Hox clusters and bilaterian phylogeny

Guillaume Balavoine,* Renaud de Rosa, and Andr�ee Adoutte

Centre de G�een�eetique Mol�eeculaire du CNRS, UPR 3167, Gif-sur-Yvette, France

Received 26 November 2001

Abstract

A large Hox cluster comprising at least seven genes has evolved by gene duplications in the ancestors of bilaterians. It probably

emerged from a mini-cluster of three or four genes that was present before the divergence of cnidarians and bilaterians. The

comparison of Hox structural data in bilaterian phyla shows that the genes of the anterior part of the cluster have been more

conserved than those of the posterior part. Some specific signature sequences, present in the form of signature residues within the

homeodomain or conserved peptides outside the homeodomain, constitute phylogenetic evidence for the monophyly of protostomes

and their division into ecdysozoans and lophotrochozoans. These conserved motifs may provide decisive arguments for the

phylogenetic position of some enigmatic phyla. � 2002 Elsevier Science (USA). All rights reserved.

Keywords: Urbilateria; Metazoan phylogeny; Hox cluster

1. Introduction

Molecular phylogeny has brought some major ad-vances in resolving the tree of metazoans. The use of 18SrRNA sequences in particular has confirmed some tra-ditional ideas based on morphology or embryology,such as the monophyly of the bilaterians (or triplo-blasts). On the other hand, surprising results have beenobtained with the same molecule: the division of thebilaterians into three branches, i.e., the deuterostomesand two related protostome clades, the ecdysozoans andthe lophotrochozoans (Aguinaldo et al., 1997; Halanychet al., 1995; review: Adoutte et al., 2000). This has led tothe unexpected demise of a traditionally recognizedgrouping, the Articulata (annelids + arthropods). Twophyla of simply organized worms, the platyhelminthsand the nematodes, which were usually considered earlyoffshoots of the metazoan tree are also found locatedinside the lophotrochozoan and ecdysozoan branches ofthe tree, respectively.Hox genes are present in all metazoans except spon-

ges. In bilaterians, they are usually grouped into acluster of genes called the Hox cluster. Hox genes code

for related homeodomain transcription regulators.During the embryonic development, these regulators areinvolved in patterning some aspects of the anterior–posterior axis, such as the morphology of segments inDrosophila or the morphology of axial mesoderm de-rivatives in mammals. Hox genes generally respect thecolinearity rule: they are expressed along the body axisin the same order as they are found in the cluster. Ex-tensive samplings of Hox genes and functional studieshave shown that the Hox cluster, colinear expression,and the function in regionalizing the anterior–posterioraxis have originated very early in metazoan history(Carroll, 1995).

In this article, we want to show what the presentknowledge of the diversity of the Hox family in severalphyla tells us about how the Hox cluster evolved inmetazoans. We will also explain how Hox data supportone of the most unexpected and most contentious resultof 18S phylogeny: the division of protostomes intolophotrochozoans (or trochozoans) and ecdysozoans.

2. Reconstituting the ancestral bilaterian cluster

Fig. 1 gives a summary of the most complete Hoxdata known in various phyla of bilaterians. Under-standing the evolution of the Hox cluster involves the

Molecular Phylogenetics and Evolution 24 (2002) 366–373

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E-mail address: guillaume.balavoine@cgm.cnrs-gif.fr (G. Balavo-

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reconstitution of the state of this structure in the lastcommon ancestor of bilaterians, the Urbilateria (De-Robertis and Sasai, 1996). For this purpose, one mustidentify what genes in extant taxa have been inheritedfrom a common ancestral gene in the urbilaterian clus-ter. These genes are called orthologous because theyhave been produced by an event of speciation while theother genes (paralogous genes) in the cluster are con-nected to them by events of tandem duplications. Sev-eral types of phylogenetic clues can be used from Hoxgenes sequences:

• The presence of key signature residues inside thehomeodomain sequence. Homeodomains of ortholo-gous Hox proteins tend to be strongly conserved.Building phylogenetic trees using the homeodomainsequences allows the recognition of closely relatedand therefore likely orthologous genes (de Rosaet al., 1999). At the level of the sequences, strong clus-tering in the trees is due to the existence of ‘‘signa-ture’’ residues, i.e., residues that are shared atcertain positions by orthologous proteins but are veryuncommon in paralogous proteins. Fig. 2 shows the

Fig. 1. Distribution of Hox genes in bilaterians. The tree on the left shows phylogenetic relationships according to the 18S rDNA. Horizontal black

lines indicate available mapping data. Vertical bars delineate orthologous genes. Question marks signal an uncertain orthology assignment. Short

PCR fragments are represented with a grey box line. The orthologous relationships specific for either ecdysozoans or trochozoans are indicated by

colors. The data used for this figure are referenced in the references section. The platyhelminths situation is a compilation of data (Balavoine and

Telford, 1995; Bayascas et al., 1997; Nogi and Watanabe, 2001). Other works not cited in the text are Damen et al. (1998), spider; Garcia-Fern�aandez

and Holland (1994), amphioxus; Grenier et al. (1997), onychophoran; Callaerts et al. (2002), squid; Martinez et al. (1999), Sea urchin.

G. Balavoine et al. / Molecular Phylogenetics and Evolution 24 (2002) 366–373 367

signature residues in the homeodomains of some tro-chozoan and ecdysozoan-specific sequences.

• Conserved ‘‘para-peptides’’. What we call para-pep-tides are short peptides present just adjacent to thehomeodomain (Fig. 2). They are not present in allHox proteins but when present, they can show spe-cific sequences for a given group of orthologousgenes, thus helping in resolving the gene phylogeny.We will see that the conservation of these peptidescan also be restricted to certain groups of related spe-

cies. These peptides can appear at the amino extrem-ity (N-para-peptide) or at the carboxy extremity (C-para-peptide) of the homeodomain. Such peptidesseem to be involved in the functional specificity ofthe protein (Chauvet et al., 2000).

• The position of the gene in the cluster is not an abso-lute criterion since inversion of large fragments ofDNA can happen. It can however provide an usefulinformation in cases where the sequences leave sev-eral possibilities of assignment. Unfortunately, very

Fig. 2. Alignment of the homeodomains and para-peptides of some bilaterian Hox genes showing the signature residues (shadowed sites) for some

superphylum-specific groups of orthologous sequences. Some of these signature residues are found sporadically in other genes. The residues found in

the majority consennus of Ubd-A-type para-peptides are indicated in white. Species abbreviations are: Nvi Nereis virens, Lan Lingula anatina, Lsa

Lineus sanguineus, Dja Dugesia japonica, Dme Drosophila melanogaster, Bfl Branchiostoma floridae, Csa Cupiennius salei, Hro Helobdella robusta,

Hme Hirudo medicinalis, Dor Dicyema orientale, Pca Priapulus caudatus, Aka Acanthokara kaputensis, Pvu Patella vulgata, Cel C. elegans.

368 G. Balavoine et al. / Molecular Phylogenetics and Evolution 24 (2002) 366–373

few Hox clusters have been mapped to date and nomapping data are available in trochozoans.

Using the combination of the structural arguments, wefind useful to divide the cluster of the bilaterians in threeparts, anterior, central, and posterior. Though this di-vision in parts is arbitrary as long as phylogeny is con-cerned, it is based on graduality in the conservationdisplayed by the genes across bilaterians.• The anterior part of the cluster contains all the genes

from lab to Scr in the fruit fly and all the genes fromparalogy groups 1–5 in vertebrates. This is the part ofthe cluster whose evolution is best understood withthe available data. Five genes (Hox1–Hox5) werepresent in the ancestral bilaterian cluster. In some ex-tant bilaterians (such as Nereis, the amphioxus or thevertebrates), these five genes have been inherited withfew changes. However, three types of modificationshave affected these genes in other animals: gene loss,duplication, or rapid sequence divergence correlatedwith dramatic functional changes. Gene loss has pre-sumably happened in the case of the sea urchin (lossof the Hox4 orthologue) (Cameron et al., 2000) andmost notably inside the Hox-C and Hox-D clustersof the mammalians (loss of several genes). A case ofduplication of an ancestral gene is illustrated by thelox6/lox18 couple in the leech (Kourakis and Martin-dale, 2001). These two genes are orthologous to theHox4 ancestral gene. One of the genes has retaineda Hox-like function (lox6) while the function of theother gene (lox18) appears to have changed. Themost dramatic case of functional divergence is foundwith the zerkn€uullt (zen and z2) and bicoid genes of thefly. These genes are found in the Antennapedia clus-ter between pb (Hox2) and Dfd (Hox4) at the place ofa missing Hox3 gene. Evidence collected from otherinsects and arthropods has now convincingly estab-lished that these three genes have derived from a sin-gle Hox3 gene present in the ancestor of thearthropods (Dearden and Akam, 1999; Stauber etal., 1999). The homeotic-like function of this genehas been lost in the lineage leading to the holometab-olous insects and new function as an early morphoge-netic gradient as well as in specifying extra-embryoniccells have been acquired in the same lineage. The du-plications that gave rise to the three genes of Dro-sophila melanogaster apparently happened withinthe dipteran lineage (Stauber et al., 2002).

• The central part generally contains three genes indeuterostomes and four genes in most protostomes.However, the orthology relationships between distantphyla are much less clear than in the anterior part.The sequences of the homeodomains of the genes ofthe central part are in general very similar to eachother. Building phylogenetic trees with the homeodo-mains typically gives an almost complete lack of res-olution. It is mostly the presence of parapeptides that

allows the delineation of orthology relationships spe-cific to the ecdysozoans or the trochozoans, detailedin the next part of the paper. According to these rela-tionships, the last ancestor of the protostomes al-ready had at least two central genes. The mostundeterminate situation occurs in the deuterostomes.Three central genes are present in sea urchins andcephalochordates, as well as three clear paralogygroups in vertebrates (Hox6–Hox8). It is thereforemost likely that three central genes were already pres-ent in the deuterostome ancestor rather than a singlegene that, through independent duplications, wouldhave given the same number of genes in each lineage.Nevertheless, the sequences have not retained anytrace of this early origin.

• In the current state of knowledge, the posterior part ofthe cluster contains quite disparate numbers of genesin different species from one (Abd-B) in the fruitfly tosix in the amphioxus (Ferrier et al., 2000). There isan important discrepancy between protostomes, inwhich at most two posterior genes have been foundand the deuterostomes, in which at least three genesare present. This discrepancy should not however betaken as definitive. Only two complete Hox clustershave been sequenced to date in the protostomes (thefruitfly and the nematode Caenorhabditis elegans)and the posterior genes are notoriously more difficultto clone using the classical degenerate PCR primers.We cannot therefore exclude that additional posteriorgenes remain to be discovered in protostome speciesalready sampled. Once again, as in the central group,it is not possible to trace orthology relationships be-tween posterior genes all accross the bilaterians. It isonly inside the trochozoans and ecdysozoans thatclear cut relationships can be recognised as is detailedbelow. The multiple genes present in the deuterosto-mes are all rather divergent from each other and noresolution is obtained when building a phylogenetictree (de Rosa et al., 1999; Ferrier et al., 2000). It is thusdifficult to tell whether these genes have appeared in-dependently by gene duplications or whether theyhave been inherited from a posterior part of the clusterthat contained already several genes in the last deuter-ostome ancestor. For this reason, it is impossible toclaim that these multiple posterior genes are a sharedderived trait of the deuterostomes.

From the above description, we can conclude that theancestor of bilaterians already had a cluster of at leastseven Hox genes (five anterior, one central, and oneposterior). This minimal number is rather unlikely sinceit necessitates many additional duplication events inseparate lineages to give the extant number of genes invarious species. Since most species contain 10 genes ormore and orthology relationships may have been blur-red by sequence divergence especially in the central part,it seems more likely that the Urbilateria had around 10

G. Balavoine et al. / Molecular Phylogenetics and Evolution 24 (2002) 366–373 369

Hox genes or more. The situation is a bit more clear forthe protostomes. Fig. 3 gives an illustration of the twohypotheses for reconstructing the ancestral protosto-mian cluster. This cluster probably comprised between 8and 11 genes.

The presence of a large Hox cluster of maybe 10genes in the Urbilateria shows that most of the geneduplications that gave rise to the cluster happened be-fore the divergence of bilaterians. Put together with themultiple gene conservations found in comparative de-velopmental biology and also the comparison of thegenomes completely sequenced to date, we arrive to theidea that the Urbilateria had a genome almost as com-plex as modern bilaterians (vertebrates excluded). Whendid this complexity of the Hox family develop? Theanswer comes when comparing bilaterian Hox geneswith the ones known in more basal metazoan phyla, i.e.,sponges and cnidarians. In sponges, no Hox gene isknown despite repeated efforts to find them (Manueland Le Parco, 2000; Seimiya et al., 1994). In cnidarians,where many independent studies have been carried outon hydrozoans (Gauchat et al., 2000; Kuhn et al., 1996)as well as anthozoans (Finnerty, 1998), few Hox genesare present and they are generally not closely related tothe bilaterian ones. Recent analyses nevertheless suggestthat a small Hox mini-cluster of maybe three or fourgenes (as well as a sister Para-Hox mini-cluster; Brookeet al., 1998)) probably existed in the last common an-cestor of cnidarians and bilaterians (Finnerty andMartindale, 1999; Kourakis and Martindale, 2000;Schierwater and Desalle, 2001). The very first gene du-plications creating the cluster must have occurred beforethe divergence of cnidarians from the bilaterians butmost of the duplications leading to the large bilateriancluster have occurred after this divergence.

3. Hox sequences and protostome phylogeny

3.1. The ‘‘Ubd-A’’ peptide supports the monophyly of theprotostomes

The Ubd-A peptide is constituted of a core of eightwell-conserved residues located at the carboxy-end of

the homeodomain in the Ubx and abd-A genes of thearthropods, as well as in two trochozoan genes, provi-sionnally called lox2 and lox4 after the leech genes ini-tially identified (Fig. 1). The peptide is found exclusivelyin some central protostome genes. Nothing closely res-sembling the Ubd-A peptide occurs in any deuterostomecentral gene or cnidarian Hox gene. Most of the pro-tostome phyla investigated have already shown Ubd-Acarrying genes. Only the nemertean Lineus sanguineuslacks these genes (Kmita-Cunisse et al., 1998) but theHox screen in this animal is probably far from exhaus-tive (Balavoine et al., unpublished results). The Ubd-Apeptide may have originated in a central gene in anancestor of the protostomes after the protostome/deut-erostome divergence and this gene may eventually haveduplicated itself in the trochozoans and ecdysozoans.An alternative possibility is that this peptide was presentin the last bilaterian ancestor and that it (or the genescarrying it) was lost very early in the lineage of thedeuterostomes. This is the reason why we consider theevidence for the monophyly of the protostomes rela-tively weaker than the evidence for trochozoan or ec-dysozoan monophyly.

3.2. Five gene sequences support the existence of thetrochozoans

Three central genes, provisionally called lox5, lox2,and lox4, as well as two posterior genes, post-1 and post-2 can be identified accross the trochozoans on the basisof their distinctive sequences (Fig. 1). The homeodo-mains of the lox5-type genes (found in annelids, nem-erteans, platyhelminths, brachiopods, and dicyemids)are not strikingly distinctive but they all share a stronglyconserved C-parapeptide that undoubtedly shows thatthey are orthologous. This Lox5 peptide has been foundonly in lophotrochozoans so far. The Lox2 proteinfound in annelids, molluscs and brachiopods, bears aUbd-A peptide (in annelids and molluscs at least) likeLox4, Ubx, and abd-A. In addition, it also has a dis-tinctive homeodomain sequence (Balavoine, 1998) thatshows its orthology accross the trochozoans. The lox4gene found in annelids, platyhelminths, molluscs, andbrachiopods also has a sequence that distinguishes it

Fig. 3. Two opposite hypotheses for the origin of the protostome Hox diversity. On the left, it is considered that no additional gene duplication has

happened inside the cluster. On the right, the ancestral cluster is kept to a minimal eight genes and additional duplications happened in both the

ecdysozoan and trochozoan lineages. See text for more details.

370 G. Balavoine et al. / Molecular Phylogenetics and Evolution 24 (2002) 366–373

from lox2, Ubx, and abd-A type genes. Comparing thesequences of the Ubd-A-type genes does not indicatewhether the lox2/lox4 pair of trochozoans and the Ubx/abd-A pair of ecdysozoans have been produced by in-dependent duplications (Fig. 3). Finally, two posteriorgenes, post-1 and post-2 have very distinctive homeod-omain sequences. post-2 is found to date in annelids,nemerteans, platyhelminths, molluscs, and brachiopodsand was surely present in the last ancestor of troch-ozoans. post-1 has been found only in annelids, mol-luscs, and brachiopods. The presence of post-1 in othertrochozoans remains to be checked.

3.3. Three gene sequences support ecdysozoan monophyly

Two central genes, Ubx and abd-A, and a posteriorgene, Abd-B have closely related sequences in arthro-pods and onychophorans, that distinguish them readilyfrom the other bilaterian posterior genes. An Ubx-like aswell as an Abd-B-like sequence are found in the pria-pulid Priapulus caudatus (de Rosa et al., 1999). Thesefindings confirms the affinity of these marine wormswith arthropods as was already found with 18S rRNAphylogeny. A single Abd-B sequence is found in thenematode C. elegans. This is the only gene that supportsthe ecdysozoan affinity of the nematodes. Both the pri-apulid and the nematode have a second posterior gene(Pca-HB4 and nob-1, respectively). Though these twogenes are not closely related, this raises the possibilitythat the ancestral ecdysozoan may have had more thanone posterior gene. Whether the derived egl-5 in C.elegans is originally a central or a posterior gene is un-clear. It is noteworthy that for a longtime, only fourHox genes were known in C. elegans. This was usuallytaken as one of the signs of the ‘‘primitiveness’’ ofnematodes. The discovery of two additional posteriorgenes by genomic sequencing as well as the reevaluationof the nematode phylogenetic position now clearly pointout that C. elegans bears a ‘‘degenerate’’ cluster. It hascertainly lost several genes (notably some anterior ones)and the remaining cluster has been split in three separatepieces.

3.4. The nature of Hox gene synapomorphies

How strong are these Hox sequence evidence? Anideal situation for deciphering phylogenetic affinitiesusing Hox genes (or other clustered genes) would be tofind some unambiguous events of gene duplicationsthat happened during the history of bilaterians. Sincesuch duplication is highly unlikely to happen twice andto give convergent pairs of sister genes, finding con-served duplicated genes in different phyla would be avery strong indication that these phyla are related toeach other. However, unambiguous cases of Hox geneduplication are rare in the data presently available.

When they exist, such as the likely triplication of anancestral Hox3 into bicoid, zen, and z2 found in the fly,they are intra-phylum events. Even the massive eventsof whole cluster duplications known in vertebrates haveoccurred after the divergence of vertebrates from otherchordates. Independent duplications, as explainedabove, could have happened in the case of the lox2/lox4genes of trochozoans and Ubx/abd-A genes of ecdyso-zoans but this question is not resolved in gene trees. Itis probable that most of the genes mentioned above aspotential evidence of protostomian affinities are notindependent duplicates. This means that orthologues ofUbd-A genes could indeed exist in deuterostomes otthat orthologues of lox5 could exist in arthropods(Telford (2000a) actually proposes that ftz is a lox5orthologue) but that these affinities have been blurredat the level of the sequence by a long divergence time.The characters that are used for advocating trochozoanor ecdysozoan monophyly are therefore not sharedgenes but shared signature residues in the gene se-quences, as explained by Telford (2000b). It is the ac-quisition of these signature residues in certain genes,unlikely to have happened convergently, that consti-tutes synapomorphies for certain groupings of phyla. Inthis sense, Hox sequences can be considered in a cla-distic framework of analysis and not only according to‘‘global similarity.’’ The possibility remains howeverthat some of these similarities are plesiomorphic, i.e.,they were present in the last protostome ancestor andwere lost later in some lineages thus leading to the falseconclusion that the phyla where they remain arerelated. This could be the case if, for example, theLox5-type peptide was present in the last protostomeancestor and lost in an ancestor of the ecdysozoans. Tothis kind of reasoning, we can oppose the argument ofcongruence: in the trochozoans, we have five gene se-quences that support the same conclusion in favor ofmonophyly and most importantly, there is no sequencethat supports an alternative hypothesis. It appears ex-tremely unlikely that all five types of sequences bearprotostome plesiomorphies that have disappeared inecdysozoans. The same reasoning goes for the ecdyso-zoans but here the evidence is slimmer: two potentialsignature sequences for the ecdysozoans and three forthe panarthropods (arthropods+ onychophorans + tar-digrades). In this interpretation, we essentially do nottake into account gene losses for two reasons: first, thePCR technique used for recovering Hox sequences can‘‘miss’’ some genes and second, Hox gene losses areprobably too frequent in the bilaterian tree for thembeing used as phylogenetic evidence at the supra-phy-lum level.

In conclusion, we think that the Hox evidence, puttogether with the indications of the 18S rRNA, nowmake a very strong case for the existence of trochozoansand ecdysozoans.

G. Balavoine et al. / Molecular Phylogenetics and Evolution 24 (2002) 366–373 371

4. Some perspectives

The above conclusion are based on data which arestill incomplete for some phyla. Complete sequencing ofthe entire Hox cluster is now technically at hands andwill possibly allow the discovery of genes that could notbe recovered with the classic PCR techniques. In thisperspective, concerted efforts should be made to ear-mark for genomics programs not only developmentalbiology model species but also species relevant for theirphylogenetic position. The presence of the potentialsynapomorphies mentioned above should be checked ina few key phyla that are good candidates according tomorpho-anatomic or molecular data to be members ofthe two great protostomian clades. Tardigrades andnematomorphs among ecdysozoans, and sipunculids,bryozoans and rotifers among trochozoans are obviousgroups that should show confirmation of the Hox sig-natures.Hox signatures can provide a first hint at the position

of some problematical phyla that have been puzzlingzoologists for a long time. For example, Chaetognathsshow a combination of deuterostome-like (enterocoelictrimeric coelom) and protostome-like (ventral nervoussystem) characters. The 18S rRNA of chaetognaths hasbeen affected by rapid substitution rates that does notpermit accurate phylogenetic analysis. Gastrotrichs andgnathostomulids (Littlewood et al., 1998) are other in-triguing animals that do not fit readily in the proposedprotostome dichotomy.

There are some problems that Hox genes apparentlywill not help to resolve. The monophyly of deuterosto-mes is well accepted by the community of zoologists.However, the relatively rich set of Hox structural datathat we now have in deuterostomes does not revealanything comparable to the protostomian Hox signa-tures for them. Another point that the Hox data may beunable to resolve is the potential existence of a sister-group to bilaterians. This question is discussed by Tel-ford (2000b) on the intriguing problem of the position ofthe acoels. Acoels are very simply organized bilateriansthat have been generally considered to be belonging withflatworms in the phylum platyhelminthes. Hox datacollected by several teams on triclad planarians havestrongly suggested that platyhelminths are not an earlybranch of the bilaterians but are members of thetrochozoans together with other phyla that share withthem spiral cleavage of the egg and a ciliated larva.However, in a paper dealing with 18S rRNA, Ruiz-Trillo et al. (1999) support the view that acoels are notplatyhelminths and occupy a place as a sister group ofbilaterians. This result, if true, would give acoels astrategic place in the evolutionary tree, especially inreference with the old theory that bilaterians are de-scended from a flatworm-like ancestor. These 18S phy-logeny results have been contested on the basis of EF1-a

sequences (Berney et al., 2000), which have in turn beendisputed in another article (Littlewood et al., 2001). Ifacoels are indeed related to platyhelminths and thereforepart of the trochozoan assemblage (the opinion of theauthors of this article), an analysis of the Hox genecontent of their genome may reveal some of the fivetrochozoan signature sequences. However, whether theacoels truly are basal to the bilaterians cannot be provenby the mere absence of these signatures. As discussedearlier, a huge gap separates the cnidarians, the closestknown bilaterian sister-group, from bilaterians in termsof Hox gene diversity and it now appears unlikely that‘‘intermediate’’ organisms can provide some informa-tion on the major period of Hox gene expansion.

Acknowledgments

The authors thank Rob DeSalle and Bernd Schier-water for inviting GB to the ESEB meeting. The Ado-utte lab is financially supported by the CNRS, theMinist�eere de la Recherche, the Fondation de la Re-cherche M�eedicale and Institut Franc�ais de la Biodiver-sit�ee.

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