JOURNAL OF EXPERIMENTAL ZOOLOGY (MOL DEV EVOL) 285:63�75 (1999)

© 1999 WILEY-LISS, INC.

Isolation of Hox Genes From the ScyphozoanCassiopeia xamachana: Implications for the EarlyEvolution of Hox Genes

KERSTIN KUHN, BRUNO STREIT, AND BERND SCHIERWATER*Zoological Institute, Department of Ecology and Evolution, J.W. Goethe-Universität, D-60054 Frankfurt, Germany

ABSTRACT The isolation of Hox genes from two cnidarian groups, the Hydrozoa and Anthozoa,has sparked hypotheses on the early evolution of Hox genes and a conserved role for these genesfor defining a main body axis in all metazoan animals. We have isolated the first five Hox genes,Scox-1 to Scox-5, from the third cnidarian class, the Scyphozoa. For all but one gene, we reportfull-length homeobox plus flanking sequences. Four of the five genes show close relationship topreviously reported Cnox-1 genes from Hydrozoa and Anthozoa. One gene, Scox-2, is an unam-biguous homologue of Cnox-2 genes known from Hydrozoa, Anthozoa, and also Placozoa. Basedon sequence similarity and phylogenetic analyses of the homeobox and homeodomain sequencesof known Hox genes from cnidarians, we suggest the presence of at least five distinct Hox genefamilies in this phylum, and conclude that the last common ancestor of the Recent cnidarianclasses likely possessed a set of Hox genes representing three different families, the Cnox-1,Cnox-2, and Cnox-5 families. The data presented are consistent with the idea that multiple du-plication events of genes have occurred within one family at the expense of conservation of theoriginal set of genes, which represent the three ancestral Hox gene families. J. Exp. Zool. (Mol.Dev. Evol.) 285:63�75, 1999. © 1999 Wiley-Liss, Inc.

Hox genes represent a large class of transcrip-tion factors that are characterized by a 60 aminoacid sequence, the homeodomain, encoded by thehomeobox (Scott et al., �89; Gehring, �93). Theseregulatory genes play important roles in patternformation and cell-fate speciation (McGinnis andKrumlauf, �92; Lawrence and Morata, �94). In thepast 15 years, Hox genes have been detected inmost major phyla of higher (triploblastic) animals(for review, see Duboule, �94), and more recentlyalso in basal diploblastic animals (e.g., Schier-water et al., �91; Schummer et al.; �92; Shenk etal., �93; Miller and Miles, �93; Aerne et al., �95;Kuhn et al., �96; Finnerty and Martindale, �97;Schierwater and Kuhn, �98). In all animals inves-tigated so far, Hox genes are arranged in clusters(e.g., Beeman et al., �89; Kenyon and Wang, �91;Krumlauf, �92, �94; Salser and Kenyon, �94). Sinceno Hox genes have been found in the Protozoa,these genes seem to be characteristic for meta-zoans and likely have played an important rolefor the successful radiation of metazoan animals.Thus, understanding the evolution of metazoanbauplans also will depend on an understandingof the evolution of Hox genes.

Based on conserved amino acid residues in the

homeodomain and the conserved chromosomal or-ganization of Hox genes in triploblastic animals,the genes have been divided into distinct Hox fami-lies (Gehring et al., �94), which might have evolvedearly in metazoan evolution (Cribbs et al., �92;Kappen et al., �93; Miller and Miles, �93; Gehringet al., �94; Bürglin, �94; Balavoine and Telford, �95;Raff, �96). Different models have been proposed todescribe hypothetical ancestral Hox gene clustersfor arthropods and vertebrates (e.g., Holland, �90,�92; Schubert et al., �93; Carroll, �95; Holland andGarcia-Fernàndez, �96) as well as for nematodesand arthropods (e.g., Bürglin and Ruvkun, �93; Val-entine et al., �96). Based on information from triplo-blastic animals, different suggestions have beenmade for the type of an ancestral Hox gene clusterpresent in the diploblastic ancestor of the tri-ploblasts (Slack et al., �93; Schubert et al., �93; Val-entine, �96; Zhang and Nei, �96). All existing ideasremain highly speculative since, in contrast to

Grant sponsor: Eurogentec (Seraing, Belgium); Grant sponsor:Deutsche Forschungsgemeinschaft; Grant number; DFG Schi 277/10-1.

*Correspondence to: Bernd Schierwater, Technische Universität BAFreiberg, Leipziger Str. 29, D-09599 Freiberg, Germany. E-mail:Schierwater@ zoology.uni-frankfurt.de

Received 26 May 1998; Accepted 3 December 1998.

64 K. KUHN ET AL.

triploblastic animals (e.g., Duboule, �94), we havevery incomplete information on Hox genes fromdiploblastic animals. At present, two putative Hoxgene fragments have been reported from thePorifera (Degnan et al., �95), one Hox gene has beenisolated from Placozoa (Schierwater and Kuhn, �98),and a larger number (mostly homeobox fragments)is known from two cnidarian classes, the Hydro-zoa and Anthozoa (Murtha et al., �91; Schierwateret al., �91; Miles and Miller, �92; Schummer et al.,�92; Naito et al., �93; Shenk et al., �93; Aerne at al.,�95; Kuhn et al., �96; Finnerty and Martindale, �97).It was shown that several distinct Hox families doexist in hydrozoans and anthozoans (Naito et al.,�93; Kuhn et al., �96; Finnerty and Martindale, �97),while no data are available yet for the thirdcnidarian class, the Scyphozoa (we follow here thetraditional view of three classes in the phylumCnidaria, e.g., Kaestner, �65, Barnes, �80). The num-ber and types of Hox gene families present indiploblast animals and their relationships to Hoxfamilies from triploblastic animals have been con-troversially discussed (e.g., Kuhn et al., �96 Fin-nerty and Martindale, �97). We believe that theexisting controversies derive from insufficient em-pirical data, including the use of very short, par-tial homeobox or homeodomain sequences inphylogenetic analyses.

Since a number of Hox genes from the Hydro-zoa and Anthozoa did not reveal any clear pat-tern of the number and type of Hox gene familiespresent in the cnidarians, we performed a com-prehensive survey for Hox genes in the thirdcnidarian class, the Scyphozoa. We report the firstHox genes from Scyphozoa and discuss the pres-ence of five of these genes in Cassiopeia xama-chana in a phylogenetic context with known Hoxgenes from other diploblastic animals. The datashow that at least five distinct Hox families mayexist in diploblasts, and that within one familyup to four Hox genes can be found in a single ge-nome. Within the cnidarians, the number of Hoxgene families seems to differ between majorgroups, while multiple and independent duplica-tion events likely have increased the number ofgenes within certain Hox gene families.

MATERIALS AND METHODS

Amplification and cloning ofhomeobox fragments

Polyps of the scyphozoan Cassiopeia xamachanaderived from a Florida strain collected and main-tained by Dietrich K. Hofmann (Bochum, Ger-

many). We maintain polyp clones of this strain inartificial seawater under our standard culture con-ditions described earlier (Schierwater, �89). Wholegenomic DNA was isolated from mature polyps andtotal RNA from whole tissue of different develop-mental (including budding) stages of the polyps,according to Kuhn et al. (�96). First-strand cDNAwas synthesized with an oligo dT-Adapter-primer(Frohmann et al., �88), using M-MLV reverse tran-scriptase (Superscript, Gibco, BRL) and total RNAas template, following the manufacturer�s proto-col. Different sets of degenerate primers were usedto amplify homeobox gene fragments from bothgenomic DNA and cDNA. PCR reactions werecarried out using the Goldstar Taq polymerase(Eurogentec) and the amplification conditions ofKuhn et al. (�96). Two different templates, genomicand cDNA, were chosen because homeoboxes maybe interrupted by introns and because we experi-enced that some Hox genes are only amplified fromone or the other template. The primers HoxA/HoxB(Murtha et al., �91) amplify a 77 bp fragment fromposition 62 to 138 of the homeobox of Hox genes,whereas the primer set HoxE/HoxF (Pendleton etal., �93) amplifies a 82 bp fragment spanning posi-tions 63 to 144. Amplification products of the ex-pected size of 166 bp and 146 bp (homeoboxfragments plus primer lengths), respectively, werecloned into the pGEM-T Vector (Promega), accord-ing to the manufacturer�s instructions. DNA mini-preparations of single clones were performed asdescribed in Sambrook et al. (�89) and sequencedin both directions by means of cycle sequencing us-ing the Boehringer Mannheim Dig-Taq-Sequenc-ing kit (cf., Kuhn et al., �96).

Rapid amplification of cDNA ends

Using sequence information from the homeoboxfragments, 5′RACE and 3′RACE (Frohmann et al.,�88) were performed to amplify cDNA sequencesas described in Kuhn et al. (�96). For all genes,we obtained multiple amplification products,which were screened for the target gene by meansof high stringency Southern analyses using thehomeobox fragments as probes (for details, seeKuhn et al., �96). Positive 5′- and 3′RACE frag-ments were cloned and sequenced.

Tree construction

To detect potential genealogical relationshipsbetween different Hox genes, we used two com-monly used tree-building algorithms, maximumparsimony (Swofford, �90) and Neighbor-Joining(Saitou and Nei, �87). Both algorithms were ap-

EARLY EVOLUTION OF HOX GENES 65

plied to homeobox (i.e., DNA), as well as homeo-domain (i.e., protein) sequences. We used the soft-ware packages PAUP (Version 3.1, Swofford, �91)and MEGA (Version 1.01, Kumar et al., �93).

RESULTS

Isolation and sequencing ofhomeobox fragments

We isolated five Hox genes from the scyphozoanCassiopeia xamachana. Using different sets of de-generate primers and both genomic DNA and firststrand cDNA, we amplified different homeoboxfragments (Table 1). In sequencing a total of 38clones from 14 separate PCR reactions, five dif-ferent Hox genes were found and named Scox-1 �Scox-5 (Scyphozoan homeobox). Scox-1, Scox-4,and Scox-5 were amplified with both primers sets,HoxE/HoxF (Pendleton et al., �93) and HoxA/HoxB(Murtha et al., �91), whereas Scox-2 and Scox-5were only amplified with the HoxE/F primer set.Scox-1, Scox-2, and Scox-4 were amplified fromboth templates, cDNA and genomic DNA, whereasScox-3 was amplified from genomic DNA and Scox-2 from cDNA only. Via 3′- and 5′-RACE we couldidentify the full-length homeoboxes, and at least230 bp of the 5′ flanking region plus full length3′-ends of the cDNA from Scox-1, Scox-2, Scox-3,and Scox-4 (Fig. 1). For Scox-5 only, the full-length5′ end of the homeobox, plus flanking sequence,could be characterized.

Classification of scyphozoan Hox genes

Comparisons of the identified scyphozoan home-odomains with most closely related full-lengthHox class homeodomains from other diploblasticanimals are summarized in Table 2. Knowncnidarian homeodomains that do not belong to theHox class showed less sequence similarities (datanot shown).

To place the known cnidarian homeobox genesinto groups of putative relatedness, we arrangedthe homeodomains into Cnox (Cnidarian home-obox) families based on sequence similarities (see

Fig. 2). As can be seen in the alignment, the dif-ferent Cnox-families exhibit characteristic aminoacid residues at different positions. For example,the Cnox-2 family can be characterized by Ile-4(i.e., isoleucin in position 4 of the homeodomain),Thr-6, Ala-7, Ser-10, Leu-13, Gln-21, Asn-22, Ser-27, Leu-29, Gln-33, Ala-36, Met/Ile-37, Asp-39,Lys-43, Val-45, Val-54, Asp-59, and Lys-60. Theproposed Hox-families are named and numberedaccording to Hox genes identified from the hydro-zoan E. dichotoma (Kuhn et al., �96), since no un-equivocal nomenclature exists for Hox genes fromcnidarians. This way we can distinguish at least8 families. Three main families, Cnox-1, Cnox-2and Cnox-5 family; three families that seem to berelated to the former; and two families that con-tain the derived Cnox-3 and Cnox-4 genes fromthe hydrozoan Eleutheria dichotoma (Kuhn et al.,�96). The alignment shown in Fig. 2 provides anoverview of all cnidarian Hox genes identified thusfar, including the five new Hox genes from C.xamachana (Scox-1 to -5).

Based on sequence similarities and character-istic amino acid residues, the Scox genes can beclassified within existing Cnox-families. All genescan be grouped either to the Cnox-1 or Cnox-2 fam-ily known from the Hydrozoa because of cumgrano salis �unambiguous� sequence identities inthe homeodomain (Fig. 2). For example, Scox-2from C. xamachana is 100% identical to Cnox-2from the hydrozoan Eleutheria dichotoma (andother hydrozoans) and, thus, possesses all aminoacid residues typical for the Cnox-2 family (e.g.,Ile-4, Thr-6, Ala-7, Ser-10, Leu-13, etc.). Scox-1and Scox-3 from C. xamachana are 70% identicalto Cnox-1 from E. dichotoma, and 68% identicalto each other (Table 2). Furthermore, Scox-3 ex-hibits typical amino acid residues for Cnox-1 genes(e.g., His-23, Phe-24, and Glu-29) and, thus, is in-cluded in the Cnox-1 family. Scox-1, which is also70% identical to Cnox-1 genes but lacks the typi-cal amino acid residues at position 23 and 24, wasgrouped in a �Cnox-1 related� family. Members ofthis family possess only some characteristic amino

TABLE 1. The isolation of new Hox gene fragments depends on both the primer set and the type of DNA template used1

Cassiopeiaxamachana Scox-1 N Scox-2 N Scox-3 N Scox-4 N Scox-5 N

DNA HoxE/F 1 HoxE/F 2 HoxE/F 1 HoxE/F 5HoxA/B 1 1 HoxA/B 16 HoxA/B 2

cDNA HoxE/F 5 HoxE/F 3 � HoxE/F 1 HoxE/F 11For example, most Scox genes were amplified only with the primer set HoxE/F, Scox-3 only from cDNA, and Scox-5 only from genomic DNAtemplates. N is the number of clones sequenced.

66 K. KUHN ET AL.

EARLY EVOLUTION OF HOX GENES 67

Fig. 1. Hox genes isolatedfrom the scyphozoan Cassiopeiaxamachana. Shown are partialcDNA and deduced amino acidsequences; the homeodomainsare underlined. Note that forScox-4 and -5 the 5′-ends outsidethe homeobox must contain PCRor sequencing errors, since stopcodons appear before a start me-thionine is found (this does not,however, affect any conclusionsdrawn in the text). All sequenceshave been submitted to Genbank(accession numbers are: Bank It250680/696/699/701/705 AF 124-591/2/3/4/5). See text for furtherexplanations.

68 K. KUHN ET AL.

acid residues for the Cnox-1 family. Scox-4 andScox-5 seem to be more distantly related to knownhomeobox genes from other cnidarians (see Table2), but show highest sequence similarities to otherScox genes; they are 68% identical to each otherand to Scox-1. The Scox-4 and Scox-5 genes weregrouped into the Cnox-1 related family togetherwith Scox-1 and Anthox4. As shown in Fig. 2, Leu-21, and Tyr-22 are two of the characteristic aminoacid residues for this group. No members of theCnox-5 or Cnox-5 related families could be identi-fied in the scyphozoan Cassiopeia xamachana.

The classification of partial homeodomains(Anthox5, Cnox-5-Hm, Cnox-1-Cv, and Cnox-4-Hm)should be taken with caution, since it is basedupon a few amino acid residues only. Anthox5 andCnox-5-Hm seem also to belong to one family andshow highest sequence similarity to members ofthe Cnox-2 family. Similarly, Cnox-1-Cv and Cnox-4-Hm seem to belong to one family, and both genesexhibit some typical amino acids of the Cnox-5family (Fig. 2).

Phylogenetic analysis of diploblastHox genes

In an attempt to detect phylogenetic patternsin the diploblast Hox gene sequences, whichwould be useful for the grouping of these genesinto distinct families, we applied Neighbor-Join-ing (Saitou and Nei, �87) and maximum parsi-mony (Swofford, �90) analyses to the homeoboxand homeodomain sequences. Maximum parsi-mony analysis of full-length and partial cni-darian homeobox sequences revealed a singleshortest tree (Fig. 3a) that supports the previousgrouping of cnidarian Hox genes into at least fivedifferent groups (cf., Fig. 3a), although Cnox-4

groups closer to the Cnox-5 family in this treethan in all the others. A very similar tree wasalso obtained by Neighbor-Joining analysis of thehomeobox sequences (data not shown). Further-more, when using the deduced amino acid se-quences of the homeodomains in the analysis, thetopology of the resulting Neighbor-Joining treealso suggests the existence of at least five mainfamilies (Fig. 3b). The DNA and protein treesmainly differ with respect to branching orderswithin the main groups. The separation of themain families, however, remains the same.

When Hox gene sequences from other diploblas-tic animals, from sponges and the placozoanTrichoplax adhaerens, are included in the analy-sis, the overall picture drawn above changes onlyslightly (Fig. 4a, b). The main difference relatesto the placement of the Cnox-2 related family,which, if non-cnidarian diploblast Hox genes areincluded, does no longer cluster as a sister groupto the Cnox-2 family (Fig. 4). The grouping ofgenes to different families, as well as the numberof families, remains the same as seen above (Fig.3). The Trox-2 gene from Trichoplax adhaerens,formerly described as a Cnox-2 homologue (Schier-water and Kuhn, �98), groups to the Cnox-2 fam-ily in all analyses (Figs. 3, 4). One gene fromsponges, SpoxH1, groups to the Cnox-5 family(Fig. 4), while the grouping of the other, SpoxH2,is ambiguous (Fig. 4). Thus, all analyses supportthe existence of at least five main homeobox fami-lies (Cnox-1 to Cnox-5) in cnidarians. The treesgive no clear answer, however, whether the so-called �related� families should be regarded asseparate families (which would increase the totalnumber of Cnox families to 8) or as part of theexisting Cnox-1, Cnox-2, and Cnox-5 families.

TABLE 2. Percent sequence identity of the isolated Hox gene homeodomains from Cassiopeia xamachana with full-lengthhomeodomains from other hydrozoans (Cnox) and an anthozoan (antpC)1

C. xamachana

Scox-1 Scox-2 Scox-3 Scox-4 Scox-52

E. dichotomaCnox-1 70 45 70 58 62Cnox-2 53 100 62 60 45

H. vulgaris: Cnox-23 53 98 62 58 45E. dichotoma: Cnox-5 57 55 60 57 49P. carnea: Cnox-1 57 52 58 52 55H. magnipapillata: Cnox-4 53 52 55 55 47A. formosa: antpC 55 60 63 52 511References: Acropora formosa (Miller and Miles, �93); Hydra magnipapillata (Naito et al., �93); Hydra vulgaris (Shenk et al., �93); Podocorynecarnea (Aerne et al., �95); Eleutheria dichotoma (Kuhn et al., �96). Note that the sequence identity scores per se are not reliable indicators ofdegree of relatedness (cf. Figs. 3�5, and Schierwater and Kuhn, �98).2Percent sequence identity is based on the comparison of 47 amino acid residues.3Cnox2 from H. vulgaris and Chlorohydra viridissima (Schummer et al., �92) are identical.

EARLY EVOLUTION OF HOX GENES 69

Fig. 2. Assignment of partial and full-length home-odomain sequences of Hox genes from diploblastic animals,including the five new scyphozoan genes (Scox-1 to -5 fromCassiopeia xamachana, underlined), to different Cnox fami-lies based on diagnostic amino acid residues. H = Hydrozoa,S = Scyphozoa, A = Anthozoa, P = Placozoa. Sequences fromthe literature are: Eleutheria dichotoma: Cnox-1-Ed to Cnox-5-Ed (Kuhn et al., �96); Hydra vulgaris: Cnox-1-Hv to Cnox-3-Hv (Shenk et al., �93); Hydra magnipapillata: Cnox-1-Hmto Cnox-5-Hm (Naito et al., �93); Sarsia sp.: SAox3 to SAox3(Murtha et al., �91); Metridium senile and Nematostella

vectensis: Anthox and Anthox-Nv (Finnerty and Martindale,�97); Chlorohydra viridissima: Cnox1-Cv and Cnox2-Cv(Schummer et al., �92); Trichoplax adhaerens: Trox-2 (Schier-water and Kuhn, �98); Hydractinia symbiolongicarpus: Cnox-2-Hs (Schierwater et al., �91); Podocoryne carnea: Cnox1-Pc(Aerne et al., �95); Acropora formosa: Antp-C (Miles andMiller, �92). Sequences are aligned to the Antp gene fromDrosophila. The suggested assignment of five main familiesplus three related families is also supported by parsimonyand Neighbor-Joining analyses (see Figs. 3 and 4). See textfor further explanations.

70 K. KUHN ET AL.

Fig. 3. Parsimony analysis of the home-oboxes (a), and Neighbor-Joining analysis of thehomeodomains (b), of cnidarian Hox genes (seeFig. 2). Figure 3a is the single shortest tree ofa heuristic search (100 random additions) us-ing the branch swapping-algorithm NNI andunweighted DNA characters (tree length = 831).Figure 3b is the result of a Neighbor-Joininganalysis using p-distances. The trees are rootedagainst the outgroup Cnox3 from Chlorohydraviridissima, a homeobox gene that does not be-long to the Hox-class (Schummer et al., �92).Both trees support the existence of the threemain plus their related families: Cnox-1 plusCnox-1 related, Cnox-2 plus Cnox-2 related,Cnox-5 plus Cnox-5 related family. The maingroupings, and also the isolated position of theCnox-3-Ed and Cnox-4-Ed genes, are concordantwith the grouping based on diagnostic aminoacid residues shown in Fig. 2.

EARLY EVOLUTION OF HOX GENES 71

Fig. 4. Parsimony analysis of thehomeoboxes (a), and Neighbor-Join-ing analysis of the homeodomains(b), of diploblast Hox genes, i.e., fromcnidarians (Fig. 2), sponges and Pla-cozoa. Figure 4a is the result of aheuristic search (100 random ad-ditions) using the branch swapping-algorithm NNI and unweighted DNAcharacters (50% majority rule consen-sus tree of three equally parsimonioustrees; tree length = 913). Figure 4b isthe result of a Neighbor-Joining analy-ses using p-distances. The trees arerooted against the triploblast outgroupgene even scipped, eve. (Bürglin, �94).In both trees the non-cnidarian Hoxgenes, Trox-2 from Placozoa and Spox-H1 from Porifera (Degnan et al., �95)group within the formerly establishedmain families Cnox-2 and Cnox-5, re-spectively. The placement of the par-tial poriferan SpoxH2 sequence isinconsistent, however.

72 K. KUHN ET AL.

Four of the five Scox genes from C. xamachana,Scox-1, -3, -4, and -5, belong to the Cnox-1 or itsrelated family. Within the cnidaria, this is thehighest number of Hox genes belonging to thesame family found in a single genome. The Scox-2 gene is an unambiguous homologue of the Cnox-2 genes known from a variety of cnidarians andalso from the Placozoa. No putative homologuesof any other gene family could be found.

DISCUSSION

Classification of cnidarian homeoboxes

The full-length homeobox sequences from the scy-phozoan Cassiopeia xamachana allow the classifi-cation of homeoboxes in different cognate groupsor families. Based on sequence similarity, conser-vation of characteristic amino acid residues, andphylogenetic analysis the scyphozoan, Hox geneScox-2 undoubtedly is a member of the Cnox-2 genefamily (or �class� by some authors) known from dif-ferent cnidarians and the placozoan Trichoplaxadhaerens. The other Scox genes seem to belong tothe Cnox-1 or its related family. All phylogeneticanalyses support this grouping. These findings arerelevant to the discussion of the early evolution ofHox genes in diplobastic animals, in particular withrespect to the number and type of Hox gene fami-lies present in diploblastic animals before the ori-gin of the triploblast Hox gene cluster.

Based on our analysis, we suggest the presenceof at least five different Cnox families in the Re-cent cnidarians. If one regards the Cnox-1, -2, and-5 related families as separate families, therewould be at least eight of such cognate groups. Ina recent analysis, Finnerty and Martinale (�97)suggest the presence of five to six such families,based on the phylogenetic relationships of antho-zoan and hydrozoan Hox genes. The proposed clas-sification of cnidarian Hox genes is similar to theone proposed here, although the other authors didnot include the Cnox-3 and Cnox-4 gene from thehydrozoan Eleutheria dichotoma (Kuhn et al., �96)in their analyses.

The existence and composition of the three mainCnox families seems to be unquestioned, while theso-called �related� families may either be treatedas separate families or as members within thethree main families (Naito et al., �93; Finnerty andMartinale, �97). The hydrozoan Cnox4-Hm andCnox1-Cv and the anthozoan Anthox4 gene havebeen classified as separate families of unknownphylogenetic relationship to other families (Naitoet al., �93; Finnerty and Martindale, �97). After

including the first scyphozoan Hox genes, it seemsclear that the anthozoan Anthox4 belongs to theCnox-1 family, which also includes the scyphozoanScox-1, -2, and Scox-4 genes, while Cnox1-Cv andCnox4-Hm seem to be related to genes of the Cnox-5 family.

Evolution of cnidarian homeobox genes

As shown in Figs. 3 and 4, one may postulatethat at least three main Hox families exist incnidarians. Homeobox genes of the Cnox-1 and theCnox-2 family have been identified in the cnidar-ian classes Hydrozoa, Anthozoa, and Scyphozoa,whereas homeobox genes of the Cnox-5 family haveonly been detected in hydrozoans and anthozoans(Fig. 5). We do not know yet whether a Cnox-5

Fig. 5. Evolution of the three main Cnox families inCnidaria. (a) This figure summarizes our current knowledgeof the presence of representative genes of the Cnox-1 to Cnox-5 families in the different cnidarian groups. This distributionis congruent with the assumption that the cnidarian ancestorpossessed a Cnox-1, Cnox-2, and a Cnox-5 homologue; the de-rived Scyphozoa possibly lost their Cnox-5 homologue (but si-multaneously increased the number of Cnox-1 genes) (b). Nodata are available for the Cubozoa yet, which traditionally havebeen grouped within the Scyphozoa (e.g., Barnes, �80). The basalposition of the Anthozoa is according to Bridge et al. (�92, �95).The drawing of the genes as a cluster is strictly hypothetical.Further explanations are given in the text.

EARLY EVOLUTION OF HOX GENES 73

homologue is indeed absent in C. xamachana, andpossibly all other scyphozoans, or whether it es-caped our survey. The first idea seems plausible,since the Scyphozoa represent a derived groupwithin the cnidarians (Bridge et al., �92, �95) and asecondary loss of a Cnox-5 homologue in this classmay have occurred. The alternative explanationwould be that a Cnox-5 homologue escaped our sur-vey. The observed relationship between the num-ber of new fragments identified and the numberof clones generated and sequenced (Fig. 6), how-ever, does not support the latter explanation. Allidentified Scox genes were found within the first12 out of 38 clones sequenced, i.e., the graphreaches a stable plateau at 5 �new fragments� (Fig.6). Given that the different clones analyzed de-rived from independent PCR reactions using dif-ferent DNA templates and primer sets, the graphin Fig. 6 suggests that there might be not morethan five Scox genes present in C. xamachana.

Independent of whether the Scyphozoa possessgenes of the Cnox-5 family or not, we may con-clude that Hox genes of at least the Cnox-1, Cnox-2, and Cnox-5 families were present before theradiation of the different cnidarian classes. Basedon morphological characters, mt-DNA structure,and ribosomal DNA-sequences, it seems clear thatthe Anthozoa represent the most basal cnidarians(Bridge et al., �92, �95), which already possess rep-resentatives of these three families (Fig. 5). Whilewe find only a single Hox gene for the Cnox-2 andCnox-5 family in one genome, there is more thanone gene of the Cnox-1 family present in Anthozoa(Anthox1-Nv and Anthox1a-Nv) and Hydrozoa(Cnox1-Hv and Cnox3-Hv). If one includes the

Cnox-1 �related� genes, this would also be truefor Scyphozoa; and the number of Hox geneswithin a single family raises to four. As the mostparsimonious interpretation, one would assumethat at least two ancestral Cnox-1 genes existedin the common ancestor of all cnidarians. The lessparsimonious interpretation would assume thatmultiple and independent duplication events ofthe Cnox-1 gene occurred in the different cnidar-ian groups. Independent of when the duplicationevent of an ancestral Cnox-1 gene happened, thepresence of four Cnox-1 and Cnox-1-related Hoxgenes in the scyphozoan Cassiopeia xamachanasuggests that in this group, at least one or twoadditional duplication events must have occurred.

The isolation of an unambiguous Cnox-2 homo-logue from the placozoan T. adhaerens (Trox-2,Schierwater and Kuhn, �98), is the first finding ofa Cnox gene homologue outside the cnidaria. TheCnox-2 family might represent an ancestral Hoxgene family, which might be ancestral to all Hoxgenes (cf., Schierwater and Kuhn, �98). If this werethe case, one would expect to find Cnox-2 homo-logues also in the other diploblastic phyla, thePorifera and the Ctenophora. At present, however,only two putative Hox genes are known fromPorifera, SpoxH1 and SpoxH2 from Haliclona sp.(Degnan et al., �95), which show no indication ofgrouping to the Cnox-2 family. In the phylogeneticanalyses, the Trox-2 gene clusters, as expected,within the Cnox-2 family, and the SpoxH1 frag-ment show relationship to genes of the Cnox-5 fam-ily (Fig. 4). The grouping of SpoxH2 remainsunclear. In the DNA tree (Fig. 4a), SpoxH2groups within the Cnox-1 family, while in theamino acid tree (Fig. 4b) the SpoxH2 fragmentgroups to the Cnox-5 family. The latter obser-vation possibly is the result of the limitationsof using partial homeobox sequences in phylo-genetic analyses, and we suggest that full-length homeobox sequences might be requiredto resolve the phylogenetic relationships ofSpoxH2 to other diploblast Cnox genes.

At the moment, however, we can only postulatethat three different Cnox families likely werepresent in the hypothetical common ancestor ofall cnidarians. Based on the phylogenetic analy-ses of the available scyphozoan, hydrozoan, andanthozoan Hox genes, these three families includeCnox-1, -2, and -5 genes. Given the problem thatonly partial homeobox fragments are available formany diploblast Hox genes and the uncertaintyof phylogenetic relationships between the diplo-blast phyla (e.g., Wainright et al., �93; Smothers

Fig. 6. The increase in the number of different Hox genes,identified as a function of the number of clones sequenced(see Materials and Methods). Scox-1 to -5 are the reportedgenes from the scyphozoan Cassiopeia xamachana, and Cnox-1 to -5 are from the hydrozoan Eleutheria dichotoma. Thegraph suggests that there might be not more than 5 differ-ent Hox genes present in C. xamachana (see text).

74 K. KUHN ET AL.

et al., �94; Bridge et al., �95; Pawlowski et al., �96),any further speculations about the Hox gene rep-ertoire of a putative cnidarian ancestor would bepremature.

In our opinion, the discussion of the existenceof an ancestral Hox cluster in diploblastic ani-mals, containing an anterior class gene (labial andproboscipedia type), one middle class gene (Antptype), and one posterior class gene (Abd-B type),is very speculative. Phylogenetic analyses of diplo-and triploblastic homeobox sequences (includinga high proportion of short homeobox fragmentsonly) are believed to support the existence of anancestral Hox gene cluster according to thezootype concept (cf., Finnerty and Martindale,�97). On the other hand, analyses of characteris-tic residues in full-length homeodomains do notreadily support this view and rather raise ques-tions about how to assign homology betweenknown Hox genes from diplo- and triploblasticanimals (Kuhn et al., �96; Schierwater and Kuhn,�98). In our view, to resolve the origin of thetriploblast Hox genes it will be crucial to resolvethe early evolution of Hox genes in diploblasticanimals. Clearly we need to increase both our ef-forts to obtain: (a) more full-length homeobox se-quences of those diploblast Hox genes we haveonly partial information of; and (b) comparativeinformation from sponges and ctenophores. Par-allel the phylogenetic relationships between thebasal metazoan phyla need to be resolved.

ACKNOWLEDGMENTS

We acknowledge comments from Thomas Städler,Andrea Ender, Heike Hadrys, and Rob DeSalle, andtechnical assistance from Margret Ruppert. We aregrateful to Professor Dr. Dietrich Hofmann for pro-viding the animal material.

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