BACKGROUND
most genes belong to gene families, which have emerged through consecutive cycles of gene duplications during evolution  <cit> .
with the availability of entire genome sequences, much progress has been made towards the understanding of gene duplication dynamics  <cit>  and the evolutionary forces responsible for the retention of a proportion of duplicate genes, such as neo-functionalization  <cit>  and sub-functionalization  <cit> , both at the level of gene expression patterns  <cit>  and protein sequence evolution  <cit> .
however, further investigation is required to understand how genomic processes, such as gene duplications and losses, result in higher-level co-ordinated molecular events, such as the emergence of novel signal transduction pathways, which in turn give rise to phenotypic innovations, such as novel organs, developmental phases, or body plans.
to approach this question from a comparative genomics viewpoint, we focus herein on the emergence and evolution of the transforming growth factor-β  pathway within the animal kingdom.
this pathway has been recognized as one of the fundamental and versatile metazoan signal transduction engines, with central roles in development, organogenesis, stem-cell control, immunity, and cancer  <cit> .
a concise description of the human pathway has been deposited by the authors of this article in the reactome  <cit>  knowledge base  <cit> .
the cellular core of all tgf-β superfamily pathways consists of cell surface receptors, called type i and type ii serine-threonine kinase receptors, and intracellular smad proteins  <cit> .
the latter constitute the actual signal transduction engine of the pathway  <cit> .
there are eight known smads in the human genome, classified as: two tgf-β sensu stricto  and three bone morphogenetic protein -type  receptor-activated smads , one common mediator smad , and two inhibitory smads .
these eight genes are highly similar in sequence and are evidently results of multiple gene duplications of unknown origins.
while the functional differences between the three biochemical classes of smads are well known, their evolutionary history, the characteristics of the ancestral smad molecule, and the selection forces behind the retention of multiple subtypes of r- and i-smads are poorly understood.
in humans, we encounter five distinct type ii receptors and seven distinct type i receptors  <cit> .
the functional receptor unit is a hetero-tetramer of two type ii receptors with two type i receptors, in which upon binding of the ligand the type ii receptors phosphorylate the type i receptors, while the latter phosphorylate and activate r-smads.
analysis of the receptor genes, so far has been limited to a few species, namely humans, rodents, african clawed frog , fruit fly  and the free-living roundworm caenorhabditis elegans  <cit> .
mammalian genomes encode up to  <dig> tgf-β related ligands, d. melanogaster seven and c. elegans five   <cit> .
however, we do not focus on tgf-β related proteins in this study, as these sequences are rather diverged  rendering them difficult to analyse in multiple genomes using a unified computational pipeline.
we refer the interested reader to a review by herpin et al  <cit> .
the most prevalent mode of extracellular modulation of tgf-β signalling is by means of soluble antagonists, called ligand traps, such of those of the chordin and gremlin family  <cit> .
bambi is another important negative regulator of tgf-β signalling, related to tgf-β family type i receptors but lacking an intracellular kinase domain  <cit> .
bmp signalling gradients, modulated by chordin, have been found to induce dorsoventral axis formation in the spemann organizer  <cit> .
thus, traditionally, the tgf-β pathway had been thought to have evolved in the context of dorsoventral patterning, and thus be present only in bilateria.
this view has been recently challenged by the discovery of the functional pathway in multiple cnidarians  <cit> .
furthermore, the origin of animals themselves is only now being understood .
on the basis of mitochondrial dna sequence comparison, the choanoflagellates have been identified as the closest single-celled animal relatives  <cit>  while the placozoan trichoplax adhaerens, the so-called tablet animal  <cit> , has been placed at the root of animal phylogeny  <cit> .
however, some authors regard sponges as earlier diverging than placozoans  <cit> .
regardless of the relative position of placozoans and sponges, the critical step of transformation to multicellulararity must have been accompanied by the development of adhesion molecules, extracellular matrix proteins , and cell-to-cell communication.
it is essential to identify the critical signalling pathways, in particular those involved in control of development, cellular differentiation and body plan formation  <cit> .
such comparisons will not only shed light on metazoan origins, and advance the field of evo-devo, but will also help us understand the fundamental functional motifs that underlie interwoven signal transduction networks of higher animals, with impact on human health.
it was reported previously that atypical dauer pathway smads could be found in free-living bacterial feeding nematodes of the genus rhabditis   <cit> .
the dauer  is a survival and dissemination form, formed by all rhabditoid nematodes, an alternative to the active third stage larvae .
dauers are induced by environmental stress factors, such as lack of food, overcrowding, or high temperature.
the dauer pathway  is of high general interest, as it has been linked with aging  <cit> , biodiversity  <cit>  and the development of parasitism in nematodes  <cit> .
however, the origins of the dauer pathway smads had been previously unknown.
RESULTS
tgf-β pathway gene content across taxa
using the full genome sequences of  <dig> species , we performed a comparative analysis of the tgf-β pathway genes, focusing on smads and receptors.
the first obvious observation is that the tgf-β pathway genes do not exist in protozoans but are universally present in metazoans.
this leads to the first important conclusion that the tgf-β pathway genes evolved rapidly and to a high degree of complexity with the first known animal species.
table  <dig> provides an overview of the pathway content in high-coverage genomes under study.
<dig> for details, see figure 3
inhibitory co-receptor bambi, although probably present in urbilateria, appears to be frequently lost, such as in ecdysozoa, tunicates, and helobdella
‡ species-specific tgf-β-r-smad duplication
‡‡ species-specific co-smad duplication
‡‡‡ leech helobdella has a modified pathway: with an additional co-smad  and a distinct set of ligand traps
Ω including a retrogene of bmpr1a of unverified functionality: ensg <dig> , ensmmug <dig> , ensrnog <dig>
the table indicates the numbers of genes in each family.
data sources: ensembl  <dig> , treefam  <dig> , jgi , wormbase , flybase
smads and receptors in bilateria – point of divergence  analysis
as a general rule, three functional classes of smads  are present in all extant species and the reconstructed ancestral genomes.
at least one type ii receptor and multiple type i receptors can be detected, and the ancestral bilaterian repertoire can be inferred as consisting of two type ii receptors and three type i receptors.
detailed observations are provided below, starting with the oldest point of divergence .
ecdysozoan pod
two r-smads , one co-smad and one i-smad are consistently present in  <dig> drosophila species, and apis mellifera, and thus can be inferred to have existed in the ancestral genome of the ecdysozoan pod.
drosophila species and apis mellifera also contain two type ii receptors and three type i receptors.
nematode genomes contain additional diverged smads  but these were excluded from figure  <dig> and figure s <dig>  and analysed separately, because of the special evolutionary status of the dauer pathway .
urochordate pods
two sea squirts  possess at least two r-smads , one co-smad and one i-smad.
additional smads can be detected, but these do not cluster with smads observed in the vertebrata, and thus represent lineage-specific duplications absent in the genome of the ancestral vertebrate.
the ancestral bilaterian tgf-β receptor repertoire is expanded to three type ii receptors: this is the first example of a bilaterian tgf-β receptor duplication, mapping to chordates in figure s <dig> , which is propagated through vertebrates.
teleost fish pod
the pod of the teleosts is the first vertebrate pod and also the first pod which can be inferred to possess all eight subtypes of smads present in extant mammalian genomes .
additional lineage-specific r-, co- and i-smads could also be detected in extant teleost fishes.
this stimulated further detailed analysis of teleost fish sequences .
all type ii and type i receptors have been duplicated, in many cases multiple times .
some of the progeny genes are common to all vertebrates, several are unique to teleost fishes, and a few are species-specific.
amphibian pod
amphibians are represented only by one genome – xenopus tropicalis.
xenopus laevis was not used, as this species is now widely regarded as tetraploid.
xenopus tropicalis possesses a distinct set of nine smads, with two co-smads, two genes for smad <dig>  and no ortholog of smad <dig>  the additional co-smad does not appear to be a lineage-specific duplication, as it groups with added genes in teleost fishes, suggesting that it may represent a gene deriving directly from the 2r event, lost in other vertebrates.
aves pod
similar to mammals, the single available avian genome  contains genes for all five r-smads, two i-smads, and five type ii and seven type i receptors.
curiously, no co-smad was detected in the chicken genome .
manual querying of the ensembl database annotation of the chicken genome  confirmed that there are no available co-smad gene predictions.
however, this is a genomic artifact.
a representative chicken co-smad cluster, gga.
<dig> containing  <dig> est sequences, was found within the ncbi unigene collection  <cit> .
furthermore, examination of synteny with human revealed a large missing sequence region in the chicken genome, which includes orthologs of the extensive gene neighbourhood of the human co-smad.
this example underlines the need for caution in interpretation of putative losses suggested by genome sequences from individual species.
mammalian pods
all extant placental mammalian genomes consistently contain a well-characterized set of eight classic mammalian smads.
an additional diverged co-smad sequence  was also detected in the marsupial mammal monodelphis domestica.
interesting observations can be made regarding alternative splicing of the tgf-β pathway genes in the mammals.
for example, alternative splicing of smad <dig> and smad <dig>  inferred from dbest, can be traced back to the origin of vertebrates, suggesting a profound functional significance .
the anti-mullerian hormone type ii receptor  is developed in placental mammals, expanding the receptor repertoire to five type ii and seven type i receptors.
retroposed copies of bmpr1a, of unknown functional significance, can also be detected in primates and rodents .
origin of dauer pathway smads: duplication, neo-functionalization and accelerated evolution
the phylogenetic relationship between d. melanogaster, and c. elegans smads was investigated in further detail .
in c. elegans, there exist a set of smads controlling the sma/mab pathway , and a set of smads of the dauer pathway   <cit>  that were all consistently detected.
functionality of one additional gene tag- <dig> has not been established.
our sequence tree  differs significantly from previously published trees  <cit>  in which dpsmads cluster together, not allowing for resolution into proper functional classes or reconstruction of evolutionary origins.
comparison of branch lengths indicates that all dpsmads have been evolving much faster than their counterparts in the sma/mab pathway  – a finding suggestive of positive selection acting upon dpsmads.
indeed, accelerated protein sequence change is confirmed by the analysis of ka/ks ratios between pairs of orthologs in c. briggsae and c. elegans .
accordingly, all ka/ks ratios for known dauer pathway genes in this comparison are higher than ratios for the remaining genes.
the average values are  <dig>  and  <dig> , respectively.
iv: <dig> .
<dig> kbp
iv: <dig> .
<dig> kbp
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<dig> kbp
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<dig> kbp
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<dig> kbp
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<dig> kbp
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<dig> kbp
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<dig> kbp
comparison of tgf-β ligands, receptors and smads in c. elegans and c. briggsae reveals conservation of exon number and chromosomal location for both the sma/mab and dauer pathways.
however, high ka/ks ratios for genes of the dauer pathway  indicate that it evolved faster since the two species diverged.
data retrieved from wormbase .
* modified nei-gojobori  model with pairwise deletion and assuming transition/transversion ratio of  <dig>
tgf-β pathway gene duplication in teleost fishes
we have also analyzed the smads present in zebrafish , medaka , fugu  and the green spotted puffer , in comparison with eight human genes representative of vertebrates .
it is clear that smads underwent duplications early in teleost fishes, followed by additional lineage-specific duplications.
interestingly, two of the additional smad <dig> genes in tetraodontidae possess a non-classic protein domain: gstent <dig> and sinfrut <dig> are predicted to harbour the haem peroxidase domain , which might be utilised in signalling response to oxidative stress.
additional lineage-specific duplications of tgf-β receptors can also be detected in these teleost fish species .
what types of novel functions are linked with multiple duplicated smads and tgf-β pathway receptors in teleost fishes remains to be elucidated.
phylogenetic analyses in basal metazoans and lophotrochozoans
the tree in figure  <dig> shows the repertoire of smads in nematostella vectensis and trichoplax adhaerens, in connection with the reconstruction of ancestral metazoan duplications which resulted in the formation of a complete signalling pathway  in these early diverging animals.
it is also worth noting that nematostella and trichoplax contain genes for both receptor classes: type i and type ii .
however, trichoplax, unlike nematostella, does not appear to harbour an ortholog of wit: taput is the only type ii receptor found in trichoplax and is likely to correspond to the ancestral type ii receptor.
furthermore, while tasax and tatkv are clear orthologs of corresponding fly genes, tababo branches out deeper in the tree and may correspond to the ancestral type i receptor.
the bayesian tree in figure s <dig>   demonstrates that the familiar pattern of four smads grouped into three functional classes can be also observed in comparatively poorly investigated lophotrochozoans .
the bayesian tree in figure s <dig>   shows two amphimedon r-smads  which are the only smads we have detected in genomic traces available for this demosponge.
species codes can be accessed in table  <dig>
discussion
the growing number of sequenced genomes provides a relatively wide coverage of the animal genome space.
this makes it possible to reconstruct ancestral developmental signalling pathways, and to retrace the ancient evolutionary events which led to their emergence and modulation, in particular gene duplications, instances of sub- and neo-functionalization, and gene losses.
herein, we focus on the gene set constituting the fundamental building blocks of a major component of the animal developmental toolkit – the tgf-β pathway.
we have examined in detail the gene content of the tgf-β pathway in extant genomes of different metazoan phyla, where high-coverage genomic data are available .
smads are of particular interest, as they constitute the core engine of the tgf-β signal transduction machinery.
we have estimated the origin of different types of smads by examining extant genomes and inferring ancestral genes .
we justify somewhat anthropocentric approach of the pod analysis by the high significance of the tgf-β pathway in human health and disease, which drives substantial proportion of research in the field.
on the lineage of pods leading to human, the smads clearly appear to have gone through a major wave of duplications, fitting well with the 2r hypothesis of two-fold genome duplication at the base of vertebrates  <cit> .
additional duplications occurred along the teleost fish lineage, in congruence with the hypothesis of a teleost fish-specific genome duplication – fsgd  <cit> .
diversification of type i and type ii receptors has also followed the course agreeable with the 2r hypothesis, with multiple additional duplications in teleost fishes .
pod analysis  shows that the core pathway  expanded dramatically and permanently at the base of vertebrates.
table  <dig> demonstrates that this expansion correlates well with the increase of complexity of regulatory networks associated with the extended pathway, such as ligand traps of the chordin and gremlin family.
the same is true of many transcriptional co-activators, and target genes – particularly those in the concurrently developed active immune system, as well as the endocytic regulators that control smad signalling, sara and endofin, which emerged through the duplication of a single ancestral gene .
analysis of the c. elegans genome revealed atypical smads belonging to sma/mab and dauer pathways.
our phylogenetic tree indicates that daf- <dig> is an r-smad, daf- <dig> a co-smad and tag- <dig> an i-smad .
sma- <dig> and sma- <dig> are likely duplicates of the ancestral bmp r-smad, as they both contain the characteristic rqdvts motif of the l <dig> loop.
conversely, daf- <dig> and daf- <dig> might be duplicates of the ancestral tgf-β r-smad, although daf- <dig> is too divergent to allow firm conclusions.
sma- <dig> and daf- <dig> share a similar pattern of multiple splice variants, which together with the tree topology suggests that they derive from the ancestral co-smad via a gene duplication event.
comparative analysis revealed that sma/mab and dauer pathway content is identical between c. elegans and c. briggsae, with strong conservation of the overall gene structure and synteny .
this proves that all the relevant genes already existed in the last common ancestor of the two rhabditoid species.
although similar in morphology, c. briggsae and c. elegans are rather distant relatives in evolutionary time: the two species split roughly  <dig> million years ago  <cit> .
analysis of lengths of protein branches  is indicative of accelerated evolution of daf- <dig>   <dig> and  <dig>  additionally, analysis of ka/ks ratios between pairs of orthologs in c. briggsae and c. elegans suggests that the dauer pathway evolved faster since the two species diverged .
the average ka/ks ratio for dauer pathway orthologs is  <dig>  versus  <dig>  for non-dauer tgf-β pathway genes.
thus, the initial duplications and neo-functionalization occurred early in nematode evolution, but have been followed by further change in separate rhabditoid lineages, as different species experienced slightly different selection pressures for entry and persistence in their dauer forms.
for example, c. elegans, unlike c. briggsae, is strongly induced to form dauers at temperatures higher than 26°c  <cit> .
overall, the dauer pathway represents an interesting example of rapidly evolving pathway neo-functionalization, developed as a lineage-specific adaptation towards the colonization of the environmental niche of the soil.
the crucial question about the taxonomic origin of the tgf-β pathway has not been categorically answered yet.
herein, we have identified tgf-β pathway components in t. adhaerens, the representative of the early diverging phylum placozoa, and the demosponge amphimedon queenslandica  <cit> .
choanoflagellata are the closest unicellular relatives of animals  <cit>  and possess some genes linked to metazoan development, for example a receptor tyrosine kinase – mbrtk <dig>  <cit> .
however, we have not been able to detect smads, tgf-β receptors, ligands, sara, chordin or gremlin in the genome of the unicellular choanoflagellate m. brevicollis  <cit> , or more distantly related protozoans volvox carteri and naegleria gruberi.
this indicates that the appearance of the tgf-β pathway was intrinsically linked to the emergence of earliest animals, and the pathway may thus be regarded as a key feature of the metazoan life forms.
it is also rather striking that such an early diverging animal as trichoplax already possesses the complete functional pathway, including multiple smads, receptors, and ligands, as well as orthologs of chordin, gremlin and sara.
we hypothesize that the single primeval common mediator/receptor activated smad functioned as a homo-dimer , and possessed the universal functionality of r-smads and the co-smad; i.e.
it could be phosphorylated by the receptor/ligand complex, shuttle to the nucleus, interact with transcriptional co-activators via the mh <dig> domain and bind dna via the mh <dig> domain.
as the number of ligands and receptors grew, the primeval smad duplicated and, through sub-functionalization, gave rise to two separate r-smads which from then on interact with non-overlapping sets of receptors .
one of the r-smads duplicated again  giving rise to a co-smad which enhanced the set of regulatory protein interactions, and possibly provided a way of integrating the signals from the two r-smad channels through competition for the available pool of co-smad molecules.
the critical role of co-smad bioavailability is also suggested by its low duplicability; in the great majority of species there is only one co-smad .
xenopus laevis is the notable exception having two genes xsmad4a and xsmad4b  <cit> , but they are differentially expressed both in embryos and adult tissues.
the fast diverging i-smad was the last addition to the pathway .
it neofunctionalized to create a controlling negative feedback loop; i-smad transcription is induced by the pathway, the protein can bind the activated receptor complex, but lacking a terminal phosphorylation motif it does not propagate the signal.
since it is not being used, over time the mh <dig> of the i-smad converted to a vestigial domain.
it will be interesting to see if future genome projects of basal animals and closest extant unicellular relatives of animals  <cit>  will provide a proof of our single smad hypothesis.
mammalian tgf-β type ii receptors are listed in the first row.
type i receptors which make functional complexes with each type ii receptor are then listed, followed by the r-smads that the type i receptor activates.
the ligands activating each receptor-smad group are then listed in a cumulative fashion.
CONCLUSIONS
the emergence of the tgf-β pathway coincided with appearance of the first animal species, and was most likely linked with duplications of the single primeval common mediator/receptor activated smad.
this resulted in the creation of the ancestral eumetazoan repertoire of four smads, forming the basis of the pathway in the placozoa, the cnidaria, the arthropoda, and in the lophotrochozoa.
after application of a formal speciation and duplication inference algorithm, we conclude that the diversification of smads and receptors in chordates is parsimoniously explained according to the 2r model, with additional rounds of duplications in teleost fishes.
the nematoda posses a heavily modified pathway which evolution has been marked by accelerated sequence change.
our multi-genome comparison and ancestral inference approach has implications extending beyond the tgf-β pathway.
origins of other developmental signalling pathways, for example hox and hedgehog, are also being investigated using phylogenomic approaches  <cit> .
results obtained for all developmental signalling pathways should be integrated and compared with paleontological records and molecular clock data, to identify the molecular nature and timing of all major changes in the shared animal developmental toolkit  <cit> , including those which gave rise to vertebrate innovations  <cit> .

 most genes belong to gene families, which have emerged through consecutive cycles of gene duplications during evolution. with the availability of entire genome sequences, much progress has been made towards the understanding of gene duplication dynamics and the evolutionary forces responsible for the retention of a proportion of duplicate genes, such as neo-functionalization and sub-functionalization, both at the level of gene expression patterns and protein sequence evolution. however, further investigation is required to understand how genomic processes, such as gene duplications and losses, result in higher-level co-ordinated molecular events, such as the emergence of novel signal transduction pathways, which in turn give rise to phenotypic innovations, such as novel organs, developmental phases, or body plans. to approach this question from a comparative genomics viewpoint, we focus herein on the emergence and evolution of the transforming growth factor-β pathway within the animal kingdom. this pathway has been recognized as one of the fundamental and versatile metazoan signal transduction engines, with central roles in development, organogenesis, stem-cell control, immunity, and cancer. a concise description of the human pathway has been deposited by the authors of this article in the reactome knowledge base. the cellular core of all tgf-β superfamily pathways consists of cell surface receptors, called type i and type ii serine-threonine kinase receptors, and intracellular smad proteins. the latter constitute the actual signal transduction engine of the pathway. there are eight known smads in the human genome, classified as: two tgf-β sensu stricto and three bone morphogenetic protein -type receptor-activated smads, one common mediator smad, and two inhibitory smads. these eight genes are highly similar in sequence and are evidently results of multiple gene duplications of unknown origins. while the functional differences between the three biochemical classes of smads are well known, their evolutionary history, the characteristics of the ancestral smad molecule, and the selection forces behind the retention of multiple subtypes of r- and i-smads are poorly understood. in humans, we encounter five distinct type ii receptors and seven distinct type i receptors. the functional receptor unit is a hetero-tetramer of two type ii receptors with two type i receptors, in which upon binding of the ligand the type ii receptors phosphorylate the type i receptors, while the latter phosphorylate and activate r-smads. analysis of the receptor genes, so far has been limited to a few species, namely humans, rodents, african clawed frog, fruit fly and the free-living roundworm caenorhabditis elegans. mammalian genomes encode up to tgf-β related ligands, d. melanogaster seven and c. elegans five. however, we do not focus on tgf-β related proteins in this study, as these sequences are rather diverged rendering them difficult to analyse in multiple genomes using a unified computational pipeline. we refer the interested reader to a review by herpin et al. the most prevalent mode of extracellular modulation of tgf-β signalling is by means of soluble antagonists, called ligand traps, such of those of the chordin and gremlin family. bambi is another important negative regulator of tgf-β signalling, related to tgf-β family type i receptors but lacking an intracellular kinase domain. bmp signalling gradients, modulated by chordin, have been found to induce dorsoventral axis formation in the spemann organizer. thus, traditionally, the tgf-β pathway had been thought to have evolved in the context of dorsoventral patterning, and thus be present only in bilateria. this view has been recently challenged by the discovery of the functional pathway in multiple cnidarians. furthermore, the origin of animals themselves is only now being understood. on the basis of mitochondrial dna sequence comparison, the choanoflagellates have been identified as the closest single-celled animal relatives while the placozoan trichoplax adhaerens, the so-called tablet animal, has been placed at the root of animal phylogeny. however, some authors regard sponges as earlier diverging than placozoans. regardless of the relative position of placozoans and sponges, the critical step of transformation to multicellulararity must have been accompanied by the development of adhesion molecules, extracellular matrix proteins, and cell-to-cell communication. it is essential to identify the critical signalling pathways, in particular those involved in control of development, cellular differentiation and body plan formation. such comparisons will not only shed light on metazoan origins, and advance the field of evo-devo, but will also help us understand the fundamental functional motifs that underlie interwoven signal transduction networks of higher animals, with impact on human health. it was reported previously that atypical dauer pathway smads could be found in free-living bacterial feeding nematodes of the genus rhabditis. the dauer is a survival and dissemination form, formed by all rhabditoid nematodes, an alternative to the active third stage larvae. dauers are induced by environmental stress factors, such as lack of food, overcrowding, or high temperature. the dauer pathway is of high general interest, as it has been linked with aging, biodiversity and the development of parasitism in nematodes. however, the origins of the dauer pathway smads had been previously unknown.  tgf-β pathway gene content across taxa using the full genome sequences of species, we performed a comparative analysis of the tgf-β pathway genes, focusing on smads and receptors. the first obvious observation is that the tgf-β pathway genes do not exist in protozoans but are universally present in metazoans. this leads to the first important conclusion that the tgf-β pathway genes evolved rapidly and to a high degree of complexity with the first known animal species. table provides an overview of the pathway content in high-coverage genomes under study. for details, see figure 3 inhibitory co-receptor bambi, although probably present in urbilateria, appears to be frequently lost, such as in ecdysozoa, tunicates, and helobdella ‡ species-specific tgf-β-r-smad duplication ‡‡ species-specific co-smad duplication ‡‡‡ leech helobdella has a modified pathway: with an additional co-smad and a distinct set of ligand traps Ω including a retrogene of bmpr1a of unverified functionality: ensg, ensmmug, ensrnog the table indicates the numbers of genes in each family. data sources: ensembl, treefam, jgi, wormbase, flybase smads and receptors in bilateria – point of divergence analysis as a general rule, three functional classes of smads are present in all extant species and the reconstructed ancestral genomes. at least one type ii receptor and multiple type i receptors can be detected, and the ancestral bilaterian repertoire can be inferred as consisting of two type ii receptors and three type i receptors. detailed observations are provided below, starting with the oldest point of divergence. ecdysozoan pod two r-smads, one co-smad and one i-smad are consistently present in drosophila species, and apis mellifera, and thus can be inferred to have existed in the ancestral genome of the ecdysozoan pod. drosophila species and apis mellifera also contain two type ii receptors and three type i receptors. nematode genomes contain additional diverged smads but these were excluded from figure and figure s and analysed separately, because of the special evolutionary status of the dauer pathway. urochordate pods two sea squirts possess at least two r-smads, one co-smad and one i-smad. additional smads can be detected, but these do not cluster with smads observed in the vertebrata, and thus represent lineage-specific duplications absent in the genome of the ancestral vertebrate. the ancestral bilaterian tgf-β receptor repertoire is expanded to three type ii receptors: this is the first example of a bilaterian tgf-β receptor duplication, mapping to chordates in figure s, which is propagated through vertebrates. teleost fish pod the pod of the teleosts is the first vertebrate pod and also the first pod which can be inferred to possess all eight subtypes of smads present in extant mammalian genomes. additional lineage-specific r-, co- and i-smads could also be detected in extant teleost fishes. this stimulated further detailed analysis of teleost fish sequences. all type ii and type i receptors have been duplicated, in many cases multiple times. some of the progeny genes are common to all vertebrates, several are unique to teleost fishes, and a few are species-specific. amphibian pod amphibians are represented only by one genome – xenopus tropicalis. xenopus laevis was not used, as this species is now widely regarded as tetraploid. xenopus tropicalis possesses a distinct set of nine smads, with two co-smads, two genes for smad and no ortholog of smad the additional co-smad does not appear to be a lineage-specific duplication, as it groups with added genes in teleost fishes, suggesting that it may represent a gene deriving directly from the 2r event, lost in other vertebrates. aves pod similar to mammals, the single available avian genome contains genes for all five r-smads, two i-smads, and five type ii and seven type i receptors. curiously, no co-smad was detected in the chicken genome. manual querying of the ensembl database annotation of the chicken genome confirmed that there are no available co-smad gene predictions. however, this is a genomic artifact. a representative chicken co-smad cluster, gga. containing est sequences, was found within the ncbi unigene collection. furthermore, examination of synteny with human revealed a large missing sequence region in the chicken genome, which includes orthologs of the extensive gene neighbourhood of the human co-smad. this example underlines the need for caution in interpretation of putative losses suggested by genome sequences from individual species. mammalian pods all extant placental mammalian genomes consistently contain a well-characterized set of eight classic mammalian smads. an additional diverged co-smad sequence was also detected in the marsupial mammal monodelphis domestica. interesting observations can be made regarding alternative splicing of the tgf-β pathway genes in the mammals. for example, alternative splicing of smad and smad inferred from dbest, can be traced back to the origin of vertebrates, suggesting a profound functional significance. the anti-mullerian hormone type ii receptor is developed in placental mammals, expanding the receptor repertoire to five type ii and seven type i receptors. retroposed copies of bmpr1a, of unknown functional significance, can also be detected in primates and rodents. origin of dauer pathway smads: duplication, neo-functionalization and accelerated evolution the phylogenetic relationship between d. melanogaster, and c. elegans smads was investigated in further detail. in c. elegans, there exist a set of smads controlling the sma/mab pathway, and a set of smads of the dauer pathway that were all consistently detected. functionality of one additional gene tag- has not been established. our sequence tree differs significantly from previously published trees in which dpsmads cluster together, not allowing for resolution into proper functional classes or reconstruction of evolutionary origins. comparison of branch lengths indicates that all dpsmads have been evolving much faster than their counterparts in the sma/mab pathway – a finding suggestive of positive selection acting upon dpsmads. indeed, accelerated protein sequence change is confirmed by the analysis of ka/ks ratios between pairs of orthologs in c. briggsae and c. elegans. accordingly, all ka/ks ratios for known dauer pathway genes in this comparison are higher than ratios for the remaining genes. the average values are and, respectively. iv:. kbp iv:. kbp v:. kbp v:. kbp v:. kbp v:. kbp iii:. kbp unassigned ii:. kbp ii:. kbp iii:. kbp iii:. kbp iv:. kbp iv:. kbp iii:. kbp iii:. kbp iii:. kbp iii:. kbp iii:. kbp iii:. kbp i:. kbp i:. kbp iv:. kbp iv:. kbp x:. kbp x:. kbp i:. kbp i:. kbp ii:. kbp ii:. kbp comparison of tgf-β ligands, receptors and smads in c. elegans and c. briggsae reveals conservation of exon number and chromosomal location for both the sma/mab and dauer pathways. however, high ka/ks ratios for genes of the dauer pathway indicate that it evolved faster since the two species diverged. data retrieved from wormbase. * modified nei-gojobori model with pairwise deletion and assuming transition/transversion ratio of tgf-β pathway gene duplication in teleost fishes we have also analyzed the smads present in zebrafish, medaka, fugu and the green spotted puffer, in comparison with eight human genes representative of vertebrates. it is clear that smads underwent duplications early in teleost fishes, followed by additional lineage-specific duplications. interestingly, two of the additional smad genes in tetraodontidae possess a non-classic protein domain: gstent and sinfrut are predicted to harbour the haem peroxidase domain, which might be utilised in signalling response to oxidative stress. additional lineage-specific duplications of tgf-β receptors can also be detected in these teleost fish species. what types of novel functions are linked with multiple duplicated smads and tgf-β pathway receptors in teleost fishes remains to be elucidated. phylogenetic analyses in basal metazoans and lophotrochozoans the tree in figure shows the repertoire of smads in nematostella vectensis and trichoplax adhaerens, in connection with the reconstruction of ancestral metazoan duplications which resulted in the formation of a complete signalling pathway in these early diverging animals. it is also worth noting that nematostella and trichoplax contain genes for both receptor classes: type i and type ii. however, trichoplax, unlike nematostella, does not appear to harbour an ortholog of wit: taput is the only type ii receptor found in trichoplax and is likely to correspond to the ancestral type ii receptor. furthermore, while tasax and tatkv are clear orthologs of corresponding fly genes, tababo branches out deeper in the tree and may correspond to the ancestral type i receptor. the bayesian tree in figure s demonstrates that the familiar pattern of four smads grouped into three functional classes can be also observed in comparatively poorly investigated lophotrochozoans. the bayesian tree in figure s shows two amphimedon r-smads which are the only smads we have detected in genomic traces available for this demosponge. species codes can be accessed in table discussion the growing number of sequenced genomes provides a relatively wide coverage of the animal genome space. this makes it possible to reconstruct ancestral developmental signalling pathways, and to retrace the ancient evolutionary events which led to their emergence and modulation, in particular gene duplications, instances of sub- and neo-functionalization, and gene losses. herein, we focus on the gene set constituting the fundamental building blocks of a major component of the animal developmental toolkit – the tgf-β pathway. we have examined in detail the gene content of the tgf-β pathway in extant genomes of different metazoan phyla, where high-coverage genomic data are available. smads are of particular interest, as they constitute the core engine of the tgf-β signal transduction machinery. we have estimated the origin of different types of smads by examining extant genomes and inferring ancestral genes. we justify somewhat anthropocentric approach of the pod analysis by the high significance of the tgf-β pathway in human health and disease, which drives substantial proportion of research in the field. on the lineage of pods leading to human, the smads clearly appear to have gone through a major wave of duplications, fitting well with the 2r hypothesis of two-fold genome duplication at the base of vertebrates. additional duplications occurred along the teleost fish lineage, in congruence with the hypothesis of a teleost fish-specific genome duplication – fsgd. diversification of type i and type ii receptors has also followed the course agreeable with the 2r hypothesis, with multiple additional duplications in teleost fishes. pod analysis shows that the core pathway expanded dramatically and permanently at the base of vertebrates. table demonstrates that this expansion correlates well with the increase of complexity of regulatory networks associated with the extended pathway, such as ligand traps of the chordin and gremlin family. the same is true of many transcriptional co-activators, and target genes – particularly those in the concurrently developed active immune system, as well as the endocytic regulators that control smad signalling, sara and endofin, which emerged through the duplication of a single ancestral gene. analysis of the c. elegans genome revealed atypical smads belonging to sma/mab and dauer pathways. our phylogenetic tree indicates that daf- is an r-smad, daf- a co-smad and tag- an i-smad. sma- and sma- are likely duplicates of the ancestral bmp r-smad, as they both contain the characteristic rqdvts motif of the l loop. conversely, daf- and daf- might be duplicates of the ancestral tgf-β r-smad, although daf- is too divergent to allow firm conclusions. sma- and daf- share a similar pattern of multiple splice variants, which together with the tree topology suggests that they derive from the ancestral co-smad via a gene duplication event. comparative analysis revealed that sma/mab and dauer pathway content is identical between c. elegans and c. briggsae, with strong conservation of the overall gene structure and synteny. this proves that all the relevant genes already existed in the last common ancestor of the two rhabditoid species. although similar in morphology, c. briggsae and c. elegans are rather distant relatives in evolutionary time: the two species split roughly million years ago. analysis of lengths of protein branches is indicative of accelerated evolution of daf- and additionally, analysis of ka/ks ratios between pairs of orthologs in c. briggsae and c. elegans suggests that the dauer pathway evolved faster since the two species diverged. the average ka/ks ratio for dauer pathway orthologs is versus for non-dauer tgf-β pathway genes. thus, the initial duplications and neo-functionalization occurred early in nematode evolution, but have been followed by further change in separate rhabditoid lineages, as different species experienced slightly different selection pressures for entry and persistence in their dauer forms. for example, c. elegans, unlike c. briggsae, is strongly induced to form dauers at temperatures higher than 26°c. overall, the dauer pathway represents an interesting example of rapidly evolving pathway neo-functionalization, developed as a lineage-specific adaptation towards the colonization of the environmental niche of the soil. the crucial question about the taxonomic origin of the tgf-β pathway has not been categorically answered yet. herein, we have identified tgf-β pathway components in t. adhaerens, the representative of the early diverging phylum placozoa, and the demosponge amphimedon queenslandica. choanoflagellata are the closest unicellular relatives of animals and possess some genes linked to metazoan development, for example a receptor tyrosine kinase – mbrtk. however, we have not been able to detect smads, tgf-β receptors, ligands, sara, chordin or gremlin in the genome of the unicellular choanoflagellate m. brevicollis, or more distantly related protozoans volvox carteri and naegleria gruberi. this indicates that the appearance of the tgf-β pathway was intrinsically linked to the emergence of earliest animals, and the pathway may thus be regarded as a key feature of the metazoan life forms. it is also rather striking that such an early diverging animal as trichoplax already possesses the complete functional pathway, including multiple smads, receptors, and ligands, as well as orthologs of chordin, gremlin and sara. we hypothesize that the single primeval common mediator/receptor activated smad functioned as a homo-dimer, and possessed the universal functionality of r-smads and the co-smad; i.e. it could be phosphorylated by the receptor/ligand complex, shuttle to the nucleus, interact with transcriptional co-activators via the mh domain and bind dna via the mh domain. as the number of ligands and receptors grew, the primeval smad duplicated and, through sub-functionalization, gave rise to two separate r-smads which from then on interact with non-overlapping sets of receptors. one of the r-smads duplicated again giving rise to a co-smad which enhanced the set of regulatory protein interactions, and possibly provided a way of integrating the signals from the two r-smad channels through competition for the available pool of co-smad molecules. the critical role of co-smad bioavailability is also suggested by its low duplicability; in the great majority of species there is only one co-smad. xenopus laevis is the notable exception having two genes xsmad4a and xsmad4b, but they are differentially expressed both in embryos and adult tissues. the fast diverging i-smad was the last addition to the pathway. it neofunctionalized to create a controlling negative feedback loop; i-smad transcription is induced by the pathway, the protein can bind the activated receptor complex, but lacking a terminal phosphorylation motif it does not propagate the signal. since it is not being used, over time the mh of the i-smad converted to a vestigial domain. it will be interesting to see if future genome projects of basal animals and closest extant unicellular relatives of animals will provide a proof of our single smad hypothesis. mammalian tgf-β type ii receptors are listed in the first row. type i receptors which make functional complexes with each type ii receptor are then listed, followed by the r-smads that the type i receptor activates. the ligands activating each receptor-smad group are then listed in a cumulative fashion.  the emergence of the tgf-β pathway coincided with appearance of the first animal species, and was most likely linked with duplications of the single primeval common mediator/receptor activated smad. this resulted in the creation of the ancestral eumetazoan repertoire of four smads, forming the basis of the pathway in the placozoa, the cnidaria, the arthropoda, and in the lophotrochozoa. after application of a formal speciation and duplication inference algorithm, we conclude that the diversification of smads and receptors in chordates is parsimoniously explained according to the 2r model, with additional rounds of duplications in teleost fishes. the nematoda posses a heavily modified pathway which evolution has been marked by accelerated sequence change. our multi-genome comparison and ancestral inference approach has implications extending beyond the tgf-β pathway. origins of other developmental signalling pathways, for example hox and hedgehog, are also being investigated using phylogenomic approaches. results obtained for all developmental signalling pathways should be integrated and compared with paleontological records and molecular clock data, to identify the molecular nature and timing of all major changes in the shared animal developmental toolkit, including those which gave rise to vertebrate innovations. 
the question of how genomic processes, such as gene duplication, give rise to co-ordinated organismal properties, such as emergence of new body plans, organs and lifestyles, is of importance in developmental and evolutionary biology. we conclude that the diversification of the tgf-β pathway can be parsimoniously explained according to the 2r model, with additional rounds of duplications in teleost fishes. after an investigation of genomes, we show that the emergence of the tgf-β pathway coincided with appearance of the first known animal species. we subsequently retrace duplications in ancestral genomes on the lineage leading to humans, as well as lineage-specific duplications, such as those which gave rise to novel smads and receptors in teleost fishes. our results challenge the view of well-conserved developmental pathways. herein, we focus on the diversification of the transforming growth factor-β pathway – one of the fundamental and versatile metazoan signal transduction engines. finally, we investigate duplications followed by accelerated evolution which gave rise to an atypical tgf-β pathway in free-living bacterial feeding nematodes of the genus rhabditis. the tgf-β signal transduction engine has expanded through gene duplication, continually adopting new functions, as animals grew in anatomical complexity, colonized new environments, and developed an active immune system.
 the question of how genomic processes, such as gene duplication, give rise to co-ordinated organismal properties, such as emergence of new body plans, organs and lifestyles, is of importance in developmental and evolutionary biology. herein, we focus on the diversification of the transforming growth factor-β pathway – one of the fundamental and versatile metazoan signal transduction engines.  after an investigation of genomes, we show that the emergence of the tgf-β pathway coincided with appearance of the first known animal species. the primordial pathway repertoire consisted of four smads and four receptors, similar to those observed in the extant genome of the early diverging tablet animal. we subsequently retrace duplications in ancestral genomes on the lineage leading to humans, as well as lineage-specific duplications, such as those which gave rise to novel smads and receptors in teleost fishes. we conclude that the diversification of the tgf-β pathway can be parsimoniously explained according to the 2r model, with additional rounds of duplications in teleost fishes. finally, we investigate duplications followed by accelerated evolution which gave rise to an atypical tgf-β pathway in free-living bacterial feeding nematodes of the genus rhabditis.  our results challenge the view of well-conserved developmental pathways. the tgf-β signal transduction engine has expanded through gene duplication, continually adopting new functions, as animals grew in anatomical complexity, colonized new environments, and developed an active immune system. 