino acid changes atdiagnostic sites versus their position within the paired domain(Fig. 5C) shows that 6 of the 9 contacts by diagnostic amino acidsoccur at minimal frequencies. As the diagnostic sites probably


influence the selection of DNA target sites, they may affect the precisestructure of the paired domain and thereby the binding of other aminoacids to the DNA binding site and/or determine directly or indirectlythe interaction of the paired domains with other transcription factors,such as Ets proteins (Fitzsimmons et al., 1996), thus influencing theselection of DNA target sites, regardless of whether these act asenhancers or silencers (Eberhard et al., 2000). Some of the diagnosticsite changes may also compensate for each other. For example, R64interacts with D20 (Xu et al., 1999), which is reversed in the paireddomain of PaxD but not Pax3/7 (Fig. 5A). Considering the paireddomain DNA interaction model derived from crystallographic studies(Xu et al., 1999), we note that the diagnostic sites generally cluster inregions oriented away from the DNA, as expected if the sites wouldinteract with other transcription factors (Garvie et al., 2001). Inagreement with this view, paired domain proteins of the samesubfamily can largely substitute for each other's functions in vivo andhence recognize the same target sites (Li and Noll, 1994; Xue and Noll,1996; Bouchard et al., 2000). To test the significance of the diagnosticpaired domain sites, it will be important to determine the target sitesof different subfamilies of paired domains as well as the amino acidswithin paired domains that mediate interactions with other transcrip-tional regulators important for target site selection.

Acknowledgments

This work has been supported by a grant from the JeffressMemorial Trust (to A.H.) and the Swiss National Science Foundationgrant 3100-56817 (to M.N.) and by the Kanton Zürich, Switzerland.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ydbio.2010.03.010.

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Fig. S1. Southern blot analysis of Chalinula loosanoffi genomic DNA with probes specific for PaxB and Six1/2. Genomic DNA (1 μg each) from C. loosanoffi was digested with different various restriction enzymes (Ba, BamHI; Bg, BglII; P, PstI), subjected to agarose gel elec-trophoresis, denatured, and transferred and fixed to a Nylon membrane (Hybond, Amersham), which was subsequently hybridized with probes for Cl-Six1/2 (320 bp spanning the Six and Six-type homeodomain; DIG-labeled) and Cl-PaxB (450 bp spanning the paired domain; 32P-labeled). After removal of non-hybridized probes, the hybridized DNA bands were detected by standard procedures. The positions of DNA length standards (in kb) are indicated in the left and right margins.

ClSix ClPaxB

23.1

9.4

6.6

4.4

2.32.0

BgPBa P

1.0

2.0

3.0

4.05.06.08.0

10.0

HD OPShort Name Accession Info Species, Gene Phylum Subphylum/Class 1 10 20 30 40 50 60 70 80 90 10

0

110

120

128 1 10 20 30 40 50 60

Cl PaxB GQ985310 Chalinula loosanoffi PaxB Porifera Demospongiae E Q G G V N Q L G G L F V N G R P L P D P I R R R I V E L A Q N G I R P C D I S R Q L R V S H G C V S K I L G R F Y E T G S V K P G I I G - G S K P K - - - - - V A T P K V V I K I E E F K R D N P S I F A W E I R D R L L T E K I C N K A N V P S V S S I N R I V R S R A P Q P F L - - K E L T Q Y Q E Q Q L E L E F S N T H Y P - D P S S V - N V L A Q K L D L P D K V I E E W F T Y H R R S L I T S T Y S Q L S I L Q TAq PaxB ACA04746 Amphimedon queenslandica PaxB Porifera Demospongiae G . . . . . . . . . V . . . . . . . . . I . . . K . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - . . . . . - - - - - . . . . . . . N . . . D Y . . E . . . . . . . . . . . . . . . . G . . . . V . . . . . . . . . . . . . T . . K T E G T - - R K . . P F . I H . . . . S Y N T S . . . - A R P T . - K Q . . S L . . . . E S T . . S . . A E . . Q M S N N A S L G H F . . . . .Ef PaxB BAA36346 Ephydatia fluviatilis PaxB Porifera Demospongiae G . . . . . . . . . . . . . . . . . . E S . . . K . . . . S . . . V . . . . . . . . . . . . . . . . . . . . . . Y . . . . . I . . . V . . - . . . . . - - - - - . . . S . . . L . . . D Y . Q E . . . . . . . . . . . . . . Q D G V . D . V . . . . . . . . . . . . . T . . E G D N T - - P T . L D A . V H E . . R S L G D C A . . - . T A T . - Q D . . C R . G . T E G Q . Q S . L K A R Q P . P A P W GTa PaxB AAY68376 Trichoplax adhaerens PaxB Placozoa Trichoplax G H V T I . . . . . V . E . . . . . R E A . . . H . . Q . . . S . V . . . . . . . . . . . . . . . . . . . . C . Y . Q . . . . S . . . . . - . . . . . - - - - - . . . . T . . D . . A . Y . . N . S T . . . . . . . E K . . G D . . . D A S . . . . . . . . . . . . . . K V L R R N R - - T M F . D E . I K K . . D I . K S . Q . . - . V Y T R - E E . . S . I G . S E A R V Q V . . S N R . A K W R K E G . R I N D L . G ITa PaxB2 gb|ABGP01000723.1; 129179 - 130530 Trichoplax adhaerens PaxB2 Placozoa Trichoplax G . . . . . . . . . V . I . . . . . . N Y . . S . . . D . . K . . V . . . . . . . R . L . . . . . . . . . . . . Y . . . . . . R . . T . . - . . . . . - - - - - . . . . T . . A . . . Q . . H E . . . . . . . . . . E K . I N . . V . K D E T . . . . . . . . . V L . H . . . R R N R - - T T F . G K . L E E . . K A . Q I N Q . . - E V N . R - E D . . K Q V A . S E A R V Q V . . S N R . A K W R R A QAj PaxB BAF56220 Anthopleura japonica PaxB Cnidaria Anthozoa G H . . . . . . . . V . . . . . . . . . G V . Q . . . . . . . S . V . . . . . . . . . . . . . . . . . . . . C . . . . . . . I . . . V . . - . . . . . - - - - - . . . . G . . N . . A . Y . M S . . T M . . . . . . . . . . S . N V . S P D . . . . . . . . . . . . . N . I M S R R R - - T T F . D D . I D K . . R V . E K . . . . - . V F T R - E E . . . Q V N . S E - - - - V . Y S N R . A K W R K E G . . I S G L . G IAm PaxB AAF64460 Acropora millepora PaxB Cnidaria Anthozoa G H . . . . . . . . . . . . . . . . . . V V . S . . . D . . . S . V . . . . . . . . . . . . . . . . . . . . C . . . . . . . I . . . V . . - . . . . . - - - - - . . . G P . . N . . A . Y . . N . . T M . . . . . . . . . . S . G V . S T D . . . . . . . . . . . . . N . I V R R Q R - - T T F S G E . I E . . . K T . E K . . . . - . V F T R - E K . . . D V . . S E A R . Q V . . S N R . A K W R K Q E . . I N G . . G MNv PaxB AAW29067 Nematostella vectensis PaxB Cnidaria Anthozoa G H . . . . . . . . V . . . . . . . . . V V . Q . . . . . . . S . V . . . . . . . . . . . . . . . . . . . . C . . . . . . . I . . . V . . - . . . . . - - - - - . . . G N . . T . . A . Y . L A . . T M . . . . . . . . . . S . G V . T S D . . . . . . . . . . . . . N . I S R R Q R - - T N F . D E . I E K . . K V . E K . . . . - . V F T R - E E . . . Q V N . S E A R . Q V . Y S N R . A K W R K E GPc PaxB CAB61522 Podocoryne carnea PaxB Cnidaria Hydrozoa G H . . . . . . . . V . . . . . . . . E . V . . K . . . . . H Q . V . . . . . . . . . . . . . . . . . . . . . . Y . . . . . I R . . V . . - . . . . . - - - - - . . . . S . . T . . A . Y . I H . . T M . . . . . . E . . . N D Q . . D V E S . . . . . . . . . . . . N . L G R R V R - - T T F S A D . K I A . . Q A . E K . P . . - . A V Q R - E Q I . I R S Q I . E A R V Q V . . S N K . A K . R R Q D . . V N G . . G ICc PaxB1 AAF91372 Cladonema californicum PaxB1 Cnidaria Hydrozoa N H . . . . . . . . I . . . . . . . . E H . . . K . . D . S S Q . V . . . . . . . E . . . . . . . . . . . . . . . . . . . . I R . . V . . - . . . . . - - - - - . . . . S . I Q . . A . Y . T Q . . T M . . . . . . E C . . N D N . . D A E S . . . . . . . . . . . . N . I S R R V R - - T T F S N E . K K E . . K A . E K . P . . - . A . Q R - E Q I S I . S Q I . E Q R V Q V . . S N K . A K . R R Q GHyma PaxB XP_002159617 Hydra magnipapillata PaxB Cnidaria Hydrozoa N H . . I . . . . . T . . . . . . . I E . V . . K . . . . . H Q . V . . . . . . . . . . . . . . . . . . . . S . . . . . . . . R . . V . . - . . . . . - - - - - . . . . S . . A . . Q . Y . Q H . . T M . . . . . . . K . . S . Q . . D S D S . . . . . . . . . . . . N . L M R R V R - - T T F S L E . R R A . . D A . E K . P . . - . A E Q R - E E I S I Q C . . . E P R V Q V . . S N K . A K . R R Q D . . I S G . . G MHyl PaxB AAB58291 Hydra littoralis PaxB Cnidaria Hydrozoa N H . . I . . . . . T . . . . . . . I E . V . . K . . . . . H Q . V . . . . . . . . . . . . . . . . . . . . S . . . . . . . . R . . V . . - . . . . . - - - - - . . . . S . . A . . Q . Y . Q H . . T M . . . . . . . K . . S . Q . . D S D S . . . . . . . . . . . . N . L M R R V R - - T T F S L E . R R A . . D A . E K . P . . - . A E Q R - E E I S I Q C . . . E P R V Q V . . S N K . A K . R R Q D . . I S G . . G MTc PaxB AAQ17211 Tripedalia cystophora PaxB Cnidaria Cubozoa S H . . . . . . . . V . . . . . . . . E Q V . . . . . . . . H Q . V . . . . . . . . . . . . . . . . . . . . . . Y . . . . . I . . . . . . - . . . . . - - - - - . . . . G . . S . . A . Y . . A . . T M . . . . . . . . . . Q D S V . S Q E . . . . . . . . . . . . . N . I N R K N R - - Y N F . P E . T D L . . Q L . E K . P . . - . A T T R - E E I . K . T N . S E A R V Q V . . S N R . A K M R K Q D . . I T G . . G I

Smp Pax2a CAZ31418 Schistosoma mansoni Pax2a Platyhelminthes Trematoda G H . . I . . . . . V . . . . . . . . N Q V . Q Q . . Q . . N Q N V . . . . . . . . . . . . . . . . . . . . . . Y . . . . . I R . . V . . - . . . . . - - - - - . . . A S . . E A . C K Y . E . . . T M . . . . . . . . . . K D Q . . T V E . . . . . . . . . . . . . N K D I K R . H - - H S S . M S N G S I N G R I Y D . . N . . - E V . Y S - F P S N D N M N N T L N S L Q Y S Q F T P Y L N R N Y D ESm Pax2a SmedGD Contig v31.020951; 10683 - 11117 Schmidtea mediterranea Pax2a Platyhelminthes Turbellaria R H . . . . . . . . . . . . . . . . . N S V . Q Q . . . . . H R . V . . . . . . . . . . . . . . . . . . . . S . . . . . . . I . . . V . . - . . . . . - - - - - . . . . E . . E A . C K Y . A E . . T M . . . . . . . M . . N D R V . S Q E . . . . . . . . . . . . . N K VBm Pax2 XP_001899226 Brugia malayi Pax2 Nematoda Chromadorea S H T . . . . . . . V . . . . . . . . . H . . N . . . . . . H Q . V . . . . . . . . . . . . . . . . . . . . . . Y . . . . . I R . . V . . - . . . . . - - - - - . . . . . . . Q T . A A Y . . A . . T M . . . . . . E K . . E . R V . D S E . . . . . . . . . . . . . N K S . . I N E L . G FCe Pax2 AAA93452 Caenorhabditis elegans Pax2 Nematoda Chromadorea S H T . . . . . . . V . . . . . . . A . T V . A Q . . . M S . H . T . . . . . . . . . K . . . . . . . . . . . . Y . S . . . . R . . V . . - . . . . . - - - - - . . . . R . . E C . A G Y . . A . . T M . . . . . . Q K . I E D Q . . G E E . . . . . . . . . . . . . N K S H . I N G L . G .Cap Pax2a jgi|Capca1|119889|e_gw1.819.21.1 Capitella capitata Pax2a Annelida Polychaeta ? H . . . . . . . . V . . . . . . . . . V V . Q . . . . . . H Q . V . . . . . . . . . . . . . . . . . . . . . . Y . . . . . I . . . V . . - . . . . . - - - - - . . . . . . . E A . C K Y . . E . . T M . . . . . . . . . . S . A V . D Q E . . . . . . . . . . ? ? ? ? ? ?Pd Pax258 CAD43608 Platynereis dumerilii Pax2/5/8 Annelida Polychaeta ? H . . . . . . . . V . . . . . . . . . V V . T . . . . . . H Q . V . . . . . . . . . . . . . . . . . . . . . . Y . . . . . . R . . V . . - . . . . . - - - - - . . . . . . . G A . C K Y . . E . . T M . . . . . . . . . . A . G V . D Q E . . . . . . . . . . . . . N K . . T I M G . . G PApis Pax2 XP_001122481 Apis mellifera Pax2 Arthropoda Hexapoda S H . . . . . . . . V . . . . . . . . . V V . Q . . . . . . H S . V . . . . . . . . . . . . . . . . . . . . S . Y . . . . . F . A . V . . - . . . . . - - - - - . . . . P . . E A . A N Y . . . . . T M . . . . . . . . . . A . G . . S Q D . . . . . . . . . . ? ? ? ? ? ?Aphid Pax2 XM_001943385 Acyrthosiphon pisum Arthropoda Hexapoda G H . . . . . . . . V . . . . . . . . . L V . Q . . . . . . H . . V . . . . . . . . . . . . . . . . . . . . S . Y . . . . . F . A . V . . - . . . . . - - - - - . . . A P . . D S . A N Y . . E . . T M . . . . . . . . . . A . G V . T Q D . . . . . . . . . . . . . N K . I K R E R S Q V H Y N G D A S L Y S N . W P G K W C L K D E H K L F S E L N . A G V T S G G G T S P F Y D A N Q T A F S A . P L . . I N G . . G IDm Pax2 AAF59385 Drosophila melanogaster Pax2 Arthropoda Hexapoda G H . . . . . . . . V . . . . . . . . . V V . Q . . . . . . H . . V . . . . . . . . . . . . . . . . . . . . S . Y . . . . . F . A . V . . - . . . . . - - - - - . . . . P . . D A . A N Y . . E . . T M . . . . . . . . . . A . A . . S Q D . . . . . . . . . . . . . N K . G K R Q R M - S T Y S G D . - L Y T N I W S G K W C I K D . H K L L A E L G N L T A S T G N C P A T Y Y E A S N - G F S T . P I . . I N G . . G ITrib Pax2 XM_962948 Tribolium castaneum Pax2 Arthropoda Hexapoda G H . . . . . . . . V . . . . . . . . . V V . Q . . . . . . H . . V . . . . . . . . . . . . . . . . . . . . S . Y . . . . . F . A . V . . - . . . . . - - - - - . . . . P . . D A . A N Y . . E . . T M . . . . . . . . . . A . G . . S Q D . . . . . . . . . . . . . N K . L K R Q R - - S Q Y N G D . - L Y S N . W S . K W S I K D E H K L L S E L G G A G T T T G Q T - - G Y Y D . H G - G F P S V G V . . I N G . . G ISk Pax2a FF472144 Saccoglossus kowalevskii Pax2a Hemichordata Enteropneusta S H . . . . . . . . V . . . . . . . . . V V . Q . . . D . . H S . V . . . . . . . . . . . . . . . . . . . . . . Y . . . . . I . . . V . . - . . . . . - - - - - . . . . . . . D . . A D Y . . Q . . T M . . . . . . . . . . A . A V . D N E . . . . . . . . . . . . . N K MPl Pax258a AAB70245 Paracentrotus lividus Pax2a Echinodermata Eleutherozoa S H . . . . . . . . V . . . . . . . . . V V . Q . . . D . . H S . V . . . . . . . . . . . . . . . . . . . . . . Y . . . . . I R . . V . . - . . . . . - - - - - . . . . . . . S . . A D Y . . Q . . T M . . . . . . . . . . A . G V . E . D . . . . . . . . . . . . . N K L M K T N S - - G A F S P H . I E A M . K A C N P S L . . - . V Y R K - D I A Q D G R G Q V G M P . S S A S E . . H A H . E A Q R . . I N G . . G IPl Pax258b AAB70246 Paracentrotus lividus Pax2b Echinodermata Eleutherozoa D A E . . . . . . . C . A . . . . . . I Q . . Q Q . . . . . R E . V . . . . . . . . . K . . . . . . . . . . V . . . . . . . I . . . V . . - . . . . . - - - - - . . . G E . . N . . A D Y . . . . . T . . . . . . . . . . . S . N V . V . D T . . . . . . . . . . L . N K ISp Pax258a NW_001342565.1; 92378 - 95887 Strongylocentrotus purpuratus Pax2/5/8a Echinodermata Eleutherozoa G H . . . . . . . . V . . . . . . . . . V V . Q . . . D . . H S . V . . . . . . . . . . . . . . . . . . . . . . Y . . . . . I R . . V . . - . . . . . - - - - - . . . . . . . S . . A D Y . . Q . . T M . . . . . . . . . . A . G V . E . D . . . . . . . . . . . . . N K LSp Pax258b AAB70247 Strongylocentrotus purpuratus Pax2/5/8b Echinodermata Eleutherozoa D A E . . . . . . . C . A . . . . . . T Q . . T Q . . . . . R E . V . . . . . . . . . K . . . . . . . . . . V . . . . . . . I . . . V . . - . . . . . - - - - - . . . R D . . D . . A D Y . . . . . T . . . . . . . . . . . S . S . . G . E T . . . . . . . . . . L . N K ISp Pax258c XR_026135.1 Strongylocentrotus purpuratus Pax2/5/8c Echinodermata Eleutherozoa C H . . . . . . . . V . . . . . . . . . V V . Q . . . D . . H S . V . . . . . . . . . . . . . . . . . . . . . . Y . . . . . I R . . V . . - . . . . . - - - - - . . . . . . . S . . A D Y . . Q . . T M . . . . . . . . . . A . G V . E . D . . . . . . . . . . . . . N K LCi Pax258 BAC41498 Ciona intestinalis Pax2/5/8 Chordata Tunicata G H . . . . . . . . V Y . . . . . . . . Q V . Q Q . . D Q . H I . V . . . . . A . . . . . . . . . . . . . . A . Y . . . . . I R . . V . . - . . . . . - - - - - . . . . R . . E . . C D Y . . Q . . T M . . . . . . . . . . S . G . . D H D . . . . . . . . . . . . . N K .Pm Pax258 CAB96396 Phallusia mammilata Pax2/5/8 Chordata Tunicata G H . . I . . . . . V Y . . . . . . . . Q . . Q Q . . D Q . H H . V . . . . . A . . . . . . . . . . . . . . A . Y . . . . . I R . . V . . - . . . . . - - - - - . . . . . . . E . . C . Y . . Q . . T M . . . . . . . . . . G . Q . . D Q D . . . . . . . . . . . . . N K .Od Pax258 AAW24012 Oikopleura dioica Pax2/5/8 Chordata Tunicata S S S . I . . . . . N Y . . . . . . A L D T . E I . R L A K . G . V . . . . . . . . . . . . . . . . . . . . T . . E . . . D I . . . V . . - . . . . . - - - - - . . . . E . . N . . T . Y . H A . . T M . . . . . . Q Q . I D D R V . L . D . . . . . . . . . . . . . . Y SOd Pax258a AAY27073 Oikopleura dioica Pax2/5/8a Chordata Tunicata G N . . . . . . . . M Y . . . . . . . . . V . Q Q . . D M . . R . V . . . . . A . . . . . . . . . . . . . . A . . . . . . . I R . . V . . - . . . . . - - - - - . . . . H . . N . . C . Y . . A . . T M . . . . . . . . . . S . N V . S Q E . . . . . . . . . . . . . N K .Hr Pax258 BAA28833 Halocynthia roretzi Pax2/5/8 Chordata Tunicata G H . . I . . . . . V Y . . . . . . . . . V . Q T . . D . . H Q . V . . . . . A . . . . . . . . . . . . . . A . Y . . . . . I . . . V . . - . . . . . - - - - - . . . . R . . E . . C . Y . . Q . . T M . . . . . . . . . . V . C . . D T E . . . . . . . . . . . . . D K .Bf Pax2a AAC12733 Branchiostoma floridae Pax2a Chordata Cephalochordata G H . . . . . . . . V Y . . . . . . . . V V . H . . . . . . H Q . V . . . . . . . . . . . . . . . . . . . . R . Y . . . . . I . . . V . . - . . . . . - - - - - . . . . . . . E . . A . Y . . Q . . T M . . . . . . . . . . A . G . . D N D T . . . . . . . . . . . . N K . . E Q K R - - S T F . P D . L E A . . Q A . N R G . . . T . . F N R - D N M S N . V . . S Q T R V Q D V K P S I S C . T T S V A . . I N G . . G IDr Pax2a NP_571259 Danio rerio Pax2a Chordata Vertebrata R H . . . . . . . . V . . . . . . . . . V V . Q . . . . . . H Q . V . . . . . . . . . . . . . . . . . . . . . . Y . . . . . I . . . V . . - . . . . . - - - - - . . . . . . . E . . A . Y . . Q . . T M . . . . . . . . . . A . G V . D N D T . . . . . . . . . . I . T K V R K H L R A - D A F . . Q . L E A . D R V . E R P S . . - . V F P T S E H I K P E Q A N E Y S - L P A L N P G L D E V K P S L S . . I N G . . G IDr Pax2b NP_571715 Danio rerio Pax2b Chordata Vertebrata R H . . . . . . . . V . . . . . . . . . V V . Q . . . . . . H Q . V . . . . . . . . . . . . . . . . . . . . . . Y . . . . . I . . . V . . - . . . . . - - - - - . . . . . . . D . . A D Y . . Q . . T M . . . . . . . . . . A . G . . D N D T . . . . . . . . . . I . T K V R K H L R A - D A F . . Q . L E A . D R V . E R P A F . - . V F P T S E H I K P E Q A S E Y S - L P A L N . G L D E V K P S L S . . I N G . . G IMm Pax2 NP_035167 Mus musculus Pax2 Chordata Vertebrata R H . . . . . . . . V . . . . . . . . . V V . Q . . . . . . H Q . V . . . . . . . . . . . . . . . . . . . . . . Y . . . . . I . . . V . . - . . . . . - - - - - . . . . . . . D . . A . Y . . Q . . T M . . . . . . . . . . A . G . . D N D T . . . . . . . . . . I . T K V R K H L R A - D T F . . Q . L E A . D R V . E R P S . . - . V F Q A S E H I K S E Q G N E Y S - L P A L T P G L D E V K S S L S . . I N G . . G IMm Pax5 NP_032808 Mus musculus Pax5 Chordata Vertebrata G H . . . . . . . . V . . . . . . . . . V V . Q . . . . . . H Q . V . . . . . . . . . . . . . . . . . . . . . . Y . . . . . I . . . V . . - . . . . . - - - - - . . . . . . . E . . A . Y . . Q . . T M . . . . . . . . . . A . R V . D N D T . . . . . . . . . . I . T K V R K Q M R G - D L F . . Q . L E V . D R V . E R Q . . S - . I F T T T E P I K P E Q T T E Y S A M A S L A G G L D D M K A N L . . . I S G . . G IMm Pax8 NP_035170 Mus musculus Pax8 Chordata Vertebrata G H . . L . . . . . A . . . . . . . . E V V . Q . . . D . . H Q . V . . . . . . . . . . . . . . . . . . . . . . Y . . . . . I R . . V . . - . . . . . - - - - - . . . . . . . E . . G D Y . . Q . . T M . . . . . . . . . . A . G V . D N D T . . . . . . . . . . I . T K V R K H L R T - D T F S . H H L E A . . C P . E R Q . . . - E A Y A S P S H T K G E Q G - L Y P - L P L L N S A L D D G K A . L . . . I N G . . G IAm PaxA AAC15713 Acropora millepora PaxA Cnidaria Anthozoa G P . . . . . . . . V . . . . . . . . . Y M . H . . . . . . H C . V . . S E . . . . . L . . . . . . . . . . . . Y . . . . . . R . . A . . - . . . . . - - - - - . . . . R . . S . . L A Y . E . . . C . . . . . . . N N . . S D G V . D . S . . . . . . . . . . . L . N A .Nv PaxA AAW29066 Nematostella vectensis PaxA Cnidaria Anthozoa G P . . . . . . . . V . . . . . . . . . Y M . H . . . . . . H C . V . . S E . . . . . L . . . . . . . . . . . . Y . . . . . . R . . A . . - . . . . . - - - - - . . . . . . . S . . L . Y . D K . . C . . . . . . . N N . . A D G V . D . T . . . . . . . . . . . L . N S .Hyma PaxA XM_002155868 Hydra magnipapillata PaxA Cnidaria Hydrozoa G P . . . . . . . . V . . . . . . . . . Y M . H . . . . . . H C . V . . S E . . . . . L . . . . . . . . . . . . Y . . . . . . R . . A . . - . . . . . - - - - - . . . . . . . C R . V K L . E E . . C M . . . . . . N S . . A . G . . D N G . . . . . . . . . . . L . N H .Hyl PaxA AAB58290 Hydra littoralis PaxA Cnidaria Hydrozoa D P . . . . . . . . V . . . . . . . . . Y M . H . . I . . . . C . V . . S E . . . . . L . . . . . . . . . . . . Y . . . . . . R . . A . . - . . . . . - - - - - . . . . . . . C R . V K L . E E . . C M . . . . . . N S . . A . G . . D N G . . . . . . . . . . . L . N H .Aj PaxC BAF56221 Anthopleura japonica PaxC Cnidaria Anthozoa N H . . M . . . . . A . . . . . . . . . Y . . H . . I Q . . A Y . V . . . E . . . R . L . . . . . . . . . . . . Y . . . . . I R . . S . . - . . . . . - - - - - . . . . P . . S . . L Q Y . Q Q . . T . . . . . . . . . . V E . G . . D R E . T . . . . . . . . . L . N K . L R R N R - - T T F S P D . L E M . . K . . E K S . . . - . V A T R - E D . . N . I . M S E A R V Q V . . S N R . A K W R R H QAm PaxC AAC15711 Acropora millepora PaxC Cnidaria Anthozoa S H . . I . . . . . P . . . . . . . . . Y . . H . . . Q . . A C . V . . . E . . . R . L . . . . . . . . . . . . . . . . . . I R . . S . . - . . . . . - - - - - . . . . P . . N . . V Q Y . Q Q . . T . . . . . . . . . . V E . G V . D R E . T . . . . . . . . . L . N K . I R R N R - - T T F S P E . L E M . . K . . E K S . . . - . V A T R - E E . . S . I . M S E A R V Q V . . S N R . A K W R R H QNv PaxC AAW29068 Nematostella vectensis PaxC Cnidaria Anthozoa G H . . L . . . . . A . . . . . . . . . Y . . H . . I Q . . T Y . V . . . E . . . C . L . . . . . . . . . . . . Y . . . . . I R . . S . . - . . . . . - - - - - . . . . P . . N . . L Q Y . Q Q . . T . . . . . . . . . . V E . G . . D R D . T . . . . . . . . . L . N K . L R R N R - - T T F . P D . L E M . . K . . E K S . . . - . V A T R - E E . . N . I . M S E A R V Q V . . S N R . A K W R R H QCap Poxn jgi|Capca1|166874|estExt_Genewise1Plus.C_2990020 Capitella capitata Poxn Annelida Polychaeta G . T . . . . . . . V . . . . . . . . . S V . . . . . . . . L L . V . . . . . . . . . L . . . . . . . . . . T . . . . . . . I . . . A . . - . G . . . - - - S R . . . . Y . . K R . L A . . Q S . . . L . . . . . . . Q . . A L G . . D H Q S I . . . . . . . . . L . N T P F . I D K . . K -Dm Poxn NP_476686 Drosophila melanogaster Poxn Arthropoda Hexapoda G . A . . . . . . . V . . . . . . . . . C V . . . . . D . . L C . V . . . . . . . . . L . . . . . . . . . . T . . . . . . . I R . . S . . - . . . T . - - - - Q . . . . T . . K . . I R L . E E . S G M . . . . . . E Q . Q Q Q R V . D P S S . . . I . . . . . . L . N S G . . I E D L . K KApis Poxn XP_001123097/001122746 Apis mellifera Poxn Arthropoda Hexapoda G . A . . . . . . . V . . . . . . . . . C V . Q . . . Q . . L V . V . . . . . . . . . L . . . . . . . . . . T . . . . . . . I R . . S . . - . . . T . - - - - Q . . . . T . . K . . L R M . Q E Q . T M . . . . . . E Q . A R Q G A . D P Q S L . . . . . V . . . L . G G G . . I E E L . K KTrib Poxn XP_973036 Tribolium castaneum Poxn Arthropoda Hexapoda R . A . . . . . . . V . . . . . . . . . C V . . . . . . . . L L . V . . . . . . . . . L . . . . . . . . . . T . . . . . . . I R . . S . . - . . . T . - - - - Q . . . . T . . K . . L R . . Q E . . G M . . . . . . E Q . I A Q R V . E P H . I . . . . . V . . . L . N S G . . I E E L . K KNasv Poxn XP_001606282 Nasonia vitripennis Poxn Arthropoda Hexapoda G . A . . . . . . . V . . . . . . . . . C V . Q K . . Q . . L M . V . . . . . . . . . L . . . . . . . . . . T . . . . . . . I R . . S . . - . . . T . - - - - Q . . . . S I . K . . L R M . Q E . . T . . . . . . . E Q . A R . G - . D P Q . L . . . . . . . . . L . G N G . . I E E L . K KAg Poxn EAA10320 Anopheles gambiae Poxn Arthropoda Hexapoda G . A . . . . . . . V . . . . . . . . . I V . . . . . . . . L M . V . . . . . . . . . L . . . . . . . . . . T . . . . . . . I R . . S . . - . . . T . - - - - Q . . . . T . . K . . L R . . Q E . . G M . . . . . . . Q . . S Q R . . D P N T I . . . . . V . . . L . N G GSk Poxn2 FF670552 Saccoglossus kowalevskii Poxn2 Hemichordata Enteropneusta G L N . L P . . . . M . L . . . . . . . . L . H . . . . . . H C . . . . . . . . . . . L I . . . S . . . M . . . Y . . M . A I R . . T . . - . . . . . - - - - - . . . . E . . S . . . L Y . Q E . . T . . . . . . . . . . I S . G V . T N S T . . . . . . . . . . L . N . .Sk Poxn ABD97270 Saccoglossus kowalevskii Poxn Hemichordata Enteropneusta G . A . . . . . . . V . . . . . . . . . F . . A . . . . . . H L . V . . . . . . . . . L . . . . . . . . . . T . Y . . . . . I R . . N . . - . . . . . - - - - - . . . . L . . K . . L Q Y . Q E . . . . . . . . . . . K . . Q . R V . D E N T I . . . . . . . . . L . N S T H . I E D . . N QSp Poxn XP_785245 Strongylocentrotus purpuratus Poxn Echinodermata Eleutherozoa G . A . . . . . . . V . . . . . . . . V C V . A . . . . . . . L . V . . . . . . . . . L . . . . . . . . . . S . Y . . . . . I A . . N . . - . . . . . - - - - - . . . . L . . K . . M S Y . . K . . . . . . . . . . . . . . R . R V . D . N T I . . I . . . . . . I . N G T P . . G I . S P DSm Pax6a SmedGD Contig v31.001769; 10313 - 19424 Schmidtea mediterranea Pax6a Platyhelminthes Turbellaria G H S . I . . . . . M . . . . . . . . . M T . Q . . I . . S . S . A . . . . . . . I . Q . . N . . . . . . . C . Y . . . . . I . . K A . . - . . . . R - - - - - . . . N T . . R . V T I Y . Q E S . . M . . . . . . . . . . Q D G V . . Q D . L . . I . . . . . . L . . L . S . R S R - - T S F . N D . I N L . . K . . E R . . . . - . V F . R - E K . S . N . K V A E T R . Q V . . S N R . A K W R R E ESm Pax6b SmedGD Contig v31.002519 ; 10514 - 12190 Schmidtea mediterranea Pax6b Platyhelminthes Turbellaria G H S . I . . . . . M . . . . . . . . . S T . Q . . . . . . H S . A . . . . . . . I . Q . . N . . . . . . . C . Y . . . . . I R . K A . . - . . . . R - - - - - . . . S S . . S . . A A Y . . E C . . . . S . . . . . . . . Q . G V . . Q D . I . . . . . . . . V L . . L S L . R N R - - T S F S T D . L D S . . K . . E R . . . . - . V F A R - E K . . D . I S . . E A R . Q V . . S N R . A K W R R E ESmp Pax6b CAZ36187 Schistosoma mansoni Pax6b Platyhelminthes Trematoda R H S . . . . . . . M . . . . . . . . . T T . Q . . I . . . H S . A . . . . . . . I . Q . . N . . . . . . . C . Y . . . . . I R . K A . . - . . . . R - - - - - . . . N L . . L . . A H Y . . E C . . . . . . . . . . . . . Q . G . . T T E . I . . . . . . . . V L . N V C N P L N H - - I P I E L K K K E I T R S . . E R . . . . - . L I I R - E Q . . E S M L I . E S R . Q V . . S N R . A K W R R E VSmp Pax6a CAZ37096 Schistosoma mansoni Pax6a Platyhelminthes Trematoda G H S . . . . . . . M . . . . . . . . . T T . Q . . I . . . H S . A . . . . . . . I . Q . . N . . . . . . . C . Y . . . . . I R . K A . . - . . . . R - - - - - . . . N A . . T . . D I Y . . E C . . . . . . . . . . . . . Q . G V . T P D . I . . . . ? ? ? ? ? ? ? ? ? ?Gt Pax6 CAA09227 Girardia tigrina Pax6 Platyhelminthes Turbellaria G H S . I . . . . . I . . . . . . . . . V T . Q . . I . . S . S . A . . . . . . . I . Q . . N . . . . . . . C . Y . . . . . I . . K A . . - . . . . R - - - - - . . . N T . . R . V T I Y . Q E S . . M . . . . . . . . P . Q D G V . . Q D . L . . I . . . . . . L . . L . S . R S R - - T S F . N D . I N L . . K . . E R . . . . - . V F . R - E K . S . N . K V A E T R . Q V . . S N R . A K W R R E EBm Pax6 XM_001898985 Brugia malayi Pax6 Nematoda Chromadorea G H T . . . . . . . V . . . . . . . . . S T . Q K . . D . . H Q . A . . . . . . . I . Q . . N . . . . . . . C . Y . . S . T I R . R A . . - . . . . R - - - - - . . . V S . C D . . . S Y . . E Q . . . . . . . . . . K . . H . . V . S P D T I . . . . ? ? ? ? ? ? ? ? ? ?Ce Pax6 NP_001024570 Caenorhabditis elegans Pax6 Nematoda Chromadorea G H T . . . . . . . V . . . . . . . . . A T . Q . . . D . . H K . C . . . . . . . L . Q . . N . . . . . . . C . Y . . S . T I R . R A . . - . . . . R - - - - - . . . S D . . E . . . D Y . . . Q . . . . . . . . . . K . . A D N . . . N E T I . . . . . . . . V L . N L . L . R N R - - T S F . . V . I E S . . K . . E R . . . . - . V F A R - E R . . . . I Q . . E A R . Q V . . S N R . A K W R R E ELs Pax6 CAA64847 Lineus sanguineus Pax6 Nemertea Anopla G H S . . . . . . . V . . . . . . . . . S T . Q . . . . . . H S . A . . . . . . . I . Q . . N . . . T . . . . . Y . . . . . I R . R A . . - . . . . R - - - - - . . . . E . . G . . A H Y . . E C . . . . . . . . . . . . . S D A V . . Q D . I . . . . . . . . V L . N L . L . R N R - - T S F . N A . I E A . . K . . E R . . . . - . V F A R - E R . . . . I . . . E A R . Q V . . S N R . A K W R R E ECap Pax6 jgi,Capca1, scaffold 29; 62333 - 60613 Capitella capitata Pax6 Annelida Polychaeta G H S . . . . . . . V . . . . . . . . . S T . Q . . . . . . H S . A . . . . . . . I . Q . . N . . . . . . . . . Y . . . . . I R . R A . . - . . . . R - - - - - . . . N D . . G R . A Q Y . . E C . . . . . . . . . . . . . S . G C . . Q D . I . . . . . . . . V L . N L T L . R N R - - T S F . T Q . I E E . . K . . E K . . . . - . V F A R - E R . . . . . . . . E A R . Q V . . S N R . A K W R R E EPd Pax6 CAJ40659 Platynereis dumerilii Pax6 Annelida Polychaeta G H S . . . . . . . V . . . . . . . . . S T . Q . . . . . . H S . A . . . . . . . I . Q . . N . . . . . . . . . Y . . . . . I R . R A . . - . . . . R - - - - - . . . . E . . N . V A Q Y . . E C . . . . . . . . . . . . . S . G V . . Q D D I . . . . . . . . V L . N L . L . R N R - - T S F . N A . I E A . . K . . E R . . . . - . V F T R - E R . . K . F . I D E T R . Q V . . S N R . A K W R R E EDm Ey AAF59318 Drosophila melanogaster Ey Arthropoda Hexapoda C H S . . . . . . . V . . G . . . . . . S T . Q K . . . . . H S . A . . . . . . . I . Q . . N . . . . . . . . . Y . . . . . I R . R A . . - . . . . R - - - - - . . . A E . . S . . S Q Y . . E C . . . . . . . . . . . . . Q . N V . T N D . I . . . . . . . . V L . N L . L . R N R - - T S F . N D . I D S . . K . . E R . . . . - . V F A R - E R . . G . I G . . E A R . Q V . . S N R . A K W R R E EDm Toy AAD31712 Drosophila melanogaster Toy Arthropoda Hexapoda G H S . I . . . . . V Y . . . . . . . . S T . Q K . . . . . H S . A . . . . . . . I . Q . . N . . . . . . . . . Y . . . . . I . . R A . . - . . . . R - - - - - . . . T P . . Q . . A D Y . . E C . . . . . . . . . . . . . S . Q V . . S D . I . . . . . . . . V L . N L . L . R N R - - T S F S N E . I D S . . K . . E R . . . . - . V F A R - E R . . D . I G . . E A R . Q V . . S N R . A K W R R E EApis Pax6b XP_394648.3 Apis mellifera Pax6b Arthropoda Hexapoda G H S . . . . . . . V Y . . . . . . . . S T . Q K . . . . . H S . A . . . . . . . I . Q . . N . . . . . . . . . Y . . . . . I . . R A . . - . . . . R - - - - - . . . T P . . N . . A D Y . . E C . . . . . . . . . . . . . Q . G V . . N D . I . . . . . . . . V L . N L .Apis Pax6a XP_001120031.1 Apis mellifera Pax6a Arthropoda Hexapoda G H S . . . . . . . V . . G . . . . . . S T . Q K . . . . . H S . A . . . . . . . I . Q . . N . . . . . . . . . Y . . . . . I R . R A . . - . . . . R - - - - - . . . A E . . S . . S L Y . S E C . . . . . . . . . . . . . Q . G V . T N D . I . . . . . . . . V L . N L .Trib Pax6 NM_001110437 Tribolium castaneum Pax6 Arthropoda Hexapoda G H S . . . . . . . V . . G . . . . . . S T . Q K . . . . . H S . A . . . . . . . I . Q . . N . . . . . . . . . Y . . . . . I R . R A . . - . . . . R - - - - - . . . A E . . S . . S S Y . . E C . . . . . . . . . . . . . Q . G V . T N D . I . . . . . . . . V L . N L . L . R N R - - T S F . N D . I D S . . K . . E R . . . . - . V F A R - E R . . A . I G . . E A R . Q V . . S N R . A K W R R E ESk Pax6a gnl|ti|1728074750 Saccoglossus kowalevskii Pax6a Hemichordata Enteropneusta G H S . L . . I . . V V . . . . . I . . S A . Q Q . . . . . H S . A S . R . . . . I . Q . . N A . . . T . . A . Y S . . . . I R . R A . . - . . N . S - - - - - . . . . P . . G . . A Q . . . E C . . . . Q . . . S . . . . Q . Q V R T Q D . I . . . . . . ? ? ? ? ? ? ? ? L . R N R - - T S F . . . . I E T . . K . . E R . . . . - . V F A R - E R . . . . I . . . E A R . Q V . . S N R . A K W R R E ESk Pax6 AAP79294 Saccoglossus kowalevskii Pax6 Hemichordata Enteropneusta G H S . . . . . . . V . . . . . . . . . S T . Q K . . . . . H S . A . . . . . . . I . Q . . N . . . . . . . . . Y . . . . . I R . R A . . - . . . . R - - - - - . . . . P . . G . . A Q . . . E C . . . . . . . . . . . . . Q . Q V . T Q D . I . . . . . . . . V L . T L . L . R N R - - T S F . . . . I E T . . K . . E R . . . . - . V F A R - E R . . . . I . . . E A R . Q V . . S N R . A K W R R E ESp Pax6 NW_001350912.1; 237948 - 238301 Strongylocentrotus purpuratus Pax6 Echinodermata Eleutherozoa G H S . . . . . . . V . . . . . . . . . S T . Q K . . . . . H S . A . . . . . . . I . Q . . N . . . . . . . . . Y . . . . . I R . R A . . - . . . . R - - - - - . . . . H . . T R . A H Y . . E C . . . . . . . . . . . . . A . . . . . Q E . I . . . . ? ? ? ? ? ? ? ? ? ?Pl Pax6 AAA75363 Paracentrotus lividus Pax6 Echinodermata Eleutherozoa G H S . . . . . . . V . . . . . . . . . S T . Q K . . . . . H S . A . . . . . . . I . Q . . N . . . . . . . . . Y . . . . . I R . R A . . - . . . . R - - - - - . . . . H . . T R . A H Y . . E C . . . . . . . . . . . . . A . . . . . Q E . I . . . . . . . . V L . N L . L . R N R - - T S F . A Q . I E E . . K . . E R . . . . - . V F A R - E R . . . . I . . . E A R . Q V . . S N R . A K W R R E EBf Pax6 CAA11368 Branchiostoma floridae Pax6 Chordata Cephalochordata R H S . . . . . . . V . . D . . . . . . S T . Q K . . . . . H Q . A . . . . . . . L . Q . . N . . . . . . . . . Y . . . . . I R . R A . . - . . . . R - - - - - . . . . E . . A . . A Q . . . E C . . . . . . . . . . . . . S . G . . T N E . I . . . . . . . . V L . N L . L . R N R - - T S F . . E . I E A . . K . . E R . . . . - . V F A R - E R . . A . I . . . E A R . Q V . . S N R . A K W R R E ECi Pax6 BAB85207 Ciona intestinalis Pax6 Chordata Tunicata G H S . M . . . . . M . . . . . . . . . S . . Q K . . . F . H . . A . . . . . . . I . Q . . N . . . . . . . A . Y . . . . T I R . R A . . - . . . . R - - - - - . . . . E . . N . . A S Y . . E C . . . . . . . . . . . . . N . G . . . N D . I . . . . . . . . V L . N L N L . R N R - - T S F . . I . V E A . . K . . E R . . . . - . V F A R - E R . . T . I . . . E A R . Q V . . S N R . A K W R R E EOd Pax6 AAY27075 Oikopleura dioica Pax6 Chordata Tunicata G H S . . . . . . . A . . . . . . . . . S T . Q K . . . . . H S . A . . . . . . . I . Q . . N . . . . . . . A . Y . . . . . I . . R A . . - . . . . R - - - - - . . . . E . . N . . A D Y . . E C . . . . . . . . . . . . I . . N V . . S D . I . . . . . . . . V L . N F Q L . R N R - - T S F . . Q . I E S . . S . . E R . . . . - . V F A R - E R . . T . I G . . E A R . Q V . . S N R . A K W R R E EPm Pax6 CAA71094 Phallusia mammilata Pax6 Chordata Tunicata G H S . . . . . . . M . . . . . . . . . S . . Q K . . . F . H . . A . . . . . . . I . Q . . N . . . . . . . A . Y . . . . T I R . R A . . - . . . . R - - - - - . . . . Q . . N . . A M Y . . E C . . . . . . . . . . . . . N . A V . . A E . I . . . . . . . . V L . N L N L . R N R - - T S F S . E . V E A . . K . . E R . . . . - . V F A R - E R . . S . I . . . E A R . Q V . . S N R . A K W R R E EDr Pax6a AAH66722 Danio rerio Pax6a Chordata Vertebrata S H S . . . . . . . V . . . . . . . . . S T . Q K . . . . . H S . A . . . . . . . I . Q . . N . . . . . . . . . Y . . . . . I R . R A . . - . . . . R - - - - - . . . . E . . G . . A Q Y . . E C . . . . . . . . . . . . . S . G V . T N D . I . . . . . . . . V L . N L . L . R N R - - T S F . . E . I E A . . K . . E R . . . . - . V F A R - E R . . A . I . . . E A R . Q V . . S N R . A K W R R E EDr Pax6b CAA44867 Danio rerio Pax6 Chordata Vertebrata S H S . . . . . . . V . . . . . . . . . S T . Q K . . . . . H S . A . . . . . . . I . Q . . N . . . . . . . . . Y . . . . . I R . R A . . - . . . . R - - - - - . . . . E . . G . . A Q Y . . E C . . . . . . . . . . . . . S . G V . T N D . I . . . . . . . . V L . N L . L . R N R - - T S F . . E . I E A . . K . . E R . . . . - . V F A R - E R . . A . I . . . E A R . Q V . . S N R . A K W R R E EMm Pax6 NP_038655 Mus musculus Pax6 Chordata Vertebrata S H S . . . . . . . V . . . . . . . . . S T . Q K . . . . . H S . A . . . . . . . I . Q . . N . . . . . . . . . Y . . . . . I R . R A . . - . . . . R - - - - - . . . . E . . S . . A Q Y . . E C . . . . . . . . . . . . . S . G V . T N D . I . . . . . . . . V L . N L . L . R N R - - T S F . . E . I E A . . K . . E R . . . . - . V F A R - E R . . A . I . . . E A R . Q V . . S N R . A K W R R E EMm Pax4 AAC40046 Mus musculus Pax4 Chordata Vertebrata G L S S . . . . . . . . . . . . . . . L D T . Q Q . . Q . . I R . M . . . . . . . S . K . . N . . . . . . . . . Y . R . . V L E . K C . . - . . . . R - - - - - L . . . A . . A R . A Q L . D E Y . A L . . . . . Q H Q . C . . G L . T Q D K A . . . . . . . . V L . A L Q S H R N R - - T I F S P G . A E A . . K . . Q R G Q . . - . S V A R - G K . . A A T S . . E D T V R V . . S N R . A K W R R Q ENv PaxD AAW29069 Nematostella vectensis PaxD Cnidaria Anthozoa G . . K I . . . . . V . I . . K . . . R . L . L . . I . . . R L . V . . S . . . . R . . . . . . . . . . . . N K . H . . . . . E . . A A I T T F . R R - - - - - D I S . A . L N . . . . Y V F E Q . D . . S . . . . . . . . K D . L . S . C . . . . L E A V S N V I K T C . . R R S R - - T R F . V S . T D E . . R A . R K . . . . - . I Y A R - E E . . . R . G . S E A R V Q V . . S N R . A R . R K E RNv PaxD2 ABI30248 Nematostella vectensis PaxD2 Cnidaria Anthozoa G . . R . . . . . . V . I . . . . . . L V L . K Q . I . . . . L . V . . . . . . . R . . . . . . . . . . . . Y . . Q Q . . . I E . . A . A G S . T . R - - - - - N V . . E I E E . . D . Y R . E . . G M . S . . V . . . . V K D N V . S R C T . . . L A A . S Q V L K N . I Q R R S R - - T K F . S K . V D E . . K A . L K . Q . . - . V Y T R - E E . . . R . N . T E A R V Q V . . S N R . A R . R K K K S . S Y . . A S INv PaxD3 ABI30249 Nematostella vectensis PaxD3 Cnidaria Anthozoa G P K K . . . . . . G Y . . . K . . . R E . . I Q . I . . . R S . V . . . E . . . R . K I T . . . I . . L . A K . Q K . . . L E . . T V Y - K G R . R - - - - - . V . S Q I E K . . D Q Y R A E Q . G . . S . . . . . Q . . Q . G . . D R S E . . . L . . . S . . . K R K V T R R T R - - T R F . S Q . L H V . Q S . . T R N P . . - T L E E R - K E . . R Y . C V C E S R V Q V . . S N K . A Q D K R R .Am PaxD AAF64461 Acropora millepora PaxD Cnidaria Anthozoa G . . K . . . . . . V . I . . . . . . K L L . W K . I . . . . M . V . . . . . . . . . . . . . . . . . . . . C . Y Q . . . T . D . . . V . - L N R . R - - - - - D V . . E I E N . . D Q . R K E . S G . . S . . V . . . . . R . N . . S . S T . . . L G A . S Q . L K . K I Q R R S R - - T K F . H A . L N A . . K A . Q K . Q . . - . V Y T R - E E . . H R . S . T E A R V Q V . . S N R . A R . R K K DAj PaxD BAF56222 Anthopleura japonica PaxD1 Cnidaria Anthozoa G . . R . . . . . . V . I . . . . . . K M L . V . . I . . . . L . V . . S . . . . K . . . . . . . . . . . . C . Y Q . . . . I E . . A S T S P . . . R - - - - - D V . . D I E Q . . . . Y C I E . . G V . S C . V . E . . I R . G . . . R L T . . T L G D . S Q V L K . . I Q R R S R - - T K F S S A . V D E . . K A . L K . Q . . - . V Y T R - E E . . H R . K . T E A R V Q V . . S N R . A R M R K H E . . I Y N . . S LSm Pax3 SmedGD Contig v31.002646; 40530-45970 Schmidtea mediterranea Pax3 Platyhelminthes Turbellaria G P . R L . . . . . . . I . . . . . . K E K . I Q . I D . . L . . . . . . E . . . K . . . . . . . . . . . . H . Y . Q . . N . N . . F L * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ? R R S R - - T S F L D W . V D L . . N Y . C . N Q . . - I L N E R - Q N . S S R T K . . E S K . Q V . . S N R . A R W R K Q MSmp Pax3 CAZ31346, CAZ31347 Schistosoma mansoni Pax3 Platyhelminthes Trematoda G . . R . . . . . . M . I . . . . . . Y E T . L K . . . . S N D . . . . . . . . . . . K . . . . . . . . . . Q . Y S . . . . . S . . A T . - . A R * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * G R R N R - - T T F S E K . V E F . . S A . Q H . . . . - . L Q L R - E T . . Y Q T N . . E C K . Q V . . S N R . A R W R K Q IBm Pax3a XP_001900160 Brugia malayi Pax3a Nematoda Chromadorea G . . R . . . . . . V . I . . . . . . Q H . . L K . . . M . T . . V K . . H . . . . . . . . . . . . . . . . Y . Y A . . . . . S . . Q . . - . N - . R - - - S R K T I L A L E E H . D R I R Q G Q . A . N . H . . . L M . I E K G . . S R S . A . T I . . . H K Y M . L D R T R R N R - - T S F . . E . L E I . . A A . K A N T . . - . Q E L R - E R . . V A T K . D E G K . Q V . . S N R . A R C R K . LBm Pax3 XM_001900123 Brugia malayi Pax3 Nematoda Chromadorea G . . R I . . . . . V . I . . . . . . Q H . . I K . I . M . S . . . K . . H . . . . . . . . . . A . . . . . N . Y V . . . . . S . . Q . . - . N - . R - - - S R L . I Q A . E K H . L A L . E K C . . L C . S . L . S C . I E Q E . . S A E . A . T . . . . . . ? ? ? ? ? ?Ce Pax3 NP_496189 Caenorhabditis elegans Pax3 Nematoda Chromadorea G . . R . . . . . . V . I . . . . . . I H V . H A . I S M . K K . . K . . H . . . . . K . . . . A . . . . . N . Y A . . . . I S . . Q . . - . . - . R - - - A R L T V Q A . E K E . L I A C D E . . Q M S . A . L . . W . I H K D . . T . G . A . T . P A . K . L I G N K G S R R N R - - T S F . A E . L D V . . N A . R A D T . . - H A N A R - E S I S K E T G . S E E K . M T . . S N R . A R C R K N M . . I D . . . G ICap Pax37 jgi|Capca1|44697|gw1.698.8.1 Capitella capitata Pax3/7 Annelida Polychaeta G . . R . . . . . . V . I . . . . . . N H . . L K . I . M . S Q . V . . . V . . . T . . . . . . . . . . . . Q . Y Q . . . . I R . . S . . - . . . . R - - - - - . . . . D . E D R . H D L . K E . . G . . S . . . . . . . . K D G V . D R S S . . . . . . . S . V L . . H L Q R R S R - - T T F S A D . L E H . . K A . D R . . . . - . I Y T R - E E . . . R S G . T E A R V Q V . . S N R . A R W R K Q MApis Pax3a gb|AADG05005077.1| 33326 - 24406 Apis mellifera Pax3a Arthropoda Hexapoda G . . R . . . . . . V . I . . . . . . N H . . L K . . . M . A A . V . . . V . . . . . . . . . . . . . . . . N . Y Q . . . . I R . . V . . - . . . . R - - - - - . . . . E . E A R . . . Y . . . . . G . . S . . . . . . . I K . G . . D R T S A . . . . A . S . L L . G . D Q R R S R - - T T F . A H . L D E . . R A . E R T Q . . - . I Y T R - E E . . . R T K . T E A R . Q V . . S N R . A R . R K Q LApis Pax3b XP_394847.3 Apis mellifera Gsbn Arthropoda Hexapoda G . . R . . . . . . V . I . . . . . . N H . . L K . . . M . A A . V . . . V . . . . . . . . . . . . . . . . N . Y Q . . . . I R . . V . . - . . . . R - - - - - . . . . E . E A R . . D M . . Q . . G . . S . . . . E K . I K D G . . D . . S A . . . . . . S . L L . G P T Q R R S R - - T T F . G E . L E . . . A A . H R . Q . . - . V Y T R - E E . . . . T N . T E A R V Q V . . S N R . A R . R K Q LApis Pax3c XP_394848.3 Apis mellifera Gsbn2 Arthropoda Hexapoda G . . R M . . . . . V . I . . . . . . N H . . L K . . . M . A A . V . . . V . . . . . . . . . . . . . . . . N . Y Q . . . . I R . . V . . - . . . . R - - - - - . . . . E I E A R . . . M . . . . . G . . S . . . . E K . V K . G F T D - - - P . . . . . . S . L L . G G R Q R R S R - - T T F . G E . L E . . . T A . Q R A Q . . . . V Y A R - E E . . . R T G . T E A R . Q V . . S N R . A R . R K H ADm Prd AAB59221 Drosophila melanogaster Prd Arthropoda Hexapoda G . . R . . . . . . V . I . . . . . . N N . . L K . . . M . A D . . . . . V . . . . . . . . . . . . . . . . N . Y Q . . . . I R . . V . . - . . . . R - - - - - I . . . E I E N R . . . Y . . S S . G M . S . . . . E K . I R . G V . D R S T A . . . . A . S . L . . G . D Q R R C R - - T T F S A S . L D E . . R A . E R . Q . . - . I Y T R - E E . . . R T N . T E A R . Q V . . S N R . A R . R K Q HDm Gsb AAF47315 Drosophila melanogaster Gsb Arthropoda Hexapoda G . . R . . . . . . V . I . . . . . . N H . . . Q . . . M . A A . V . . . V . . . . . . . . . . . . . . . . N . . Q . . . . I R . . V . . - . . . . R - - - - - . . . . D I E S R . . . L . Q S Q . G . . S . . . . A K . I E A G V . D . Q . A . . . . . . S . L L . G S S Q R R S R - - T T F S N D . I D A . . R I . A R . Q . . - . V Y T R - E E . . . S T G . T E A R V Q V . . S N R . A R . R K Q L H . I D G . . G GDm Gsbn AAL49215 Drosophila melanogaster Gsbn Arthropoda Hexapoda G . . R . . . . . . V . I . . . . . . N H . . L K . . . M . A S . V . . . V . . . . . . . . . . . . . . . . N . Y Q . . . . I R . . V . . - . . . . . - - - - - . T S . E I E T R . D . L R K E . . . . . S . . . . E K . I K . G F A D - - - P . . T . . . S . L L . G S D Q R R S R - - T T F . A E . L E A . . R A . . R . Q . . - . V Y T R - E E . . . T T A . T E A R . Q V . . S N R . A R . R K H S . T I N G . . G GTrib Pax3a NM_001077622 Tribolium castaneum Pax3/7a Arthropoda Hexapoda G . . R . . . . . . V . I . . . . . . N H . . L K . . . M . A . . . . . . V . . . . . . . . . . . . . . . . N . Y Q . . . . I R . . V . . - . . . . R - - - - - . . . . E . E N R . . Q Y . . E . . . . . S . . . . . . . V K . G . . D R S T A . . . . A . S . L L . G K G Q R R S R - - T T F . A H . L D E . . K A . E R . Q . . - . I Y T R - E E . . . R T K . T E A R . Q V . . S N R . A R . R K Q ITrib Pax3b XM_969092 Tribolium castaneum Pax3/7b Arthropoda Hexapoda G . . R . . . . . . V . I . . . . . . N H . . L K . . . M . A A . V . . . V . . . . . . . . . . . . . . . . N . Y Q . . . . I R . . V . . - . . . . R - - - - - . . . . E I E T R . . Q M . K E . . T . . S . . . . E . . I K . G V T D - - - P . . . . . . S . L L . G G G Q R R S R - - T T F S G E . L E A . . R A . . R . Q . . - . V Y T R - E E . . . Q T G . T E A R . Q V . . S N R . A R . R K H VTrib Pax3c XM_969119 Tribolium castaneum Pax3/7c Arthropoda Hexapoda G . . R . . . . . . V . I . . . . . . N H . . L K . . . M . A A . . . . . V . . . . . . . . . . . . . . . . N . Y Q . . . . I R . . V . . - . . . . R - - - - - . . . L E . E A R . . Q L . K E E . Q . . S . . . . . . . I K . G . . D . N S A . . . . . . S . L L . G G R Q R R S R - - T T F . G E . L E A . . R A . G R . Q . . - . V Y T R - E E . . . . T K . T E A R V Q V . . S N R . A R . R K Q LOd Pax37a AAW24013 Oikopleura dioica Pax3/7a Chordata Tunicata G . . R . . . . . . V . I . . . . . . N H . . H K . . . M . A M . . . . . V . . . . . . . . . . . . . . . . C . Y Q . . . . I . . . S T . - . . . . R - - - - - I . S . E F E K R . L D I Q K E . . G V . S . . . . E K . . K . G Q M D R . A . . . . . C . S . . L . . H G Q R R S R - - T T F . A E . L E E . . K C . E R . . . . - . I Y T R - E E . . . R T K . T E A R V Q V G . S N R . A R G G K Q VOd Pax37b AAW24014 Oikopleura dioica Pax3/7b Chordata Tunicata ? ? ? ? M . . . . . V . I . . . . . . T H . . H K . . . M . A M . V . . . V . . . N . . . . . . . . . . . . C . Y Q . . . . I . . . S . . - . N . T . - - - - - G P Q . E I E E . . L Q Y S S E . S G . . S . . L . E M . I K N G D . E R S T A . . . . T . S . T L . A H G Q R R S R - - T T F S A N . L D E . . K C . E R . . . . - . I Y T R - E E . . G R T G . S E A R V Q V . . S N R . A R W R K Q MOd Pax37c AAW24015 Oikopleura dioica Pax3/7c Chordata Tunicata N . A R L . . . . . A . . . . . . . S L E T . E K . . Q M S . M . . . . . V . . . D . . . . . . . . . . . . S . . N K . . Q I . . . A S . - . . . R R - - - - S N V S K E H E Y L . V . Y R K Q F A - - Y . . . M . E E M V K R G - - - V Q K . . P . D Q . K . V L . A K G . R R A R - - S N F . . A . V E A . . G A V I K . . . . - . V Y V R - E E . S A L T G M T E N R . Q I . . S N R . A R Y R K Q COd Pax37d AAW24016 Oikopleura dioica Pax3/7d Chordata Tunicata N S A K L . . . . . A . . . . . . . S L E T . E K . . Q M S . M . V . A . I . . . D . . . . . . . . . . . . T . . N K . . E I . . . A S . - . . . R R - - - - S S V S N E N E C L . V . L R Q Q F A - - Y . . . L . E E M V K R G - - - A K K . . . . D Q . K . V L . A K GHr Pax37 BAA12289 Halocynthia roretzi Pax3/7 Chordata Tunicata G . . R . . . . . . V . I . . . . . . N H . . H K . . . M . A H . . . . . V . . . . . . . . . . . . . . . . C . Y Q . . . . I . . . A . . - . . . . . - - - - - . T N S E I E S . . . Q Y . K . S . . M . S . . . . . Q . I K . G L . D R S S A . T . . A . S . . L . . K G Q R R S R - - T T F S A D . L E E . . R C . E R . . . . - . I Y T R - E E . . . R T R . T E A R V Q V . . S N R . A R W R K Q MDr Pax3a AAH76069 Danio rerio Pax3a Chordata Vertebrata T P L . . . . . . . V . I . . . . . . N H . . H K . . . M . H H . . . . . V . . . . . . . . . . . . . . . . C . Y Q . . . . I R . . A . . - . . . . . - - - - Q S T . . D . E K . . . . Y . . E . . G M . S . . . . . K . . K D G . . D R N . . . . . . . . S . M L . C K F Q R R S R - - T T F . A E . L E E . . R A . E R . . . . - . I Y T R - E E . . . R A K . T E A R V Q V . . S N R . A R W R K Q A H . I D G . . G DDr Pax3b ACN88554 Danio rerio Pax3b Chordata Vertebrata G . . R . . . . . . V . I . . . . . . N H . . H K . . . M . H H . . . . . V . . . . . . . . . . . . . . . . C . Y Q . . . . I R . . A . . - . T . S . - - - - - T T S . D . E K R V . . Y . . E . . G . . S . . . . . K . . K . G . . D R N . . . T . . . . S . . M . G . S Q R R S R - - T T F . A D . L E E . . R A . E R . . . . - . I Y T R - E E . . . R A K . T E A R V Q V . . S N R . A R W R K Q A H . I E G . Q A DDr Pax7a AAI63580 Danio rerio Pax7a Chordata Vertebrata G . . R . . . . . . V . I . . . . . . N H . . H K . . . M . H H . . . . . V . . . . . . . . . . . . . . . . C . Y Q . . . . I R . . A . . - . . . . R - - - - Q . . . . D . E K R . . . Y . . E . . G M . S . . . . . K . . K D G V . D R G T . . . . . . . S . V L . A . F Q R R S R - - T T F . A E . L E E . . K A . E R . . . . - . I Y T R - E E . . . R T K . T E A R V Q V R Y V H L . Y L Q C M T L H . I D G . . G DDr Pax7b ACN88553 Danio rerio Pax7b Chordata Vertebrata G . . R . . . . . . V . I . . . . . . N H . . H K . . . M . H H . . . . . V . . . . . . . . . . . . . . . . C . Y Q . . . . I R . . A . . - . . . . R Q T Q K Q . . . . D . E K R . . . Y . . E . . G M . S . . . . . K . . K D G V . D R S T . . . . . . . S . V L . A . F Q R R S R - - T T F . A E . L E E . . K A . E R . . . . - . I Y T R - E E . . . R T K . T E A R V Q V . . S N R . A R W R K Q A H . I D G . . G DMm Pax3 NP_032807 Mus musculus Pax3 Chordata Vertebrata G . . R . . . . . . V . I . . . . . . N H . . H K . . . M . H H . . . . . V . . . . . . . . . . . . . . . . C . Y Q . . . . I R . . A . . - . . . . . - - - - Q . T . . D . E K . . . . Y . . E . . G M . S . . . . . K . . K D A V . D R N T . . . . . . . S . . L . . K F Q R R S R - - T T F . A E . L E E . . R A . E R . . . . - . I Y T R - E E . . . R A K . T E A R V Q V . . S N R . A R W R K Q A H . I D G . . S EMm Pax7 NP_035169 Mus musculus Pax7 Chordata Vertebrata G . . R . . . . . . V . I . . . . . . N H . . H K . . . M . H H . . . . . V . . . . . . . . . . . . . . . . C . Y Q . . . . I R . . A . . - . . . . R - - - - Q . . . . D . E K . . . . Y . . E . . G M . S . . . . . . . . K D G H . D R S T . . . . . . . S . V L . I K F Q R R S R - - T T F . A E . L E E . . K A . E R . . . . - . I Y T R - E E . . . R T K . T E A R F Q V . . S N R . A R W R K Q A H . I D G . . G DBm Pax1 XM_001895370 Brugia malayi Pax1 Nematoda Chromadorea S S I E I . . . . . E . I . . . . . . L R L . Y K . I . . T K D . Y . . . . . . . . . K I . . . . I . . . . S ? Y A . Y . T . M . . T V . - . . . . R - - - - - . T . . A . . E Y . N F L . L R Q . R . . . . . . . E Q . . R N . . . Q . N K F A I ? ? ? ? ? ? ? ? ? ? ? ? Ce Pax1 AAA96181 Caenorhabditis elegans Pax1 Nematoda Chromadorea K T A E . . . . . . V . . . . . . . . F E M . C K . . . . S R Q . T . . . . . . . . . K I . . . . . . . . . T . . S . N . T I M . . T . . - . . R . R - - - - - . T . . . . . E Y . R S L . . S D . G . . . . . . . . . . I S A D . . D R . . L . . . . . . S . . L . N K NCap Pax1 jgi|Capca1|64892|gw1.112.75.1 Capitella capitata Pax1 Annelida Polychaeta A Y . E . . . . . . M . . . . . . . . N T V . L . . . . . . . L . V . . . . . . . . . K . . . . . . . . . . A . Y N . . . . I L . . T . . - . . . . R - - - - - . T . . . . . D S . R V Y . D K D . G . . . . . . . . K . . A D R . . D . F . . . . . . . . S . . L . N K IDm Poxm ABI31147 Drosophila melanogaster Poxm Arthropoda Hexapoda Q Y . E . . . . . . V . . . . . . . . N A T . M . . . . . . R L . . . . . . . . . . . . . . . . . . . . . . A . Y H . . . . I L . . A . . - . . . . R - - - - - . T . . . . . N Y . R . L . Q R D . G . . . . . . . . . . . S . G . . D . T . . . . . . . . S . . L . N K LApis Pax1 XP_001121954.1 Apis mellifera Pax1 Arthropoda Hexapoda Q Y . E . . . . . . V . . . . . . . . N A V . L . . . . . . . L . . . . . . . . . . . . . . . . . . . . . . A . Y H . . . . I L . . A . . - . . . . R - - - - - . T . . . . . Q Y . K Q L . L K D . G . . . . . . . . . . . S D G V . D . Y . . . . . . . . S . . L . N K VTrib Pax1 XM_970385 Tribolium castaneum Pax1 Arthropoda Hexapoda Q Y . E . . . . . . V . . . . . . . . N A V . L . . . . . S . V . . . . . . . . . . . . . . . . . . . . . . A . Y H . . . . I L . . A . . - . . . . R - - - - - . T . . . . . A Y . K D L . Q K D . G . . . . . . . . . . . S D G . . D . Y . . . . . . . . S . . L . N K ISk Pax19 ABI31622 Saccoglossus kowalevskii Pax1/9 Hemichordata Enteropneusta T F . E . . . . . . V . . . . . . . . N G . . L . . . . . . . L . . . . . . . . . . . . . . . . . . . . . . A . Y N . . . . I L . . A . . - . . . . R - - - - - . T . . T . . K H . K . Y . . M D . G . . . . . . . . . . I A D N V . D . Y . . . . . . . . S . . L . N K VSp Pax19 XP_783154 Strongylocentrotus purpuratus Pax1/9 Echinodermata Eleutherozoa T F . E . . . . . . V . . . . . . . . N A . . L . . . . . . . L . . . . . . . . . . . . . . . . . . . . . . A . Y N . . . . I L . . A . . - . . . . R - - - - - . T . . N . . K . . R . Y . Q R D . G . . . . . . . . K . . A . G V . D . Y . . . . . . . . S . . L . N K IOd Pax19 AAW24011 Oikopleura dioica Pax1/9 Chordata Tunicata G G . E M . . . . . H . . . . . . . . N H T . T K . . . M . R E . T . . . . . . . R . . . . . . . . . . . . Q . Y H D . . . I L . . S . . - . . . . R - - - - - . T . . Q I . N . . R S Y . . L D . G M . . . . . . . L . I E D . V . D T N S A . . . . . . S . . L . N K IHr Pax19 BAA74830 Halocynthia roretzi Pax1/9 Chordata Tunicata T F . E . . . . . . I . . . . . . . . N T . . L . . . . . . . L . . . . . . . . . . . . . . . . . . . . . . A . Y N . . . . I L . . A . . - . . . . R - - - - - . T . . S . . N A . K D Y . I R D . G . . . . . . . . . . . S D C . . D . Y . . . . . . . . S . . L . N K ICi Pax19 BAA74829 Ciona intestinalis Pax1/9 Chordata Tunicata T F . E . . . . . . V . . . . . . . . N A L . L . . I . . . . L . . . . . . . . . . . . . . . . . . . . . . A . Y N . . . . I L . . A . . - . . . . R - - - - - . T . . G . . N A . K D Y . V R D . G . . . . . . . . . . . S D A V . D . Y . . . . . . . . S . . L . N K IDr Pax9 NP_571373 Danio rerio Pax9 Chordata Vertebrata A F . E . . . . . . V . . . . . . . . N A . . L . . . . . . . L . . . . . . . . . . . . . . . . . . . . . . A . Y N . . . . I L . . A . . - . . . . R - - - - - . T . . N . . K H . R T Y . Q R D . G . . . . . . . . . . . A D G V . D . F . L . . . . . . S . . L . N K IMm Pax1 NP_032806 Mus musculus Pax1 Chordata Vertebrata T Y . E . . . . . . V . . . . . . . . N A . . L . . . . . . . L . . . . . . . . . . . . . . . . . . . . . . A . Y N . . . . I L . . A . . - . . . . R - - - - - . T . . N . . K H . R D Y . Q G D . G . . . . . . . . . . . A D G V . D . Y . . . . . . . . S . . L . N K IMm Pax9 NP_035171 Mus musculus Pax9 Chordata Vertebrata A F . E . . . . . . V . . . . . . . . N A . . L . . . . . . . L . . . . . . . . . . . . . . . . . . . . . . A . Y N . . . . I L . . A . . - . . . . R - - - - - . T . . T . . K H . R T Y . Q R D . G . . . . . . . . . . . A D G V . D . Y . . . . . . . . S . . L . N K ICw PaxA BAF56224 Coeloplana willeyi PaxA Ctenophora Typhlocoela R R I . . . . . . . A . K . . . . . . E D . . K . . . D . . H F . V . . . . . . . . . . . . . . . . . . . . . . Y S D . . . I L . . N . . - . . . . . - - - - - . . . . E . . A I . S . Y . . R . . . M . . . . . . . . . . M D G M P S N - K . . . A . T . . . . L . N K VCw PaxB BAF56225 Coeloplana willeyi PaxB Ctenophora Typhlocoela R R V . . . . . . . S . R . . . . . . . E V . Q K . . . . . . H . V . . . . . . . . . . . . . . . . . . . . . . . T D . . . . M . . A . . - . . . . R - - - - - . . . . E . . A V . . R Y . N A . . . M . . . . . . . . . . L D G M . P N - K . . . A . T . . . . L . . K SSp Pax258d XP_001183514 Strongylocentrotus purpuratus Pax2d Echinodermata Eleutherozoa G H . . I . . . . . I . A . . . . . . I H . . H . . L . . . H L . L . . . . . . . . . L . . . . . . . . . . S . . A . . . . I L . . A . . - . . . . R - - - - - . S . . A . . N R . A Q Y . Q E . S . M . . . . . . E . . . L D G . . S . E T L . . . . . . . . . L . N C SSk Pax2b Contig1: gnl|ti|1672812677-, gnl|ti|1870559133+, gnl|ti|1741092241-, gnl|ti|1723739128-, gnl|ti|1708837087+, gnl|ti|1708837092+, gnl|ti|1671137469-, gnl|ti|1799254196+ Saccoglossus kowalevskii Pax2b Hemichordata Enteropneusta G . . . I . . . . . I . A . . . . . . F H T . H . . L . . . H L . L . . . . . . . . . L . . . . . . . . . . S . Y S . . . . I L . . A . . - . . . . R - - - - - . S . . E . I E R . S D Y . H E . S . M . . . . . . E . . . I . G V . T . E . L . . . . . . . . . L . N T TBm Pax3b XM_001900049 Brugia malayi Pax3b Nematoda Chromadorea A R T . T . . . . . I Y A . . . . . . V H L . E . . I Q M . A S . T K ? . Q . . . . . . I . . . . . . . . . S . Y R D . . . I R . . K . . - . . . . . - - - - - K S L . Q . . T A . A I Y . H C R . . M Y S . . . . S . . I S D G V . S A ? . . . . I . . . S . ? ? ? ? ? ?Cap Pax2b jgi|Capca1|115154|e_gw1.532.36.1 Capitella capitata Pax2b Annelida Polychaeta G G K N I . . Y . R I . T . . . . . . E H L . V Q . L Q . . L Q . V . . . E . . . . . Q . . . . . . . . . . N . Y R K . . . I N . . Q . . - . . . . . - - - - - . T . . D . . S R V R Q Y . I E . . Q M . . . . . . Q . . . D D N . . C E K . I . . I . . . . . . I . D K SSm Pax2b SmedGD Contig v31.000344; 41669 - 42202 Schmidtea mediterranea Pax2b Platyhelminthes Turbellaria N G Q N I . . Y . R C . T . . . . . . E E L . L E . L K . . M E N V . . . . . . . . . C . . . . . . . . . . N . Y V K . . . I N . . Q . . - . . . . . - - - - - . T . . D I . D R V R I Y . L N . . Q . Y . . . . . Q K . I E D Q V . T E Q S A . . I . . . . . . I . D K GSm Pax2c SmedGD Contig v31.000050; 146518 - 146881 Schmidtea mediterranea Pax2c Platyhelminthes Turbellaria A G Q N . . . Y . R V . T . . . . . . E D L . L K . L K M . M E N V . . . E . . . . . Q . . . . . . . . . . N . Y S R . . . I N . . Q . . - . . . . . - - - - - . T . . E I . S L V K S Y R E K . . Q . . . . . . . Q . . V D . G A . T E R S A . . I . . . . . . I . D K G

PD

Fig. S2. Alignment of paired domains with cognate homeodomains and octapeptides of 136 Pax genes. These paired domains, with the exception of that of Aphid Pax2 and the two partial paired domains including only a PAI subdomain, were used for theconstruction of the phylogenetic tree shown in Fig. 4. A few uncertain amino acids are indicated by question marks. All other features are as explained in the legends to Figs. 2 and 5A. The eight paired domains listed at the bottom could not be assignedunambiguously to a paired domain subfamily, neither by the phylogenetic tree (Fig. 4) nor on basis of the paired domain sites diagnostic for subfamilies (Figs. 5A, B). According to the latter criterion, the two paired domains of Coeloplana willeyi are clearlyclosest to the PaxB/2/5/8 subfamily with 13 and 15 differences at sites diagnostic for subfamilies, those of Sp Pax258d and Sk Pax2b most similar to PaxB and Poxn subfamilies with 17 or 18 differences, and those of Bm Pax3b and Cap Pax2b closest to thePaxB and Pax2/5/8 subfamily with 20 and 22 differences, respectively, out of the 41 diagnostic sites (Figs. 5A, B). The two paired domains of Sm Pax2b and Sm Pax2c exhibit differences at ≥ 60% of the diagnostic sites with regard to all subfamilies. Sincenone of these eight Pax genes in triploblasts encode a homeodomain, they are probably considerably diverged members of the Pax2/5/8 or Poxn subfamilies. The Platyhelminthean Sm Pax2b and Sm Pax2c have no intron at the site diagnostic for Poxn closeto the 4 helix (Fig. 1C) and hence do not belong to the Poxn subfamily.

Short name Accession Info Species, Gene Phylum Subphylum/Class 1 20 40 60 80 100

118 1 20 40 60

Cl Six12 GQ985311 Chalinula loosanoffi Six1/2 Porifera Demospongiae S Q E Q V A C V C D V L Q Q S G N I E R L A R F L W S L P - - - - A C E Q I Q K N E S V L K A K A L I A F H Q - - - - G N F P E L Y R I I E L N S F T - P E S H P K M Q Q L W L Q A H Y I E A E R L R - G K P L G A V G K Y R I R R K F P L P R T I W D G E E T S Y C F K E K S R V V L R Q W Y T K N P Y P S P R E K R Q L A E Q T G L T T T Q V S N W F K N R R Q R D R A S EAq Six12 JGI Reniera sp WGS contig 11951; 115263-117244 (Larroux et al., 2008) Amphimedon queenslandica Six1/2 Porifera Demospongiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - - - . . . . . . . . . . . . . . . . . . . . . . - - - - . . . Q . . . . . . . T . N . S - . D . . . . . . . . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A .Ef SixC BAF56226 Ephydatia fluviatilis SixC Porifera Demospongiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - - - . . . . . . . . . . . . . . R . . . . . . . - - - - . . . A . . . . . . . N . N . A - . D . . . . . . . . . . . . . . . . . . . V . - A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A .Sc SixC BAF56227 Sycon calcaravis SixC Porifera Calcarea T L . . L . L I T E Y I V L T K D V A H . E . Y . I . . . - - - - N . P R L . S H . . I . I . . . K V . Y . A G C S T . D . K R . . H . L . T E T . S - E R . F . R L . E M . T N . . . K . . . . Q . D . . . . . . . . . . . . . . . Y . F . . N . . . . . . . N . . . . . . . . A M . . T R . E . S . . . . . Q Q . K E . . . A . E . S V . . . . . . . . . . . . . . . . A DTa Six12 XP_002117378 Trichoplax adhaerens Six1/2 Placozoa Trichoplax . T D . F . S . . N I . L . R N H . D . . . T . . . . . . - - - - P N D E L K V . Q N I . L . R . T V . Y . . - - - - H . . E . . . Q L L . N Y P . S - S . F . . . L . E . . K E . . . L . E K Q S . - . . E . D . . T . . . V . K . Y . . . L . . S . . . K I T . S . . . S . . K M . V E Y . Q R . . . . T S E . . A I I . . A A S . . K V . . . . . . . . K . . . . . . K SCw SixC BAF56229 Coeloplana willeyi SixC Ctenophora Typhlocoela T . . . . . . . . E . . S . G . . M . . . . . . . . . . . - - - - S . D H L H . . . . . . . . . . V V . . . R - - - - . . . K . . . Q . L . N . . . S - A N N . . . L . S I . . K . . . M . . . K . . - . R S . . . . . . . . V . K . . . . . . . . . . . D . . . . . . . . . . . T . . . D . . A H . . . . . . . . . . E . . . A . . . . . . . . . . . . . . . . . . . . . A .Aj SixC BAF56229 Anthopleura japonica SixC Cnidaria Anthozoa T P . . . . . . . E . . . . . . D . . . . G . . . . . . . - - - - E . . T . . . . . . . . . . . . I V . . . N - - - - . . . Q . . . . L L . S S N . S - . A . . . . L . S . . . K . . . L . . . K . . - . R . . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . N I . . E . . S H . . . . . . . . . . E . . . N . . . . . . . . . . . . . . . . . . . . . A .Nv Six12 DS469735 Nematostella vectensis Six1/2 Cnidaria Anthozoa T P . . . . . . . E . . . . . . D . . . . G . . . . . . . - - - - E . . T . . . . . . . . . . . . I V S . . . - - - - Q . . Q . . . . . L . N . N . S - . N A . . . L . S . . . K . . . M . . . K . . - . R . . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . N I . . E . . S H . . . . . . . . . . E . . . G . . . . . . . . . . . . . . . . . . . . . A .Nv Six12b XP_001634996 Nematostella vectensis Six1/2b Cnidaria Anthozoa . M . . I I S . . E C . . N . . . . . . . . . . . . . . . - - - K D S . E . H A C . T I . V . . . V V . . . . - - - - N . . K . . . S . L . S R K . Q - R S E . E . L . C . . R T . . . . . . . . V . - . R . . . . . . . . . V . . . . . . . . . . . . . . . . T . . . . . . . . S I . N K A . V D S . . . T . . . . H E . . K M . D . . V . . . . . . . . . K . . . V . . A .Cr Six12 AAT11873 Cladonema radiatum Six1/2 Cnidaria Hydrozoa T P . . I . . . . E . . E . . . . . D . . S . . . . . . . - - - - S Y D D V Y T T . . . . V . . C V V . . . . - - - - . . L Q . . . H . . . N . N . . - Q Q Y . T . L . M . . . R G . . . . . . K I . - . R . . . . . . . . . V . . . Y . . . . . . . . . . . . . . . . . . . . . A I . . D . . S R . . . . . . . . . K E . S Q G . . . S . . . . . . . . . . . . . . . . . A .Pc Six12 AAT11871 Podocoryne carnea Six1/2 Cnidaria Hydrozoa T P . . . . . . . E . . E . . . . . D . . . . . . . . . . - - - - N Y D D V Y A . . . . . V . . S V V . . . . - - - - . . L Q . . . H . . . N . N . . - Q N . . S . L . M . . . K . . . M . . . K I . - . R . . . . . . . . . V . . . H . . . . . . . . . . . . . . . . . . . . . A . . . D . . . R . . . . . . . . . K E . S . G . . . S . . . . . . . . . . . . . . . . . A .Dj Six12 CAD89530 Dugesia japonica Six1/2 Platyheminthes Turbellaria T . . . . . . . . E . . E N G . . . D . . . L . I . . . . - - - - P . Q . L . T . . . . . T . . . A V . . . R - - - - Q . . K . . . . . L . S Y T . S - . H N . Y . L . A . . . . . . . . . E . K I K - . R S . . . . A . . . . . . . Y . . . . . . . . . . . . . . . . . . . . . A . . . . . . L H . . . . . . . . . K D . . . M . S . . . . . . . . . . . . . . . . . . . A .Gt Six12 CAB89515 Girardia tigrina Six1/2 Platyheminthes Turbellaria T . . . . . . . . E . . E N G . . . D . . . L . I . . . . - - - - P . Q . L . T . . . . . T . . . A V . . . R - - - - Q . . K . . . . . L . S Y T . S - . H N . Y . L . A . . . . . . . . . E . K I K - . R S . . . . A . . . . . . . Y . . . . . . . . . . . . . . . . . . . . . A . . . . . . L H . . . . . . . . . K D . . . M . S F . . . . . . . . . . . . . . . . . . A .Pd Six2 CAC86663 Platynereis dumerilii Six2 Annelida Polychaeta T . . . . . . . . E . . . . G . . . . . . . . . . . . . . - - - - . . . H L H . . . . . . . . . . V V . . . R - - - - . . . K . . . K L L . S H Q . S - . H N . . . L . A . . . K . . . . . . . K . . - . R . . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . T . . . E . . A H . . . . . . . . . . E . . . A . . . . . . . . . . . . . . . . . . . . . A .Dm so NP_476733 Drosophila melanogaster sine oculis Arthropoda Hexapoda T . . . . . . . . E . . . . A . . . . . . G . . . . . . . - - - - Q . D K L . L . . . . . . . . . V V . . . R - - - - . Q Y K . . . . L L . H H H . S - A Q N . A . L . A . . . K . . . V . . . K . . - . R . . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . S . . . D . . S H . . . . . . . . . . D . . . A . . . . . . . . . . . . . . . . . . . . . A .Apis Six2 XP_396811 Apis mellifera Six2 Arthropoda Hexapoda T . . . . . . . . E . . . . A . S V . . . G . . . . . . . - - - - . . T R L H R H . . . . . . . . I V . . . R - - - - . H . K . . . . . L . S H T . S - . H N . Q . L . A . . . K . . . . . . . . . . - . R . . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . S . . . D . . A T . . . . . . . . . . E . . . S . . . . . . . . . . . . . . . . . . . . . A .Nasv Six2 XP_001600428 Nasonia vitripennis Six2 Arthropoda Hexapoda T . . . . . . . . E . . . . A . S V . . . . . . . . . . . - - - - E . A R L R . . . . . . . . Q . V V . . . H - - - - . . . K . . . Q . L . S . T . S - S H N . N . L . L . . . K . . . . . . . . . . - . R . . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . L . . . K . . D T . . . . . . . . . Q E . . . T . . . . . . . . . . . . . . . . . . . . . . .Tc so XP_972167 Tribolium castaneum Six2 Arthropoda Hexapoda T . . . . . . . . E . . . . A . . V . . . G . . . . . . . - - - - . . D K L H N . . . . . . . . . I V . . . R - - - - . . . K . . . K . L . S H Q . S - . H N . A . L . A . . . K . . . . . . . . . . - . R . . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . S . . . D . . S H . . . . . . . . . . E . . D A . . . . . . . . . . . . . . . . . . . . . A .Sp Six12 XP_781551 Strongylocentrotus purpuratus Six1/2 Echinodermata Eleutherozoa T . . . . . . . . E . . . . . . . . . . . G . . . . . . . - - - - . . . H L H . . . . . . . . . . I V . . . R - - - - . . . R . . . K . L . S . N . S - . H N . . . L . A . . . K . . . . . . . K . . - . R . . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . S I . . E . . S H . . . . . . . . . . E . . . A . . . . . . . . . . . . . . . . . . . . . A .Ci Six12 AB210682 Ciona intestinalis Six1/2 Chordata Tunicata T . . . . . . . . E . . . . G . . . . . . . . . . . . . . - - - - G . . H L H . . . . . . . . . . V V . . . R - - - - . . . R . . . K . . . N . N . S - E H N . A . L . . . . . K S . . . . . . K . . - . R . . . . . . . . . V . . . . . . . . S . . . . . . . . . . . . . . . . A . . . D . . . H . . . . . . . . . . E . . . G . . . . V . . . . . . . . . . . . . . . . A .Od Six12 AAZ23140 Oikopleura dioica Six1/2 Chordata Tunicata T I . . I S . . . . . . . K . S A . D . . S . . I . . . . - - - - N . . V L . . H . A . . . . R . V V N . . R - - - - . . . R D . . K V L . S H T . S - . . N . S . L . . . . . K . . . . . . . K . . - . R . . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . A . . . D . . . H . . . . . . . . . . E . . . A . . . . V . . . . . . . . . . . . . . . . A .Od Six12a AAW23092 Oikopleura dioica Six1/2a Chordata Tunicata . I . . I I . L L E . . E T . Q . G . K . Q E . . M R . . - - - - K S . E L E N . . A I . . . . . T A . . F R - - - - . D . R . . . K . L . S R Q Y S - . A F . D R L . . . . . K . . . . . . . K V . - . R . . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . A . . . D . . L . S . . . . . . . . . E . . . M . D . . V . . . . . . . . . . . . . E . . A .Dr Six1 AAO83592 Danio rerio Six1 Chordata Vertebrata T . . . . . . . . E . . . . G . . L . . . G . . . . . . . - - - - . . D H L H . . . . . . . . . . V V . . . R - - - - . . . R . . . K . L . S H Q . S - . H N . . . L . . . . . K . . . . . . . K . . - . R . . . . . V . . . V . . . . . . . . . . . . . . . . . . . . . . . . . G . . . E . . . H . . . . . . . . . . E . . . A . . . . . . . . . . . . . . . . . . . . . A .Dr Six2 NP_001122206 Danio rerio Six2 Chordata Vertebrata T . . . . . . . . E . . . . G . S . . . . G . . . . . . . - - - - . . . H L H . . . . . . . . . . V V . . . R - - - - . . . R . . . K V L . S H Q . S - . H N . . . L . . . . . K . . . . . . . K . . - . R . . . . . . . . . V . . . . . . . . S . . . . . . . . . . . . . . . . C . . K E . . . H . . . . . . . . . . E . . . A . . . . . . . . . . . . . . . . . . . . . A .Mm Six1 Q62231 Mus musculus Six1 Chordata Vertebrata T . . . . . . . . E . . . . G . . L . . . G . . . . . . . - - - - . . D H L H . . . . . . . . . . V V . . . R - - - - . . . R . . . K . L . S H Q . S - . H N . . . L . . . . . K . . . V . . . K . . - . R . . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . G . . . E . . A H . . . . . . . . . . E . . . A . . . . . . . . . . . . . . . . . . . . . A .Mm Six2 AAH68021 Mus musculus Six2 Chordata Vertebrata T . . . . . . . . E . . . . G . . . . . . G . . . . . . . - - - - . . . H L H . . . . . . . . . . V V . . . R - - - - . . . R . . . K . L . S H Q . S - . H N . A . L . . . . . K . . . . . . . K . . - . R . . . . . . . . . V . . . . . . . . S . . . . . . . . . . . . . . . . S . . . E . . A H . . . . . . . . . . E . . . A . . . . . . . . . . . . . . . . . . . . . A .Hs Six1 CAA62974 Homo sapiens Six1 Chordata Vertebrata T . . . . . . . . E . . . . G . . L . . . G . . . . . . . - - - - . . D H L H . . . . . . . . . . V V . . . R - - - - . . . R . . . K . L . S H Q . S - . H N . . . L . . . . . K . . . V . . . K . . - . R . . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . G . . . E . . A H . . . . . . . . . . E . . . A . . . . . . . . . . . . . . . . . . . . . A .Hs Six2 AAK16581 Homo sapiens Six2 Chordata Vertebrata T . . . . . . . . E . . . . G . . . . . . G . . . . . . . - - - - . . . H L H . . . . . . . . . . V V . . . R - - - - . . . R . . . K . L . S H Q . S - . H N . A . L . . . . . K . . . . . . . K . . - . R . . . . . . . . . V . . . . . . . . S . . . . . . . . . . . . . . . . S . . . E . . A H . . . . . . . . . . E . . . A . . . . . . . . . . . . . . . . . . . . . A .

Ta Six36 XP_002117377 Trichoplax adhaerens Six3/6 Placozoa Trichoplax . V H . . . S . . E A . E S . . D . . . . S . . . . . . . S T L D G Y T N L L N H D A I . R . R . V V . Y . . - - - - . H Y R . . . G . . . N H R . P - K D F . G . L . H M . . E . . . R . . . K . . - . R S . . P . D . . . . . K . Y . . . V . . . . . . Q K T H . . . . . T . N L . . E . . L R D . . . N . G K . . E . . N A . . . . P . . . G . . . . . . . . . . . . A ACw SixA BAF56232 Coeloplana willeyi SixA Ctenophora Typhlocoela . V . . . . S . . . S . E A . . D . D . . . . . . . . . . L S - - Q M . E F N . . . K I . R S R . V V S . . R - - - - Q D . R . . . S . . . N C R . K - K S . . E . L . Y . . N E . . . M . . . K . . - . R . . . . . D . . . V . K . . . . . . . . . . . K I Q N H . . . . . . . N I . K E . . S . . . . . . . H T . . E . . D A A . . . P . . . . . . . . . . . . . . . . A INv Six36 XP_001625159 Nematostella vectensis Six3/6 Cnidaria Anthozoa . A H . I . Q . . E T . E E . . D V . . . . . . . . . . . V A P G T L . A L G . H . . . . R . R . I V . . . M - - - - . . . R D . . H . L . T H R . . - R . . . A . L . A M . . E . . . Q . . . . . . - . R . . . P . D . . . V . K . . . . . . . . . . . . Q K T H . . . . R T . S L . . E . . L Q D . . . N . T K . . E . . Q A . . . . P . . . G . . . . . . . . . . . . A AAj SixA BAF56228 Anthopleura japonica SixA Cnidaria Anthozoa . P Q . I . Q . . E T . E E . . D V . . . . . . . . . . . V A P G T L . A L S . H . . . . R . R . I V . . . M - - - - . . . R D . . H . L . S H R . . - K . . . A . L . A M . . E . . . Q . . . . . . - . R . . . P . D . . . V . K . . . M . . . . . . . . Q K T H . . . . R T . S L . . E . . L Q D . . . N . T K . . E . . Q A . . . . P . . . G . . . . . . . . . . . . A AHm SixA BAF56230 Hydra magnipapillata SixA Cnidaria Hydrozoa . S . . I I K . . E T . E E C . D . . . . S . . . . . . . N T P Y I R N L . N N . . T I . R S R S M V . . . N - - - - R H . E . . . F . L . H F R . G - K K F . S . . . A I . . E . . . . . . . Q . . - . R . . . P . D . . . V . K R . . . . . . . . . . . Q K T H . . . . R T . K H . . E F . L E D . . . . . S K . . E . . D L . H . . P . . . G . . . . . . . . . . . . A APc Six36 AAT11872 Podocoryne carnea Six3/6 Cnidaria Hydrozoa . A . . I S K . . E T . E E C . D . . . . S . . . . . . . N N R E V R . L . N S . . T I . R S R . V V . . . N - - - - S H . H . . . Y . L . H F R . N - K K . . G . L . A I . . E . . . L . . . . . . - . R . . . P . D . . . V . K R . . . . . . . . . . . Q K A H . . . . R T . K L . . E F . L Q D . . . . . S K . . D . . D A . H . . P . . . G . . . . . . . . . . . . A ACr Six36 AAT11874 Cladonema radiatum Six3/6 Cnidaria Hydrozoa . A D . I V K . . E T . E E C . D V . . . S . . . . . . . S N R D V S . L V N T . . T . . R S R . . V . . N N - - - - H H . H . . . Y . L . H F R . S - K K . . S . . . A M . . E . . . . . . . . . . - . R . . . P . D . . . V . K R . . . . . . . . . . . Q K T H . . . . R T . K L . . E F . L Q D . . . . . S K . . D . . N A . H . . P . . . G . . . . . . . . . . . . A AGt Six3 AAN77127 Girardia tigrina Six3 Platyheminthes Turbellaria . A D . I T K . . E T . E E A . D . D . . S . . . . . . . S F N A L W . S L S R R . . I Q R . R . . V . . . V - - - - . . . R . . . N L . . K . R . . - K A . . S . L . A . . . E . . . Q . . . . . . - . R S . . P . D . . . V . K . . . M . . . . . . . . Q K T H . . . . R T . N L . . E C . L D D . . . N . S K . . . . . S A . . . . P . . . G . . . . . . . . . . . . A APd Six3 CAR66435 Platynereis dumerilii Six3 Annelida Polychaeta T P Q . . . Q . . E T . E E . . D V . . . G . . . . . . T A N P M . . . A L N . . . . I . R . R C . V . . . T - - - - . . . K D . . H . L . N H K . S - R D . . A . L . A M . . E . . . Q . . . . . . - . R . . . P . D . . . V . K . . . . . . . . . . . . Q K T H . . . . R T . N L . . E . . L Q D . . . N . T K . . E . . Q A . . . . P . . . G . . . . . . . . . . . . A ADm Six3 AAD39863 Drosophila melanogaster Six3 Arthropoda Hexapoda . A A . . E I . . K T . E D . . D . . . . . . . . . . . . V A L P N M H E . L N C . A . . R . R . V V . Y . V - - - - . . . R . . . A . . . N H K . . - K A . Y G . L . A M . . E . . . . . . . K . . - . R S . . P . D . . . V . K . . . . . P . . . . . . Q K T H . . . . R T . S L . . E . . L Q D . . . N . T K . . E . . K A . . . N P . . . G . . . . . . . . . . . . A ASp Six3 XP_781696 Strongylocentrotus purpuratus Six3 Echinodermata Eleutherozoa . P T . I . S . . E T . E E . . D . . . . . . . . . . . . V A P G T . . A L S . . . . . . R . R . V V S . . . - - - - . . Y R . . . H . L . N H R . . - K D . . A . L . A M . . E . . . Q . . . K . . - . R . . . P . D . . . V . K . . . . . . . . . . . . Q K T H . . . . R T . S L . . E . . L Q D . . . N . T K . . E . . Q A . . . . P . . . G . . . . . . . . . . . . A AOd Six36a AAW23094 Oikopleura dioica Six3/6a Chordata Tunicata A V Q H . I N I . S T . E D C . D . D . . G Q Y . . . . . A L P A I L . A L S . . . F L I R . R . V V . . K . - - - - . . Y R . . . A L . . S R R . S - N I H . A . L . A . . . E . . . G . . . A A . - . R . . . P . D . . . V . K . H . F . S . . . . . . Q K . H . . . . R T . N T . . E S . L . D . . . N . S R . . E . . . A . A . . P . . . G . . . . . . . . . . . . A AOd Six36b AAW23095 Oikopleura dioica Six3/6b Chordata Tunicata . P A S . S Q L . A . . E E T . D F D . . . . . . . . . . A L P P I L D A L A N D . T L . R . R . V V . Y . . - - - - . . . R . M . . . V . S K R . S - K V H . S . L . E . . . E . . . G . . . A T . - . R S . . P . D . . . . . K . Y . . . . . . . . . . Q K . H . . . . R T . T L . . E S . I . D . . . N . T K . . E . . . . . N . . P . . . G . . . . . . . . . . . . A ACiSix36 BAE06688 Ciona intestinalis Six3/6 Chordata Tunicata . P S . I . T . . E S . . E . . D . . . . . . . . . . . . A A P G V L . V L N T . . V . . R . R . I V . . . . - - - - . H Y R D . . A . L . T H R . . E K D . . G . L . A M . . E . . . Q . . A K . . - . R . . . P . D . . . . . K . . . . . . . . . . . . Q K T H . . . . R T . S L . . E . . L R D . . . N . S K . K E . . H A . . . . P . . . G . . . . . . . . . . . . A ADr Six3 AAC27448 Danio rerio Six3 Chordata Vertebrata . P . . . . S . . E T . E E T . D . . . . G . . . . . . . V A P G . . . A . N . H . . I . R . R . V V . . . T - - - - . . . R D . . H . L . N H K . . - K D . . G . L . A M . . E . . . Q . . . K . . - . R . . . P . D . . . V . K . . . . . . . . . . . . Q K T H . . . . R T . S L . . E . . L Q D . . . N . S K . . E . . Q A . . . . P . . . G . . . . . . . . . . . . A ADr Six6 AAC27449 Danio rerio Six6 Chordata Vertebrata . A . . . . S . . E T . E E T . D . . . . G . . . . . . . V A P G . . D A . N . H . . I Q R . R . V V . Y . T - - - - . S . R . . . H . L . T H K . . - K D . . G . L . A M . . E . . . Q . . . K . . - . R . . . P . D . . . V . K . . . . . . . . . . . . Q K T H . . . . R T . G L . . E . . L Q D . . . N . S K . . E . . Q A . . . . P . . . G . . . . . . . . . . . . A AMm Six3 AAH94426 Mus musculus Six3 Chordata Vertebrata . P . . . . S . . E T . E E T . D . . . . G . . . . . . . V A P G . . . A . N . H . . I . R . R . V V . . . T - - - - . . . R D . . H . L . N H K . . - K . . . G . L . A M . . E . . . Q . . . K . . - . R . . . P . D . . . V . K . . . . . . . . . . . . Q K T H . . . . R T . S L . . E . . L Q D . . . N . S K . . E . . Q A . . . . P . . . G . . . . . . . . . . . . A AMm Six6 AAD48911 Mus musculus Six6 Chordata Vertebrata . P Q . . . G . . E T . E E . . D V . . . G . . . . . . . V A P A . . . A L N . . . . . . R . R . I V . . . G - - - - . . Y R . . . H . L . N H K . . - K . . . A . L . A . . . E . . . Q . . . K . . - . R . . . P . D . . . V . K . . . . . . . . . . . . Q K T H . . . . R T . H L . . E . . L Q D . . . N . S K . . E . . Q A . . . . P . . . G . . . . . . . . . . . . A AHs Six3 CAB42539 Homo sapiens Six3 Chordata Vertebrata . P . . . . S . . E T . E E T . D . . . . G . . . . . . . V A P G . . . A . N . H . . I . R . R . V V . . . T - - - - . . . R D . . H . L . N H K . . - K . . . G . L . A M . . E . . . Q . . . K . . - . R . . . P . D . . . V . K . . . . . . . . . . . . Q K T H . . . . R T . S L . . E . . L Q D . . . N . S K . . E . . Q A . . . . P . . . G . . . . . . . . . . . . A AHs Six6 AAH69413 Homo sapiens Six6 Chordata Vertebrata . P Q . . . G . . E T . E E . . D V . . . G . . . . . . . V A P A . . . A L N . . . . . . R . R . I V . . . G - - - - . . Y R . . . H . L . N H K . . - K . . . A . L . A . . . E . . . Q . . . K . . - . R . . . P . D . . . V . K . . . . . . . . . . . . Q K T H . . . . R T . N L . . E . . L Q D . . . N . S K . . E . . Q A . . . . P . . . G . . . . . . . . . . . . A A

Cw SixB1 BAF56233 Coeloplana willeyi SixB1 Ctenophora Typhlocoela T A G . . . . . . E A . L . . . . . K . . . A . . . . . . - - - C H D S N L M N . . . . M . . R . E V . . N N - - - - . . . S . V . . . L G S R N . S - . N . . . . L . . . . . K S . . . . . . T A . - . R . . . . . D . . . . . . . Y . . . N . . . . . . . . . . . . . . . . . N R . . E . . A Q . K . . . . H . . . . . . . S . . . S L . . . . . . . . . . . . . . . . A .Nv Six45 XP_001625507 Nematostella vectensis Six4/5 Cnidaria Anthozoa T A D . . . . I . . A . F . E . D . K . . S Q . . L . I . - - - - Q E D L Q N . S . . L . . . R . M V . . . R - - - - . C Y Q . V . N . L . N . K . D - T S . . E F L . C . . Y K . . . S . G . K . . - . R S . S . . D . F . . . K . S . . . N . . S . . . K . I . F . . . . V . T . . K E C . E H K K . . T L K . . . V I . T . . N . . L R . . R . . . R . . . H . . . I . SNv Six45a XP_001623134 Nematostella vectensis Six4/5a Cnidaria Anthozoa . A D . . . . . . . A . R . A . D . . . . S . . . . . . . - - - - P D D L L N G S . . . . . . R . I V S . . R - - - - . R Y R . V . N . L . T . E . D - . S . . E L L . C . . Y K . . . S . . . K . . - . R S . . . . D . . . . . . . . . . . . . . . . . . . . V . . . . . . A . A A . K D C . E Q . K . . T . Q . . . L I . K . . N . . L K . . . . . . . . . . . . . . I P SCr Six45 AAT11875 Cladonema radiatum Six4/5 Cnidaria Hydrozoa T I . . I D . . . . . . T . . Q D F D T . . K . . . . . . - - - - V N D L V N G S . C . . . . R . H V F L . . - - - - S R Y K . . . S L L . T H K . S - S D L . Q L . . . M . H D . . . S . . . K V . - . R . . . . . E . . . H H . . Y . . . . . . . . . . . . I . . . . . . . . Q M . . E . . E . . K . . T . Q D . . L . . K R . E . . L V . . . . . . . . . . . . . K P Q NHm SixB BAF56231 Hydra magnipapillata SixB Cnidaria Hydrozoa T Y D . I D . . . E A . I . . Q D F N T . . K . . . . I . - - - - R N D I V R N S . H . V . . . . H V . M Y . - - - - E R Y R . . . N . L . N H K . K - S . N . . I L . . M . H D . . . L D . . K M . - . R . . . . . E . . . . . . . Y . . . . . . . . . . . . V . . . . . . . . Q . . . D . . E N . K . . T . Q D . . I . . K R . E . . L V . . . . . . . . . . . . . K P Q SDm Six4 AAD39864 Drosophila melanogaster Six4 Arthropoda Hexapoda . T D . I Q . M . E A . . . K . D . . K . T T . . C . . . - - - - P S . F F K T . . . . . R . R . M V . Y N L - - - - . Q . H . . . N L L . T H C . S - I K Y . V D L . N . . F K . . . K . . . K V . - . R . . . . . D . . . L . K . Y . . . K . . . . . . . . V . . . . . . . . N A . K D C . L T . R . . T . D . . K T . . K K . . . . L . . . . . . . . . . . . . . . T P QSp Six4 XP_ 781616 Strongylocentrotus purpuratus Six4 Echinodermata Eleutherozoa . A Q . . V . . . E A . R . E . . . D . . . . . . . T . . - - - - . D . T L . N D . T . . R . R . A V . Y . . - - - - . H Y K . . . N L L Q N H N . N - . A F . T E L . D . . Y . . . . K . S . K . . - . R . . . . . D . . . . . . . H . . . . . . . . . . . M A . . . . . . . . N M . K E C . K Q . R . . T . D . . . N . . K V . . . . M . . I . . . . . . . . . . . K L P MCiSix45 BAE06689 Ciona intestinalis Six4/5 Chordata Tunicata T L D . . S . I . Q D . L . R R Q . D C . S S . . V T . . - - - - K H L L Y G A . . N M . . . R . . V . . K . - - - - R K . T D . . Q L L . S H T . S - . S N . K L L . N . . Y S . . . A . . . K A . - . R . . . . . D . . . . . . . . S . . . . . . . . . . M V . . . . . . . . L A . K E C . K . . K . . T . D D . . H . . . D . . . S I L . . . . . . . . . . . . . . S P QOd Six45 AAW23096 Oikopleura dioica Six4/5 Chordata Tunicata T P N . I . . . . N . . M E K . D Y . K . T K . M L . . . - - - - N D K S L Y Q . . D . V R . Q C V A L . . I - - - - N D . K T . . H Q L . S Q H . A - T . H . Q F L . E . . Y K . . . L . V Q . M . - N R . . . . . D . . . . . . R . . . . . . . . . . . H . I . . . . . . . . N . . K T S . H R . R . . . Q E . R . R . . . L . . . S M V . . . . . . . . . . . . E . V P PDr Six41 BAB18513 Danio rerio Six4.1 Chordata Vertebarata . P . . . . . . . E A . M . G . . V D . . . . . . . . . . - - - - Q S D L L R G . . . I . . . Q . I V . . . H - - - - A R Y Q . . . C . L . N H . . S - . S N . S S L . D M . Y K . R . T . . . K A . - . R . . . . . D . . . L . . . Y . . . . . . . . . . . . V . . . . . R . . N A . K D M . K R . R . . . . A . . . N . . K M . . . S L . . . . . . . . . . . . . . . N P SMm Six4 NP_035512 Mus musculus Six4 Chordata Vertebarata . P D H . . . . . E A . . . G . . L D . . . . . . . . . . - - - - Q S D L L R G . . . L . . . R . . V . . . . - - - - . I Y . . . . S . L . S H . . E - S A N . . L L . . . . Y K . R . T . . . . A . - . R . . . . . D . . . L . . . . . . . . . . . . . . . . V . . . . . . . . N A . K E L . K Q . R . . . . A . . . H . . K I . . . S L . . . . . . . . . . . . . . . N P SMm Six5 BAA11824 Mus musculus Six5 Chordata Vertebarata . P . . . . . . . E A . L . A . H A G . . S . . . G A . . - - - - P A . R L R G S D P . . R . R . . V . . Q R - - - - . E Y A . . . Q L L . S R P . P - A A H . A F L . D . Y . R . R . H . . . . A . - . R A . . . . D . . . L . K . . . . . K . . . . . . . . V . . . . . R . . A A . K A C . R G . R . . T . D . . . R . . T L . . . S L . . . . . . . . . . . . . . . T G THs Six4 NP_059116 Homo sapiens Six4 Chordata Vertebarata . P D H . . . . . E A . . . G . . L D . . . . . . . . . . - - - - Q S D L L R G . . . L . . . R . . V . . . . - - - - . I Y . . . . S . L . S H . . E - S A N . . L L . . . . Y K . R . T . . . . A . - . R . . . . . D . . . L . . . . . . . . . . . . . . . . V . . . . . . . . N A . K E L . K Q . R . . . . A . . . H . . K I . . . S L . . . . . . . . . . . . . . . N P SHs Six5 NP_787071 Homo sapiens Six5 Chordata Vertebarata . P . . . . . . . E A . L . A . H A G . . S . . . G A . . - - - - P A . R L R G S D P . . R . R . . V . . Q R - - - - . E Y A . . . . L L . S R P . P - A A H . A F L . D . Y . R . R . H . . . . A . - . R A . . . . D . . . L . K . . . . . K . . . . . . . . V . . . . . R . . A A . K A C . R G . R . . T . D . . . R . . T L . . . S L . . . . . . . . . . . . . . . T G A

SD HD

Fig. S3. Alignment of Six domains and adjacent Six-type homeodomains of 62 Six genes, which were used for the construction of the phylogenetic tree shown in Fig. 6. All other features are as explained in the legend to Fig. 3B.

Origin of Pax and Six gene families in sponges: Single ... · queenslandica (Larroux et al., 2008)....

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Transcript of Origin of Pax and Six gene families in sponges: Single ... · queenslandica (Larroux et al., 2008)....