Evolutionary history of the iroquois/Irx genes in metazoans
© Kerner et al; licensee BioMed Central Ltd. 2009
Received: 23 September 2008
Accepted: 15 April 2009
Published: 15 April 2009
The iroquois (iro/Irx) genes encode transcriptional regulators that belong to the TALE superclass of homeodomain proteins and have key functions during development in both vertebrates and insects. The Irx genes occur in one or two genomic clusters containing three genes each within the Drosophila and several vertebrate genomes, respectively. The similar genomic organization in Drosophila and vertebrates is widely considered as a result of convergent evolution, due to independent tandem gene duplications. In this study, we investigate the evolutionary history of the Irx genes at the scale of the whole metazoan kingdom.
We identified in silico the putative full complement of Irx genes in the sequenced genomes of 36 different species representative of the main metazoan lineages, including non bilaterian species, several arthropods, non vertebrate chordates, and a basal vertebrate, the sea lamprey. We performed extensive phylogenetic analyses of the identified Irx genes and defined their genomic organizations. We found that, in most species, there are several Irx genes, these genes form two to four gene clusters, and the Irx genes are physically linked to a structurally and functionally unrelated gene known as CG10632 in Drosophila.
Three main conclusions can be drawn from our study. First, an Irx cluster composed of two genes, araucan/caupolican and mirror, is ancestral to the crustaceans+insects clade and has been strongly conserved in this clade. Second, three Irx genes were probably present in the last common ancestor of vertebrates and the duplication that has given rise to the six genes organized into two clusters found in most vertebrates, likely occurred in the gnathostome lineage after its separation from sea lampreys. Third, the clustered organization of the Irx genes in various evolutionary lineages may represent an exceptional case of convergent evolution or may point to the existence of an Irx gene cluster ancestral to bilaterians.
Gene duplication clearly plays an important role in generating molecular diversity. In some cases, these duplications arise through the duplication of entire chromosomes or large chromosomal regions. In other cases, duplications appear as tandem copies of genes, which form clusters of evolutionarily related genes. Two evolutionary questions are raised by the latter situation. When did these clusters form and what are the evolutionary forces that act to maintain these genes clustered? One of the most intensively studied cases of clustered genes is that of the Hox genes . As complete genome sequences become available, we can begin to track the evolutionary history of many other clusters of evolutionarily related genes. One gene family which displays genomic linkage of unknown functionality and origin is the iroquois (iro/Irx) family.
The Irx genes encode transcription factors that are involved in many developmental processes in metazoans [2, 3]. The Irx proteins belong to the TALE (three aminoacid loop extension) superclass of homeodomain proteins and are characterized by the presence, in addition to the homeodomain, of two conserved specific domains of unknown functions, named "IRO A" and "IRO box" [3–5]. Within the TALE superclass of homeobox genes, the Irx genes appear closely related to the mohawk (Mkx, also known as iroquois-like, Irxl) genes which encode proteins with a homeodomain similar to that of the Irx proteins, but lack the Irx-specific domains and harbour other conserved domains (the "MKX A", "MKX B" and "MKX C" domains) [5–7].
Three Irx genes – araucan, caupolican, and mirror – have been identified in Drosophila, and they form a gene complex (the so-called iroquois complex, Iro-C) that is involved, during larval development and metamorphosis, in the formation of sense organs (including the eyes), in the specification of the dorsal part of the adult thorax and in the patterning of the wing veins, as well as in the segmentation of the body during embryonic development [2, 3]. Six Irx genes, organized into three-gene complexes (IrxA which contains the Irx1, Irx2 and Irx4 genes and IrxB which contains the Irx3, Irx5 and Irx6 genes) have been isolated in mammals and have been shown to have key roles during development, e.g. in neurogenesis and in the patterning of the heart [2, 3]. Orthologs of these genes have been found in other vertebrates, such as the zebrafish in which 11 Irx genes are organized into four clusters [8, 9]. A single Irx gene has been identified in the nematode Caenorhabditis elegans , but its function has not been characterized. Irx genes have also been identified in a few other species, such as sponges, but they have not been studied at the functional level [e.g. [5, 10–12]].
The presence of several Irx genes and their organization into three-gene complexes in both vertebrates and Drosophila have raised several questions about the evolutionary history of these genes. First, are the Drosophila and vertebrates clusters homolog, already present in the last common ancestor of these species (this ancestor is known as Urbilateria as it is the ancestor of all bilaterians, the animals displaying a bilateral symmetry)? Comparisons of the vertebrates and Drosophila Irx genes suggest that the vertebrate genes are more similar to one another than to their Drosophila counterpart, suggesting that the gene duplications that have given rise to the Drosophila and vertebrate complexes occurred independently and therefore that these complexes do not derive from an ancestral cluster present in Urbilateria [e.g. [3, 13, 14]]. However, this conclusion is based on a very small sampling of species and establishing a firmly-based scenario for Irx genes evolution in bilaterians would require a broader sampling of the species in which these genes are characterized. Such a study has been recently published and the occurrence of independent duplications has been advocated by the authors of this study . Second, what is the origin of the 2 or 4 complexes observed in vertebrates? Several lines of evidence indicate that the two clusters found in mammals (and other vertebrates such as birds) derive from an ancestral Irx cluster as a consequence of a chromosomal duplication event [3, 13, 14]. Indeed, Irx1, Irx2 and Irx4 are most similar to Irx3, Irx5 and Irx6, respectively, suggesting that Irx1/Irx3, Irx2/Irx5 and Irx4/Irx6 are pairs of paralogs. Furthermore, the organization of the clusters, including the orientation of the transcription of the genes, is very similar and paralogous genes flank the clusters in both mouse and zebrafish. In this latter (and in other teleosts), the two additional clusters that are found, would have been generated by a teleosts-specific genome duplication (whose occurrence is now well documented; e.g. [8, 9, 15]). It is, however, not clear when the ancestral vertebrate cluster was established and when the suggested chromosomal duplication occurred. Answering these questions requires data from more basal vertebrates and non-vertebrate chordates and non-chordate deuterostomes.
In a previously published study, Irimia et al.  reported the existence of more than one Irx gene in several metazoan genomes and these genes were in most cases clustered. A phylogenetic analysis of these genes suggests that these genes were produced by several independent duplications. In this article, we significantly extend this analysis by retrieving and analyzing, at the phylogenetic and genomic levels, the Irx genes encoded by several newly-sequenced metazoan genomes. We confirmed that in most species there are several Irx genes and that these genes are clustered. We performed multiple phylogenetic analyses of these sequences with up-to-date phylogenetic methods. Taken together, our data suggest two possible alternative evolutionary scenarios for the evolution of Irx genes in animals: either several independent tandem duplications events have occurred in the different bilaterian lineages and selective pressures independently acted in each of these lineages to maintain the genes clustered (in agreement with ), or a complex of Irx genes is ancestral to bilaterians and has been conserved in most species, but differential evolutionary rates have obscured the orthology relationships between genes from the different bilaterian lineages. Both hypotheses are supported by parts of the data and we do not think we can favor one of the scenarios over the other one.
Results and discussion
Identification of the Irx and Mkx genes from the fully-sequenced genomes of 32 metazoan species
When reported on the species phylogeny, the numbers of identified Irx and Mkx genes indicate contrasting trends in the evolution of these two families (Figure 1). First, bona fide Mkx genes cannot be found in non-bilaterian species in contrast to Irx genes which are found, at least in cnidarians and the placozoan Trichoplax, as well as probably in sponges. We are therefore faced with two alternative hypotheses: either the Mkx genes are ancestral to metazoans and have been lost in the analysed non-bilaterian species, or the Mkx genes may represent an innovation of bilaterians and could be considered as bilaterian-specific divergent Irx genes. Second, while Mkx genes are found in both protostomes and deuterostomes (and are therefore likely to be already present in Urbilateria), several unrelated species (13 out of 36) lack the Mkx gene, indicating several independent events of gene loss. In contrast, Irx genes are found in all studied species indicating strong evolutionary pressures to conserve these genes. Third, while Mkx genes are usually found as a single gene in each species, we found several Irx genes in most cases (27 out of 36 metazoan species, 26 out of 32 if we only consider bilaterians). This indicates that the Irx (but not the Mkx) gene family evolution has been shaped by gene duplications. We further studied these gene duplications by phylogenetic analyses and characterizing the genomic organization of the Irx genes.
Phylogenetic analyses of the Irx genes suggest the occurrence of many independent duplication events in protostomes and deuterostomes
We next separately analyzed protostome and deuterostome Irx genes, as it allowed us to construct phylogenetic trees based on alignments which include many more aminoacid residues than when all metazoan sequences are considered. We excluded from our analyses the most divergent Irx sequences (those from Helobdella, Ciona, Oikopleura, and the different nematode species) in order to maximize the size of the unambiguously aligned portion of the proteins.
Phylogenetic analyses of the protostome Irx genes suggest the presence of two ancestral Irx genes in arthropods and lophotrochozoans
The two other protostome monophyletic groups concern lophotrochozoan sequences (Figure 4): one group includes three Irx genes from the limpet Lottia and one gene identified in an EST collection of the mussel Mytilus and therefore indicates the occurrence of duplications specific to molluscs. The other group includes Irx genes from three distantly-related species, the annelid Capitella (1 gene), the mollusc Lottia (1 gene), and the flatworm Schmidtea (2 genes). The other Irx genes from Capitella (2 genes), Lottia (3), and Schmidtea (2) do not cluster together (Figure 4). Our interpretation of this phylogenetic tree is that there were two Irx genes in the last common ancestor of the three aforementioned lophotrochozoan species and that one of the paralogs in each evolutionary lineage underwent highly divergent evolution (in such a way that these paralogs do not cluster in the phylogenetic trees).
Since an ancestral two gene situation is found for both arthropods and lophotrochozoans, it is therefore conceivable that the presence of two Irx genes may be ancestral to protostomes, but that differential evolutionary rates have obscured the orthology relationships between genes from the arthropod and lophotrochozoan lineages. We however have to note that a single Irx gene is found in several different nematode species (Figure 1) which belong, together with arthropods, to the ecdysozoans, one of the two main protostome branches. If our hypothesis of an ancestral two gene situation in protostomes is true, we therefore have to consider that one or several Irx gene losses have occurred in the nematode lineage. This is not unconceivable as it is known that strongly-conserved genes in bilatarians have been lost in nematodes, for example several Hox genes , and our study points to the loss of the Mkx genes in all the studied nematode species. We can however clearly not exclude that the presence of a single Irx gene in nematodes may represent the ancestral state in protostomes and that independent gene duplications occurred in arthropods and lophotrochozoans.
Phylogenetic analyses of the deuterostome Irx genes indicate the presence of a single Irx cluster of at least 2 genes in the last common ancestor of present-day vertebrates and suggest gene losses in non vertebrate deuterostomes
We also studied Irx genes from urochordates and cephalochordates. Unfortunately, the Irx genes from the urochordates (the two Ciona species and Oikopleura) are very divergent and when included in the phylogenetic analyses, they perturb the overall topology of the trees and do not cluster with vertebrate sequences (not shown). The phylogenetic tree shown in Figure 5 therefore contains only the Irx genes from the cephalochordate Branchiostoma (amphioxus), as well as the only two Irx genes known from non-chordate deuterostomes, the single Irx gene encoded by the genome of the echinoderm Strongylocentrotus and the single Irx gene cloned (other Irx genes may exist) in the hemichordate Saccoglossus (hemichordates and echinoderms form a monophyletic group – the Ambulacria – within deuterostomes). The 3 Branchiostoma Irx genes strongly cluster together (and therefore derive from Branchiostoma-specific duplications) and with the Irx2/Irx5 group (statistical supports are not strong, but this clustering is found with all methods). Similarly, the Ambulacria Irx genes strongly cluster together and with the Irx4/Irx6 group. The fact that the Branchiostoma Irx genes, on one hand, and the Ambulacria Irx genes, on the other hand, cluster with different vertebrate Irx gene (Irx2/Irx5 and Irx4/Irx6 groups, respectively) suggest that there were at least two Irx genes in the last common ancestor of the deuterostomes, like in prostostomes. The fact that a third independent group (Irx1/Irx3) exists in vertebrates may even suggest an ancestral situation where three Irx genes form a cluster in deuterostomes (Irx1/Irx3, Irx2/Irx5 and Irx4/Irx6). In these views, we have to consider that the two or three ancestral genes would have been conserved in vertebrates (and subsequently duplicated), but one or two of them were independently lost in Ambulacria (Irx4/Irx6 remained) and cephalochordates (Irx2/Irx5 remained and was subsequently duplicated).
Organization of the Irx genes in clusters is a general rule in bilaterians
These data about the genomic organization of the Irx genes can be interpreted in two different ways. The simplest and most parsimonious explanation is that a cluster of at least two Irx genes (+CG10632) is ancestral to bilaterians and has been conserved in this evolutionary lineage, like what has been observed for other homeobox gene clusters, such as the Hox, ParaHox and NK clusters [1, 17]. This hypothesis is supported by our analyses that suggest the presence of at least 2 genes in the last common ancestor of each investigated lineage, lophotrochozoans, arthropods, and deuterostomes. One plausible and parsimonious interpretation of these analyses is that this situation might be ancestral to bilaterians. This view is, however, not supported by the phylogenetic analyses of the Irx genes at the scale of the bilaterians, which suggest independent gene duplications in the different bilaterian lineages (see previous sections). The hypothesis of cluster of Irx genes already present in the last common ancestor of bilaterians would require that we postulate that the phylogenetic trees do not show the real relationships between the Irx genes from the different bilaterian lineages, which might be a consequence of differential rates of evolution in these lineages. This hypothesis is also not supported by the presence of a single Irx gene in several different nematode species – we would have to postulate that one or more Irx genes have been lost in the nematode lineages.
The second possibility is that the duplications of the Irx genes have occurred independently and in all cases there have been pressures to maintain the physical linkage of the duplicated genes. This explanation already proposed by Irimia et al.  is in agreement with the phylogenetic analyses, but is faced with one major problem, explaining why in several independent lineages there have been similar pressures to keep the duplicated genes in clusters. Indeed, it is easy to explain that following tandem gene duplications there could be, in some rare cases, molecular events that lead to phenomenon such as shared regulatory regions or global gene regulation, favouring cluster maintenance, while in most other cases, the duplicated genes would be, after some time, dispersed in the genome, as it is observed for most multigenic families. It is much more difficult to understand why, in the case of the Irx genes, there would have been systematic events leading to cluster maintenance after numerous instances of gene duplications, unless postulating some particular properties of the Irx genomic region that would, by itself, favor the conservation of the physical link between the duplicated genes. The existence of an ancestral cluster of a single Irx gene and CG10632 may represent such a property, constraining the duplicated Irx genes to remain associated with CG10632 and therefore with each others. This remains nevertheless to be proven and does not explain everything, as for example why the CG10632 gene has never been duplicated while the Irx genes would have duplicated so many times (Figure 6, Additional file 5).
We present here a large-scale phylogenomic analysis of the Irx and Mkx genes that extends a previously published study on the evolution of Irx genes in metazoans. Several main conclusions can be drawn from our study. First, an Irx cluster composed of two genes, araucan/caupolican and mirror, is ancestral to the crustaceans+insects clade and has been strongly conserved in this clade. Second, 3 Irx genes organized into a cluster were probably present in the last common ancestor of vertebrates and the duplication that has given rise to the six groups of genes (organized into 2 clusters) found in most vertebrates occurred in the gnathostome lineage after its separation from sea lampreys. Third, several Irx genes organized into clusters are found in many different bilaterian species representing various evolutionary lineages. This unexpected feature can be explained in two opposite ways. Our analyses were unable to discriminate between these two possibilities. The first possibility is that there was an ancestral Irx cluster composed of two or three genes in Urbilateria, and that there have been structural and/or functional constraints that maintained this organization in the various bilaterian lineages. However, the genes constituting these clusters would have a differential evolution which would hide their orthology relationships. Furthermore, additional genes duplications and losses would have also occurred in some lineages. The second possibility is that the different Irx clusters have been independently acquired, implying numerous independent tandem duplications and pressures to maintain the physical linkage of duplicated genes in several different lineages. In this view, the Irx genes would be specially prone to duplication events and/or retention of functional paralogs over long evolutionary times. With the currently available data, we do not think it is possible to favor one of the explanations over the other one.
Retrieval of the Irx and Mkx sequences
Irx and Mkx gene sequences were retrieved using TBLASTN and BLASTP algorithms  on the current assembly and the predicted proteins (if available) of the genomes of the species indicated in Figure 1, using the BLAST servers dedicated to these species (Doe Joint Genome Institute, Baylor College of Medicine, Flybase, Genome Sequencing Center, and Ensembl) or the National Center for Biotechnology Information (NCBI) BLAST server (Genomic BLAST databases) [19–24]. Additional BLAST searches were also performed against the NCBI protein and EST databases in order to identify Irx and Mkx genes in additional species whose genomes are not completely sequenced. Aminoacid sequences were subsequently predicted using Geneid, Genscan, and TBLASTN against the NCBI nr protein database [18, 25, 26]. All the sequences we have isolated are available upon request. Species abbreviations used in the present article are: Acypis = Acyrthosiphon pisum (pea aphid – insect); Aedaeg = Aedes aegypti (yellow fever mosquito – insect); Ampque = Amphimedon queenslandica (demosponge); Anogam = Anopheles gambiae (mosquito – insect); Apimel = Apis mellifera (honey bee – insect); Bommor = Bombyx mori (silkworm – insect); Braflo = Branchiostoma floridae (amphioxus – cephalochordate); Brarer = Brachydanio rerio (zebrafish – vertebrate); Caeele = Caenorhabditis elegans (nematode); Calvic = Calliphora vicina (Blue blowfly – insect); Capsp1 = Capitella sp I (annelid); Culpipqui = Culex pipiens quinquefasciatus (mosquito – insect); Dappul = Daphnia pulex (water flea – crustacean); Dromel = Drosophila melanogaster (fruitfly – insect); Galgal = Gallus gallus (chick – vertebrate); Helera = Heliconius erato (Red Passion Flower butterfly – insect); Homsap = Homo sapiens (vertebrate); Hydmag = Hydra magnipapillata (cnidarian); Lotgig = Lottia gigantea (limpet – mollusk); Musmus = Mus musculus (mouse – vertebrate); Mytcal = Mytilus californianus (mussel – mollusk); Nasvit = Nasonia vitripennis (parasitoid wasp – insect); Nemvec = Nematostella vectensis (sea anemone – cnidarian); Pedhumcor = Pediculus humanus corporis (human body lice – insect); Petmar = Petromyzon marinus (Sea lamprey – vertebrate); Sackow = Saccoglossus kowalevskii (hemichordate); Schmed = Schmidtea mediterranea (Planarian – platyhelminthes); Spofru = Spodoptera frugiperda (fall armyworm – insect); Strpur = Strongylocentrotus purpuratus (purple sea urchin – echinoderm); Subdom = Suberites domuncula (demosponge); Tetnig = Tetraodon nigroviridis (pufferfish – vertebrate); Triadh = Trichoplax adhaerens; Tricas = Tribolium castaneum (red flour beetle – insect); Xentro = Xenopus tropicalis (vertebrate).
Multiple alignments were performed with Clustal W  using the ClustalW web server at the Bioinformatics Center of the Kyoto University  and they were subsequently manually improved. Handling of the multiple alignments was done using SEAVIEW . Unweighted maximum-parsimony (MP) and neighbour-joining (NJ) reconstructions were performed with the PAUP 4.0 program . NJ analyses were done using the BioNJ algorithm  and 10,000 bootstrap replicates. MP analyses were performed with the following settings: heuristic search of over 250 bootstrap replicates; MAXTREES set at 3000, and other parameters set at default values. Maximum likelihood (ML) analyses were performed with PHYML . PHYML analyses were performed using the WAG amino-acid substitution model , the frequencies of amino acids being estimated from the data set, and rate heterogeneity across sites being modelled by two rate categories (one constant and eight g-rates). The amino acid substitution model was chosen using ModelGenerator . Statistical support for the different internal branches was assessed by bootstrap resampling (500 bootstrap replicates), as implemented in PHYML . Bayesian inference was performed using the Markov chain Monte Carlo method as implemented in the MRBAYES (version 3) package [35, 36]. We used the WAG substitution frequency matrix  with among-sites rate variation modelled by means of a discrete g distribution with four equally probable categories. Two independent Markov chains were run, each containing from 1,500,000 to 3,000,000 Monte Carlo steps (depending on the number of steps required to get chain convergence). One out of every 250 trees was saved. The trees obtained in the two runs were meshed and the first 25% of the trees were discarded as 'burnin'. Marginal probabilities at each internal branch were taken as a measure of statistical support. All the alignments and the trees are available upon request. Phylogenetic relationships between the species used in this study (as depicted in Figure 1) are based on [38–42].
We are extremely grateful to the Department of Energy (DoE) Joint Genome Institute, the Baylor College of Medicine (BCM-HGSC), the J. Craig Venter Institute, the Genome Sequencing Center (Whashington University in St Louis, School of Medicine), and the National Human Genome Research Institute for sequencing the genomes of the different species used in this study and for making these sequences publicly available. We are also very grateful to the scientists who set up and led these projects. We are also grateful to Ashleigh Fritz for helpful comments on the manuscript and to anonymous reviewers for helpful suggestions. This work was supported by grants from the Agence National de la Recherche and the Ministère Français de la Recherche, through its ACI 'Jeunes chercheurs'(MV). P.K. holded a “Bourse pour Docteur-Ingénieur” from the CNRS and is now supported by the Université Paris Diderot – Paris 7.
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