BACKGROUND
since the initial description of the drosophila segmentation gene runt over a decade ago  <cit> , a small family of runt domain  genes has been described and extensively analyzed in several species.
the  <dig> amino acid long runt domain is very highly conserved and is readily identifiable computationally.
rd transcription factors are developmental regulators involved both in cell proliferation and differentiation, and their disruption usually leads to disease  <cit> .
in humans, three different rd genes were identified  <cit>  and named according to various schemes, currently standardized by the human gene symbols runx <dig>  runx <dig> and runx <dig>  runx genes have two promoters   <cit>  separated by a long intron; the proximal promoter  is always located within a large cpg island  <cit> .
extensive alternative splicing giving rise to many isoforms has been described for all runx genes  <cit> .
orthologs of all three human runx genes were identified in mouse.
a single rd gene was described in xenopus, presumed to be orthologous to runx <dig>  <cit> .
an experimental search for rd genes in fugu showed the existence of a fugu ortholog of human runx <dig>  and suggested the existence of a single additional rd gene in fugu  <cit> , while a computational search of the fugu genomic sequence revealed three runx genes  <cit> .
four rd genes were identified in drosophila  <cit> , while a single rd gene exists in c. elegans  <cit> , sea urchin and amphioxus  <cit> .
based on these data, current thought on the evolution of the rd gene family posits that a single rd gene was present in the common ancestor of chordates  <cit> , and this ancestral gene triplicated during early vertebrate evolution, giving rise to the modern runx gene complement.
the proposed mechanism of expansion involved large-scale genomic duplications, identifiable today as large paralogous segments  <cit> .
the proper identification of true orthology relationships is often helpful for inferring gene function and translating knowledge between model organisms and more complex species.
under the current model, simple orthology relationships should be expected among vertebrate runx genes, but their functional relationship to the ancestral rd gene is unknown.
the single known rd gene in c. elegans has been shown to be required for the formation of a functional gut; this role has been claimed to be conserved with mouse runx <dig>  <cit> .
the current availability of genome drafts for several vertebrate species, including homo sapiens, mus musculus, rattus norvegicus, canis familiaris, gallus gallus, takifugu rubripes, tetraodon nigroviridis, danio rerio and xenopus tropicalis, allows us to explore a comprehensive set of vertebrate rd genes and characterize their genomic environments, shedding light on the structure and evolution of this important gene family.
RESULTS
the fugu genome has at least four runt domain genes
our search for rd genes in the fugu draft yielded four distinct genomic scaffolds , each containing a single, complete rd gene.
each scaffold had one or more sequence gaps, some within the rd genes, others between them and their neighbors.
we employed a directed sequencing approach to obtain the additional sequence needed to close the gaps in these four scaffolds and to improve sequence quality.
sizes are expressed in kb.
n.o.
: no ortholog.
we studied the four scaffold sequences using the gestalt workbench  <cit>  and constructed hypothetical gene structures for the fugu rd genes by maximizing similarity to known vertebrate rd proteins.
three of the four rd genes found in the fugu genome have clear one-to-one similarity relationships with the three mammalian runx genes .
they have been assumed to be their orthologs  <cit> ; we call them frrunx <dig>  frrunx <dig> and frrunx <dig> .
their genomic structures are similar to those of their human counterparts, but their sizes have evolved differently.
runx <dig> is the smallest of the three human runx genes, while in fugu frrunx <dig> is the largest , and frrunx <dig> is significantly larger than frrunx <dig>  frrunx <dig> has acquired an additional intron  <cit>  that is not present in human runx <dig> or in any other rd gene.
this intron is just  <dig> bp long, has canonical splice signals, and is in phase  <dig> with respect to the protein reading frame, at the beginning of the runt domain.
an additional intron has been described at the 5' end of the coding region, yielding a short form that would be locally non-homologous to the other rd genes  <cit> .
a detailed comparison of human runx <dig> and frrunx <dig> has been published  <cit> .
in both human and in fugu, runx <dig> has the highest g+c content of the rd genes, while the g+c content of runx <dig> differs significantly between the two species .
the fugu runt gene
in addition to the three runx genes, the fugu genome has a fourth and more divergent runt-domain gene, that we named frrunt.
frrunt is an extremely compact gene, spanning just  <dig> kb of genomic sequence .
based on sequence analysis only, frrunt appears to have two promoters, with an intron separating the hypothetical distal promoter  and first exon from the main body of the gene.
this intron is usually very long in runx genes.
it is indeed the longest intron observed in frrunt, but it is nevertheless very short, spanning just  <dig> bp.
there is a local concentration of cpg dinucleotides 200– <dig> bp upstream of exon  <dig> , suggesting that an incipient cpg island might function as a proximal promoter .
the g+c content is not elevated in this area, in similarity to the cpg islands of the fugu runx genes .
the main body of the gene is split into five exons, separated by much shorter introns , all of which have canonical splice signals.
the longest predicted frrunt product is  <dig> amino acids long, in contrast with the  <dig> aa,  <dig> aa and  <dig> aa observed for frrunx <dig>  frrunx <dig> and frrunx <dig>  respectively.
the small number of exons in frrunt leaves little room for alternative splicing by exon skipping, without compromising functionally important domains of the protein.
the overall compactness of the gene makes the incorporation of yet undetected exons improbable.
several cryptic splice sites within the exons could enable splicing variants altering exon length.
frrunt is exceptional in that the length of the runt domain  varies from the universally conserved  <dig> amino acids, due to the introduction of an asparagine residue after position  <dig> in the rd .
this appears to be the first report of such a mutation within this highly conserved domain.
we also noted that the rd sequence of the tunicate oikopleura dioica  has an insertion at the same position .
comparison to the published structure of this domain  <cit>  shows that this variable region is located in loop l <dig>  opposite the dna-binding region, i.e.
in the location least likely to disrupt the structure of the protein.
a surprising observation is that frrunt is "interlocked" with frsupt3h , the gene orthologous to the human transcription factor supt3h  <cit> : the first exon of frrunt is located within the first intron of frsupt3h, and vice-versa.
in the human genome, though, supt3h is interlocked with runx <dig>  as shown in  <cit> .
we discuss the puzzles posed by these differences in genomic organization below.
four rd genes in tetraodon and in zebrafish
a genome draft of another pufferfish species, tetraodon nigroviridis, has been released  <cit> .
a computational search into this draft reveals four rd genes with clear orthologous relationships with the four fugu rd genes.
we call them tnrunx <dig>  tnrunx <dig> and tnrunx <dig>  and tnrunt .
the rd portion of tnrunt has been deposited in the embl database ; in this work we report the complete gene structure of tnrunt, which is similar to that of frrunt.
in further similarity with the fugu genomic organization, tnrunt is "interlocked" with the tetraodon ortholog of supt3h .
the three runx gene pairs are conserved between fugu and tetraodon  and display a larger percentage of protein identity than nucleotide identity, indicating a prevalence of conservative substitutions.
in contrast, tnrunt is only  <dig> % identical to frrunt at the protein level, less than their nucleotide identity.
indeed, a striking series of non-synonymous mutations has created a highly divergent segment  including the n-terminus of the runt domain .
the strong and unexpected divergence is not the result of local low sequence quality, as tested by examining the relevant tetraodon entries from the ncbi trace archive, and by resequencing the fugu gene.
the differences between tnrunt and frrunt do not modify the ig-like β-sandwich core of the runt domain  <cit> , which is highly conserved as expected.
therefore, the structural integrity of the runt domain appears not to be compromised.
we can only speculate that the extensive variation in its n-terminus may reflect species-specific constraints.
sizes are expressed in kb.
asterisks denote minimal sizes due to the presence of gaps within the genes; the gene structure for tnrunx <dig> is incomplete at its 3' end.
we also searched for rd genes in the genome draft of the zebrafish, danio rerio  <cit> .
orthologs of runx1- <dig> are present, but no ortholog of frrunt could be found.
this could be due to the incompleteness of the draft sequence.
on the other hand, there are two copies of runx <dig>  runx2a and runx2b, which have been shown to have somewhat different patterns of expression  <cit> .
we next analyze the phylogenetic relationships between all the observed rd genes.
phylogenetic distribution of runt domain genes
we performed exhaustive computational searches for rd genes, and in particular for potential orthologs of frrunt, using the available drafts of the human, chimp, mouse, rat, dog, chicken and frog genomes.
in all cases, we identified three clear matches corresponding to orthologs of the three runx genes.
none of these genomes included a potential ortholog of the fugu/tetraodon runt gene.
in principle, such orthologs might be found in the future within current sequencing gaps or heterochromatic regions, but considering the virtually finished human genome, and the combined coverage of all the genome drafts, we can infer that the runt gene is absent in mammals, and probably in all tetrapods.
using representative protein sequences of the runx and runt genes , we reconstructed a molecular tree  showing the relationship between the three runx proteins, frrunt and tnrunt, and the runt proteins of ciona intestinalis and branchiostoma floridae .
in this analysis, the frrunt protein is nearly equidistant from the three runx proteins and amphioxus runt .
in comparison, the identity level between the runx proteins is 90%–98% in the same region , and they are 90%–95% identical to amphioxus runt.
therefore, while the amphioxus runt protein is very closely related to the vertebrate runx, the pufferfish runt proteins are significantly more divergent.
percentages of identity between the human and fugu runt domains.
a difficulty has been documented in phylogenetic reconstruction of gene families with anciently duplicated genes  <cit> , in which saturation of frequently-mutating amino acids leads to erroneous "outgroup topologies".
in these incorrect topologies, the duplication event appears to be more ancient than supported by the data, which in turn suggest the existence of lineage-specific gene loss events.
we tested our phylogenetic reconstruction using the program asatura  <cit> , which identifies and suppresses saturated amino acids, thereby correcting the affected tree topology.
this analysis did not modify the location of frrunt and tnrunt in our reconstruction, suggesting that mutational saturation is not causing the observed divergence age of the pufferfish runt genes.
in this protein-level comparison of the conserved runt domains, the pufferfish runt proteins appear to be surprisingly ancient, predating the divergence between craniates  and cephalochordates .
on the other hand, when comparing the nucleotide sequences of the first exon from each human and fugu runt domain gene, we observed that frrunt is more closely related to runx <dig> , suggesting the possibility of a recombination event between these genes .
we also studied the relationships between the runx <dig>  runx <dig> and runx <dig> orthologs in several vertebrates and found the species trees to be largely as expected .
one of the two zebrafish runx <dig> protein sequences  appeared to be slightly more closely related to tetrapod runx <dig> genes than to the other runx <dig> genes in fish species, including zebrafish runx2b .
we tested this result using asatura  <cit> , and found it to be an artifact of mutational saturation: in the corrected tree , runx2a is more closely related to the other fish runx <dig> genes.
comparative genomics of runt domain genes
four rd genes have been identified in drosophila  <cit> , more similar to each other than to the vertebrate runx genes: they represent an independent family expansion in insects.
the four drosophila rd genes are all linked on chromosome x. moreover, three of these genes are clustered within a  <dig> kb region.
there is no linkage of rd genes, however, in the human genome: each gene is on a different chromosome.
their genomic environments usually show some conservation: the three human runx genes are followed by clic genes in the complementary strand , and linked to members of the dscr <dig> family.
the rd genes appear not to be clustered in the fugu genome, though this conclusion is limited by the fragmentary nature of the current genome draft.
all four fugu rd genes are flanked by at least one non-rd gene on each side.
fugu runx genes are followed by clic genes except for frrunx <dig>  but a clic gene is located ~ <dig> kb upstream of frrunx <dig> and in the same orientation.
this organization could have arisen by an inversion event in the fugu lineage.
to ensure a misassembly did not cause this apparent inversion, we performed a 3x shotgun sampling of the bac clone oml <dig>  which spans the range 1– <dig> of scaffold  <dig>  this quality control step failed to uncover any misassemblies, and confirmed the genomic organization observed in this fugu scaffold.
frrunt does not appear to be linked to any clic gene, and no dscr <dig> family members can be discerned near any of the fugu rd genes.
when studying the wider genomic environments of fugu and human rd genes, we observed significant synteny conservation, in agreement with the observations reported for chromosome x genes  <cit> .
indeed, when comparing each of the genes neighboring fugu runx genes to the human genome, we find that their orthologs tend to be located in the corresponding human chromosome, e.g.
most of the genes linked to frrunx <dig> have orthologs on human chromosome  <dig>  where human runx <dig> resides .
some inversion events can be inferred, e.g.
one involving the genes phip and irak1bp <dig> and another involving mut.
gene order and orientation has changed, and intergenic distances have changed drastically, but the overall gene synteny is largely preserved, lending support to the assignments of orthology between the human and fugu runx gene pairs.
an exception to the conservation of synteny involves the genes cdc5l and supt3h, which in the human genome are found immediately upstream of runx <dig>  but in fugu are instead located upstream of frrunt, including the first-exon interlocking with supt3h mentioned earlier.
evidence points at a larger duplication in fishes, encompassing at least cdc5l, supt3h, runx <dig>  clic <dig>  enpp <dig> and enpp <dig>  followed by differential gene loss.
one duplicate copy would have retained frrunx <dig>  clic <dig> and enpp <dig> , while the other copy  would have retained cdc5l, supt3h, a second rd gene, a second copy of clic <dig>  and enpp <dig>  the presence of remnants of supt3h upstream of frrunx <dig>  <cit> , and of two copies of runx <dig> in zebrafish, lends further support to this hypothesis.
thus, comparative genomic analysis of human, fugu and zebrafish suggests that frrunt may be a derivative form of a duplicated runx <dig> gene.
however, this contradicts the conclusions from phylogenetic analysis or the protein sequences; we discuss this contradiction below.
a new family of follistatin-like genes
we find several fugu genes for which no human ortholog can be discerned , among them frrunt.
immediately downstream to frrunt we identified a novel gene  from the follistatin family, most closely related to fstl <dig>  a detailed computational search for additional sequences of this family identified two novel, large human follistatin-like genes, which we called fstl <dig> and fstl <dig>  we then found clear orthologs for both in the fugu genome .
the genes in this family share a kazal-type cysteine-rich domain  and a calcium-binding ef-hand domain .
having characterized the complete gene family in both human and fugu, we performed a phylogenetic reconstruction based on the conserved kazal-type domain .
frfstl <dig> appears to have no ortholog in the human genome, nor could we identify a potential ortholog in the mouse and frog genomes.
this suggests that frfstl <dig> was also lost in the tetrapod lineage.
it is reasonable to hypothesize that the neighboring frrunt and frfstl <dig> genes were lost in a single deletion event.
discussion
we have taken advantage of the availability of genomic drafts for several vertebrate species, including the finished human genome, to identify the orthologs of all currently known runt-domain  genes, as well as a novel member of this small gene family.
both pufferfish species  have four rd genes; since these genomes are only available as draft assemblies, additional rd genes might be found when the finished genomic sequences are made available.
the function of the novel frrunt/tnrunt gene is currently unknown.
based on the phylogenetic analysis, this novel gene appears to represent an ancestral form of the rd family in vertebrates, subsequently lost in the tetrapod lineage.
it is therefore surprising that its gene structure, and not that of runx <dig> as in humans, is interlocked with the supt3h gene.
based on the comparative genomics analysis alone, one could hypothesize that frrunt is simply a derivative form of runx <dig>  i.e.
the ortholog of zebrafish runx2a.
in this case, though, one would expect frrunt to be more similar to frrunx <dig> than it is to either frrunx <dig> or frrunx <dig>  but in terms of amino acid sequence similarity, it appears to be equidistant from the three runx genes.
this discrepancy might be explained by invoking accelerated evolution of the pufferfish runt genes, perhaps as a lineage-specific adaptation.
typically, nucleotide sequences diverge much faster than amino acid sequences, and the first exons of rd genes are significantly less conserved than the runt domain itself, on which we based our phylogenetic analysis.
therefore, we find it hard to sustain that, while the first exon of frrunt maintains its nucleotide similarity to the first exon of runx <dig>  the amino acid sequence of the  runt domain itself has diverged at such an accelerated pace.
furthermore, we found this not to be an artifact of mutational saturation  <cit> .
a similar situation is observed for the neighboring fstl <dig> gene: parsimony considerations could lead one to assume that fstl <dig> is a fish-specific duplicate of fstl <dig>  though contradicting the phylogenetic reconstruction of the evolution of this gene family .
this situation could again be explained by assuming accelerated evolution of fstl <dig>  but we consider this to be a remarkable coincidence.
the conundrum is whether frrunt is an ancestral form, or is derived from runx <dig>  both hypotheses contradict part of the available data.
we propose here a third hypothesis, in the form of an evolutionary history : an ancestral rd gene duplicated in chordates, after divergence from sea urchin, which has a single rd gene  <cit> .
one of the two resulting rd genes became the runx family founder, which expanded by triplication, and one of the three runx genes  became interlocked with supt3h.
after the teleost/tetrapod divergence, a regional duplication in teleosts created a second copy of runx <dig> and its neighboring genes.
in the tetrapod lineage, the ancestral runt gene was lost, in conjunction with fstl <dig>  in the pufferfish lineage, the runt gene replaced most of runx2a, perhaps by recombination .
this is supported by the clear similarity between the first exons of frrunt and runx <dig>  the copy of supt3h interlocked with runx2b, apparently superfluous, is being lost by gradual degradation, and only small fragments of it remain  <cit> .
this scenario is compatible with all the data observed.
while it posits a small number of additional evolutionary events, it does not involve highly improbable events like the accelerated evolution of a normally highly conserved protein structural domain.
interestingly, the first duplication event could correspond to the first round of vertebrate genome tetraploidization  <cit> .
a second round of tetraploidization in the ancestral vertebrate could have produced a set of four paralogous runt domain genes, and a hypothetical gene conversion event may have led to the current complement of three runx genes .
conversion between paralogous copies of genes derived from tetraploidization events has been demonstrated  <cit> .
under the proposed scenario, none of the three extant runx genes in mammals represents the ancestral vertebrate rd form.
rather, these derivative genes coexisted with an additional runt gene and still do so in teleost genomes.
the single known rd gene in amphioxus is more similar to the vertebrate runx genes than it is to frrunt.
it is possible, therefore, that cephalochordates  have a second rd gene, short and divergent enough to escape experimental detection by dna hybridization  <cit> .
why was one of the ancestral rd genes lost in the tetrapod lineage?
the three runx genes bind to the same dna motif and modify the expression of target genes through recruitment of transcriptional modulators, which are also shared  <cit> ; functional differences between the three runx genes are attained by way of tightly regulated spatiotemporal expression patterns.
we hypothesize that the ancestral runt gene became inessential to amniotes by functional reprogramming of the remaining three runx genes.
its loss would therefore represent an example of evolution by reduction in complexity.
in pufferfishes, the hypothesized recombination event would have placed the runt gene under the regulatory control of the former runx2a promoter.
the viability of such a sudden regulatory change would in turn suggest a significant level of functional redundancy among the rd genes.
CONCLUSIONS
we identified a fourth runt domain gene in the fugu genome, which appears to represent either a pufferfish-specific, fast-evolving derivative of runx <dig>  or a direct descendant of the ancestral chordate runt gene.
we find the latter hypothesis more reasonable.
this novel gene evolved in parallel with the vertebrate runx genes, and while it has been preserved in pufferfishes, it appears to have been lost entirely in tetrapods.
this suggests that the ancestral vertebrate was more complex than previously suspected.
by studying a very limited set of fugu genomic regions, namely the scaffolds related to rd genes, we have identified seven apparently functional fugu genes that are absent from the human genome , and were probably lost early in tetrapod history.
in the process of identifying relevant homologs for one of these genes , we have identified a new family of follistatin-like genes in the human genome.
phylogenetic analysis of the rd protein sequences led to results that contradict those derived from comparative genomics, but we showed that the two could be reconciled into a coherent evolutionary model.
these results underscore the importance of obtaining complete genomic sequences of strongly divergent vertebrates, and the value to be derived by performing detailed and integrated analyses of their gene complements.

 since the initial description of the drosophila segmentation gene runt over a decade ago, a small family of runt domain genes has been described and extensively analyzed in several species. the amino acid long runt domain is very highly conserved and is readily identifiable computationally. rd transcription factors are developmental regulators involved both in cell proliferation and differentiation, and their disruption usually leads to disease. in humans, three different rd genes were identified and named according to various schemes, currently standardized by the human gene symbols runx runx and runx runx genes have two promoters separated by a long intron; the proximal promoter is always located within a large cpg island. extensive alternative splicing giving rise to many isoforms has been described for all runx genes. orthologs of all three human runx genes were identified in mouse. a single rd gene was described in xenopus, presumed to be orthologous to runx. an experimental search for rd genes in fugu showed the existence of a fugu ortholog of human runx and suggested the existence of a single additional rd gene in fugu, while a computational search of the fugu genomic sequence revealed three runx genes. four rd genes were identified in drosophila, while a single rd gene exists in c. elegans, sea urchin and amphioxus. based on these data, current thought on the evolution of the rd gene family posits that a single rd gene was present in the common ancestor of chordates, and this ancestral gene triplicated during early vertebrate evolution, giving rise to the modern runx gene complement. the proposed mechanism of expansion involved large-scale genomic duplications, identifiable today as large paralogous segments. the proper identification of true orthology relationships is often helpful for inferring gene function and translating knowledge between model organisms and more complex species. under the current model, simple orthology relationships should be expected among vertebrate runx genes, but their functional relationship to the ancestral rd gene is unknown. the single known rd gene in c. elegans has been shown to be required for the formation of a functional gut; this role has been claimed to be conserved with mouse runx. the current availability of genome drafts for several vertebrate species, including homo sapiens, mus musculus, rattus norvegicus, canis familiaris, gallus gallus, takifugu rubripes, tetraodon nigroviridis, danio rerio and xenopus tropicalis, allows us to explore a comprehensive set of vertebrate rd genes and characterize their genomic environments, shedding light on the structure and evolution of this important gene family.  the fugu genome has at least four runt domain genes our search for rd genes in the fugu draft yielded four distinct genomic scaffolds, each containing a single, complete rd gene. each scaffold had one or more sequence gaps, some within the rd genes, others between them and their neighbors. we employed a directed sequencing approach to obtain the additional sequence needed to close the gaps in these four scaffolds and to improve sequence quality. sizes are expressed in kb. n.o.: no ortholog. we studied the four scaffold sequences using the gestalt workbench and constructed hypothetical gene structures for the fugu rd genes by maximizing similarity to known vertebrate rd proteins. three of the four rd genes found in the fugu genome have clear one-to-one similarity relationships with the three mammalian runx genes. they have been assumed to be their orthologs; we call them frrunx frrunx and frrunx. their genomic structures are similar to those of their human counterparts, but their sizes have evolved differently. runx is the smallest of the three human runx genes, while in fugu frrunx is the largest, and frrunx is significantly larger than frrunx frrunx has acquired an additional intron that is not present in human runx or in any other rd gene. this intron is just bp long, has canonical splice signals, and is in phase with respect to the protein reading frame, at the beginning of the runt domain. an additional intron has been described at the 5' end of the coding region, yielding a short form that would be locally non-homologous to the other rd genes. a detailed comparison of human runx and frrunx has been published. in both human and in fugu, runx has the highest g+c content of the rd genes, while the g+c content of runx differs significantly between the two species. the fugu runt gene in addition to the three runx genes, the fugu genome has a fourth and more divergent runt-domain gene, that we named frrunt. frrunt is an extremely compact gene, spanning just kb of genomic sequence. based on sequence analysis only, frrunt appears to have two promoters, with an intron separating the hypothetical distal promoter and first exon from the main body of the gene. this intron is usually very long in runx genes. it is indeed the longest intron observed in frrunt, but it is nevertheless very short, spanning just bp. there is a local concentration of cpg dinucleotides 200– bp upstream of exon, suggesting that an incipient cpg island might function as a proximal promoter. the g+c content is not elevated in this area, in similarity to the cpg islands of the fugu runx genes. the main body of the gene is split into five exons, separated by much shorter introns, all of which have canonical splice signals. the longest predicted frrunt product is amino acids long, in contrast with the aa, aa and aa observed for frrunx frrunx and frrunx respectively. the small number of exons in frrunt leaves little room for alternative splicing by exon skipping, without compromising functionally important domains of the protein. the overall compactness of the gene makes the incorporation of yet undetected exons improbable. several cryptic splice sites within the exons could enable splicing variants altering exon length. frrunt is exceptional in that the length of the runt domain varies from the universally conserved amino acids, due to the introduction of an asparagine residue after position in the rd. this appears to be the first report of such a mutation within this highly conserved domain. we also noted that the rd sequence of the tunicate oikopleura dioica has an insertion at the same position. comparison to the published structure of this domain shows that this variable region is located in loop l opposite the dna-binding region, i.e. in the location least likely to disrupt the structure of the protein. a surprising observation is that frrunt is "interlocked" with frsupt3h, the gene orthologous to the human transcription factor supt3h: the first exon of frrunt is located within the first intron of frsupt3h, and vice-versa. in the human genome, though, supt3h is interlocked with runx as shown in. we discuss the puzzles posed by these differences in genomic organization below. four rd genes in tetraodon and in zebrafish a genome draft of another pufferfish species, tetraodon nigroviridis, has been released. a computational search into this draft reveals four rd genes with clear orthologous relationships with the four fugu rd genes. we call them tnrunx tnrunx and tnrunx and tnrunt. the rd portion of tnrunt has been deposited in the embl database; in this work we report the complete gene structure of tnrunt, which is similar to that of frrunt. in further similarity with the fugu genomic organization, tnrunt is "interlocked" with the tetraodon ortholog of supt3h. the three runx gene pairs are conserved between fugu and tetraodon and display a larger percentage of protein identity than nucleotide identity, indicating a prevalence of conservative substitutions. in contrast, tnrunt is only % identical to frrunt at the protein level, less than their nucleotide identity. indeed, a striking series of non-synonymous mutations has created a highly divergent segment including the n-terminus of the runt domain. the strong and unexpected divergence is not the result of local low sequence quality, as tested by examining the relevant tetraodon entries from the ncbi trace archive, and by resequencing the fugu gene. the differences between tnrunt and frrunt do not modify the ig-like β-sandwich core of the runt domain, which is highly conserved as expected. therefore, the structural integrity of the runt domain appears not to be compromised. we can only speculate that the extensive variation in its n-terminus may reflect species-specific constraints. sizes are expressed in kb. asterisks denote minimal sizes due to the presence of gaps within the genes; the gene structure for tnrunx is incomplete at its 3' end. we also searched for rd genes in the genome draft of the zebrafish, danio rerio. orthologs of runx1- are present, but no ortholog of frrunt could be found. this could be due to the incompleteness of the draft sequence. on the other hand, there are two copies of runx runx2a and runx2b, which have been shown to have somewhat different patterns of expression. we next analyze the phylogenetic relationships between all the observed rd genes. phylogenetic distribution of runt domain genes we performed exhaustive computational searches for rd genes, and in particular for potential orthologs of frrunt, using the available drafts of the human, chimp, mouse, rat, dog, chicken and frog genomes. in all cases, we identified three clear matches corresponding to orthologs of the three runx genes. none of these genomes included a potential ortholog of the fugu/tetraodon runt gene. in principle, such orthologs might be found in the future within current sequencing gaps or heterochromatic regions, but considering the virtually finished human genome, and the combined coverage of all the genome drafts, we can infer that the runt gene is absent in mammals, and probably in all tetrapods. using representative protein sequences of the runx and runt genes, we reconstructed a molecular tree showing the relationship between the three runx proteins, frrunt and tnrunt, and the runt proteins of ciona intestinalis and branchiostoma floridae. in this analysis, the frrunt protein is nearly equidistant from the three runx proteins and amphioxus runt. in comparison, the identity level between the runx proteins is 90%–98% in the same region, and they are 90%–95% identical to amphioxus runt. therefore, while the amphioxus runt protein is very closely related to the vertebrate runx, the pufferfish runt proteins are significantly more divergent. percentages of identity between the human and fugu runt domains. a difficulty has been documented in phylogenetic reconstruction of gene families with anciently duplicated genes, in which saturation of frequently-mutating amino acids leads to erroneous "outgroup topologies". in these incorrect topologies, the duplication event appears to be more ancient than supported by the data, which in turn suggest the existence of lineage-specific gene loss events. we tested our phylogenetic reconstruction using the program asatura, which identifies and suppresses saturated amino acids, thereby correcting the affected tree topology. this analysis did not modify the location of frrunt and tnrunt in our reconstruction, suggesting that mutational saturation is not causing the observed divergence age of the pufferfish runt genes. in this protein-level comparison of the conserved runt domains, the pufferfish runt proteins appear to be surprisingly ancient, predating the divergence between craniates and cephalochordates. on the other hand, when comparing the nucleotide sequences of the first exon from each human and fugu runt domain gene, we observed that frrunt is more closely related to runx, suggesting the possibility of a recombination event between these genes. we also studied the relationships between the runx runx and runx orthologs in several vertebrates and found the species trees to be largely as expected. one of the two zebrafish runx protein sequences appeared to be slightly more closely related to tetrapod runx genes than to the other runx genes in fish species, including zebrafish runx2b. we tested this result using asatura, and found it to be an artifact of mutational saturation: in the corrected tree, runx2a is more closely related to the other fish runx genes. comparative genomics of runt domain genes four rd genes have been identified in drosophila, more similar to each other than to the vertebrate runx genes: they represent an independent family expansion in insects. the four drosophila rd genes are all linked on chromosome x. moreover, three of these genes are clustered within a kb region. there is no linkage of rd genes, however, in the human genome: each gene is on a different chromosome. their genomic environments usually show some conservation: the three human runx genes are followed by clic genes in the complementary strand, and linked to members of the dscr family. the rd genes appear not to be clustered in the fugu genome, though this conclusion is limited by the fragmentary nature of the current genome draft. all four fugu rd genes are flanked by at least one non-rd gene on each side. fugu runx genes are followed by clic genes except for frrunx but a clic gene is located ~ kb upstream of frrunx and in the same orientation. this organization could have arisen by an inversion event in the fugu lineage. to ensure a misassembly did not cause this apparent inversion, we performed a 3x shotgun sampling of the bac clone oml which spans the range 1– of scaffold this quality control step failed to uncover any misassemblies, and confirmed the genomic organization observed in this fugu scaffold. frrunt does not appear to be linked to any clic gene, and no dscr family members can be discerned near any of the fugu rd genes. when studying the wider genomic environments of fugu and human rd genes, we observed significant synteny conservation, in agreement with the observations reported for chromosome x genes. indeed, when comparing each of the genes neighboring fugu runx genes to the human genome, we find that their orthologs tend to be located in the corresponding human chromosome, e.g. most of the genes linked to frrunx have orthologs on human chromosome where human runx resides. some inversion events can be inferred, e.g. one involving the genes phip and irak1bp and another involving mut. gene order and orientation has changed, and intergenic distances have changed drastically, but the overall gene synteny is largely preserved, lending support to the assignments of orthology between the human and fugu runx gene pairs. an exception to the conservation of synteny involves the genes cdc5l and supt3h, which in the human genome are found immediately upstream of runx but in fugu are instead located upstream of frrunt, including the first-exon interlocking with supt3h mentioned earlier. evidence points at a larger duplication in fishes, encompassing at least cdc5l, supt3h, runx clic enpp and enpp followed by differential gene loss. one duplicate copy would have retained frrunx clic and enpp, while the other copy would have retained cdc5l, supt3h, a second rd gene, a second copy of clic and enpp the presence of remnants of supt3h upstream of frrunx, and of two copies of runx in zebrafish, lends further support to this hypothesis. thus, comparative genomic analysis of human, fugu and zebrafish suggests that frrunt may be a derivative form of a duplicated runx gene. however, this contradicts the conclusions from phylogenetic analysis or the protein sequences; we discuss this contradiction below. a new family of follistatin-like genes we find several fugu genes for which no human ortholog can be discerned, among them frrunt. immediately downstream to frrunt we identified a novel gene from the follistatin family, most closely related to fstl a detailed computational search for additional sequences of this family identified two novel, large human follistatin-like genes, which we called fstl and fstl we then found clear orthologs for both in the fugu genome. the genes in this family share a kazal-type cysteine-rich domain and a calcium-binding ef-hand domain. having characterized the complete gene family in both human and fugu, we performed a phylogenetic reconstruction based on the conserved kazal-type domain. frfstl appears to have no ortholog in the human genome, nor could we identify a potential ortholog in the mouse and frog genomes. this suggests that frfstl was also lost in the tetrapod lineage. it is reasonable to hypothesize that the neighboring frrunt and frfstl genes were lost in a single deletion event. discussion we have taken advantage of the availability of genomic drafts for several vertebrate species, including the finished human genome, to identify the orthologs of all currently known runt-domain genes, as well as a novel member of this small gene family. both pufferfish species have four rd genes; since these genomes are only available as draft assemblies, additional rd genes might be found when the finished genomic sequences are made available. the function of the novel frrunt/tnrunt gene is currently unknown. based on the phylogenetic analysis, this novel gene appears to represent an ancestral form of the rd family in vertebrates, subsequently lost in the tetrapod lineage. it is therefore surprising that its gene structure, and not that of runx as in humans, is interlocked with the supt3h gene. based on the comparative genomics analysis alone, one could hypothesize that frrunt is simply a derivative form of runx i.e. the ortholog of zebrafish runx2a. in this case, though, one would expect frrunt to be more similar to frrunx than it is to either frrunx or frrunx but in terms of amino acid sequence similarity, it appears to be equidistant from the three runx genes. this discrepancy might be explained by invoking accelerated evolution of the pufferfish runt genes, perhaps as a lineage-specific adaptation. typically, nucleotide sequences diverge much faster than amino acid sequences, and the first exons of rd genes are significantly less conserved than the runt domain itself, on which we based our phylogenetic analysis. therefore, we find it hard to sustain that, while the first exon of frrunt maintains its nucleotide similarity to the first exon of runx the amino acid sequence of the runt domain itself has diverged at such an accelerated pace. furthermore, we found this not to be an artifact of mutational saturation. a similar situation is observed for the neighboring fstl gene: parsimony considerations could lead one to assume that fstl is a fish-specific duplicate of fstl though contradicting the phylogenetic reconstruction of the evolution of this gene family. this situation could again be explained by assuming accelerated evolution of fstl but we consider this to be a remarkable coincidence. the conundrum is whether frrunt is an ancestral form, or is derived from runx both hypotheses contradict part of the available data. we propose here a third hypothesis, in the form of an evolutionary history: an ancestral rd gene duplicated in chordates, after divergence from sea urchin, which has a single rd gene. one of the two resulting rd genes became the runx family founder, which expanded by triplication, and one of the three runx genes became interlocked with supt3h. after the teleost/tetrapod divergence, a regional duplication in teleosts created a second copy of runx and its neighboring genes. in the tetrapod lineage, the ancestral runt gene was lost, in conjunction with fstl in the pufferfish lineage, the runt gene replaced most of runx2a, perhaps by recombination. this is supported by the clear similarity between the first exons of frrunt and runx the copy of supt3h interlocked with runx2b, apparently superfluous, is being lost by gradual degradation, and only small fragments of it remain. this scenario is compatible with all the data observed. while it posits a small number of additional evolutionary events, it does not involve highly improbable events like the accelerated evolution of a normally highly conserved protein structural domain. interestingly, the first duplication event could correspond to the first round of vertebrate genome tetraploidization. a second round of tetraploidization in the ancestral vertebrate could have produced a set of four paralogous runt domain genes, and a hypothetical gene conversion event may have led to the current complement of three runx genes. conversion between paralogous copies of genes derived from tetraploidization events has been demonstrated. under the proposed scenario, none of the three extant runx genes in mammals represents the ancestral vertebrate rd form. rather, these derivative genes coexisted with an additional runt gene and still do so in teleost genomes. the single known rd gene in amphioxus is more similar to the vertebrate runx genes than it is to frrunt. it is possible, therefore, that cephalochordates have a second rd gene, short and divergent enough to escape experimental detection by dna hybridization. why was one of the ancestral rd genes lost in the tetrapod lineage? the three runx genes bind to the same dna motif and modify the expression of target genes through recruitment of transcriptional modulators, which are also shared; functional differences between the three runx genes are attained by way of tightly regulated spatiotemporal expression patterns. we hypothesize that the ancestral runt gene became inessential to amniotes by functional reprogramming of the remaining three runx genes. its loss would therefore represent an example of evolution by reduction in complexity. in pufferfishes, the hypothesized recombination event would have placed the runt gene under the regulatory control of the former runx2a promoter. the viability of such a sudden regulatory change would in turn suggest a significant level of functional redundancy among the rd genes.  we identified a fourth runt domain gene in the fugu genome, which appears to represent either a pufferfish-specific, fast-evolving derivative of runx or a direct descendant of the ancestral chordate runt gene. we find the latter hypothesis more reasonable. this novel gene evolved in parallel with the vertebrate runx genes, and while it has been preserved in pufferfishes, it appears to have been lost entirely in tetrapods. this suggests that the ancestral vertebrate was more complex than previously suspected. by studying a very limited set of fugu genomic regions, namely the scaffolds related to rd genes, we have identified seven apparently functional fugu genes that are absent from the human genome, and were probably lost early in tetrapod history. in the process of identifying relevant homologs for one of these genes, we have identified a new family of follistatin-like genes in the human genome. phylogenetic analysis of the rd protein sequences led to results that contradict those derived from comparative genomics, but we showed that the two could be reconciled into a coherent evolutionary model. these results underscore the importance of obtaining complete genomic sequences of strongly divergent vertebrates, and the value to be derived by performing detailed and integrated analyses of their gene complements. 
three runt domain genes have been described in each vertebrate genome, but only one in other chordates. we consider alternative explanations for an apparent contradiction between the phylogenetic data and the comparison of the genomic neighborhoods of human and fugu runt domain genes. we hypothesize that an ancient runt locus was lost in the tetrapod lineage, together with frfstl a member of a novel family of follistatin-like genes. our results suggest that the runt domain family may have started expanding in chordates much earlier than previously thought, and exemplify the importance of detailed analysis of whole-genome draft sequence to provide new insights into gene evolution. the runt domain transcription factors are key regulators of developmental processes in bilaterians, involved both in cell proliferation and differentiation, and their disruption usually leads to disease. analysis of the genome draft of the fugu pufferfish reveals the existence of a fourth runt domain gene, frrunt, in addition to the orthologs of human runx runx and runx the tiny frrunt packs six exons and two putative promoters in just kb of genomic sequence. in the human genome, supt3h is instead interlocked with runx frrunt has no detectable ortholog in the genomes of mammals, birds or amphibians. therefore, the common ancestor of vertebrates has been thought to have had a single runt domain gene.
 the runt domain transcription factors are key regulators of developmental processes in bilaterians, involved both in cell proliferation and differentiation, and their disruption usually leads to disease. three runt domain genes have been described in each vertebrate genome, but only one in other chordates. therefore, the common ancestor of vertebrates has been thought to have had a single runt domain gene.  analysis of the genome draft of the fugu pufferfish reveals the existence of a fourth runt domain gene, frrunt, in addition to the orthologs of human runx runx and runx the tiny frrunt packs six exons and two putative promoters in just kb of genomic sequence. the first exon is located within an intron of frsupt3h, the ortholog of human supt3h, and the first exon of frsupt3h resides within the first intron of frrunt. the two gene structures are therefore "interlocked". in the human genome, supt3h is instead interlocked with runx frrunt has no detectable ortholog in the genomes of mammals, birds or amphibians. we consider alternative explanations for an apparent contradiction between the phylogenetic data and the comparison of the genomic neighborhoods of human and fugu runt domain genes. we hypothesize that an ancient runt locus was lost in the tetrapod lineage, together with frfstl a member of a novel family of follistatin-like genes.  our results suggest that the runt domain family may have started expanding in chordates much earlier than previously thought, and exemplify the importance of detailed analysis of whole-genome draft sequence to provide new insights into gene evolution. 