The purpose of this study was to identify the repertoire of chemosensory receptors for olfaction in the sea lamprey draft genome and infer the evolution of those gene families in the chordate lineage. All known chemosensory receptors are 7-transmembrane G-protein coupled receptors, but vary considerably in overall sequence and exon structure, consistent with an ability to recognize diverse ligands . This study identifies 59 intact single-exon chemosensory receptor genes in the sea lamprey genome that belong to three CR families: V1R-like, OR and TAAR families. These genes share many characteristics of gnathostome single-exon CR genes. Sea lamprey CR genes identified in this study are similar in structure and size to those described in higher vertebrates [3, 4, 8]. We found several contigs that each contain highly homologous members of the same CR families, which is consistent with findings in gnathostome genomes where CR genes often cluster in specific chromosomal regions . Furthermore, of the 59 intact CR genes from our Petromyzon marinus genome Draft Assembly, we have identified a set of seventeen in the cDNA from olfactory epithelia of mature and parasitic-stage adult sea lamprey using 454 sequencing. RT-PCR and in situ hybridization confirmed that expression of representative OR and TAAR are largely limited to odorant receptor neurons and testis, which is consistent with expression of single-exon CR genes in higher vertebrates [1–3]. The observed life history-specific expression of CR genes may be adaptive for different olfactory-mediated behaviours demonstrated in the sea lamprey, as parasites display search behaviours for detection of prey odor and adults respond specifically to migratory and mating pheromones . It is likely that the majority of the 59 predicted intact genes described in this study are functional CR genes for olfaction in the sea lamprey.
Our data indicate that all three families of single exon CR genes were present in the most recent common ancestor of jawless and jawed vertebrates (Figure 1). Both the OR and TAAR genes are numerous and diverse in the genomes of sea lamprey and higher vertebrates [7, 9], supporting an expansion prior to the most recent common ancestor of jawed and jawless vertebrates. Our search for predicted genes from the draft genomes of Stronglyocentrotus
http://www.hgsc.bcm.tmc.edu/blast.hgsc, Ciona and Branchiostoma
http://genome.jgi-psf.org/euk_cur1.html using lamprey gene queries and subsequent phylogenetic analyses suggest that OR, TAAR and V1R gene families had not expanded before the rise of vertebrates, and that OR and V1R receptor families are vertebrate-specific, with TAAR specific to chordates. This is consistent with findings from the genomes of the cephalochordate Branchiostoma  and the ascidian Ciona , both possessing trace amine receptors (11 and 14 TAAR, respectively), but neither of which have been shown to possess V1R pheromone or olfactory receptors. A recent report has inferred a much more ancient origin for ORs, placing them in the common ancestor of vertebrates and cephalochordates, while placing the origin of TAARs in the common ancestor of vertebrates . These results are not in agreement with the BLAST results presented here, nor with the available genome-scale analyses of cephalochordates and urochordates [11, 14]. A large-scale phylogenetic analysis of available ORs, TAARs and biogenic amine receptors would address this discrepancy. However, given the expanse of time involved in the evolution of these ancestral molecules and the striking variation observed within OR and TAAR gene families, resolution of these discrepancies may require more data than BLAST searches, alignments and phylogenetic inference can provide. Specific ligand-receptor interactions and structural analysis of ligand-binding domains in these receptors should provide critical data regarding homology and convergence in the chemosensory receptor repertoires of deuterostomes.
Analysis of the genome of Strongylocentrotus purpuratus has offered strong evidence of an elaborate chemosensory system in echinoderms . Sea urchin rhodopsin-type GPCR repertoires indicate that these large chemosensory receptor subfamilies expanded in echinoderms in a lineage-specific manner and were derived from biogenic amine receptors [28–30]. Using lamprey TAAR and OR to query the GLEAN predicted peptides in the Strongylocentrotus genome produced a non-redundant list of 63 serotonin-, octopamine-, histamine and adreno-receptors. Thirteen of these "best hit" receptors that were identified as potential TAARs were included in our phylogenetic analysis. These formed a well-supported clade distinct from single-exon vertebrate chemosensory genes [see Additional File 2]. These results support the hypothesis that chemosensory trace-amine receptors evolved independently at least twice in the deuterostome lineage.
The 28 intact lamprey TAAR genes identified in our study cluster into a well resolved group, in agreement with the 21 TAAR genes identified from a previous sea lamprey draft assembly that form a monophyletic clade . Other than in a few teleost groups, the number of TAAR genes did not expand dramatically in tetrapods . In contrast, OR gene repertoires in both teleosts and tetrapods experienced dynamic expansion before and after these two vertebrate groups diverged. The sea lamprey ORs cluster most closely to the intact η, κ, and θ OR subfamilies described by Niimura and Nei . These OR subfamilies are represented but not expanded in either teleosts or tetrapods .
It is interesting that V1R-like genes, a remarkably diverse CR family in tetrapods , did not expand extensively in either the sea lamprey or teleost genomes. Sea lamprey V1R-like genes cluster in NJ analysis with expressed V1R genes described in Danio  [see Additional File 3]. The presence of V1R-like genes in lamprey and our phylogenetic analysis indicate that this family arose early in the vertebrate lineage, before the agnathan-gnathostome divergence (Figure 1), but did not expand dramatically until vertebrates began exploiting terrestrial habitats . Further, the V1R family likely arose after the separation of cephalochordate and urochordate lineages which do not possess identifiable V1R-like sequences in their current draft genomes. The early divergence of V1R in the vertebrate lineage is further demonstrated by the phylogenetic signal observed between the lamprey, teleost and mammalian V1Rs [see Additional File 3] and the need to use an appropriate protein substitution matrix to detect similar homologies across greater than 300 million years of evolutionary time . During the successful diversification in the teleost lineage, the V1R family did not undergo the dramatic expansions that are observed in the genomes of terrestrial vertebrates , nor did V1R expand during additional rounds of genome duplication in teleosts . Our results indirectly support a previous study that the V1R family expanded when vertebrates adapted to the terrestrial environment .
The most surprising, and arguably the most interesting, results from this study are the lack of V2R-like sequences identifiable from the currently available genomic resources for the sea lamprey. The V2R family is believed to encode receptors for detection of water borne compounds [8, 10]. This family is characterized by rapid gene turnover and lineage specific phylogenetic clustering , and is most expanded in fish and frogs . We anticipated that this family would be represented and expanded in the lamprey genome. Our search for potential V2R orthologs in the current sea lamprey draft genome, and those of other groups [11, 13, 14], did not produce an identifiable full or partial V2R candidate, nor were any V2R-like transcripts found in olfactory organ EST library (18,815 transcripts), filtered 454 sequences (373,391 transcripts) from adult and parasite olfactory organ, or in the NCBI Trace database for lamprey (19,177,230 traces). It is also not likely that our search parameters were too stringent, as our queries using V2R sequences returned metabotropic glutamate receptors (2 intact genes, 13 pseudogenes) and calcium sensing receptors (2 intact genes) that are most similar to known V2R receptors. Additionally, searches of urochordate and cephalochordate genomes for potential V2R genes have identified no putative V2R sequences [11, 14]. These results from urochordate, cephalochordate, and the current sea lamprey draft genomes, as well as olfactory organ transcriptome data suggest that the V2R gene family arose and diversified in the lineage leading to teleosts after the separation of jawed and jawless vertebrates. This provides evidence regarding the timing of the V2R expansion and its' potential role in the successful radiation of the modern vertebrate lineage.
The limited repertoire of CR genes in the lamprey genome, in both the number of gene families and the restricted degree of expansion in each CR family, accounts for the limited number of odorants that are detected by lamprey. The 59 intact CR genes described in this study likely represent a large portion of the complete CR repertoire of sea lamprey, for two reasons. Extensive and stringent alignment and mapping analysis using lamprey ESTs indicate that the assembled contiguous sequences represent a minimum of 76 percent of the sea lamprey genome. The majority of lamprey ESTs that do not map to the assembly appear to be non-coding RNAs from those same repetitive regions that have confounded efforts to assemble larger scaffolds. Moreover, the CR transcripts detected in olfactory organ cDNA by 454 sequencing analysis all mapped to the draft genome, suggesting that 76% coverage provides a cautious estimate for coverage of CR families. Therefore, the complete sea lamprey CR repertoire is likely much smaller than those of teleost fish and tetrapods. Electrophysiological studies indicate that the sea lamprey can detect all of the classes of odorants that are detected by teleosts ; the actual number of individual odorant compounds within each odorant class that are detectable by the sea lamprey, however, is much reduced .
The lack of identifiable V2R homologs in the sea lamprey genome provides a perspective on development of CR early in the vertebrate lineage that leads to the dynamic and vagile life histories that characterize modern vertebrates. Similar to cephalochordates and urochordates, all extant agnathan species spend the largest portion of their life cycles, or their entire life, amongst the benthos. The diversification of CR genes in jawed vertebrates, including the advent of the V2R gene family, likely corresponded to an increased ability in ancient vertebrates to locate food and mates while exploiting novel habitats and avoiding potential threats. In retrospect, this was one of several critical early developments in our lineage that lead ultimately to the diversity of modern vertebrates.