BACKGROUND
the evolution of the secretin family of brain-gut peptides remains enigmatic despite being some of the first endocrine factors ever identified.
for example, in  <dig> the ground breaking experiments of bayliss and starling with dog intestinal extracts set off the search for the active principal and, by  <dig>  secretin  had been isolated and sequenced  <cit> .
currently,  <dig> peptides belonging to the secretin family have been isolated in humans and include; sct, vasoactive intestinal peptide , pituitary adenylate cyclase-activating polypeptide , peptide histidine methionine , pacap-related peptide , growth hormone-releasing hormone , glucagon , glucagon-like peptide  and glucose-dependent insulinotropic peptide   <cit> .
members of the secretin family share significant structural and conformational homology and their key metabolic and developmental functions in human make them of considerable pharmacological interest.
members of class  <dig> g-protein coupled receptors , bind and are activated by the secretin family members  and specific peptide-receptor pairs have been identified in representatives of different vertebrate classes.
class  <dig> gpcrs is a larger family of receptors and also includes members of the metazoan adhesion  and insect methuselah  families and secretin family gpcrs  are proposed to descend from the adhesion receptors prior to protostome-deuterostome divergence  <cit> .
in protostomes  and early deuterostomes such as ciona and amphioxus, immunohistochemical  approaches using antisera raised against various mammalian secretin family members suggest they possess similar peptides to vertebrates .
pacap-like genes  have only been reported in the tunicate, chelyosoma productum  <cit>  and partial mrnas  corresponding to the highly conserved pacap coding exon  <cit>  have been isolated in hydra magnipapillata and several protostomes and deposited in public databases  <cit> .
the existing data has been taken to indicate that an ancestral secretin family gene was probably present prior to the deuterostome-protostome divergence and most likely resembled the vertebrate pacap precursor  <cit> .
paradoxically, in protostomes with fully sequenced genomes and extensive molecular resources , genes encoding ligands homologous to members of the vertebrate secretin family have not been reported.
in contrast, secretin-like family gpcr encoding genes which share similar sequence, structure and conserved gene environment with the vertebrate members have been identified, making ligand-receptor evolution an interesting enigma  <cit> .
nucleotide , protein , immunohistochemical  and in situ
n* non-vertebrate nucleotide sequences deposited in ncbi
the present study focuses on secretin family ligands and complements previous studies aimed at identifying and characterising the evolution of family  <dig> gpcrs  <cit> .
a comparative approach which takes advantage of the wealth of information currently available  for porifera, cnidaria, protostomes, early deuterostomes and vertebrates , is undertaken to re-evaluate the origin of the secretin family in metazoa.
the sequence, gene structure and gene environment of secretin family members in vertebrates with sequenced genomes was characterised and used to search for homologue peptides, genes or genome regions in non-vertebrates.
RESULTS
vertebrate secretin family members
sequence database searches using the nucleotide and mature peptide sequences of human and zebrafish secretin family members, readily identified homologues in lamprey, teleost and tetrapod  genomes.
this is due to the high sequence conservation of the mature peptide region between the vertebrate members which facilitates their identification  .
chicken
lizard
xenopus
takifugu
tetraodon
zebrafish
stickleback
medaka
lamprey
contig/scaffold numbers are indicated when ensembl accession number are not available
dashes indicate genes not identified
the tetrapod members
in humans,  <dig> peptides encoded by six genes have been isolated.
in aves, homologues of the mammalian members have been identified and peptides and corresponding transcripts were isolated in duck , chicken  and turkey   <cit> .
in silico analysis of the chicken genome identified six genes encoding secretin family members which share similar organisation to the human homologues and comparative analysis revealed they correspond to the peptides and nucleotide precursors previously described .
searches in the reptile and amphibia genomes identified homologues for all human members with the exception of sct and it remains to be established if the failure is due to the incomplete nature of their genome assemblies or to the absence of this gene.
in reptiles, few members of this family have been reported to date.
a vip and gcg peptides were isolated from the alligator mississippiensis, the nucleotide precursor of the latter peptide reported from the heloderma suspectum  <cit>  and a prp/pacap mrna was recently characterised from the italian wall lizard podarcis sicula  <cit> .
sequence database searches on the lizard anolis carolinensis genome identified for the first time the genes encoding the reptile phi/vip , prp/pacap , ghrh , gcg/glp  and gip  .
the predicted gene organisation suggests the coding exons for the mature peptides share identical structure with other metazoan genes and to date the exon encoding the lizard glp <dig> remains to be identified.
homologues of human secretin members have previously been reported in amphibians.
vip and pacap and were isolated in rana ridibunda  <cit> ; gcg from rana catesbeiana  <cit>  and in xenopus laevis single transcripts for prp/pacap  <cit> , phi/vip, ghrh  <cit> , gcg/glp  <cit>  and gip  <cit>  have been described.
the genome of xenopus tropicalis contains secretin family homologues which share high sequence conservation with the tetrapod genes , but one difference was the presence of three glp <dig> exons  within the gcg/glp gene structure as a result of a species-specific exon duplication.
the fish members
peptides, transcripts and genes of the secretin family have also been isolated from the most diverse vertebrate clade, the teleosts.
vip was isolated from cod   <cit>  and goldfish   <cit> , pacap from the japanese stargazer   <cit>  and gcg from the channel catfish   <cit>  .
the identification of two transcripts for phi/vip in goldfish carassius auratus  <cit>  and zebrafish   and of two prp/pacap   <cit>  and gcg/glp precursors in zebrafish  <cit>  suggests they are duplicates in fish and this has been confirmed by the identification of two prp/pacap and gcg/glp genes in takifugu  and tetraodon  genomes   <cit> .
the greater number of secretin family genes identified in fish relative to tetrapods is most likely to be a result of the proposed teleost specific genome duplication and the absence of gip and ghrh gene duplicates suggests they were probably deleted  <cit> .
in common with xenopus, no homologue of human sct has been identified in fish genomes.
in the sea lamprey , a primitive vertebrate of the agnatha clade, two homologues of human proglucagon  were characterised in the genome assembly contig  <dig>  and contig  <dig> which correspond to the previously reported proglucagon i  and proglucagon ii  transcripts, respectively  <cit> .
in the present study, searches of the partially sequenced lamprey genome retrieved putative prp/pacap  and phi/vip genes .
however, ghrh, sct and gip were not identified possibly due to the present incomplete nature of its genome assembly.
sequence and gene structure comparison
sequence comparisons reveal that members of the secretin family are highly conserved and this also applies to their secondary structure which consists of a random n-terminal structure and a c-terminal alpha-helix  <cit> .
the lamprey, teleost, xenopus, reptile and chicken members are in general 50% identical in amino acid sequence with the human homologues  and two main peptide subfamilies, which share in general a maximum of 60% sequence similarity between their members were identified; i) pacap-like subfamily which includes  <dig> peptide groups  and ii) gcg-like subfamily which contains  <dig> peptide groups .
highest sequence conservation from lamprey to human  occurs within pacap, vip and gcg peptide groups which contrasts with prp and glp <dig> that are the most divergent and takifugu prp b and glp <dig> predicted peptides are only 33% and 37% identical with the human homologues.
consensus amino acids for peptide subfamilies were deduced and the overall conservation characterised.
with few exceptions,  <dig> amino acid residues h <dig>  d <dig>  f <dig>  t <dig>  y <dig>  q <dig> and l <dig> are the most abundant across the pacap-like subfamily .
the residues h <dig>  d <dig> and l <dig> are present in all peptide groups with occasional exceptions in some taxa.
however, the motif f6t <dig> is absent from the vertebrate prp sequences and is only present in tunicate prp and the residue q <dig> is absent from the sct mature peptide and y <dig> is only present in the chicken sct homologue.
this suggests that specific modifications occurred within the conserved core domain of the pacap-like subfamily members and their functional significance remains to be explored.
a similar comparison of the vertebrate gcg-like subfamily indicates that  <dig> amino acid residues h <dig>  a <dig>  g <dig>  t <dig>  f <dig>  s <dig>  d <dig>  s <dig>  l <dig>  a <dig>  k <dig>  f <dig>  v <dig>  w <dig>  and l <dig> are generally maintained across the  <dig> peptide groups.
even if taxa variability is taken into consideration, the residues g <dig>  f <dig> and f <dig> and the motifs s <dig> d <dig> and w25l <dig> are in general maintained .
peptide specific variations include for gip, h <dig> which is replaced by y, l <dig> which is replaced by m or v  and a <dig> which is replaced by k or q. in the gcg mature peptide sequence a <dig> is replaced by s and in glp <dig> with the exception of chicken and takifugu t <dig> is replaced by s. comparison of the metazoan pacap-like and gcg-like subfamily consensus sequence revealed they overlap for the residues h <dig> and f <dig> which are key amino acids in secretin gpcr binding affinity  <cit> .
this suggests that, after exon/chromosome duplication of their common ancestor exon, distinct evolutionary pressures within each subfamily occurred.
the chordate pacap shares in general 92% of amino acid sequence similarity with vip and these two peptides are the most conserved members .
in contrast, the vertebrate sct demonstrates the lowest conservation  however this may be an artefact due to the restricted number of species in which it has been characterised.
in addition, signature amino acid residues within or between peptide groups were also identified and they may reflect and support common evolutionary pathways and overlapping or specific functional roles.
this includes, the pacap y <dig> and a24a <dig> motif, the v <dig>  n <dig>  f <dig> and n <dig> for vip, the motif n7k8a <dig> and residue h <dig> within prp, the l <dig>  s <dig>  e <dig> and i <dig> for ph, and the ghrh residues n <dig>  i <dig>  q <dig> and i <dig> across the tetrapod and teleost members .
gene structure comparisons restricted to the mature peptide precursors revealed that pacap and vip exons are encoded in the same precursor as prp and ph, respectively  <cit>  .
most common amino acids to the chordate pacap and vip members includes r <dig> and k15and the motifs m17a18v <dig> and v26l <dig> and they share a maximum of 37% amino acid sequence identity with prp and ph indicating that after exon duplication considerable changes occurred.
the latter peptides have a similar sequence  which is closely related to vertebrate ghrh  with which they partition the residues a <dig>  k <dig>  l <dig>  g <dig> and the motif s18a19r <dig> and suggest a common evolutionary origin.
the sct gene has only been identified in tetrapods and the deduced peptide residues, e <dig> and q <dig> are maintained in the mammalian and chicken homologues.
the evolutionary origin of sct is still enigmatic and it is proposed to have been lost in the fish lineage  <cit> .
members of the gcg-like subfamily share at least 62% amino acid similarity and glp <dig> and glp <dig> have the highest identity .
in addition to the general sequence conservation of the gcg-like subfamily members, conserved amino acid positions within the vertebrate peptide groups were also identified .
this includes s <dig>  q <dig>  m <dig> and the motifs r17r <dig> and q20d <dig> for gcg; v <dig>  s <dig> and q <dig> for glp1; s <dig>  n <dig> and l <dig> for glp2; and y <dig> and l <dig> in gip.
the glp <dig> and glp <dig> peptides are encoded in the same precursor  as gcg with which they share 77% and 81% amino acid sequence similarity, respectively and the mature peptide coding exons are proposed to be a consequence of exon duplication events.
vertebrate gip shares 37% and 40% sequence identity with gcg and glps, respectively and studies based upon gene structure comparisons suggest the latter emerged from the same exon as gcg, however the results of sequence analysis are inconclusive and more data is required  <cit> .
the secretin members in non-vertebrates
database searches using the vertebrate nucleotide and peptide sequences of secretin family members and the duplicate urochordate chelyosoma productum prp/pacap transcripts failed to identify conserved sequence and structure homologues in genome or est databases from porifera, cnidaria, protostome and early deuterostomes .
instead, short sequence matches were identified in unrelated genes or non-annotated genome segments.
in depth analysis of the best matches, revealed homologies for the central region and c-terminal ends  of the chordate mature peptides and, when the invertebrate fragments were used to interrogate vertebrate databases, they failed to retrieve a secretin family homologue suggesting that members of this family are absent from non-vertebrate genomes.
the exception is pacap for which a highly conserved partial sequence corresponding to the exon encoding the mature peptide  has been isolated in hydra magnipapillata , in the tunicate, halocynthia roretzi  and in several protostomes such as planarian , crab , squid  and cockroach   <cit> .
database searches using the non-vertebrate pacap nucleotide or deduced peptide sequences failed to retrieve homologues from protostomes with available genome data .
moreover, searches performed in hydra magnipapillata and related species nematostella vectensis genome assemblies with the hydra pacap nucleotide or deduced peptide sequence also failed to confirm the existence of a gene encoding pacap.
in early deuterostomes, searches using the chelyosoma productum prp/pacap nucleotide or deduced peptide sequences in ciona intestinalis and ciona savignyi genomes or available tunicate ests failed to identify possible sequence homologues in urochordate.
taken together these results raise questions about the authenticity of the previously reported sequences.
an alternative strategy utilized a secretin family prototype sequence model based on the assumption that the chordate members arose from a common precursor gene which duplicated to give pacap-like and gcg-like subfamily exons .
the prototype sequence deduced in silico was h1a2d3g4t5f6t7s8d9y10s11xxl14xq16xxa19k20xf22xxw25l <dig>  and contains  <dig> conserved amino acid positions and high conservation was found for the n-terminal region.
structural characterisation using pfam analysis classified the generated prototype sequence as a hormone_ <dig> member   <cit>  and sequence similarity searches performed retrieved secretin family members in vertebrates but failed to identify potential members in non-vertebrates.
a second approach using hmm models and searching the general ncbi non-redundant  peptide and an invertebrate subset of the ncbi nucleotide and est  databases corroborated the preceding results.
phylogenetic analysis
the optimal maximum likelihood  tree with bootstrap support values higher than 50% is presented in figure  <dig>  despite the high level of sequence identity and short sequences utilized, phylogenetic analyses of the chordate mature peptides  and their corresponding nucleotide sequences resulted in similar tree topologies and suggests that members of the secretin family share a common ancestry.
in the optimal ml tree presented in figure  <dig> two clades pacap-like and gcg-like were obtained suggesting that after ancestral exon duplication two main peptide subgroups emerged and underwent distinct evolutionary trajectories.
similar tree topologies were obtained using bayesian approaches  and a pacap-like derived clade includes the peptides pacap, prp, ph, vip, ghrh and sct and a gcg-like clade the peptides gcg, glp <dig>  glp <dig> and gip.
the chelyosoma pacap deduced peptide sequences always grouped with the vertebrate peptide sequence homologues and the tunicate prps did not cluster with any particular peptide clade and in all the analysis performed tended to be more closely related to the tetrapod sct.
gene environment comparisons
the immediate gene environment of vertebrate secretin members was compared and indicates that prp/pacap, gcg/glp, and ghrh genome regions are syntenic and gene order is in general maintained .
the gcg/glp gene environment shares at least  <dig> genes , prp/pacap,  <dig> genes  and ghrh,  <dig> gene  when equivalent genome regions are compared between takifugu, xenopus, chicken and human.
the vip and gip genomic regions are poorly conserved and no gene synteny or gene order was identified .
moreover, searches also failed to identify conservation of gene linkage between the different vertebrate secretin family members.
in takifugu, the neighbouring genes of the paralogue gcg/glp  and pacap  genes were also duplicates supporting the teleost gene or genome duplication event.
searches were extended to the lamprey and lizard genomes but contiguous sequences were too small to confirm the existence of gene environment conservation.
comparison of the sct genome region between chicken  and human  revealed they are highly conserved and the order of  <dig> genes  is maintained.
in xenopus genome, the chicken and human sct flanking genes are localised in the amphibian scaffold_ <dig> and in takifugu, where a gene homologue is proposed to be absent, they are distributed in two distinct genome regions: mucdhl and drd <dig> are localised in scaffold n <dig> and the remaining genes in scaffold n <dig> suggesting the existence of a conserved gene block prior to the emergence of the tetrapod gene .
in order to identify a potential secretin family genome region in non-vertebrates, the conserved vertebrate gene environment was used to retrieve homologues in the c. elegans, drosophila and ciona genomes.
genes sharing similarity in sequence to those flanking the vertebrate gcg/glp, prp/pacap and ghrh loci were identified although the genes encoding secretin family members were absent .
in c. elegans the genes drh- <dig>  abts- <dig>  src- <dig> and m01a <dig>  map to chromosome i and are respectively homologues of the human ifih <dig> and slc4a <dig> on chromosome  <dig>  yes <dig> on chromosome  <dig> and rpn <dig> on chromosome  <dig>  in drosophila, sei and cg <dig> are localised in chromosome 2r and are the homologues of human kcnh <dig> and rpn <dig> and the fruit-fly dcr- <dig> and cg <dig> genes in chromosome 3r the correspondent in sequence of the human ifih <dig> and mettl <dig>  respectively.
in the tunicate genome, homologues of the vertebrate secretin family flanking genes were identified scattered in the ciona genome assembly.
homologues of human kcnh <dig> , ifih <dig>  and slc4a <dig>  genes which flank gcg/glp in vertebrates were identified.
the conserved gene environment of the vertebrate prp/pacap genes: yes <dig>  and mettl <dig>  genes are also present as well the putative urochordate homologue of the rpn <dig> gene  conserved in the vertebrate ghrh genome region.
in the c. elegans genome chromosome  <dig> contained homologues of the genes which flank prp/pacap, gcg/glp and ghrh in vertebrates.
this suggests that this chromosome may be the protostome genome fragment that most resembles the potential metazoan ancestral secretin genome region from which the vertebrate members emerged.
however, detailed analysis of chromosome positions  revealed they are not mapped in close proximity and do not forms a gene cluster.
moreover, no conserved linkage between the putative drosophila and the c. elegans gene homologues exists.
discussion
comparative analysis of data from phylogenetically distant organisms is a major contributor for understanding gene and gene family evolution and the role of function and regulation in this process.
the identification of gene homologues in vertebrates and early metazoan genomes provides a unique opportunity to perform comparative studies and to investigate gene family ancestries.
the secretin family is a well-studied group of peptides which activate specific receptors of family  <dig> gpcrs to bring about their pleotropic actions in vertebrates.
secretin-like family gpcrs have been identified and cloned from non-vertebrate genomes  <cit>  and their putative peptide ligands identified by immunohistochemistry  using antisera raised against the mammalian peptide homologues .
both peptides and their corresponding receptors are proposed to have arisen by gene duplication events prior to the vertebrate radiation  <cit>  and they represent an interesting model for studies of receptor-ligand evolution.
in the present study, despite extensive in silico database searches it was not possible to identify members of the secretin peptide family in non-vertebrates regardless of the report of a full-length pacap cdna in the tunicate, chelyosoma productum  <cit>  and cdna of the pacap coding exon in crab, cockroach, squid, planarian and hydra magnipapillata  <cit> .
moreover, using the partial pacap cdna previously identified in hydra magnipapillata  to interrogate its sequenced genome  <cit>  failed to identify the gene homologue.
it seems unlikely that the failure to identify pacap in non-vertebrates was related to the methodology used as a similar approach has been successfully utilized to identify the gene encoding the active nonapeptide hormone, vasotocin/vasopressin in the amphioxus genome assembly and also the gene loci in several chordates including teleosts  <cit> .
in fact, the existence of neurohypophysial hormones  in deuterostomes and protostomes has been amply confirmed by the isolation of both genes and peptides from representatives of a number of different phyla .
similarly, conserved sequence homologues of vertebrate neuropeptide y family members have been isolated from molluscs and also from fruit-fly and mosquito and gpcr ligand-receptor pairs similar to the vertebrate npy system have been characterised  <cit> .
a comprehensive in silico analysis of the fully sequenced drosophila genome identified and classified gpcrs and compared their number to putative neuropeptide ligands.
although  <dig> secretin family gpcr members were identified in the arthropod genome, only two potential ligands, corticotrophin releasing factor -related peptides and amnesiac genes, unrelated to chordate secretin peptide family members were predicted  <cit> .
in contrast, recent studies performed in molluscs  using ihc and maldi-tof/tof identified partial peptides with a similar mass to vertebrate pacap in the snail whole hemolymph and cns extracts  <cit> .
should the identity of the peptide be confirmed by sequencing taking in consideration the results of the present study a new paradigm will be required to explain secretin family evolution.
there is evidence that genes for amnesiac in drosophila and maxadilan in sand-fly might encode functional homologues of the vertebrate pacap despite their lack of sequence similarity  <cit>  and the maxadilan peptide is able to activate mammalian pac <dig> receptors in vitro  <cit> .
the activation of family  <dig> gpcrs members by the secretin family of ligands has been linked to their well conserved structure  <cit>  which comprises an alpha helix in the mid and c-terminal region and an n-terminal loop.
the region of maxadilan implicated in pac <dig> receptor activation has an identical structure to the n-terminal region of secretin family ligands and contains key amino acids involved in receptor activation .
in fact, mutation analysis with maxadilan demonstrated that despite its greater size compared to vertebrate secretin family peptides, the disruption of four conserved cysteine residues  responsible for the formation of two disulfide bonds led to loss of activity  <cit> .
taking into consideration the degree of conservation of the n-terminal ligand binding domain of the secretin family gpcrs  <cit>  and their relative promiscuity  <cit>  it is unsurprising that protostome peptides activate vertebrate receptors.
in nematode and drosophila, pdf  stimulated the homologues of vertebrate secretin gpcrs but they were not stimulated by secretin family members  <cit> .
these observations suggest that specificity of the receptor members has changed during evolution and may explain the failure to identify conserved ligands.
a general model to explain peptide ligand binding and receptor activation has emerged recently for class b gpcrs   <cit> .
the proposed mechanism, known as the "two-domain model" suggests that initial ligand-receptor interactions are mediated by the central and c-terminal peptide segments with the extracellular n-terminal receptor region and that activation occurs subsequently when the bioactive n-terminus of the ligand binds the receptor juxtamembrane domain  <cit> .
in this context, the identification in the present study of putative "signature" amino acids conserved across taxa for each peptide groups may explain differences in selection and affinity for receptors.
for example, mutation of conserved residues within the c-terminal region of vip revealed that substitution of l <dig>  decreased peptide biological activity without altering the predicted structure  <cit> .
d <dig> is conserved across the pacap-like peptides and this residue has a role in adenyl cyclase  stimulation and interacts with basic residues  in the second transmembrane helix of vpac  <cit> .
similarly, d <dig> which is conserved across the gcg-like members is essential in the activation of mammalian gcgr  <cit> .
it will be of interest in the future to study the role of unique amino acid residues/motifs identified in the present study within the n-terminal and c-terminal regions of each peptide group.
recently, a darwinian evolutionary model was proposed to explain the origin of steroid hormones and their receptors and may also explain the emergence of metazoan secretin peptide-receptor pairs.
the steroid hormones and receptors were proposed to have evolved through a molecular exploitation process in which structurally adapted receptors evolved prior to ligand emergence  <cit> .
by reconstructing the sequence of the ancestral steroid receptors  the authors verified that they are activated by hormones  that only emerged in the tetrapod lineage.
at present, evolutionary comparisons of the metazoan receptor members and secretin peptide family suggests that, receptors emerged prior to the ligands which were subsequently acquired as a consequence of genome evolution in the chordate radiation.
generally, two major gene or genome duplication events are proposed to have occurred at the origin of vertebrates and have accompanied increased organismal complexity and emergence of gene novelties  <cit> .
whilst the majority of gene duplicates were probably lost as a consequence of their functional redundancy, some were fixed in the genome by the gain of new biological functions or partitioning the function of the ancestral counterpart and this may be the case for the secretin family gpcrs and their ligands  <cit> .
the origin of the chordate secretin peptide family has been previously associated with the insect adipokinetic hormone  and akh-precursor related peptides  precursor evolution  <cit> .
despite their low sequence similarity, the arthropod akh and aprp precursor was found to share a similar gene organisation and comparable functions with vertebrate gcg and ghrh and they were proposed to have shared common origin prior to protostome-deuterostome divergence .
however, against this hypothesis is the recent demonstration that insect adk signals through a gonadotropin-releasing hormone  like receptor  and also the isolation of putative nematode akh-gnrh related precursors suggesting that the invertebrate adk members may share common evolution with the metazoan gnrh system  <cit> .
CONCLUSIONS
the present study does not confirm the results of ihc studies in the early 70's and 80's which identified putative invertebrate secretin family members using antisera against mammalian peptides.
it was not possible to identify sequence homologues of the chelyosoma productum prp/pacap peptides in the sequenced ciona genomes, although at least  <dig> putative secretin-like family gpcrs have been reported  <cit> .
the previous facts taken with i) the identification in snail of a putative pacap peptide; and ii) the activation of a secretin-like family gpcrs in nematode and drosophila by pdf but not by vertebrate secretin family members makes it difficult to establish when the peptide members emerged in the deuterostome lineage.
it is hypothesised that the emergence of the full suite of receptors and their ligands accompanied the rapid genome changes during chordate evolution.
the ancestral secretin family gene probably arose as part of an existing gene or gene fragment and via exon and gene duplication events generated the existing suite of family members .
this occurred after the emergence of the secretin family gpcrs and led to the establishment of novel and specific receptor ligand interactions that contributed to the generation of novel physiological functions.
in contrast, to other peptide families, such as npy/pyy and oxytocin/vasopressin which stimulate receptors of family  <dig> gpcrs and are highly conserved from protostomes to deuterostomes, members of the secretin family gpcrs appear to have adopted new ligands during evolution.

 the evolution of the secretin family of brain-gut peptides remains enigmatic despite being some of the first endocrine factors ever identified. for example, in the ground breaking experiments of bayliss and starling with dog intestinal extracts set off the search for the active principal and, by secretin had been isolated and sequenced. currently, peptides belonging to the secretin family have been isolated in humans and include; sct, vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide, peptide histidine methionine, pacap-related peptide, growth hormone-releasing hormone, glucagon, glucagon-like peptide and glucose-dependent insulinotropic peptide. members of the secretin family share significant structural and conformational homology and their key metabolic and developmental functions in human make them of considerable pharmacological interest. members of class g-protein coupled receptors, bind and are activated by the secretin family members and specific peptide-receptor pairs have been identified in representatives of different vertebrate classes. class gpcrs is a larger family of receptors and also includes members of the metazoan adhesion and insect methuselah families and secretin family gpcrs are proposed to descend from the adhesion receptors prior to protostome-deuterostome divergence. in protostomes and early deuterostomes such as ciona and amphioxus, immunohistochemical approaches using antisera raised against various mammalian secretin family members suggest they possess similar peptides to vertebrates. pacap-like genes have only been reported in the tunicate, chelyosoma productum and partial mrnas corresponding to the highly conserved pacap coding exon have been isolated in hydra magnipapillata and several protostomes and deposited in public databases. the existing data has been taken to indicate that an ancestral secretin family gene was probably present prior to the deuterostome-protostome divergence and most likely resembled the vertebrate pacap precursor. paradoxically, in protostomes with fully sequenced genomes and extensive molecular resources, genes encoding ligands homologous to members of the vertebrate secretin family have not been reported. in contrast, secretin-like family gpcr encoding genes which share similar sequence, structure and conserved gene environment with the vertebrate members have been identified, making ligand-receptor evolution an interesting enigma. nucleotide, protein, immunohistochemical and in situ n* non-vertebrate nucleotide sequences deposited in ncbi the present study focuses on secretin family ligands and complements previous studies aimed at identifying and characterising the evolution of family gpcrs. a comparative approach which takes advantage of the wealth of information currently available for porifera, cnidaria, protostomes, early deuterostomes and vertebrates, is undertaken to re-evaluate the origin of the secretin family in metazoa. the sequence, gene structure and gene environment of secretin family members in vertebrates with sequenced genomes was characterised and used to search for homologue peptides, genes or genome regions in non-vertebrates.  vertebrate secretin family members sequence database searches using the nucleotide and mature peptide sequences of human and zebrafish secretin family members, readily identified homologues in lamprey, teleost and tetrapod genomes. this is due to the high sequence conservation of the mature peptide region between the vertebrate members which facilitates their identification. chicken lizard xenopus takifugu tetraodon zebrafish stickleback medaka lamprey contig/scaffold numbers are indicated when ensembl accession number are not available dashes indicate genes not identified the tetrapod members in humans, peptides encoded by six genes have been isolated. in aves, homologues of the mammalian members have been identified and peptides and corresponding transcripts were isolated in duck, chicken and turkey. in silico analysis of the chicken genome identified six genes encoding secretin family members which share similar organisation to the human homologues and comparative analysis revealed they correspond to the peptides and nucleotide precursors previously described. searches in the reptile and amphibia genomes identified homologues for all human members with the exception of sct and it remains to be established if the failure is due to the incomplete nature of their genome assemblies or to the absence of this gene. in reptiles, few members of this family have been reported to date. a vip and gcg peptides were isolated from the alligator mississippiensis, the nucleotide precursor of the latter peptide reported from the heloderma suspectum and a prp/pacap mrna was recently characterised from the italian wall lizard podarcis sicula. sequence database searches on the lizard anolis carolinensis genome identified for the first time the genes encoding the reptile phi/vip, prp/pacap, ghrh, gcg/glp and gip. the predicted gene organisation suggests the coding exons for the mature peptides share identical structure with other metazoan genes and to date the exon encoding the lizard glp remains to be identified. homologues of human secretin members have previously been reported in amphibians. vip and pacap and were isolated in rana ridibunda; gcg from rana catesbeiana and in xenopus laevis single transcripts for prp/pacap, phi/vip, ghrh, gcg/glp and gip have been described. the genome of xenopus tropicalis contains secretin family homologues which share high sequence conservation with the tetrapod genes, but one difference was the presence of three glp exons within the gcg/glp gene structure as a result of a species-specific exon duplication. the fish members peptides, transcripts and genes of the secretin family have also been isolated from the most diverse vertebrate clade, the teleosts. vip was isolated from cod and goldfish, pacap from the japanese stargazer and gcg from the channel catfish. the identification of two transcripts for phi/vip in goldfish carassius auratus and zebrafish and of two prp/pacap and gcg/glp precursors in zebrafish suggests they are duplicates in fish and this has been confirmed by the identification of two prp/pacap and gcg/glp genes in takifugu and tetraodon genomes. the greater number of secretin family genes identified in fish relative to tetrapods is most likely to be a result of the proposed teleost specific genome duplication and the absence of gip and ghrh gene duplicates suggests they were probably deleted. in common with xenopus, no homologue of human sct has been identified in fish genomes. in the sea lamprey, a primitive vertebrate of the agnatha clade, two homologues of human proglucagon were characterised in the genome assembly contig and contig which correspond to the previously reported proglucagon i and proglucagon ii transcripts, respectively. in the present study, searches of the partially sequenced lamprey genome retrieved putative prp/pacap and phi/vip genes. however, ghrh, sct and gip were not identified possibly due to the present incomplete nature of its genome assembly. sequence and gene structure comparison sequence comparisons reveal that members of the secretin family are highly conserved and this also applies to their secondary structure which consists of a random n-terminal structure and a c-terminal alpha-helix. the lamprey, teleost, xenopus, reptile and chicken members are in general 50% identical in amino acid sequence with the human homologues and two main peptide subfamilies, which share in general a maximum of 60% sequence similarity between their members were identified; i) pacap-like subfamily which includes peptide groups and ii) gcg-like subfamily which contains peptide groups. highest sequence conservation from lamprey to human occurs within pacap, vip and gcg peptide groups which contrasts with prp and glp that are the most divergent and takifugu prp b and glp predicted peptides are only 33% and 37% identical with the human homologues. consensus amino acids for peptide subfamilies were deduced and the overall conservation characterised. with few exceptions, amino acid residues h d f t y q and l are the most abundant across the pacap-like subfamily. the residues h d and l are present in all peptide groups with occasional exceptions in some taxa. however, the motif f6t is absent from the vertebrate prp sequences and is only present in tunicate prp and the residue q is absent from the sct mature peptide and y is only present in the chicken sct homologue. this suggests that specific modifications occurred within the conserved core domain of the pacap-like subfamily members and their functional significance remains to be explored. a similar comparison of the vertebrate gcg-like subfamily indicates that amino acid residues h a g t f s d s l a k f v w and l are generally maintained across the peptide groups. even if taxa variability is taken into consideration, the residues g f and f and the motifs s d and w25l are in general maintained. peptide specific variations include for gip, h which is replaced by y, l which is replaced by m or v and a which is replaced by k or q. in the gcg mature peptide sequence a is replaced by s and in glp with the exception of chicken and takifugu t is replaced by s. comparison of the metazoan pacap-like and gcg-like subfamily consensus sequence revealed they overlap for the residues h and f which are key amino acids in secretin gpcr binding affinity. this suggests that, after exon/chromosome duplication of their common ancestor exon, distinct evolutionary pressures within each subfamily occurred. the chordate pacap shares in general 92% of amino acid sequence similarity with vip and these two peptides are the most conserved members. in contrast, the vertebrate sct demonstrates the lowest conservation however this may be an artefact due to the restricted number of species in which it has been characterised. in addition, signature amino acid residues within or between peptide groups were also identified and they may reflect and support common evolutionary pathways and overlapping or specific functional roles. this includes, the pacap y and a24a motif, the v n f and n for vip, the motif n7k8a and residue h within prp, the l s e and i for ph, and the ghrh residues n i q and i across the tetrapod and teleost members. gene structure comparisons restricted to the mature peptide precursors revealed that pacap and vip exons are encoded in the same precursor as prp and ph, respectively. most common amino acids to the chordate pacap and vip members includes r and k15and the motifs m17a18v and v26l and they share a maximum of 37% amino acid sequence identity with prp and ph indicating that after exon duplication considerable changes occurred. the latter peptides have a similar sequence which is closely related to vertebrate ghrh with which they partition the residues a k l g and the motif s18a19r and suggest a common evolutionary origin. the sct gene has only been identified in tetrapods and the deduced peptide residues, e and q are maintained in the mammalian and chicken homologues. the evolutionary origin of sct is still enigmatic and it is proposed to have been lost in the fish lineage. members of the gcg-like subfamily share at least 62% amino acid similarity and glp and glp have the highest identity. in addition to the general sequence conservation of the gcg-like subfamily members, conserved amino acid positions within the vertebrate peptide groups were also identified. this includes s q m and the motifs r17r and q20d for gcg; v s and q for glp1; s n and l for glp2; and y and l in gip. the glp and glp peptides are encoded in the same precursor as gcg with which they share 77% and 81% amino acid sequence similarity, respectively and the mature peptide coding exons are proposed to be a consequence of exon duplication events. vertebrate gip shares 37% and 40% sequence identity with gcg and glps, respectively and studies based upon gene structure comparisons suggest the latter emerged from the same exon as gcg, however the results of sequence analysis are inconclusive and more data is required. the secretin members in non-vertebrates database searches using the vertebrate nucleotide and peptide sequences of secretin family members and the duplicate urochordate chelyosoma productum prp/pacap transcripts failed to identify conserved sequence and structure homologues in genome or est databases from porifera, cnidaria, protostome and early deuterostomes. instead, short sequence matches were identified in unrelated genes or non-annotated genome segments. in depth analysis of the best matches, revealed homologies for the central region and c-terminal ends of the chordate mature peptides and, when the invertebrate fragments were used to interrogate vertebrate databases, they failed to retrieve a secretin family homologue suggesting that members of this family are absent from non-vertebrate genomes. the exception is pacap for which a highly conserved partial sequence corresponding to the exon encoding the mature peptide has been isolated in hydra magnipapillata, in the tunicate, halocynthia roretzi and in several protostomes such as planarian, crab, squid and cockroach. database searches using the non-vertebrate pacap nucleotide or deduced peptide sequences failed to retrieve homologues from protostomes with available genome data. moreover, searches performed in hydra magnipapillata and related species nematostella vectensis genome assemblies with the hydra pacap nucleotide or deduced peptide sequence also failed to confirm the existence of a gene encoding pacap. in early deuterostomes, searches using the chelyosoma productum prp/pacap nucleotide or deduced peptide sequences in ciona intestinalis and ciona savignyi genomes or available tunicate ests failed to identify possible sequence homologues in urochordate. taken together these results raise questions about the authenticity of the previously reported sequences. an alternative strategy utilized a secretin family prototype sequence model based on the assumption that the chordate members arose from a common precursor gene which duplicated to give pacap-like and gcg-like subfamily exons. the prototype sequence deduced in silico was h1a2d3g4t5f6t7s8d9y10s11xxl14xq16xxa19k20xf22xxw25l and contains conserved amino acid positions and high conservation was found for the n-terminal region. structural characterisation using pfam analysis classified the generated prototype sequence as a hormone_ member and sequence similarity searches performed retrieved secretin family members in vertebrates but failed to identify potential members in non-vertebrates. a second approach using hmm models and searching the general ncbi non-redundant peptide and an invertebrate subset of the ncbi nucleotide and est databases corroborated the preceding results. phylogenetic analysis the optimal maximum likelihood tree with bootstrap support values higher than 50% is presented in figure despite the high level of sequence identity and short sequences utilized, phylogenetic analyses of the chordate mature peptides and their corresponding nucleotide sequences resulted in similar tree topologies and suggests that members of the secretin family share a common ancestry. in the optimal ml tree presented in figure two clades pacap-like and gcg-like were obtained suggesting that after ancestral exon duplication two main peptide subgroups emerged and underwent distinct evolutionary trajectories. similar tree topologies were obtained using bayesian approaches and a pacap-like derived clade includes the peptides pacap, prp, ph, vip, ghrh and sct and a gcg-like clade the peptides gcg, glp glp and gip. the chelyosoma pacap deduced peptide sequences always grouped with the vertebrate peptide sequence homologues and the tunicate prps did not cluster with any particular peptide clade and in all the analysis performed tended to be more closely related to the tetrapod sct. gene environment comparisons the immediate gene environment of vertebrate secretin members was compared and indicates that prp/pacap, gcg/glp, and ghrh genome regions are syntenic and gene order is in general maintained. the gcg/glp gene environment shares at least genes, prp/pacap, genes and ghrh, gene when equivalent genome regions are compared between takifugu, xenopus, chicken and human. the vip and gip genomic regions are poorly conserved and no gene synteny or gene order was identified. moreover, searches also failed to identify conservation of gene linkage between the different vertebrate secretin family members. in takifugu, the neighbouring genes of the paralogue gcg/glp and pacap genes were also duplicates supporting the teleost gene or genome duplication event. searches were extended to the lamprey and lizard genomes but contiguous sequences were too small to confirm the existence of gene environment conservation. comparison of the sct genome region between chicken and human revealed they are highly conserved and the order of genes is maintained. in xenopus genome, the chicken and human sct flanking genes are localised in the amphibian scaffold_ and in takifugu, where a gene homologue is proposed to be absent, they are distributed in two distinct genome regions: mucdhl and drd are localised in scaffold n and the remaining genes in scaffold n suggesting the existence of a conserved gene block prior to the emergence of the tetrapod gene. in order to identify a potential secretin family genome region in non-vertebrates, the conserved vertebrate gene environment was used to retrieve homologues in the c. elegans, drosophila and ciona genomes. genes sharing similarity in sequence to those flanking the vertebrate gcg/glp, prp/pacap and ghrh loci were identified although the genes encoding secretin family members were absent. in c. elegans the genes drh- abts- src- and m01a map to chromosome i and are respectively homologues of the human ifih and slc4a on chromosome yes on chromosome and rpn on chromosome in drosophila, sei and cg are localised in chromosome 2r and are the homologues of human kcnh and rpn and the fruit-fly dcr- and cg genes in chromosome 3r the correspondent in sequence of the human ifih and mettl respectively. in the tunicate genome, homologues of the vertebrate secretin family flanking genes were identified scattered in the ciona genome assembly. homologues of human kcnh, ifih and slc4a genes which flank gcg/glp in vertebrates were identified. the conserved gene environment of the vertebrate prp/pacap genes: yes and mettl genes are also present as well the putative urochordate homologue of the rpn gene conserved in the vertebrate ghrh genome region. in the c. elegans genome chromosome contained homologues of the genes which flank prp/pacap, gcg/glp and ghrh in vertebrates. this suggests that this chromosome may be the protostome genome fragment that most resembles the potential metazoan ancestral secretin genome region from which the vertebrate members emerged. however, detailed analysis of chromosome positions revealed they are not mapped in close proximity and do not forms a gene cluster. moreover, no conserved linkage between the putative drosophila and the c. elegans gene homologues exists. discussion comparative analysis of data from phylogenetically distant organisms is a major contributor for understanding gene and gene family evolution and the role of function and regulation in this process. the identification of gene homologues in vertebrates and early metazoan genomes provides a unique opportunity to perform comparative studies and to investigate gene family ancestries. the secretin family is a well-studied group of peptides which activate specific receptors of family gpcrs to bring about their pleotropic actions in vertebrates. secretin-like family gpcrs have been identified and cloned from non-vertebrate genomes and their putative peptide ligands identified by immunohistochemistry using antisera raised against the mammalian peptide homologues. both peptides and their corresponding receptors are proposed to have arisen by gene duplication events prior to the vertebrate radiation and they represent an interesting model for studies of receptor-ligand evolution. in the present study, despite extensive in silico database searches it was not possible to identify members of the secretin peptide family in non-vertebrates regardless of the report of a full-length pacap cdna in the tunicate, chelyosoma productum and cdna of the pacap coding exon in crab, cockroach, squid, planarian and hydra magnipapillata. moreover, using the partial pacap cdna previously identified in hydra magnipapillata to interrogate its sequenced genome failed to identify the gene homologue. it seems unlikely that the failure to identify pacap in non-vertebrates was related to the methodology used as a similar approach has been successfully utilized to identify the gene encoding the active nonapeptide hormone, vasotocin/vasopressin in the amphioxus genome assembly and also the gene loci in several chordates including teleosts. in fact, the existence of neurohypophysial hormones in deuterostomes and protostomes has been amply confirmed by the isolation of both genes and peptides from representatives of a number of different phyla. similarly, conserved sequence homologues of vertebrate neuropeptide y family members have been isolated from molluscs and also from fruit-fly and mosquito and gpcr ligand-receptor pairs similar to the vertebrate npy system have been characterised. a comprehensive in silico analysis of the fully sequenced drosophila genome identified and classified gpcrs and compared their number to putative neuropeptide ligands. although secretin family gpcr members were identified in the arthropod genome, only two potential ligands, corticotrophin releasing factor -related peptides and amnesiac genes, unrelated to chordate secretin peptide family members were predicted. in contrast, recent studies performed in molluscs using ihc and maldi-tof/tof identified partial peptides with a similar mass to vertebrate pacap in the snail whole hemolymph and cns extracts. should the identity of the peptide be confirmed by sequencing taking in consideration the results of the present study a new paradigm will be required to explain secretin family evolution. there is evidence that genes for amnesiac in drosophila and maxadilan in sand-fly might encode functional homologues of the vertebrate pacap despite their lack of sequence similarity and the maxadilan peptide is able to activate mammalian pac receptors in vitro. the activation of family gpcrs members by the secretin family of ligands has been linked to their well conserved structure which comprises an alpha helix in the mid and c-terminal region and an n-terminal loop. the region of maxadilan implicated in pac receptor activation has an identical structure to the n-terminal region of secretin family ligands and contains key amino acids involved in receptor activation. in fact, mutation analysis with maxadilan demonstrated that despite its greater size compared to vertebrate secretin family peptides, the disruption of four conserved cysteine residues responsible for the formation of two disulfide bonds led to loss of activity. taking into consideration the degree of conservation of the n-terminal ligand binding domain of the secretin family gpcrs and their relative promiscuity it is unsurprising that protostome peptides activate vertebrate receptors. in nematode and drosophila, pdf stimulated the homologues of vertebrate secretin gpcrs but they were not stimulated by secretin family members. these observations suggest that specificity of the receptor members has changed during evolution and may explain the failure to identify conserved ligands. a general model to explain peptide ligand binding and receptor activation has emerged recently for class b gpcrs. the proposed mechanism, known as the "two-domain model" suggests that initial ligand-receptor interactions are mediated by the central and c-terminal peptide segments with the extracellular n-terminal receptor region and that activation occurs subsequently when the bioactive n-terminus of the ligand binds the receptor juxtamembrane domain. in this context, the identification in the present study of putative "signature" amino acids conserved across taxa for each peptide groups may explain differences in selection and affinity for receptors. for example, mutation of conserved residues within the c-terminal region of vip revealed that substitution of l decreased peptide biological activity without altering the predicted structure. d is conserved across the pacap-like peptides and this residue has a role in adenyl cyclase stimulation and interacts with basic residues in the second transmembrane helix of vpac. similarly, d which is conserved across the gcg-like members is essential in the activation of mammalian gcgr. it will be of interest in the future to study the role of unique amino acid residues/motifs identified in the present study within the n-terminal and c-terminal regions of each peptide group. recently, a darwinian evolutionary model was proposed to explain the origin of steroid hormones and their receptors and may also explain the emergence of metazoan secretin peptide-receptor pairs. the steroid hormones and receptors were proposed to have evolved through a molecular exploitation process in which structurally adapted receptors evolved prior to ligand emergence. by reconstructing the sequence of the ancestral steroid receptors the authors verified that they are activated by hormones that only emerged in the tetrapod lineage. at present, evolutionary comparisons of the metazoan receptor members and secretin peptide family suggests that, receptors emerged prior to the ligands which were subsequently acquired as a consequence of genome evolution in the chordate radiation. generally, two major gene or genome duplication events are proposed to have occurred at the origin of vertebrates and have accompanied increased organismal complexity and emergence of gene novelties. whilst the majority of gene duplicates were probably lost as a consequence of their functional redundancy, some were fixed in the genome by the gain of new biological functions or partitioning the function of the ancestral counterpart and this may be the case for the secretin family gpcrs and their ligands. the origin of the chordate secretin peptide family has been previously associated with the insect adipokinetic hormone and akh-precursor related peptides precursor evolution. despite their low sequence similarity, the arthropod akh and aprp precursor was found to share a similar gene organisation and comparable functions with vertebrate gcg and ghrh and they were proposed to have shared common origin prior to protostome-deuterostome divergence. however, against this hypothesis is the recent demonstration that insect adk signals through a gonadotropin-releasing hormone like receptor and also the isolation of putative nematode akh-gnrh related precursors suggesting that the invertebrate adk members may share common evolution with the metazoan gnrh system.  the present study does not confirm the results of ihc studies in the early 70's and 80's which identified putative invertebrate secretin family members using antisera against mammalian peptides. it was not possible to identify sequence homologues of the chelyosoma productum prp/pacap peptides in the sequenced ciona genomes, although at least putative secretin-like family gpcrs have been reported. the previous facts taken with i) the identification in snail of a putative pacap peptide; and ii) the activation of a secretin-like family gpcrs in nematode and drosophila by pdf but not by vertebrate secretin family members makes it difficult to establish when the peptide members emerged in the deuterostome lineage. it is hypothesised that the emergence of the full suite of receptors and their ligands accompanied the rapid genome changes during chordate evolution. the ancestral secretin family gene probably arose as part of an existing gene or gene fragment and via exon and gene duplication events generated the existing suite of family members. this occurred after the emergence of the secretin family gpcrs and led to the establishment of novel and specific receptor ligand interactions that contributed to the generation of novel physiological functions. in contrast, to other peptide families, such as npy/pyy and oxytocin/vasopressin which stimulate receptors of family gpcrs and are highly conserved from protostomes to deuterostomes, members of the secretin family gpcrs appear to have adopted new ligands during evolution. 
in human, members exist and sequence and functional homologues and ligand-receptor pairs have been characterised in representatives of most vertebrate classes. this event occurred after the emergence of the metazoan secretin gpcrs and led to the establishment of novel peptide-receptor interactions that contributed to the generation of novel physiological functions in the chordate lineage. conserved regions flanking secretin family members were established by comparative analysis of the takifugu, xenopus, chicken and human genomes and gene homologues were identified in nematode, drosophila and ciona genomes but no gene linkage occurred. it has not been possible to determine when the ligands evolved but it seems likely that it was after the protostome-deuterostome divergence from an exon that was part of an existing gene or gene fragment by rounds of gene/genome duplication. receptors of the secretin-like family gpcrs are present in protostomes but no sequence homologues of the vertebrate cognate ligands have been identified. the secretin family is a pleotropic group of brain-gut peptides with affinity for class g-protein coupled receptors proposed to have emerged early in the metazoan radiation via gene or genome duplications. in silico sequence comparisons failed to retrieve a non-vertebrate secretin family homologue. in contrast, secretin family members were identified in lamprey, several teleosts and tetrapods and comparative studies revealed that sequence and structure is in general maintained. however, in drosophila and nematode genes which flank vertebrate secretin family members were identified in the same chromosome. secretin-like family gpcr homologues have also been isolated in non-vertebrate genomes however their corresponding ligands have not been convincingly identified and their evolution remains enigmatic. sequence comparisons and phylogenetic analysis revealed that pacap, vip and gcg are the most highly conserved members and two major peptide subfamilies exist; i) pacap-like which includes pacap, prp, vip, ph, ghrh, sct and ii) gcg-like which includes gcg, glp glp and gip.
 the secretin family is a pleotropic group of brain-gut peptides with affinity for class g-protein coupled receptors proposed to have emerged early in the metazoan radiation via gene or genome duplications. in human, members exist and sequence and functional homologues and ligand-receptor pairs have been characterised in representatives of most vertebrate classes. secretin-like family gpcr homologues have also been isolated in non-vertebrate genomes however their corresponding ligands have not been convincingly identified and their evolution remains enigmatic.  in silico sequence comparisons failed to retrieve a non-vertebrate secretin family homologue. in contrast, secretin family members were identified in lamprey, several teleosts and tetrapods and comparative studies revealed that sequence and structure is in general maintained. sequence comparisons and phylogenetic analysis revealed that pacap, vip and gcg are the most highly conserved members and two major peptide subfamilies exist; i) pacap-like which includes pacap, prp, vip, ph, ghrh, sct and ii) gcg-like which includes gcg, glp glp and gip. conserved regions flanking secretin family members were established by comparative analysis of the takifugu, xenopus, chicken and human genomes and gene homologues were identified in nematode, drosophila and ciona genomes but no gene linkage occurred. however, in drosophila and nematode genes which flank vertebrate secretin family members were identified in the same chromosome.  receptors of the secretin-like family gpcrs are present in protostomes but no sequence homologues of the vertebrate cognate ligands have been identified. it has not been possible to determine when the ligands evolved but it seems likely that it was after the protostome-deuterostome divergence from an exon that was part of an existing gene or gene fragment by rounds of gene/genome duplication. the duplicate exon under different evolutionary pressures originated the chordate pacap-like and gcg-like subfamily groups. this event occurred after the emergence of the metazoan secretin gpcrs and led to the establishment of novel peptide-receptor interactions that contributed to the generation of novel physiological functions in the chordate lineage. 