BACKGROUND
the g protein-coupled receptor  superfamily is one of the largest families of integral transmembrane proteins in vertebrates and plays a dominant role in signal transduction in most eukaryotes.
gpcrs, which mediate most of the cellular responses through hormones, neurotransmitters and environmental stimulants are thus major drug targets, with approximately 36% of current clinical drugs targeting these receptors  <cit> .
in humans, there are around  <dig> genes coding for gpcrs, and we earlier classified them into five main grafs families: glutamate, rhodopsin, adhesion, frizzled and secretin  <cit> .
subsequent gpcr mining studies have suggested that grafs families are present in most bilaterian species  <cit> .
in addition, our earlier studies demonstrated that four of the five grafs families  are found in basal fungi, indicating that the glutamate, rhodopsin, adhesion, and frizzled families evolved before the divergence of metazoan lineages  <cit> .
although the four grafs families first evolved in the basal fungi, only a few sequences were unambiguous homologs of metazoan representatives  <cit> .
for example, only a few homologues of the rhodopsin family were found in basal fungi and rhodopsin gpcrs were not found in choanoflagellates  and filasterean capsaspora owczarzaki  <cit> .
moreover, these closest metazoan relatives are limited to only a few genes coding for adhesion and glutamate gpcr families.
these observations clearly indicate that the first large expansions of rhodopsin gpcrs, as well as other families of gpcrs, occurred at the early origins of metazoans.
this model is well supported by the recent genome release of amphimedon queenslandica , which belongs to one of the earliest diverging and oldest surviving phyletic branches of metazoa.
the draft genome as well as the transcriptome profiling of sponge indicated the presence of several rhodopsin-like gpcrs and an overall count of more than  <dig> gpcrs, including adhesion and glutamate family gpcrs  <cit> .
additional studies on some specific subsets of sponge gpcrs such as glutamate  <cit>  and frizzled  <cit>  provided further insights into the gpcr component in sponge.
taken together this suggests that the last common ancestor of metazoans possessed a complex gpcr system, perhaps with expansions within the rhodopsin family in comparison to pre-metazoan lineages like choanoflagellata  <cit>  and filasterea  <cit> .
furthermore, genome data of species that diverged after sponges provided valuable insights into the evolution of the gpcr superfamily.
previous mining of gpcrs in a cnidarian, nematostella vectensis and a placozoan, trichoplax adhaerens revealed that these pre-bilaterian metazoans contained a large gpcr repertoire with  <dig> and  <dig> gpcr coding genes, respectively  <cit> .
although several studies including genome-wide analysis of the sponge demonstrated the presence of several gpcrs, a comprehensive overview of sponge gpcr families is still lacking and their relationship to the versions found in eumetazoans and bilaterians is largely unknown.
this is important because sponges and the eumetazoans  are known to lack most of cell types found in bilaterians.
for example, sponges are simple pore bearing animals that lack gut, a nervous system and muscle, but constitute an internal network of canals and ciliated choanocyte chambers that pump water to extract food  <cit> .
placozoans  are flat animals consisting of a lower and upper epithelium, which sandwich layers of multinucleated fibre cells  <cit> .
similarly, nerves, sensory cells and muscle cells are apparently absent in placozoans.
in contrast, nematostella is regarded as one of the first animals possessing a nervous system.
in nematostella, an ectodermal and endodermal nerve net constituting of a simple and diffuse nervous system runs throughout the animal’s body  <cit> .
in order to better understand the components of the gpcr system and its evolution at the early origins of metazoa, we aimed to curate a complete set of gpcrs in sponge, as well as provide a comparative analysis with gpcrs found in eumetazoans  and bilaterians .
utilising the sponge genome, we sought to answer questions such as, 1) does one of the most ancient metazoan lineage have orthologs of mammalian gpcrs, 2) do sponges hold mammalian-like subfamily level classifications for each major grafs families, 3) are sponge gpcrs orthologous to those found in cnidarians and other pre-bilaterian lineages.
RESULTS
identification and classification of gpcrs in sponge
in order to generate a complete set of sponge gpcrs, we aligned the sponge proteome with hidden markov models  of the  <dig> families contained within the pfam database .
we retrieved all sequences that contained the pfam domains corresponding to the various gpcr families .
this initial screen identified  <dig> gpcr sequences belonging to the gpcr_a pfam clan .
these numbers are similar and comparable with previous studies where sponge gpcr sequences were identified  <cit> .
however, this initial list of gpcrs included fragments and possibly some false positives, and thus had to be refined before performing phylogenetic analysis to obtain stable and consistent topologies.
therefore, we examined these  <dig> gpcrs for the presence of seven transmembrane  helices using hmmtop and phobius servers.
to remove fragments and false positives we excluded the sequences having less than five or more than eight tm regions from the final dataset.
to cross verify the list of sponge gpcrs, we aligned each putative sponge gpcr sequence with our tagged human gpcrs using the standalone blastp program .
such stepwise processes led to the verification and categorisation of a final dataset containing  <dig> gpcrs.
a majority of these were categorised into four of the five main grafs families, including  <dig> rhodopsin ,  <dig> adhesion ,  <dig> glutamate  and three frizzled receptors.
however, we did not find secretin family receptors in the sponge genome.
it must be mentioned that an earlier report suggested that sponge has secretin family gpcrs  <cit>  possibly due to the presence of the hrm  domain in their n-termini, which is a usual characteristic of secretin gpcrs  <cit> .
however, secretin family gpcrs are mostly activated by peptide hormones, which to date have not been identified in the sponge genome  <cit> .
our finding that the sponge genome lack secretins is also consistent with earlier studies which proposed that secretin family descended from the adhesion family after the split of the cnidarians from other bilaterians and that the secretin gpcrs are a bilaterian innovation  <cit> .
considering that adhesion and secretin families belong to the same class of gpcrs  and encode a 7tm_ <dig> transmembrane domain, it is sometimes difficult to distinguish between the families and they can be wrongly assigned.
since adhesion is a parent family to secretin gpcrs and due to the lack of experimental support for the presence of secretin gpcr activity in sponges we here label these class b  receptors as adhesion family receptors.
nevertheless, the presence of an hrm domain in these adhesion gpcrs is intriguing and should prompt further experimental verifications to provide evidence for gpcr mediated hormonal activity in sponges.
interestingly, in addition to the grafs families, we identified  <dig> cyclic amp-like receptors , two intimal thickness-related-like receptors , and one lung-7tm-like receptor  in the sponge genome.
moreover, we identified a putative homolog of gpr <dig> , which in humans and other mammals is associated with ocular albinism.
in summary, the proportion of sponge gpcrs to the genome size is comparable to several other metazoans and that it also constitutes a large expansion within the rhodopsin family  <cit> .
the complete set of sponge gpcr sequences identified in this study is available in fasta format .
it must be noted here that the numbers provided in this study may vary from future predictions using subsequent genome assemblies of amphimedon, which may provide better resolution of the fragmentary sequences/regions.
to avoid possible confusion in subsequent paragraphs, a whole family is denoted using the corresponding family name in italics with an initial capital letter , while the homologs/members of a particular family are denoted as rhodopsins or adhesions or adhesion-like receptors.
phylogenetic verification of grafs topological classification
the human gpcr repertoire can be classified into five main groups  based on phylogenetic analysis  <cit> .
subsequent comparative phylogenetic studies in several vertebrate and invertebrate species have supported this classification system and established that gpcrs indeed formed distinct clusters corresponding to its five main families  <cit> .
to investigate whether the gpcrs identified in sponge also exhibit distinct phylogenetic clusters corresponding to the graf classes , we performed a bayesian phylogenetic analysis using all sponge gpcr sequences.
to test the robustness of the topology as well as to resolve the orthology relationships between the members in sponge and other bilaterians and pre-bilaterians, we expanded the dataset to include a few representative sequences from the trichoplax, nematostella and sea urchin gpcrs.
this set contained equal proportion of representatives from rhodopsin, glutamate and adhesion gpcr families.
the overall unrooted topology indicated that the rhodopsin, glutamate and adhesion gpcrs descend into separate and distinct clusters, whereas frizzled gpcrs were placed basal to the adhesion gpcr node .figure  <dig>
phylogenetic relationships of gpcrs identified in the sponge genome.
the tree topology shows distinct phylogenetic clusters belonging to the glutamate, rhodopsin, adhesion, and frizzled families of the grafs classification system.
the tree also includes a few representatives of gpcr families from other genomes.
the edges are colored according to the families, while the accession ids are colored differently for each species.
the illustration shows putative adhesion like gpcrs that lacks the conventional long n-termini.
however, these adhesion gpcrs contain the characteristic 7tm_ <dig> domain and placed basal to the major node that contains all the other adhesions included in the phylogenetic tree making.
sponge specific rhodopsin clusters  within the major rhodopsin cluster are highlighted.
rhodopsin receptor family
the human rhodopsin family gpcrs are classified into four major groups termed α-, β-, γ-, and δ that are divided into  <dig> major subfamilies.
some of these  <dig> subfamilies like amine and peptide binding rhodopsin family receptors are present and seem fairly conserved in most of the analysed bilaterians  <cit> .
similarly, in order to categorize sponge rhodopsins and explore their similarity to those found in other species, we aligned each sponge rhodopsin family sequence against a database of gpcrs containing complete repertories from human, sea urchin, trichoplax and nematostella.
the entire list of blast hits are provided in additional file  <dig>  furthermore, we performed blast searches against manually annotated and reviewed rhodopsin  gpcrs obtained from the swiss-prot database .
this reviewed list of rhodopsin gpcrs included most of the gpcrs from well characterised vertebrates, as well as from several well-known invertebrate model organisms.
from blastp search results we were unable to classify most of the sponge rhodopsin family sequences into any of the  <dig> known rhodopsin-like gpcrs subfamilies.
this is because sponge rhodopsins failed to satisfy our classification criteria that at least four of the first five hits must be from the same subfamily.
however, a few sponge rhodopsins had their best aligned hits  to beta-adrenergic receptors, serotonin and opsin family receptors, among others .
these blastp results were subsequently verified using bayesian and maximum likelihood  based phylogenetic analysis.
phylogenetic analysis using bayesian and ml methods was performed to resolve the relationships between human and sponge rhodopsin gpcr family sequences .
the results obtained from both tree building methods indicated that the largest differences between human and sponge gpcr repertoires was within the rhodopsin family.
although a few of the sponge rhodopsins were placed in the same branch containing human rhodopsins, they lacked reliable confidence value support from both tree making methods .
taken together, our results from the phylogenetic analysis suggest that the sponge rhodopsins lacked unambiguous orthologous relationships to any of the known human subfamilies .
this is consistent with the blast results that could not classify sponge rhodopsins into subfamilies.
moreover, it must be mentioned here that a few of the sponge rhodopsins had their top hits  as amine and opsin-like receptors in the blast search.
however, these sequences failed to form a coherent group with the human rhodopsin homologs.
instead, they clustered separately and are found scattered within the major node that grouped the sponge rhodopsins .
this distinctive repertoire of sponge rhodopsins had relatively high similarity between them and form five observable clusters .
here, we putatively labelled these sponge specific clusters as aqrho a to e.figure  <dig>
phylogenetic tree showing relationships between
rhodopsin
family gpcrs in sponge and human genomes.
the tree topology was inferred from bayesian analysis with a gamma correction using mrbayes software.
the phylogenetic tree is based only on the transmembrane region.
the mcmc analysis was used to test the robustness of the nodes and was supported by a non-parametric bootstrap analysis with  <dig> replicates.
the edges corresponding to human rhodopsin family gpcrs are highlighted in green.
edges containing sponge rhodopsins are highlighted in blue.
sponge specific clusters and are labeled as aqrho-a – aqrho-e .
the values indicated at major branches are posterior probability values from bayesian analysis and percentage bootstrap values from maximum likelihood analysis.
red asterisk symbol denotes sequences that have at least four of the top five hits as beta-adrenergic, serotonin and opsin family receptors  in our blast search.
however, they did not seem to form a coherent group with the human rhodopsins.
scale bars indicate phylogenetic distance as number of substitutions per site.
phylogenetic relationships between sponge rhodopsins, the eumetazoans  and sea-urchin genomes are provided in additional file  <dig>
since the phylogenetic distance between the gpcr dataset representing human and sponge was large, we wanted to investigate whether similar phylogenetic relationships existed between sponge and other closely related species.
therefore, we extended our study to three additional species having completely sequenced genomes.
this included two non-bilaterian animals from the eumetazoan lineage, the placozoan trichoplax and the cnidarian nematostella, as well as the deuterostome bilaterian, sea urchin .
the phylogenetic trees indicated a similar topology wherein sponge rhodopsins lack orthologous relationships to those found in nematostella, trichoplax and sea urchin .
adhesion receptor family
the human genome contains  <dig> adhesion receptors that phylogenetically cluster into eight main groups , with vlgr <dig> placed as an out-group.
earlier studies demonstrated that potential homologs of genes belonging to families i, iii, iv, v, viii and vlgr <dig> are present in most invertebrates, whereas families ii, vi and vii are more likely to be vertebrate innovations  <cit> .
to explore whether sponge adhesions show homologous relationships to any known adhesion gpcr groups, we included  <dig> human adhesions and all identified sponge adhesions for phylogenetic analysis.
furthermore, we included adhesions from other metazoans to explore their relationship with sponge adhesions.
phylogenetic analysis revealed that a few sponge adhesions were placed basal to the node that contained human adhesions belonging to family viii .
this tree topology was better supported when the analysis was restricted to only human and sponge adhesions .
also, the sponge adhesion sequence aq <dig> clustered basal to the node containing human group i and group ii adhesion sequences .
additionally, two more sequences from sea urchin  and nematostella  were placed in the same node containing human group i and group ii adhesions.
this implies that these sequences are putative ancestral representatives of groups i/ii .
a closer examination of the phylogenetic relationships showed that there were several adhesions from sponge, trichoplax and nematostella placed in a major node that contained human adhesions from groups vi and vii .
the remaining sponge adhesions are most likely sponge specific, since they clustered separately from any known adhesion groups.
this observation was consistent with other analysed metazoans, where most of the adhesions from sea urchin and other genomes clustered separately from the human counterparts .figure  <dig>
phylogenetic relationships between
adhesion
family gpcrs in sponge and other genomes.
the color scheme for the branches is according to species used.
the posterior probability values > <dig> ;  <dig>  - <dig>  and  <dig>  to  <dig>  are highlighted in filled green, orange and grey circles, respectively.
accession numbers for most of the sea urchin adhesions were removed from the final representation for display reasons.
human adhesion gpcr groups i to viii are heighted in grey.
another noteworthy observation was that some of the sponge specific adhesions have short n-termini and lack gpcr proteolytic site .
however, these protein transcripts contained the core 7tm_ <dig> domain region, characteristic to all adhesion gpcrs.
it is possible that these adhesions were incompletely modelled at the n-termini due to sequencing errors.
an alternative explanation is that these sponge adhesions may truly lack a gps site and the n-terminal domains, since the divergence is also reflected in the transmembrane helices that were utilized for phylogenetic tree making.
these sequences clustered separately from rest of the sponge adhesion gpcrs .
however, future experimental verification, as well as mining of adhesions in other sponge genomes, is required to confirm these attributes of sponge adhesions.
this might also clarify whether the short n-termini are more prevalent in ancient adhesion gpcrs and the long n-termini was later gained due to addition and shuffling of key domains during the course of metazoan evolution.
although, a few sponge adhesions lack a gps site and n-terminal domains, the rest show diverse domain architecture similar to that observed in other metazoan adhesions.
the gps domain, a common cleavage site for many members of this family is present in  <dig> out of  <dig> sponge adhesions.
another interesting feature was the presence of a hrm domain in at least eight of the sponge adhesions although hormones have not been reported in sponges .
intriguingly, we could also identify sponge adhesions  that harbour multiple repeats of the scavenger receptor cysteine-rich protein  domain .
to the best of our knowledge identification of srcr repeats is unique to adhesion gpcrs and it is worth mentioning that srcr repeats are often associated with immune system functions in vertebrates  <cit> .figure  <dig>
n-terminal domain architecture of a selection of sponge
adhesion
gpcrs.
the domains were identified by aligning sponge adhesions to the latest version of pfam library.
few adhesion gpcr sequences that lack n-terminal domains are not shown.
the domains shown in the figure include; 7tm: seven-transmembrane domain, duf: domain of unknown function, egf: epidermal growth factor-like domain, fn3: fibronectin type iii domain, gps: gpcr proteolytic site domain, hrm: hormone receptor domain, ig/ig_2/ig_3: immunoglobulin domains, i-set: immunoglobulin i-set domain, snare: soluble n-ethylmaleimide-sensitive factor  attachment protein  receptor domain, srcr: scavenger receptor cysteine-rich domain, v-set: immunoglobulin v-set domain.
glutamate receptor family
glutamate receptors  are crucial modulators of neurotransmission, and in humans there are  <dig> receptors consisting of eight metabotropic glutamate receptors , two gababrs, the calcium-sensing receptor , the sweet and umami taste receptors , gprc6a and seven orphan receptors  <cit> .
phylogenetic analysis of the glutamate family members from human and sponge  revealed that sponge had seven glrs homologous to human metabotropic glutamate receptors .
in addition, phylogenetic analysis revealed that two sponge glrs clustered with human gabab receptors and another three were placed on the same node containing gpr <dig> and gpr <dig>  but with a low posterior probability support .
to test the robustness of these relationships, we included glutamate gpcrs from nematostella, trichoplax and sea urchin.
an overall unrooted tree obtained from a large dataset demonstrated that among the  <dig> indentified sponge glrs, only seven are homologous to grms, while the rest were sponge specific receptors .
however, the sponge glrs that had similarity to gabab and two other orphans  failed to give stable or consistent topology in a larger dataset and clustered separately from the known glutamate receptors, suggesting they are divergent from other metazoan counterparts.figure  <dig>
phylogenetic tree showing relationship between
glutamate
family gpcrs in sponge and human.
the edges containing human glutamate family gpcrs are highlighted in green, while the edges containing sponge glutamate are highlighted in blue.
phylogenetic relationships between sponge glutamate, the eumetazoans  and sea-urchin genomes are provided in additional file  <dig>
frizzled receptor family
the sponge proteome dataset contained three full length members of the frizzled gpcr family.
a comparative phylogenetic analysis with frizzled receptors from sponge and other metazoan genomes was performed.
for the phylogenetic tree construction, we also included the closely related smoothened gpcr family members from human and other metazoans.
phylogenetic relationships revealed that sponge frizzled gpcrs are fast evolving or divergent from other metazoan counterparts  .
two sponge frizzled receptors were placed basal to the node that contained human fzd <dig>  fzd <dig> receptors.
also, one frizzled receptor each from other analysed metazoans was placed in the same node with human fzd <dig>  fzd <dig> receptors.
interestingly, one sponge frizzled-like receptor was placed basal to the smoothened receptor cluster .
this finding was consistent with a recent study that identified a smoothened receptor in sponges  <cit> .
it must be mentioned here that our initial screen for frizzled gpcrs identified  <dig> frizzled-like gpcr sequences, of which six were removed due to fragmentary models that contained less than  <dig> tm regions.
similarly, an earlier study identified eight frizzled- like gpcrs in the sponge genome  <cit> .
however, a few of these seem to be incompletely modeled and were not included in the final sponge gpcr dataset for better handling of the msa  data for subsequent phylogenetic studies.
other gpcr families
our analysis revealed that the sponge proteome dataset also contains members of other gpcr families that do not belong to the grafs classification system.
these included camp-like, intimal thickness-related receptor like , lung 7tm receptor-like and ocular albinism like  receptors.
subsequent cross-genome phylogenetic analysis was performed on these gpcr families with the corresponding family members obtained from other species .
protein sequences belonging to these gpcr families were obtained from nematostella, trichoplax, and sea urchin using pfam hmm profile based searches.
corresponding sequences from human were obtained from the swiss-prot database.
overall phylogenetic tree topology indicated the presence of gpr143-like, lung 7tm-like and intimal thickness-related receptors in sponge.
these sponge sequences clustered with their corresponding family sequences obtained from other species .
they formed separate clusters in the phylogenetic analysis with high confidence support.
phylogenetic analysis also revealed that the  <dig> camp-like receptors identified in the sponge genome form a separate cluster with high confidence support .
these  <dig> camp-like sequences contained the core region encoded by a pfam domain  characteristic to the dictyostelium camp gpcr family.
similarly, pairwise similarity search performed using these sequences as queries clearly demonstrated that camp family sequences are among the top hits.
however, sponge camp-like receptors clustered separately from the dictyostelium camp gpcr sequences, suggesting that they are quite divergent or fast evolving.
it would thus be interesting to experimentally verify whether the camp-like receptors in the sponge genome have analogous roles to the previously known functions of dictyostelium camp gpcr family.
discussion
the draft genome, as well as the transcriptome of amphimedon queenslandica , revealed the genetic complexity of this primitive animal in detail and catalogued the presence of several crucial gene families, including gpcrs and other signaling system components  <cit> .
however, a detailed curation of sponge gpcr families/subfamilies and phylogenetic comparisons with those versions found in eumetazoans and bilaterians needs to be performed to better understand the gpcr component in sponges from an evolutionary perspective.
in this study, we curated gpcrs in the sponge genome and have phylogenetically compared the receptors to those found in other metazoans.
our hmm based search approach and phylogenetic analysis demonstrates that sponge contains four of the five main grafs families, namely, rhodopsin, adhesion, glutamate and frizzled.
it is noteworthy that the sponge genome encodes one of the most ancient metazoan lineage specific expansions of the rhodopsin family of gpcrs  <cit> .
moreover, our phylogenetic analysis with pre-bilaterian metazoans homologs clearly reveals that the rhodopsin family has undergone significant diversifications in these pre-bilaterian metazoans.
possible explanations could be that they diversified due to the evolution of diverse morphological characteristics and adaptations of these species during the course of the early metazoan evolution  <cit> .
this is also evident in other gpcr families, where phylogenetic analysis revealed that most members of the adhesion and glutamate families grouped into sponge-specific clusters.
in summary, the study describes the sponge gpcr gene families in detail and our phylogenetic comparisons postulates a significantly diversified subset of gpcrs in sponge.
sponge rhodopsins
comparative phylogenetic analysis demonstrates that sponge rhodopsin family gpcrs do not share orthologous relationship with those found in eumetazoans and other bilaterians .
in addition, sponge rhodopsin-like gpcrs form five distinct clusters that are most likely sponge specific .
here, we putatively labelled these sponge specific clusters as aqrho-a to e. it must be mentioned here that several rhodopsins belonging to these sponge specific clusters are contained in the same contig region and located adjacent to each other.
several of the flanking sequences are found as many as a cluster of  <dig> to  <dig> sequences and share relatively high pairwise protein sequence identities ranging from 51% to 74%.
this suggests that the expansions of sponge rhodopsins are possibly driven by gene duplication events and it seems most likely true for other pre-bilaterian metazoans as well.
however, to further examine whether these sequences can be classified into any of the known  <dig> rhodopsin subfamilies, we performed a blastp search against the swiss-prot database.
the results obtained from the blast search showed that a few sponge rhodopsin-like gpcrs had their top hits as adrenergic, serotonin, dopamine, and opsin-like receptors .
this is in line with the draft genome report of sponge , which demonstrated the presence of serotonin and dopamine-like receptors  <cit> .
this is also consistent with a recent study that identified adrenergic-like receptors in sponges  <cit> .
although the pairwise similarity search results suggest the presence of these putative receptors, our phylogenetic analysis was unable to reveal any clear orthologous relationships of the sponge rhodopsins to the bilaterian counterparts.
a possible explanation could be that sponge rhodopsins have diverged considerably, possibly based on sponge specific physiology and behavior  <cit> .
this hypothesis is plausible because species such as trichoplax and nematostella, belonging to the eumetazoan lineage and diverged from sponges later in the metazoan species tree, contain rhodopsin-like gpcrs more similar to bilaterians than sponges.
in fact, earlier studies provided evidence that eumetazoans do contain putative orthologues for some of the amine and peptide binding receptors  <cit> .
the recent genome release of mnemiopsis leidyi suggests that ctenophores are the sister group to the rest of the extant animals, including sponges, and that components of neuronal signaling were already present in an early metazoan ancestor  <cit> .
also, the same study proposed that components of neuronal signaling have undergone major loss and gain events in pre-bilaterian lineages  <cit> .
considering these observations, it is likely that rhodopsin family gpcrs expanded independently in these species and may perform diverse functions based on the morphological characteristic of the organism  <cit> .
also, it is possible that these large expansions may have evolved to perform neuronal functions in ctenophores and cnidarians, and that this ability is secondarily lost in sponges and placozoans during the course of metazoan evolution.
nonetheless, at present it is evident that sponge rhodopsins expanded due to gene duplication events and seems to have diverged considerably from those found in other pre-bilaterians and bilaterians.
further comparative genomics, as well as developmental/neurobiological studies would be essential to understand the roles of rhodopsin family gpcrs that predated the divergence of bilateria.
sponge adhesions
the repertoire of adhesion gpcrs  in the sponge genome is one of the first expansions within the adhesion gpcr family at the roots of metazoan evolution.
in comparison to the sponge, the closest unicellular metazoan relatives such as salpingoeca rosetta and capsaspora owczarzaki contained only a few genes  coding for adhesion gpcrs.
furthermore, it must be highlighted here that our initial hmm search in the sponge genome identified a staggering  <dig> genes that encoded a 7tm_ <dig>  domain.
however, we removed  <dig> of those from our phylogenetic analysis, as they were lacking three or more helices.
therefore, it would be interesting to explore whether the subsequent improved versions of the sponge genome contain more full length adhesion gpcrs.
collectively, this may suggest that the expansions of adhesion gpcrs at the early origins of metazoans, relative to unicellular relatives, were possibly driven by the evolution of multicellularity in early metazoans since cell-cell adhesion is one of the major factors involved in driving multicellularity  <cit> .
another noteworthy observation is that a few of the sponge adhesion gpcrs are found to be phylogenetically similar to vertebrate adhesion gpcrs belonging to group i/ii .
this suggests that some of the adhesion gpcr subfamilies may have diverged early in metazoan evolution and would have later evolved or co-opted for more specialised functions, which we observe in bilaterians.
of note, previous studies suggested that group i and group ii adhesion gpcrs have potential roles in neurogenesis and migration  <cit> .
also, in contrast to rhodopsins, which mostly bind hormones and neurotransmitters, the identified ligands of adhesion gpcrs are mostly single-pass membrane proteins  <cit> .
for instance, lphn <dig>  a group i adhesion, interacts with teneurin- <dig>  flrt1/ <dig> and neurexin i-alpha & beta, which are all single-pass membrane proteins with a variety of n-terminal domains  <cit> .
a blastp search using these single-pass membrane proteins  as queries against the sponge proteome obtained few reliable hits .
interestingly, these hits contained a single tm helix at the c-terminal end, n-terminal functional domains like laminin g-like, laminin egf-like , and cadherins that are widely known to influence cell-cell adhesion, cell differentiation and migration  <cit> .
this implies that some of the sponge adhesions might interact with single pass membrane proteins to aid cell-cell adhesion in sponges.
thus, it would be interesting to explore the functional roles of ancestral group i/ii like adhesion gpcrs in sponge and eumetazoans.
although the sponge genome has a few adhesion gpcrs that are somewhat similar to vertebrate adhesions and placed basal to families i, ii and viii, most of them formed a distinct cluster and are likely to be sponge specific.
intriguingly, at least eight adhesion receptors contain a hormone receptor domain  in their n-termini, a common characteristic of secretin gpcrs .
the absence of secretin gpcrs in the sponge and the early presence of an hrm domain in adhesions supports our previous hypothesis that the secretin family descended from adhesion gpcrs in an event somewhere during the split of cnidarians  <cit> .
to the best of our knowledge, sponge adhesion-like receptors are one of the most ancient gpcrs containing a hormone-binding domain.
the hrm domain is essential for secretin gpcr activity and is conserved in all the secretin receptors.
this suggests that hrm domain containing adhesion gpcrs, found before the divergence of bilateria, may have a possible role analogous to that observed in secretin family of gpcrs  <cit> .
however, this hypothesis needs further experimental verification since the presence of the hrm domain is surprising due to the lack of hormones in the sponge genome.
another distinctive feature of the sponge adhesion gpcrs is the absence of a gps domain in as many as  <dig> of the adhesions.
it is important to note that the intra-molecular processing at a gps site in the gpcr autoproteolysis-inducing domain , proximal to the first transmembrane helix is attributed to several factors including signalling, membrane trafficking, as well as for the formation of heterodimeric gpcr complexes  <cit> .
however, the absence of a gps domain in some sponge adhesions might imply that the evolutionary requirement for the conservation of the cleavage site is not very stringent or that the gps site is more essential to those adhesions with long n-termini.
moreover, it is possible that the missing gps domains are an outcome of incomplete or missing regions in the current sponge genome draft assembly.
the subsequent draft assemblies may help provide a complete picture of adhesion gpcrs in sponges.
sponge glutamate receptor family
cross-genome phylogenetic analysis between the sponge and human  suggests that the sponge has homologous representatives for metabotropic glutamate and gaba-like receptors with a similar n-terminal domain architecture, commonly observed in bilaterian counterparts.
also, we attempted to search for components necessary for a gaba shunt, a process by which gaba is produced in animal cells  <cit> .
by homology search methods we found strong evidence for the presence of gaba-t , which catalyses the synthesis of l-glutamate and glutamate decarboxylase, which catalyzes the synthesis of gaba.
this is in line with an earlier study that identified glutamate decarboxylase in sponges  <cit> .
however, these results are not surprising because gaba and metabotropic glutamate receptor-like gpcrs were identified previously in sponges, as well as in the amoeba dictyostelium discoideum that evolved well before the divergence of metazoans  <cit> .
it is interesting to note that previous studies provided potential insights into the role of glutamate receptors and neurotransmitter glutamate in non-neuronal cells  <cit> .
for example, elliot and leys  <cit>  showed that sponges, which lack neurons, use metabotropic glutamate and gaba receptor signaling for organized contractions of the sponge canal system.
these roles of glutamate receptors are rather distinctive from the commonly known functions of glutamate gpcrs in a synaptic environment.
interestingly, there is growing evidence that challenges theories proposing the early origins of synapses and first components of a protosynapse somewhere close to the origins of cnidarians.
a current hypothesis suggests that nerve cell components evolved at the very origins of metazoans and have undergone major loss and gain events in pre-bilaterian lineages  <cit> .
also, a few studies have suggested that glutamate is found in non-excitable cells, providing insights for glutamate to function beyond its general role acting as a neurotransmitter .
therefore, the presence of glutamate gpcrs in almost all pre-bilaterians including sponges shows the dynamic nature of glutamate gpcrs, which seem to be functional both in synaptic rich and synaptic free environments that prevailed before the divergence of bilateria.
CONCLUSIONS
we present the first overall analysis of the gpcr repertoire in the sponge genome and have compared this to the eumetazoans and bilaterian versions.
in summary, the sponge gpcr repertoire contains four of the five gpcr grafs families, as well as other gpcr gene families including camp-like receptors, intimal thickness-related receptor like , lung 7tm and gpr <dig>  receptors.
on the other hand, sponge lacks many of the classical mammalian-like sensory receptors including the olfactory receptors that are widely found in several bilaterians  <cit> .
moreover, our phylogenetic comparison reveals that the sponge rhodopsin family does not share orthologous relationships with eumetazoan and bilaterian counterparts.
this might imply that subfamily level diversifications of rhodopsins, common in several bilaterians, likely became more pronounced later in the metazoan evolution, as indicated by the presence of some of the subfamilies in nematostella and trichoplax  <cit> .
nonetheless, the sponge encodes one of the first expansions of rhodopsin and adhesion family gpcrs early in metazoan evolution.
also, unexpectedly, sponge adhesions encodes hormone binding domains, although hormone-like peptides are yet to be found in sponges.
similarly, the long n-termini of a few sponge adhesions contain diverse domain architectures commonly observed in other metazoans.
in conclusion, our analysis provides a wider framework for understanding the sponge gpcrs and to relate them to versions found in other pre-bilaterians and bilaterians.
furthermore, our data set comparisons provide a platform to perform more comparative genomic studies for understanding gpcr biology and signal transduction at the early origins of multicellularity.

 the g protein-coupled receptor superfamily is one of the largest families of integral transmembrane proteins in vertebrates and plays a dominant role in signal transduction in most eukaryotes. gpcrs, which mediate most of the cellular responses through hormones, neurotransmitters and environmental stimulants are thus major drug targets, with approximately 36% of current clinical drugs targeting these receptors. in humans, there are around genes coding for gpcrs, and we earlier classified them into five main grafs families: glutamate, rhodopsin, adhesion, frizzled and secretin. subsequent gpcr mining studies have suggested that grafs families are present in most bilaterian species. in addition, our earlier studies demonstrated that four of the five grafs families are found in basal fungi, indicating that the glutamate, rhodopsin, adhesion, and frizzled families evolved before the divergence of metazoan lineages. although the four grafs families first evolved in the basal fungi, only a few sequences were unambiguous homologs of metazoan representatives. for example, only a few homologues of the rhodopsin family were found in basal fungi and rhodopsin gpcrs were not found in choanoflagellates and filasterean capsaspora owczarzaki. moreover, these closest metazoan relatives are limited to only a few genes coding for adhesion and glutamate gpcr families. these observations clearly indicate that the first large expansions of rhodopsin gpcrs, as well as other families of gpcrs, occurred at the early origins of metazoans. this model is well supported by the recent genome release of amphimedon queenslandica, which belongs to one of the earliest diverging and oldest surviving phyletic branches of metazoa. the draft genome as well as the transcriptome profiling of sponge indicated the presence of several rhodopsin-like gpcrs and an overall count of more than gpcrs, including adhesion and glutamate family gpcrs. additional studies on some specific subsets of sponge gpcrs such as glutamate and frizzled provided further insights into the gpcr component in sponge. taken together this suggests that the last common ancestor of metazoans possessed a complex gpcr system, perhaps with expansions within the rhodopsin family in comparison to pre-metazoan lineages like choanoflagellata and filasterea. furthermore, genome data of species that diverged after sponges provided valuable insights into the evolution of the gpcr superfamily. previous mining of gpcrs in a cnidarian, nematostella vectensis and a placozoan, trichoplax adhaerens revealed that these pre-bilaterian metazoans contained a large gpcr repertoire with and gpcr coding genes, respectively. although several studies including genome-wide analysis of the sponge demonstrated the presence of several gpcrs, a comprehensive overview of sponge gpcr families is still lacking and their relationship to the versions found in eumetazoans and bilaterians is largely unknown. this is important because sponges and the eumetazoans are known to lack most of cell types found in bilaterians. for example, sponges are simple pore bearing animals that lack gut, a nervous system and muscle, but constitute an internal network of canals and ciliated choanocyte chambers that pump water to extract food. placozoans are flat animals consisting of a lower and upper epithelium, which sandwich layers of multinucleated fibre cells. similarly, nerves, sensory cells and muscle cells are apparently absent in placozoans. in contrast, nematostella is regarded as one of the first animals possessing a nervous system. in nematostella, an ectodermal and endodermal nerve net constituting of a simple and diffuse nervous system runs throughout the animal’s body. in order to better understand the components of the gpcr system and its evolution at the early origins of metazoa, we aimed to curate a complete set of gpcrs in sponge, as well as provide a comparative analysis with gpcrs found in eumetazoans and bilaterians. utilising the sponge genome, we sought to answer questions such as, 1) does one of the most ancient metazoan lineage have orthologs of mammalian gpcrs, 2) do sponges hold mammalian-like subfamily level classifications for each major grafs families, 3) are sponge gpcrs orthologous to those found in cnidarians and other pre-bilaterian lineages.  identification and classification of gpcrs in sponge in order to generate a complete set of sponge gpcrs, we aligned the sponge proteome with hidden markov models of the families contained within the pfam database. we retrieved all sequences that contained the pfam domains corresponding to the various gpcr families. this initial screen identified gpcr sequences belonging to the gpcr_a pfam clan. these numbers are similar and comparable with previous studies where sponge gpcr sequences were identified. however, this initial list of gpcrs included fragments and possibly some false positives, and thus had to be refined before performing phylogenetic analysis to obtain stable and consistent topologies. therefore, we examined these gpcrs for the presence of seven transmembrane helices using hmmtop and phobius servers. to remove fragments and false positives we excluded the sequences having less than five or more than eight tm regions from the final dataset. to cross verify the list of sponge gpcrs, we aligned each putative sponge gpcr sequence with our tagged human gpcrs using the standalone blastp program. such stepwise processes led to the verification and categorisation of a final dataset containing gpcrs. a majority of these were categorised into four of the five main grafs families, including rhodopsin, adhesion, glutamate and three frizzled receptors. however, we did not find secretin family receptors in the sponge genome. it must be mentioned that an earlier report suggested that sponge has secretin family gpcrs possibly due to the presence of the hrm domain in their n-termini, which is a usual characteristic of secretin gpcrs. however, secretin family gpcrs are mostly activated by peptide hormones, which to date have not been identified in the sponge genome. our finding that the sponge genome lack secretins is also consistent with earlier studies which proposed that secretin family descended from the adhesion family after the split of the cnidarians from other bilaterians and that the secretin gpcrs are a bilaterian innovation. considering that adhesion and secretin families belong to the same class of gpcrs and encode a 7tm_ transmembrane domain, it is sometimes difficult to distinguish between the families and they can be wrongly assigned. since adhesion is a parent family to secretin gpcrs and due to the lack of experimental support for the presence of secretin gpcr activity in sponges we here label these class b receptors as adhesion family receptors. nevertheless, the presence of an hrm domain in these adhesion gpcrs is intriguing and should prompt further experimental verifications to provide evidence for gpcr mediated hormonal activity in sponges. interestingly, in addition to the grafs families, we identified cyclic amp-like receptors, two intimal thickness-related-like receptors, and one lung-7tm-like receptor in the sponge genome. moreover, we identified a putative homolog of gpr, which in humans and other mammals is associated with ocular albinism. in summary, the proportion of sponge gpcrs to the genome size is comparable to several other metazoans and that it also constitutes a large expansion within the rhodopsin family. the complete set of sponge gpcr sequences identified in this study is available in fasta format. it must be noted here that the numbers provided in this study may vary from future predictions using subsequent genome assemblies of amphimedon, which may provide better resolution of the fragmentary sequences/regions. to avoid possible confusion in subsequent paragraphs, a whole family is denoted using the corresponding family name in italics with an initial capital letter, while the homologs/members of a particular family are denoted as rhodopsins or adhesions or adhesion-like receptors. phylogenetic verification of grafs topological classification the human gpcr repertoire can be classified into five main groups based on phylogenetic analysis. subsequent comparative phylogenetic studies in several vertebrate and invertebrate species have supported this classification system and established that gpcrs indeed formed distinct clusters corresponding to its five main families. to investigate whether the gpcrs identified in sponge also exhibit distinct phylogenetic clusters corresponding to the graf classes, we performed a bayesian phylogenetic analysis using all sponge gpcr sequences. to test the robustness of the topology as well as to resolve the orthology relationships between the members in sponge and other bilaterians and pre-bilaterians, we expanded the dataset to include a few representative sequences from the trichoplax, nematostella and sea urchin gpcrs. this set contained equal proportion of representatives from rhodopsin, glutamate and adhesion gpcr families. the overall unrooted topology indicated that the rhodopsin, glutamate and adhesion gpcrs descend into separate and distinct clusters, whereas frizzled gpcrs were placed basal to the adhesion gpcr node.figure phylogenetic relationships of gpcrs identified in the sponge genome. the tree topology shows distinct phylogenetic clusters belonging to the glutamate, rhodopsin, adhesion, and frizzled families of the grafs classification system. the tree also includes a few representatives of gpcr families from other genomes. the edges are colored according to the families, while the accession ids are colored differently for each species. the illustration shows putative adhesion like gpcrs that lacks the conventional long n-termini. however, these adhesion gpcrs contain the characteristic 7tm_ domain and placed basal to the major node that contains all the other adhesions included in the phylogenetic tree making. sponge specific rhodopsin clusters within the major rhodopsin cluster are highlighted. rhodopsin receptor family the human rhodopsin family gpcrs are classified into four major groups termed α-, β-, γ-, and δ that are divided into major subfamilies. some of these subfamilies like amine and peptide binding rhodopsin family receptors are present and seem fairly conserved in most of the analysed bilaterians. similarly, in order to categorize sponge rhodopsins and explore their similarity to those found in other species, we aligned each sponge rhodopsin family sequence against a database of gpcrs containing complete repertories from human, sea urchin, trichoplax and nematostella. the entire list of blast hits are provided in additional file furthermore, we performed blast searches against manually annotated and reviewed rhodopsin gpcrs obtained from the swiss-prot database. this reviewed list of rhodopsin gpcrs included most of the gpcrs from well characterised vertebrates, as well as from several well-known invertebrate model organisms. from blastp search results we were unable to classify most of the sponge rhodopsin family sequences into any of the known rhodopsin-like gpcrs subfamilies. this is because sponge rhodopsins failed to satisfy our classification criteria that at least four of the first five hits must be from the same subfamily. however, a few sponge rhodopsins had their best aligned hits to beta-adrenergic receptors, serotonin and opsin family receptors, among others. these blastp results were subsequently verified using bayesian and maximum likelihood based phylogenetic analysis. phylogenetic analysis using bayesian and ml methods was performed to resolve the relationships between human and sponge rhodopsin gpcr family sequences. the results obtained from both tree building methods indicated that the largest differences between human and sponge gpcr repertoires was within the rhodopsin family. although a few of the sponge rhodopsins were placed in the same branch containing human rhodopsins, they lacked reliable confidence value support from both tree making methods. taken together, our results from the phylogenetic analysis suggest that the sponge rhodopsins lacked unambiguous orthologous relationships to any of the known human subfamilies. this is consistent with the blast results that could not classify sponge rhodopsins into subfamilies. moreover, it must be mentioned here that a few of the sponge rhodopsins had their top hits as amine and opsin-like receptors in the blast search. however, these sequences failed to form a coherent group with the human rhodopsin homologs. instead, they clustered separately and are found scattered within the major node that grouped the sponge rhodopsins. this distinctive repertoire of sponge rhodopsins had relatively high similarity between them and form five observable clusters. here, we putatively labelled these sponge specific clusters as aqrho a to e.figure phylogenetic tree showing relationships between rhodopsin family gpcrs in sponge and human genomes. the tree topology was inferred from bayesian analysis with a gamma correction using mrbayes software. the phylogenetic tree is based only on the transmembrane region. the mcmc analysis was used to test the robustness of the nodes and was supported by a non-parametric bootstrap analysis with replicates. the edges corresponding to human rhodopsin family gpcrs are highlighted in green. edges containing sponge rhodopsins are highlighted in blue. sponge specific clusters and are labeled as aqrho-a – aqrho-e. the values indicated at major branches are posterior probability values from bayesian analysis and percentage bootstrap values from maximum likelihood analysis. red asterisk symbol denotes sequences that have at least four of the top five hits as beta-adrenergic, serotonin and opsin family receptors in our blast search. however, they did not seem to form a coherent group with the human rhodopsins. scale bars indicate phylogenetic distance as number of substitutions per site. phylogenetic relationships between sponge rhodopsins, the eumetazoans and sea-urchin genomes are provided in additional file since the phylogenetic distance between the gpcr dataset representing human and sponge was large, we wanted to investigate whether similar phylogenetic relationships existed between sponge and other closely related species. therefore, we extended our study to three additional species having completely sequenced genomes. this included two non-bilaterian animals from the eumetazoan lineage, the placozoan trichoplax and the cnidarian nematostella, as well as the deuterostome bilaterian, sea urchin. the phylogenetic trees indicated a similar topology wherein sponge rhodopsins lack orthologous relationships to those found in nematostella, trichoplax and sea urchin. adhesion receptor family the human genome contains adhesion receptors that phylogenetically cluster into eight main groups, with vlgr placed as an out-group. earlier studies demonstrated that potential homologs of genes belonging to families i, iii, iv, v, viii and vlgr are present in most invertebrates, whereas families ii, vi and vii are more likely to be vertebrate innovations. to explore whether sponge adhesions show homologous relationships to any known adhesion gpcr groups, we included human adhesions and all identified sponge adhesions for phylogenetic analysis. furthermore, we included adhesions from other metazoans to explore their relationship with sponge adhesions. phylogenetic analysis revealed that a few sponge adhesions were placed basal to the node that contained human adhesions belonging to family viii. this tree topology was better supported when the analysis was restricted to only human and sponge adhesions. also, the sponge adhesion sequence aq clustered basal to the node containing human group i and group ii adhesion sequences. additionally, two more sequences from sea urchin and nematostella were placed in the same node containing human group i and group ii adhesions. this implies that these sequences are putative ancestral representatives of groups i/ii. a closer examination of the phylogenetic relationships showed that there were several adhesions from sponge, trichoplax and nematostella placed in a major node that contained human adhesions from groups vi and vii. the remaining sponge adhesions are most likely sponge specific, since they clustered separately from any known adhesion groups. this observation was consistent with other analysed metazoans, where most of the adhesions from sea urchin and other genomes clustered separately from the human counterparts.figure phylogenetic relationships between adhesion family gpcrs in sponge and other genomes. the color scheme for the branches is according to species used. the posterior probability values >; - and to are highlighted in filled green, orange and grey circles, respectively. accession numbers for most of the sea urchin adhesions were removed from the final representation for display reasons. human adhesion gpcr groups i to viii are heighted in grey. another noteworthy observation was that some of the sponge specific adhesions have short n-termini and lack gpcr proteolytic site. however, these protein transcripts contained the core 7tm_ domain region, characteristic to all adhesion gpcrs. it is possible that these adhesions were incompletely modelled at the n-termini due to sequencing errors. an alternative explanation is that these sponge adhesions may truly lack a gps site and the n-terminal domains, since the divergence is also reflected in the transmembrane helices that were utilized for phylogenetic tree making. these sequences clustered separately from rest of the sponge adhesion gpcrs. however, future experimental verification, as well as mining of adhesions in other sponge genomes, is required to confirm these attributes of sponge adhesions. this might also clarify whether the short n-termini are more prevalent in ancient adhesion gpcrs and the long n-termini was later gained due to addition and shuffling of key domains during the course of metazoan evolution. although, a few sponge adhesions lack a gps site and n-terminal domains, the rest show diverse domain architecture similar to that observed in other metazoan adhesions. the gps domain, a common cleavage site for many members of this family is present in out of sponge adhesions. another interesting feature was the presence of a hrm domain in at least eight of the sponge adhesions although hormones have not been reported in sponges. intriguingly, we could also identify sponge adhesions that harbour multiple repeats of the scavenger receptor cysteine-rich protein domain. to the best of our knowledge identification of srcr repeats is unique to adhesion gpcrs and it is worth mentioning that srcr repeats are often associated with immune system functions in vertebrates.figure n-terminal domain architecture of a selection of sponge adhesion gpcrs. the domains were identified by aligning sponge adhesions to the latest version of pfam library. few adhesion gpcr sequences that lack n-terminal domains are not shown. the domains shown in the figure include; 7tm: seven-transmembrane domain, duf: domain of unknown function, egf: epidermal growth factor-like domain, fn3: fibronectin type iii domain, gps: gpcr proteolytic site domain, hrm: hormone receptor domain, ig/ig_2/ig_3: immunoglobulin domains, i-set: immunoglobulin i-set domain, snare: soluble n-ethylmaleimide-sensitive factor attachment protein receptor domain, srcr: scavenger receptor cysteine-rich domain, v-set: immunoglobulin v-set domain. glutamate receptor family glutamate receptors are crucial modulators of neurotransmission, and in humans there are receptors consisting of eight metabotropic glutamate receptors, two gababrs, the calcium-sensing receptor, the sweet and umami taste receptors, gprc6a and seven orphan receptors. phylogenetic analysis of the glutamate family members from human and sponge revealed that sponge had seven glrs homologous to human metabotropic glutamate receptors. in addition, phylogenetic analysis revealed that two sponge glrs clustered with human gabab receptors and another three were placed on the same node containing gpr and gpr but with a low posterior probability support. to test the robustness of these relationships, we included glutamate gpcrs from nematostella, trichoplax and sea urchin. an overall unrooted tree obtained from a large dataset demonstrated that among the indentified sponge glrs, only seven are homologous to grms, while the rest were sponge specific receptors. however, the sponge glrs that had similarity to gabab and two other orphans failed to give stable or consistent topology in a larger dataset and clustered separately from the known glutamate receptors, suggesting they are divergent from other metazoan counterparts.figure phylogenetic tree showing relationship between glutamate family gpcrs in sponge and human. the edges containing human glutamate family gpcrs are highlighted in green, while the edges containing sponge glutamate are highlighted in blue. phylogenetic relationships between sponge glutamate, the eumetazoans and sea-urchin genomes are provided in additional file frizzled receptor family the sponge proteome dataset contained three full length members of the frizzled gpcr family. a comparative phylogenetic analysis with frizzled receptors from sponge and other metazoan genomes was performed. for the phylogenetic tree construction, we also included the closely related smoothened gpcr family members from human and other metazoans. phylogenetic relationships revealed that sponge frizzled gpcrs are fast evolving or divergent from other metazoan counterparts. two sponge frizzled receptors were placed basal to the node that contained human fzd fzd receptors. also, one frizzled receptor each from other analysed metazoans was placed in the same node with human fzd fzd receptors. interestingly, one sponge frizzled-like receptor was placed basal to the smoothened receptor cluster. this finding was consistent with a recent study that identified a smoothened receptor in sponges. it must be mentioned here that our initial screen for frizzled gpcrs identified frizzled-like gpcr sequences, of which six were removed due to fragmentary models that contained less than tm regions. similarly, an earlier study identified eight frizzled- like gpcrs in the sponge genome. however, a few of these seem to be incompletely modeled and were not included in the final sponge gpcr dataset for better handling of the msa data for subsequent phylogenetic studies. other gpcr families our analysis revealed that the sponge proteome dataset also contains members of other gpcr families that do not belong to the grafs classification system. these included camp-like, intimal thickness-related receptor like, lung 7tm receptor-like and ocular albinism like receptors. subsequent cross-genome phylogenetic analysis was performed on these gpcr families with the corresponding family members obtained from other species. protein sequences belonging to these gpcr families were obtained from nematostella, trichoplax, and sea urchin using pfam hmm profile based searches. corresponding sequences from human were obtained from the swiss-prot database. overall phylogenetic tree topology indicated the presence of gpr143-like, lung 7tm-like and intimal thickness-related receptors in sponge. these sponge sequences clustered with their corresponding family sequences obtained from other species. they formed separate clusters in the phylogenetic analysis with high confidence support. phylogenetic analysis also revealed that the camp-like receptors identified in the sponge genome form a separate cluster with high confidence support. these camp-like sequences contained the core region encoded by a pfam domain characteristic to the dictyostelium camp gpcr family. similarly, pairwise similarity search performed using these sequences as queries clearly demonstrated that camp family sequences are among the top hits. however, sponge camp-like receptors clustered separately from the dictyostelium camp gpcr sequences, suggesting that they are quite divergent or fast evolving. it would thus be interesting to experimentally verify whether the camp-like receptors in the sponge genome have analogous roles to the previously known functions of dictyostelium camp gpcr family. discussion the draft genome, as well as the transcriptome of amphimedon queenslandica, revealed the genetic complexity of this primitive animal in detail and catalogued the presence of several crucial gene families, including gpcrs and other signaling system components. however, a detailed curation of sponge gpcr families/subfamilies and phylogenetic comparisons with those versions found in eumetazoans and bilaterians needs to be performed to better understand the gpcr component in sponges from an evolutionary perspective. in this study, we curated gpcrs in the sponge genome and have phylogenetically compared the receptors to those found in other metazoans. our hmm based search approach and phylogenetic analysis demonstrates that sponge contains four of the five main grafs families, namely, rhodopsin, adhesion, glutamate and frizzled. it is noteworthy that the sponge genome encodes one of the most ancient metazoan lineage specific expansions of the rhodopsin family of gpcrs. moreover, our phylogenetic analysis with pre-bilaterian metazoans homologs clearly reveals that the rhodopsin family has undergone significant diversifications in these pre-bilaterian metazoans. possible explanations could be that they diversified due to the evolution of diverse morphological characteristics and adaptations of these species during the course of the early metazoan evolution. this is also evident in other gpcr families, where phylogenetic analysis revealed that most members of the adhesion and glutamate families grouped into sponge-specific clusters. in summary, the study describes the sponge gpcr gene families in detail and our phylogenetic comparisons postulates a significantly diversified subset of gpcrs in sponge. sponge rhodopsins comparative phylogenetic analysis demonstrates that sponge rhodopsin family gpcrs do not share orthologous relationship with those found in eumetazoans and other bilaterians. in addition, sponge rhodopsin-like gpcrs form five distinct clusters that are most likely sponge specific. here, we putatively labelled these sponge specific clusters as aqrho-a to e. it must be mentioned here that several rhodopsins belonging to these sponge specific clusters are contained in the same contig region and located adjacent to each other. several of the flanking sequences are found as many as a cluster of to sequences and share relatively high pairwise protein sequence identities ranging from 51% to 74%. this suggests that the expansions of sponge rhodopsins are possibly driven by gene duplication events and it seems most likely true for other pre-bilaterian metazoans as well. however, to further examine whether these sequences can be classified into any of the known rhodopsin subfamilies, we performed a blastp search against the swiss-prot database. the results obtained from the blast search showed that a few sponge rhodopsin-like gpcrs had their top hits as adrenergic, serotonin, dopamine, and opsin-like receptors. this is in line with the draft genome report of sponge, which demonstrated the presence of serotonin and dopamine-like receptors. this is also consistent with a recent study that identified adrenergic-like receptors in sponges. although the pairwise similarity search results suggest the presence of these putative receptors, our phylogenetic analysis was unable to reveal any clear orthologous relationships of the sponge rhodopsins to the bilaterian counterparts. a possible explanation could be that sponge rhodopsins have diverged considerably, possibly based on sponge specific physiology and behavior. this hypothesis is plausible because species such as trichoplax and nematostella, belonging to the eumetazoan lineage and diverged from sponges later in the metazoan species tree, contain rhodopsin-like gpcrs more similar to bilaterians than sponges. in fact, earlier studies provided evidence that eumetazoans do contain putative orthologues for some of the amine and peptide binding receptors. the recent genome release of mnemiopsis leidyi suggests that ctenophores are the sister group to the rest of the extant animals, including sponges, and that components of neuronal signaling were already present in an early metazoan ancestor. also, the same study proposed that components of neuronal signaling have undergone major loss and gain events in pre-bilaterian lineages. considering these observations, it is likely that rhodopsin family gpcrs expanded independently in these species and may perform diverse functions based on the morphological characteristic of the organism. also, it is possible that these large expansions may have evolved to perform neuronal functions in ctenophores and cnidarians, and that this ability is secondarily lost in sponges and placozoans during the course of metazoan evolution. nonetheless, at present it is evident that sponge rhodopsins expanded due to gene duplication events and seems to have diverged considerably from those found in other pre-bilaterians and bilaterians. further comparative genomics, as well as developmental/neurobiological studies would be essential to understand the roles of rhodopsin family gpcrs that predated the divergence of bilateria. sponge adhesions the repertoire of adhesion gpcrs in the sponge genome is one of the first expansions within the adhesion gpcr family at the roots of metazoan evolution. in comparison to the sponge, the closest unicellular metazoan relatives such as salpingoeca rosetta and capsaspora owczarzaki contained only a few genes coding for adhesion gpcrs. furthermore, it must be highlighted here that our initial hmm search in the sponge genome identified a staggering genes that encoded a 7tm_ domain. however, we removed of those from our phylogenetic analysis, as they were lacking three or more helices. therefore, it would be interesting to explore whether the subsequent improved versions of the sponge genome contain more full length adhesion gpcrs. collectively, this may suggest that the expansions of adhesion gpcrs at the early origins of metazoans, relative to unicellular relatives, were possibly driven by the evolution of multicellularity in early metazoans since cell-cell adhesion is one of the major factors involved in driving multicellularity. another noteworthy observation is that a few of the sponge adhesion gpcrs are found to be phylogenetically similar to vertebrate adhesion gpcrs belonging to group i/ii. this suggests that some of the adhesion gpcr subfamilies may have diverged early in metazoan evolution and would have later evolved or co-opted for more specialised functions, which we observe in bilaterians. of note, previous studies suggested that group i and group ii adhesion gpcrs have potential roles in neurogenesis and migration. also, in contrast to rhodopsins, which mostly bind hormones and neurotransmitters, the identified ligands of adhesion gpcrs are mostly single-pass membrane proteins. for instance, lphn a group i adhesion, interacts with teneurin- flrt1/ and neurexin i-alpha & beta, which are all single-pass membrane proteins with a variety of n-terminal domains. a blastp search using these single-pass membrane proteins as queries against the sponge proteome obtained few reliable hits. interestingly, these hits contained a single tm helix at the c-terminal end, n-terminal functional domains like laminin g-like, laminin egf-like, and cadherins that are widely known to influence cell-cell adhesion, cell differentiation and migration. this implies that some of the sponge adhesions might interact with single pass membrane proteins to aid cell-cell adhesion in sponges. thus, it would be interesting to explore the functional roles of ancestral group i/ii like adhesion gpcrs in sponge and eumetazoans. although the sponge genome has a few adhesion gpcrs that are somewhat similar to vertebrate adhesions and placed basal to families i, ii and viii, most of them formed a distinct cluster and are likely to be sponge specific. intriguingly, at least eight adhesion receptors contain a hormone receptor domain in their n-termini, a common characteristic of secretin gpcrs. the absence of secretin gpcrs in the sponge and the early presence of an hrm domain in adhesions supports our previous hypothesis that the secretin family descended from adhesion gpcrs in an event somewhere during the split of cnidarians. to the best of our knowledge, sponge adhesion-like receptors are one of the most ancient gpcrs containing a hormone-binding domain. the hrm domain is essential for secretin gpcr activity and is conserved in all the secretin receptors. this suggests that hrm domain containing adhesion gpcrs, found before the divergence of bilateria, may have a possible role analogous to that observed in secretin family of gpcrs. however, this hypothesis needs further experimental verification since the presence of the hrm domain is surprising due to the lack of hormones in the sponge genome. another distinctive feature of the sponge adhesion gpcrs is the absence of a gps domain in as many as of the adhesions. it is important to note that the intra-molecular processing at a gps site in the gpcr autoproteolysis-inducing domain, proximal to the first transmembrane helix is attributed to several factors including signalling, membrane trafficking, as well as for the formation of heterodimeric gpcr complexes. however, the absence of a gps domain in some sponge adhesions might imply that the evolutionary requirement for the conservation of the cleavage site is not very stringent or that the gps site is more essential to those adhesions with long n-termini. moreover, it is possible that the missing gps domains are an outcome of incomplete or missing regions in the current sponge genome draft assembly. the subsequent draft assemblies may help provide a complete picture of adhesion gpcrs in sponges. sponge glutamate receptor family cross-genome phylogenetic analysis between the sponge and human suggests that the sponge has homologous representatives for metabotropic glutamate and gaba-like receptors with a similar n-terminal domain architecture, commonly observed in bilaterian counterparts. also, we attempted to search for components necessary for a gaba shunt, a process by which gaba is produced in animal cells. by homology search methods we found strong evidence for the presence of gaba-t, which catalyses the synthesis of l-glutamate and glutamate decarboxylase, which catalyzes the synthesis of gaba. this is in line with an earlier study that identified glutamate decarboxylase in sponges. however, these results are not surprising because gaba and metabotropic glutamate receptor-like gpcrs were identified previously in sponges, as well as in the amoeba dictyostelium discoideum that evolved well before the divergence of metazoans. it is interesting to note that previous studies provided potential insights into the role of glutamate receptors and neurotransmitter glutamate in non-neuronal cells. for example, elliot and leys showed that sponges, which lack neurons, use metabotropic glutamate and gaba receptor signaling for organized contractions of the sponge canal system. these roles of glutamate receptors are rather distinctive from the commonly known functions of glutamate gpcrs in a synaptic environment. interestingly, there is growing evidence that challenges theories proposing the early origins of synapses and first components of a protosynapse somewhere close to the origins of cnidarians. a current hypothesis suggests that nerve cell components evolved at the very origins of metazoans and have undergone major loss and gain events in pre-bilaterian lineages. also, a few studies have suggested that glutamate is found in non-excitable cells, providing insights for glutamate to function beyond its general role acting as a neurotransmitter. therefore, the presence of glutamate gpcrs in almost all pre-bilaterians including sponges shows the dynamic nature of glutamate gpcrs, which seem to be functional both in synaptic rich and synaptic free environments that prevailed before the divergence of bilateria.  we present the first overall analysis of the gpcr repertoire in the sponge genome and have compared this to the eumetazoans and bilaterian versions. in summary, the sponge gpcr repertoire contains four of the five gpcr grafs families, as well as other gpcr gene families including camp-like receptors, intimal thickness-related receptor like, lung 7tm and gpr receptors. on the other hand, sponge lacks many of the classical mammalian-like sensory receptors including the olfactory receptors that are widely found in several bilaterians. moreover, our phylogenetic comparison reveals that the sponge rhodopsin family does not share orthologous relationships with eumetazoan and bilaterian counterparts. this might imply that subfamily level diversifications of rhodopsins, common in several bilaterians, likely became more pronounced later in the metazoan evolution, as indicated by the presence of some of the subfamilies in nematostella and trichoplax. nonetheless, the sponge encodes one of the first expansions of rhodopsin and adhesion family gpcrs early in metazoan evolution. also, unexpectedly, sponge adhesions encodes hormone binding domains, although hormone-like peptides are yet to be found in sponges. similarly, the long n-termini of a few sponge adhesions contain diverse domain architectures commonly observed in other metazoans. in conclusion, our analysis provides a wider framework for understanding the sponge gpcrs and to relate them to versions found in other pre-bilaterians and bilaterians. furthermore, our data set comparisons provide a platform to perform more comparative genomic studies for understanding gpcr biology and signal transduction at the early origins of multicellularity. 
interestingly, the sponge rhodopsin family sequences lack orthologous relationships with those found in eumetazoan and bilaterian lineages, since they clustered separately to form sponge specific groups in the phylogenetic analysis. this is evident at the family/subfamily level comparisons for most gpcr families, in particular for the rhodopsin family of gpcrs. our phylogenetic comparisons of sponge gpcrs with other metazoan genomes suggest that sponge contains a significantly diversified set of gpcrs. we also phylogenetically clarified that sponge has homologs of metabotropic glutamate and gaba receptors. however, the gpcr component of this signalling system, at the early origins of metazoans is not fully understood. furthermore, phylogenetic comparisons of sponge gpcrs with eumetazoan and bilaterian gpcrs will be essential to our understanding of the gpcr system at the roots of metazoan evolution. a few sponge adhesions clustered basal to adhesion subfamilies commonly found in most vertebrates, suggesting some adhesion subfamilies may have diverged prior to the emergence of bilateria. we present a curated list of gpcrs in the sponge genome after excluding incomplete sequences and false positives from our initial dataset of predicted gpcr sequences obtained using pfam search. phylogenetic analysis reveals that the sponge genome contains members belonging to four of the five major grafs families including glutamate, rhodopsin, adhesion and frizzled. this suggests that sponge rhodopsins diverged considerably from that found in other basal metazoans. here we aim to identify and classify gpcrs in amphimedon queenslandica, a member of an earliest diverging metazoan lineage.
 g protein-coupled receptors play a central role in eukaryotic signal transduction. however, the gpcr component of this signalling system, at the early origins of metazoans is not fully understood. here we aim to identify and classify gpcrs in amphimedon queenslandica, a member of an earliest diverging metazoan lineage. furthermore, phylogenetic comparisons of sponge gpcrs with eumetazoan and bilaterian gpcrs will be essential to our understanding of the gpcr system at the roots of metazoan evolution.  we present a curated list of gpcrs in the sponge genome after excluding incomplete sequences and false positives from our initial dataset of predicted gpcr sequences obtained using pfam search. phylogenetic analysis reveals that the sponge genome contains members belonging to four of the five major grafs families including glutamate, rhodopsin, adhesion and frizzled. interestingly, the sponge rhodopsin family sequences lack orthologous relationships with those found in eumetazoan and bilaterian lineages, since they clustered separately to form sponge specific groups in the phylogenetic analysis. this suggests that sponge rhodopsins diverged considerably from that found in other basal metazoans. a few sponge adhesions clustered basal to adhesion subfamilies commonly found in most vertebrates, suggesting some adhesion subfamilies may have diverged prior to the emergence of bilateria. furthermore, at least eight of the sponge adhesion members have a hormone binding motif in their n-termini, although hormones have yet to be identified in sponges. we also phylogenetically clarified that sponge has homologs of metabotropic glutamate and gaba receptors.  our phylogenetic comparisons of sponge gpcrs with other metazoan genomes suggest that sponge contains a significantly diversified set of gpcrs. this is evident at the family/subfamily level comparisons for most gpcr families, in particular for the rhodopsin family of gpcrs. in summary, this study provides a framework to perform future experimental and comparative studies to further verify and understand the roles of gpcrs that predates the divergence of bilaterian and eumetazoan lineages. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords neurotransmittersg protein-coupled receptorsadhesionsignal transductionporiferaeumetazoaissue-copyright-statement© the author 2014 