Tetrapod V1R-like ora genes in an early-diverging ray-finned fish species: the canonical six ora gene repertoire of teleost fish resulted from gene loss in a larger ancestral repertoire
© Zapilko and Korsching. 2016
Received: 28 October 2015
Accepted: 15 January 2016
Published: 27 January 2016
Chemical senses serve a multitude of essential functions across the animal kingdom. Vertebrates employ four GPCR families to detect odors, among them the v1r/ora gene family. The V1R family is known to evolve rapidly in the lobe-finned lineage giving rise to tetrapods, but the homologous ORA family consists of just six highly conserved genes in teleost fish, with direct orthologs in the lobe-finned fish coelacanth. Thus, the teleost repertoire of six canonical ora genes was assumed to be the ancestral feature before the divergence of ray-finned and lobe-finned fish. So far, this hypothesis has not been tested with earlier diverging ray-finned fish.
We have newly identified the complete ora gene repertoires of five teleost species, and of spotted gar, a basal ray-finned fish, using thorough data mining and extensive phylogenetic analysis. The genomes of eight further teleost species were re-analyzed for their ORA repertoires. We report that direct orthologs of the six canonical ora genes (ora1-6) were present in all newly analyzed species, with faithfully preserved exon/intron structure and mostly preserved genomic arrangement in symmetric pairs for ora1-4. In four teleost species including medaka and cave fish we observe species-specific gene duplication events. Thus, the ora gene repertoire in teleost fish is not quite as strictly conserved as previously assumed. In fact, the examination of non-synonymous vs. synonymous substitution rates (dN/dS) shows pronounced negative selection in five of the six ora genes, but also rare occurrence of positive selection in ora3 and ora6. Surprisingly, spotted gar possesses beyond the six canonical genes three additional genes, ora7-8b, orthologous to coelacanth genes v1r07-10. No orthologs for these genes were found in teleosts and cartilaginous fish.
Early diverging ray-finned fish such as the spotted gar possess several v1r-like genes previously assumed to be restricted to the lobe-finned lineage, but now found to be already present in the most recent common ancestor of lobe- and ray-finned fish. Thus, the presence of just six canonical ora genes in many teleost species is not the ancestral feature of the ray-finned lineage, but caused by loss of two ancestral genes in teleosts.
The chemical senses of animals deliver crucial information for essential tasks such as prey localization, predator evasion, reproduction and social behavior. Vertebrates possess a specialized olfactory sense, which employs four different GPCR families to detect odors. Generally, these families are characterized by a very dynamic evolution, with many gene gains and losses leading to distinctly different receptor repertoires even in closely related species [1, 2]. A notable exception is the ORA family, which consists of the same six genes in several distantly related teleost fish species, with very rare gene duplication events and a singular gene loss [3–5]. In all these species, ortholog ora genes are always more closely related than paralogs [3–5], and four of these genes have direct orthologs already in a cartilaginous fish [6, 7]. This is all the more remarkable, since the mammalian V1R family - which has originated from a single gene of the ORA family, ora1  - shows a very dynamic evolution characterized by frequent gene gains and losses even between closely neighboring species [9, 10]. V1R repertoires range between zero and close to 300 genes in tetrapod species . The recent discovery of a family of 20 v1r genes already in coelacanths [8, 12] showed this tendency towards dynamic evolution to be present early in the lobe-finned lineage (which comprises coelacanths and tetrapods).
Thus, the difference in family properties - on one hand six canonical ora genes for teleosts, which are ray-finned fish, and on the other hand highly dynamic V1R repertoires for coelacanths and tetrapods - appeared to be a difference between the ray-finned and the lobe-finned lineage. Since orthologs of all canonical ora genes were also present in coelacanths and since all V1Rs originated from the ORA1 clade, the teleost repertoire of six canonical ora genes was assumed to be the ancestral feature before the divergence of ray-finned and lobe-finned fish. So far this hypothesis has been consistent with results in eight teleost species: two cichlids (Haplochromis chilotes, Oreochromis niloticus), medaka (Oryzias latipes), two pufferfish (Takifugu rubripes, Tetraodon nigroviridis), salmon (Salmo salar), stickleback (Gasterosteus aculeatus), and zebrafish (Danio rerio) [3–5, 13, 14].
Although teleosts make up the bulk of fish species with ~29000 species , analysis of earlier diverging ray-finned fish would help to elucidate the evolution of the canonical ORA repertoire. We have investigated the ORA family of the spotted gar, Lepisosteus oculatus, a species that diverged early in the ray-finned lineage from teleosts . Furthermore we have delineated the ORA family in five newly available teleost genomes, among them cod (Gadus morhua) as an early-diverging teleost, cave fish (Astyanax mexicanus), as close relative to zebrafish, platyfish (Xiphophorus maculatus), Amazon molly (Poecilia formosa) and mummichog, a species of killifish (Fundulus heteroclitus) as sister group of medaka. Finally we have re-analyzed the eight species mentioned above and present a comprehensive view of the ORA family in 14 ray-finned fish species.
Here we report that the canonical six ora genes are present in all 14 fish genomes analyzed. We notice six species-specific, i.e. evolutionarily late gene duplications, three species-specific intron gains, and rare instances of positive selection as suggested by dN/dS analysis. Surprisingly, we identified three additional monoexonic ora genes in spotted gar, ora7-8b, which have no teleost orthologs, but form two clades with V1Rs restricted to the lobe-finned lineage. Hence we propose that the ancestral ORA repertoire of bony fish consisted of at least eight genes, ora1-8, of which two genes were lost in teleosts (ora7-8), and seven genes in mammals (ora2-8). The full repertoire of eight ancestral ora genes is present in lobe-finned coelacanth and ray-finned spotted gar, and thus the canonical six gene repertoire so characteristic for teleosts should be considered a derived feature.
The ora gene repertoire of an early-derived ray-finned fish encompasses three genes specific to the lobe-finned lineage
Previous research has shown the presence of six highly conserved, canonical ora genes in a total of eight teleost fish species ranging from zebrafish, an early-derived teleost, to several neoteleost species [3–5]. Orthologs of all six genes were identified in a lobe-finned fish [8, 12] suggesting this gene set to be the ancestral feature of lobe-finned as well as ray-finned fish. Multiple gene expansions appeared to be a feature characteristic for and restricted to the lobe-finned lineage that gave rise to tetrapods with their highly variable v1r gene repertoires [9–11].
Unexpectedly, these three novel ora genes belong to V1R clades of the lobe-finned lineage. Despite thorough data mining, no orthologs for these genes could be identified in teleosts and elephant shark. Extending the established numbering for ORAs the three genes were named ora7, ora8a and ora8b. ORA7 is a direct ortholog of the coelacanth receptor V1R07 (Fig. 1). The genes ora8a and ora8b result from a late, species-specific duplication event, and are orthologous to V1R08-10 of coelacanths. Thus, the most recent common ancestor (MRCA) of ray-finned fish and lobe-finned fish had not six, but at least eight ancestral genes, seven of which are present as single genes in coelacanths as well as spotted gar, whereas the eighth gene has undergone small independent expansions in both lineages (Fig. 1). Genes ora7 and ora8 appear to have been lost early in the teleost lineage. Ora8 is ancestral to a large cluster of amphibian v1r genes (Additional file 1), whereas ora7 appears to have been lost early in the tetrapod lineage, similar to ora2, 4-6 .
Rare species-specific gene duplications in teleost ora genes
We performed a comprehensive search of the ORA family in the genomes of 13 teleost species, five of which had not been analyzed before (cave fish, cod, Amazon molly, killifish, platyfish). Direct orthologs of all six canonical ora genes were identified in all five species (Fig. 1, Additional files 2 and 3). Our reanalysis of eight teleost genomes (zebrafish, stickleback, medaka, fugu, tetraodon, salmon, Lake Victoria cichlid, Nile tilapia) confirmed most of the previously found ora sequences [3–5, 13, 14, 18]. Tetraodon ora6 and fugu ora2 were reported as multiexonic , but in the currently available database versions a monoexonic prediction results in higher homology to orthologous genes.
We observe a duplication of the zebrafish, salmon and medaka ora3 gene, and of the cave fish and salmon ora5 gene (Figs. 1 and 2). All duplications are species-specific (Fig. 1), i.e. late evolutionary events after speciation had occurred. The duplicate genes neighbor each other in the genome, i.e. resulted from local gene duplications (Fig. 2 and Additional file 2). For zebrafish and salmon, our results are consistent with  and , respectively. Another duplicate gene reported for ORA1  was not found in the genome. On average one gene duplication per three species occurs. This is much less frequent than what is observed for the highly variable mammalian V1R repertoires, but nevertheless shows the conservation of the ora gene family size not quite as strict as initial results suggested . We have therefore investigated the degree of conservation for two other features, the exon/intron organization and genomic arrangement, and the ratio of synonymous vs. nonsynonymous mutations as a measure for the selective pressure on the six canonical ora genes.
Intron gains in the ora gene family of ray-finned fish
In previous analyses ora1-2 and ora5-6 genes were found to be generally intronless, whereas ora3 and ora4 possess three and one intron, respectively [3–5, 13, 14, 18]. Additional introns had been reported for fugu ora2, zebrafish ora4, tetraodon ora6 and salmon ora6 [3, 4].
All five newly analyzed species and Nile tilapia show a conserved exon/intron pattern: ora1-2, 5-6 are monoexonic (except cave fish ora2), ora3 has three introns, and ora4 one intron (Fig. 3). The exact borders of these introns are strictly conserved in all species, including the early-derived ray-finned spotted gar (Additional file 4), consistent with a common origin of these introns early in the evolution of the ray-finned lineage, since they are absent in the coelacanth orthologs .
Genomic arrangement of ora1/2 and ora3/4 gene pairs is not always preserved
Ora1 and ora2 are arranged head-to-head in the genomes of previously analyzed teleost species [3–5], whereas ora3 and ora4 exhibit a tail-to-tail genomic orientation [3, 5]. Interestingly the head-to-head arrangement for the ora1/ora2 gene pair is already present in the elephant shark, a cartilaginous fish, whereas the ora3/ora4 gene pair has head-to-tail orientation in this species (Fig. 2). Both features may correspond to the ancestral situation since coelacanths also show head-to-head orientation for ora1/ora2 and head-to-tail for ora3/ora4 . If so, the ora3 gene must have flipped at some point in the ray-finned lineage resulting in tail-to-tail orientation.
We found the head-to-head arrangement of the ora1/ora2 gene pair as well as the tail-to-tail arrangement of the ora3/ora4 gene pair in most, but not all of the newly analyzed species, including the early-derived spotted gar. However, in cod ora1 and ora2 appear to have lost their close genomic association, and in cave fish the same is true for ora3 and ora4 (Fig. 2). Three of the four genes are present within large contigs, so that technical reasons for the association loss appear unlikely. Again, this analysis shows a somewhat less stringent conservation of genomic features within the ora gene family than previously assumed.
Two sites in ora3 and ora6 show evidence of positive selection despite generally strong negative selection in the six canonical ora genes
The rate of nonsynonymous to synonymous (silent) nucleotide substitutions (dN/dS) is often used to estimate the selective pressure acting on particular genes. A dN/dS value below 1 is taken as evidence of negative selection, whereas dN/dS >1 is an indicator of positive selective pressure, i.e. a tendency towards diversification. Positive selection has been reported in several other chemosensory receptor gene families [8, 22, 23]. For ora genes previous analyses have yielded somewhat conflicting results. While a study of ora genes in five teleost genomes has found strong negative selection and no evidence for positive selection , similar to results with 13 salmonid species , other studies have reported positive selection in one of the ora genes in several closely related species [24, 25].
Interestingly, two positively selected sites were observed, one in an extracellular loop of ORA3 and one in an intracellular loop of ORA6 (Fig. 4). While the functional significance of these sites is so far not clear, this result again shows a less stringent conservation within the ora gene family than previously assumed, based on a considerably smaller data set .
Olfactory receptor families are among the fastest evolving gene families . In particular, the V1R family is known to rapidly evolve in tetrapods , whereas the sister group in teleost fish, the ORA family, consists of a near constant repertoire of six canonical genes , all of which are present in the MRCA of ray-finned and lobe-finned fish, but with one exception have successively been lost in the lobe-finned lineage [3, 8, 12]. Thus, the six ora genes of teleosts were assumed to constitute the ancestral feature of both lineages. Here we have re-examined this assumption by delineating the ORA family in a larger set of teleost genomes than previously available, and in particular by investigation of an early-derived ray-finned fish genome, the first non-teleost ray-finned fish genome to be analyzed.
We find the canonical six ora genes in 11 teleost species (ora1 is lost in both pufferfish ) and an early-derived ray-finned fish, but also occasional species-specific duplications of single ora genes. We also report a loss of the pairwise genomic arrangement, another characteristic feature of the ora gene family, for a single gene pair in two species. We observe two new species-specific intron gains within the ORA family. While there exists some controversy about the extent of intron dynamics in higher eucaryotes, it is generally accepted that there are very few intron gains in the vertebrate lineage . A total of three intron gains in a small family in 14 species then shows considerable intron dynamics. Such intron dynamics appears to be characteristic for olfactory receptor gene families, as it has also been reported for taar genes  and or genes . Finally, rare occurrence of positively selected sites in two ora genes again points to a somewhat more dynamic evolution of the ora gene family than previously assumed, based on a much smaller data set .
Unexpectedly we have identified three additional ora genes in spotted gar, which do possess coelacanth, but no teleost orthologs. Two of these genes, ora8a, and ora8b result from a recent gene duplication, which leaves two genes, ora7-8 as ancestral genes already present in the MRCA of ray-finned and lobe-finned fish. We conclude that these genes most likely have been lost in the teleost lineage. Thus, the unusual conservation of the six canonical ora genes in teleost fish constitutes a secondarily evolved feature of the teleost lineage.
It may be expected that the very different evolutionary dynamics in tetrapod V1Rs vs. teleost ORAs reflect a difference in function. V1Rs have been reported as pheromone receptors . So far a single ORA receptor has been deorphanized as receptor for a substance acting as reproductive pheromone . Known reproductive pheromones of teleost fish encompass steroid and prostaglandin hormones and their metabolites  and it is conceivable that this double constraint on pheromonal and hormonal quality forces such substances to evolve less rapidly  than pheromones not being constrained by a concomitant hormonal function. However, a test of this hypothesis will require considerably more information on receptor/ligand pairing for ora genes in different species than currently available.
Data mining and phylogenetic analysis
Nomenclature of species used in phylogenetic analysis
Latin species name
Mexican cave fish
Mummichog (a killifish)
Pf, Xm, Ol
Lake Victoria cichlid
Pf, Xm, Fh
Xm, Fh, Ol
Spotted green pufferfish
Japanese pufferfish (fugu)
Pf, Fh, Ol
The final inclusion criteria for candidate ora genes were firstly, a position within the ORA clade in the phylogenetic analysis, secondly, the presence of amino acid motifs characteristic for the Ora family , and thirdly, for the full length sequences, the prediction of seven trans-membrane domains. Transmembrane regions were predicted for multiple aligned sequences using PRALINE .
For phylogenetic analysis published sequences from elephant shark, African coelacanth, and two rockfish species (Table 1) were included [6, 8, 12, 25]. Sequences were aligned with MAFFT 7 , an online version of the multiple alignment tool MAFFT , using the E-INS-I strategy with the default parameters. The multiple sequence alignment was manually edited using Jalview  to remove regions with gaps in over 90 % of sequences. The phylogenetic tree was calculated using a Maximum likelihood algorithm, PhyML-aLRT with SPR setting for tree optimization and chi square-based aLRT for branch support  available online [37, 38]. Branch support above 80 % was considered significant. T2r genes of zebrafish, stickleback, spotted green pufferfish and coelacanth served as outgroup (see Additional file 3). The tree was drawn using an online version of TreeDyn .
The dN/dS ratios for the individual codons of the different ora family members were calculated using single likelihood ancestor counting (SLAC) described in , as well as the fixed effects likelihood method (FEL) that directly estimates nonsynonymous and synonymous substitution rates at each site . Both methods were used as implemented on the datamonkey server . Codon based nucleotide alignments were generated with PAL2NAL , and regions with gaps in over 90 % of sequences were removed using Gap Strip/Squeeze v2.1.0 . The salmon ORA6 sequence is incompletely predicted (three transmembrane domains are missing) and was not included in the analysis. ORA1 from cod, a full length sequence, was also excluded from the analysis due to incomplete sequencing.
Availability of supporting data
The data sets supporting the results of this article are included within the article and its additional files.
We thank Vladimir Shiriagin and Adnan Syed for technical advice and the Deutsche Forschungsgemeinschaft for financial support (grant Ko-1046/7-1 to SIK).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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