MBE Advance Access originally published online on July 18, 2007
Molecular Biology and Evolution 2007 24(9):2099-2107; doi:10.1093/molbev/msm140
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Research Articles |
Evolution of Trace Amine–Associated Receptor (TAAR) Gene Family in Vertebrates: Lineage-Specific Expansions and Degradations of a Second Class of Vertebrate Chemosensory Receptors Expressed in the Olfactory Epithelium
Division of Molecular Marine Biology, Ocean Research Institute, University of Tokyo, Nakano-ku, Tokyo, Japan
E-mail: yhashi{at}ori.u-tokyo.ac.jp.
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Abstract |
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The trace amine–associated receptors (TAARs) form a specific family of G protein–coupled receptors in vertebrates. TAARs were initially considered neurotransmitter receptors, but recent study showed that mouse TAARs function as chemosensory receptors in the olfactory epithelium. To clarify the evolutionary dynamics of the TAAR gene family in vertebrates, near-complete repertoires of TAAR genes and pseudogenes were identified from the genomic assemblies of 4 teleost fishes (zebrafish, fugu, stickleback, and medaka), western clawed frogs, chickens, 3 mammals (humans, mice, and opossum), and sea lampreys. Database searches revealed that fishes had many putatively functional TAAR genes (13–109 genes), whereas relatively small numbers of TAAR genes (3–22 genes) were identified in tetrapods. Phylogenetic analysis of these genes indicated that the TAAR gene family was subdivided into 5 subfamilies that diverged before the divergence of ray-finned fishes and tetrapods. In tetrapods, virtually all TAAR genes were located in 1 specific region of their genomes as a gene cluster; however, in fishes, TAAR genes were scattered throughout more than 2 genomic locations. This possibly reflects a whole-genome duplication that occurred in the common ancestor of ray-finned fishes. Expression analysis of zebrafish and stickleback TAAR genes revealed that many TAARs in these fishes were expressed in the olfactory organ, suggesting the relatively high importance of TAARs as chemosensory receptors in fishes. A possible evolutionary history of the vertebrate TAAR gene family was inferred from the phylogenetic and comparative genomic analyses.
Key Words: olfaction • pheromone • multigene family • comparative genomics • genome duplication
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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In vertebrates, biogenic amines play important roles in many biological activities (Deutch and Roth 1999; Lewin 2006; Zucchi et al. 2006
). The classical biogenic amines (serotonin (5-HT), noradrenaline, adrenaline, dopamine, and histamine) are well-known hormones and neurotransmitters in the central and peripheral nervous systems (e.g., Deutch and Roth 1999; Silber 1999). A second group of endogenous amines called "trace amines," including p-tyramine, ß-phenylethylamine, and tryptamine, are also thought to function as chemical messengers in vertebrates (Zucchi et al. 2006 and references therein).
The trace amine–associated receptors (TAARs) form a specific family of G protein–coupled receptors (GPCRs) that are single-exon encoded and have coding sequences of about 1 kb in length (Lindemann and Hoener 2005). TAARs were initially identified as GPCRs that respond to trace amines but not to the classical biogenic amines (Borowsky et al. 2001; Bunzow et al. 2001). Recently, it was demonstrated that TAARs function as chemosensory receptors in the olfactory epithelium in mice and perceive several volatile amines (Liberles and Buck 2006). Fifty-seven intact TAAR genes were identified in zebrafish, although only 5 functional TAAR genes have been found in humans (Gloriam et al. 2005), suggesting that the TAAR gene family repertoire differs substantially among vertebrate species. In addition, the evolutionary pattern of the TAAR gene family is characterized by lineage-specific phylogenetic clustering (Gloriam et al. 2005; Lindemann et al. 2005). Interestingly, these characteristics are very similar to those observed in the vertebrate odorant (OR) and vomeronasal (V1R, V2R) receptor gene families (Grus et al. 2005; Niimura and Nei 2005; Yang et al. 2005; Young et al. 2005; Hashiguchi and Nishida 2006).
Pheromones are defined as substances secreted to the outside of an individual and received by a conspecific individual that cause a specific reaction (Brennan and Zufall 2006). Biogenic amines and related chemicals may function as pheromones, if they are used as odor substances because biogenic amines can involve physiological changes to individuals. Indeed, several mouse TAARs respond to isoamylamine, trimethylamine, and ß-phenylethylamine, which are contained in mouse urine and thought to be sex pheromones (Liberles and Buck 2006). In the goldfish Carassius auratus, olfactory sensitivity to catecholamines (adrenaline, noradrenaline, and dopamine) and their metabolites has been confirmed, and it is thought that the release of catecholamines into water may play a role in chemical communication (Hubbard et al. 2003). In masu salmon Oncorhynchus masou masou, L-kynurenine, a metabolite of L-tryptophan, is a sex pheromone (Yambe et al. 2006). Therefore, if TAARs function as receptors for biogenic amines, the size and diversity of TAAR repertoires in various species can provide an insight into the relative complexity and species specificity of pheromone-based behaviors. Furthermore, the evolutionary processes that occurred in the TAAR gene family may reflect the evolution of chemical communication in reproduction and social interaction in vertebrates. However, the evolutionary dynamics of the vertebrate TAAR gene family are poorly understood, although the TAAR repertoires in humans, mice, rats, chickens, and zebrafish have been studied separately (Gloriam et al. 2005; Lindemann et al. 2005; Lagerstrom et al. 2006).
In this study, we identified near-complete repertoires of TAAR genes and pseudogenes in 4 teleost fishes (zebrafish Danio rerio, stickleback Gasterosteus aculeatus, fugu Takifugu rubripes, and medaka Oryzias latipes), the western clawed frog Xenopus tropicalis, the chicken Gallus gallus, 3 mammals (opossum Monodelphis domestica, mouse Mus musculus, and human Homo sapiens), and sea lamprey Petromyzon marinus from their genomic sequences and analyzed the phylogenetic relationships of TAAR genes to clarify the evolutionary dynamics of the vertebrate TAAR gene family more comprehensively. In addition, to test whether TAARs are used as chemosensory receptors in fishes, the expression of TAAR genes in the olfactory organ was examined in stickleback and zebrafish. A possible evolutionary scenario for the vertebrate TAAR gene family was inferred based on the results of these analyses.
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Materials and Methods |
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Identification of TAAR Sequences
We examined recent sequence assemblies of the sea lamprey (P. marinus-3.0, 2007), zebrafish (Zv6, 2006), medaka (Medaka 1, 2006), stickleback (BROAD S1, 2006), fugu (Fugu 4.0, 2005), the western clawed frog (JGI 4.1, 2005), chicken (WASHUC 2, 2006), opossum (monDom5, 2006), mouse (NCBI m36, 2005), and human (NCBI 36, 2005) to identify near-complete TAAR gene repertoires in each species. The genomic sequences of all species except sea lamprey are available at the ENSEMBL Web site (http://www.ensembl.org/). Sea lamprey genomic sequences are available from the Genome Sequencing Center (GSC) at the Washington University School of Medicine in St Louis, MO (http://genome.wustl.edu/).
TAAR-like sequences were identified applying a modified version of the method used to find fish vomeronasal-type odorant receptors (Hashiguchi and Nishida 2006). First, a TBlastN search was conducted with E value 10–10 against genomic data using several representative TAAR amino acid sequences known in humans and mice as queries. The obtained sequences were verified as TAARs by BlastP searches against the National Center for Biotechnology Information nonredundant (nr) database. In each of these sequences, if the best hit in this search was a previously known TAAR, it was considered a putative TAAR sequence. Several sequences showed similar amino acid identity (ca. 40%) to the known TAAR sequences and serotonin 4 (5-HT-4) receptors. Such sequences were included in the TAAR data set, and 3 known 5-HT-4 genes (from human, mouse, and zebrafish) were also used in the phylogenetic analysis.
Second, each region of Blast similarity was extended 2 kb in the 5' and 3' directions to perform a detailed prediction of TAAR-coding sequences. For each of these regions, TAAR-coding sequences were estimated based on the profile hidden Markov model (profile HMM)–based gene prediction with the program WISE2 (Birney et al. 2004). Fifteen mouse and 5 human full-length TAARs were aligned with the program ClustalW (Thompson et al. 1994) using the default settings. A profile HMM was constructed from the alignment using the HMMER software package (http://hmmer.janelia.org) and used for gene prediction. The putative vertebrate TAAR sequences obtained were classified into 2 groups, putatively functional genes and nonfunctional pseudogenes. A gene was considered putatively functional if it had a complete coding sequence. If a gene had any disruptive frameshifts or stop codons, it was considered a pseudogene. In this study, several "partial" TAAR sequences were classified as pseudogenes, particularly in fugu, medaka, and sea lamprey. Some of these may be attributable to incomplete genomic data in these species. In each species, the chromosomal positions of putative TAAR genes and pseudogenes were determined by mapping them onto a chromosome or scaffold. The list and sequences of vertebrate TAAR genes and pseudogenes are available as supplementary information (Supplementary Material online).
Phylogenetic Analysis
The deduced amino acid sequences of 268 putatively functional TAAR genes in the sea lamprey, 4 teleost fishes, the frog, chicken, and 3 mammals, 3 river lamprey Lampetra fluviatilis putative odorant receptor genes, three 5-HT-4 genes, and 2 outgroups (histamine H2 receptor and alpha-1D adrenoreceptor genes in zebrafish) were aligned using the program L-INS-i (MAFFT 5.860; Katoh et al. 2002) and slightly modified by eyes. An alignment of nucleotide sequences of these genes was constructed based on the alignment of the amino acid sequences. Phylogenetic trees were constructed using the maximum likelihood (ML) and Bayesian methods. The ML tree was searched by the genetic algorithm-based heuristic search, implemented in the program GARLI version 0.94 (http://www.bio.utexas.edu/faculty/antisense/garli/Garli.html). The assumed model of nucleotide substitution was the GTR + I + 
model. The ML tree was searched twice independently from 2 different starting trees (a Neighbor-Joining tree reconstructed by Kimura's 2-parameter distances and a random tree). The reliability of the ML tree nodes was assessed by the bootstrap method with 1,000 replications.
The Bayesian phylogenetic analysis was conducted with a parallel version of MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003). In the Bayesian analysis, we also assumed the GTR + I + model of nucleotide substitution. The Markov chain Monte Carlo process was set so that 4 chains (3 heated and 1 cold) ran simultaneously. On the basis of 2 preliminary runs with 2.0 x 106 generations, we estimated average log likelihood scores when stationary (mean lnL = –126748.37). Subsequently, we conducted the runs twice independently. After becoming stationary in the 2 runs, we continued the runs for 1.0 x 106 cycles to confirm the lack of improvement in the likelihood scores, with 1 in every 100 trees being sampled. Posterior probabilities of the phylogeny and its branches were determined from 20,000 trees pooled from the 2 runs. A Bayesian phylogenetic tree was also constructed using TAAR amino acid sequences, with almost the same procedure as used for the nucleotide data set. The assumed model of amino acid substitutions was the JTT + I + model. Posterior probabilities of the phylogeny and its branches were determined from 7,000 trees pooled from the 2 independent runs.
Gene Expression Analysis
To test whether fish TAAR genes were expressed in the olfactory organ, we conducted expression analysis of zebrafish and stickleback TAAR genes. In zebrafish, to identify the TAAR genes expressed, we conducted BlastN searches against the published zebrafish olfactory epithelium, brain, heart, and liver expressed sequence tags (ESTs), using 109 putatively functional zebrafish TAAR genes as queries. If a query sequence showed >95% nucleotide identity over 300-bp region to the best hit of the EST sequences, they were considered identical.
In the stickleback, the expression of TAAR genes in the olfactory organ was examined by reverse transcriptase–polymerase chain reaction (RT–PCR) and sequencing of amplified fragments. An adult anadromous-form stickleback (Pacific Ocean group) was collected at Akkeshi Lake, Hokkaido, Japan (Higuchi and Goto 1996). Total RNA was extracted from the lip, nose (olfactory rosette and surrounding tissues), gill raker, brain, heart, liver, skin, and muscle of the stickleback individual, using 1-ml TRIZOL reagent (Invitrogen, Carlsbad, CA). After deoxyribonuclease I treatment to remove residual genomic DNA, 0.5 µg of total RNA from each tissue was reverse-transcribed and first-strand cDNA was synthesized using TaKaRa RNA PCR kit ver. 3.0 (Takara Bio. Inc., Otsu, Shiga, Japan). Genomic DNA was also extracted from the same individual using AquaPure DNA extraction kit (BioRad Laboratories, Inc., Hercules, CA). The reverse-transcribed product from each tissue was subjected to polymerase chain reactions (PCR) with pairs of primers designed for 7 representative groups of stickleback TAAR genes (groups A–G, see Results). In addition, primers for the stickleback ß-actin gene were also designed as a positive control. Primer sequences for each group of stickleback TAAR genes are shown in supplementary table 2 (Supplementary Material online). The PCR reaction conditions were as follows: 95 °C for 2 min, 40 cycles at 95 °C for 15 s, 55 °C for 15 s, and 72 °C for 1 min, and 72 °C for 5 min. As a positive control for primer matching, a PCR was also conducted using genomic DNA as a template. In addition, as a negative control, PCR was conducted for each RNA sample without a reverse-transcribed reaction. Amplified RT–PCR products were subcloned and sequenced. Sequenced fragments were compared with the stickleback TAAR genes predicted from the genomic data by BlastN. If a fragment showed >95% nucleotide identity over a 300-bp region to the best hit of the predicted stickleback TAAR gene, they were considered identical. The stickleback TAAR partial sequences obtained in this study were submitted to the DNA Data Bank of Japan (GenBank accession numbers: AB290864–AB290880).
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Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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TAAR Gene Repertoires in Vertebrates
The numbers of putatively functional TAAR genes and pseudogenes found in each genome assembly are shown in table 1. For the mouse and humans, our survey results are in good agreement with a previous study (Lindemann et al. 2005
). Slightly more TAAR sequences occurred in the opossum compared with the mouse. More TAAR genes and pseudogenes (32–119) were found in teleost fishes, except fugu, than in mammals (8–25). Interestingly, in fishes, the number of TAAR genes was highly variable among species (table 1). The number of putatively functional TAAR genes in zebrafish found here (109 genes) was much larger than previously reported (57 genes; Gloriam et al. 2005). This difference is likely attributable to both the different genome assemblies examined and the more "strict" criteria of the previous study (Gloriam et al. 2005). In the frog and chicken, only 6 and 3 putatively functional TAAR genes were identified, respectively. In sea lamprey, 21 putatively functional genes and 17 pseudogenes were found; however, these numbers may not be accurate because the coverage of the lamprey genome assembly was not high (ca. 5.7x) and the lamprey genome may be incomplete.
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Phylogenetic Relationships of Vertebrate TAARs
The ML tree of the 268 putatively functional vertebrate TAAR genes identified in this study, 3 river lamprey putative odorant receptors (Berghard and Dryer 1998
; GenBank accession numbers: AF069546–AF069548) and three 5-HT-4 genes (GenBank accession numbers: AJ519673, NM_008313
[GenBank]
, and XM_679765) is shown in figure 1. The ML tree was searched twice independently from 2 different starting trees, and both searches provided essentially the same result (see supplementary fig. 1, Supplementary Material online). All sea lamprey TAAR genes formed a monophyletic clade with a high bootstrap probability (fig. 1). Similarly, all TAAR genes in jawed vertebrates formed a monophyletic clade, although this was not supported by >50% bootstrap probability (fig. 1). The monophyly of TAAR genes in jawed vertebrates was also supported based on the Bayesian tree by nucleotide sequences (supplementary fig. 2, Supplementary Material online) but not by the Bayesian tree based on amino acid sequences (supplementary fig. 3, Supplementary Material online). Thus, we believe that the ML tree reflects the evolution of TAAR genes.
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The ML tree also indicated that the vertebrate TAAR gene family was subdivided into 5 subfamilies that diverged before the divergence between fishes (ray-finned fishes) and tetrapods; thus we call them subfamilies I–V (fig. 1). In the ML tree, subfamilies I, III, IV, and V were supported by >50% bootstrap probabilities, although subfamily II was not. The monophyly of subfamily II was supported in the Bayesian tree from amino acid sequences (see supplementary fig. 3, Supplementary Material online) but not by the Bayesian tree from nucleotide sequences (supplementary fig. 2, Supplementary Material online).
Phylogenetic analysis revealed that the expansion of fish TAAR genes occurred mainly in subfamily I (fig. 1). In all fishes, subfamily I TAAR genes tended to form species-specific clades. Particularly in zebrafish and stickleback, substantially large species-specific phylogenetic clades were recognized. Gene prediction revealed that most subfamily I TAAR genes in the stickleback, medaka, and fugu had 1 intron in the coding region. Phylogenetic analysis indicated that this intron originated from 1 insertion event occurring in a common ancestor of these fishes (fig. 1). In mammals, most TAAR genes were found in subfamily I. Similar to fishes, mammalian subfamily I TAAR genes also tended to form species-specific clades (fig. 1). In the chicken, only 1 subfamily I TAAR gene was identified. Interestingly, in the frog, no subfamily I TAAR genes or pseudogenes were found in the genomic sequences.
Subfamily II contained several tetrapod and zebrafish TAAR genes (fig. 1). Stickleback, medaka, and fugu had no subfamily II TAAR genes. In this subfamily, the number of frog TAAR genes was slightly increased (4 genes). Subfamily III is a fish-specific subfamily and genes in this subfamily may have been lost in the common ancestor of tetrapods. In contrast, genes in subfamily IV were found in all tetrapod species examined, but no fishes had subfamily IV genes except 1 in zebrafish. It is notable that all tetrapod species examined had only 1 subfamily IV TAAR gene. TAAR genes belonging to subfamily V were found only in fishes and the frog, not in the chicken and mammals (fig. 1), suggesting that TAAR genes in subfamily V were lost in the avian and mammal lineage.
Genomic Distribution of TAAR Genes in Tetrapods and Fishes
Physical maps of the TAAR genes and pseudogenes in mammals, chickens, and frogs are shown in figure 2A. TAAR genes in humans and mice were clustered in the region of a single chromosome, as previously reported (Lindemann et al. 2005). Similar to humans and mice, opossum TAAR sequences were also located within a 600-kb region of a chromosome (chromosome 2) as a gene cluster (fig. 2A). Similarly, 3 chicken TAAR genes were located within a 100-kb region of chromosome 3. In mammals and the chicken, virtually all TAAR genes and pseudogenes were located in a single-chromosomal region, and the arrangement of genes was well conserved at the subfamily level. In the frog, 3 TAAR genes and 1 pseudogene, belonging to subfamilies II and IV, were located within a 100-kb region of scaffold_172, but 1 gene in subfamily V was found in another scaffold, scaffold_153 (fig. 2A).
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The genomic distribution of TAAR genes in fishes was more complicated. Figure 2B shows the physical maps of TAAR genes and pseudogenes in zebrafish, stickleback, and medaka. Data for fugu and the sea lamprey are not shown because their TAAR sequences were separated into many unconnected scaffolds and thus we could not reconstruct the gene clusters (see supplementary table 1, Supplementary Material online). In fishes, TAAR genes were scattered in more than 2 different chromosomal regions. In particular, subfamily I TAAR genes were located in 2 or more genomic regions. For example, in zebrafish, 16 subfamily I genes and 2 pseudogenes, 7 subfamily II genes, 8 subfamily III genes, and 1 subfamily IV gene were located within a 700-kb region in chromosome 20. One subfamily V TAAR gene was encoded in chromosome 1. In addition, 65 subfamily I genes and 4 pseudogenes were encoded within a 1-Mb region of chromosome 10. Furthermore, 1 subfamily I TAAR gene was located on chromosome 12, 9 genes on chromosome 13, and 1 gene on chromosome 15. In all fishes examined, the genomic location of 1 TAAR gene in subfamily V was distinct from those in other subfamilies.
Expression of TAAR Genes in Zebrafish and Sticklebacks
Liberles and Buck (2006) reported that 24 zebrafish TAAR genes were included in its olfactory epithelium EST sequences. In this study, we found 18 distinct zebrafish TAAR genes from the olfactory epithelium EST sequences using TBlastN searches. The zebrafish TAAR genes contained in the olfactory epithelium EST sequences are shown in figure 1 (indicated by blue arrows). A substantial number of TAAR genes in subfamilies I–III was found in zebrafish EST sequences, indicating that these genes are expressed in the olfactory epithelium; however, 1 TAAR sequence in subfamily IV and 1 in subfamily V were not found in the EST sequences. We also conducted TBlastN searches against brain, heart, and liver EST sequences in zebrafish; however, we did not find any TAAR sequences in these EST sequences. Our results suggest that zebrafish TAARs are expressed mainly in the olfactory epithelium, not other tissues.
Tissue-specific expression patterns of most TAAR genes in stickleback (groups A–G, fig. 1) were determined by RT–PCR and sequencing of amplified fragments. The results of the RT–PCR experiment are shown in figure 3. Eleven TAAR genes in stickleback were expressed in the nose. Several genes in group A (subfamily I) were expressed in the lip, nose, and brain. One gene in group D (subfamily III) and 1 gene in group F (subfamily III) were expressed specifically in the nose. Eleven TAAR genes sequenced from the amplified fragments of nose cDNA are indicated in figure 1 (blue arrows). One gene in group C (subfamily I) was expressed specifically in the brain. Genes expressed in the brain (red arrows) and lip (gray arrows) are also shown in figure 1. Expression of several group A genes was not confirmed; however, this does not mean that these genes are never expressed in any organ or any life stage. We could not detect expression of genes belonging to groups B, E, and G in any tissues examined in this experiment (fig. 3).
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Large Variation of TAAR Gene Repertoire in Vertebrate Species
In this study, we showed that the number of putatively functional TAAR genes varied dramatically among vertebrate species (table 1). The number of functional TAAR genes in fishes (ray-finned fishes), particularly in zebrafish and stickleback, was substantially larger than in tetrapods (table 1). In zebrafish and stickleback, many TAAR genes were suggested to be expressed in the olfactory organ (figs. 1 and 3). This indicates that many fish TAAR genes are used for chemosensory receptors, as in mammals (Liberles and Buck 2006
). The number of functional OR and V2R genes in fishes range from 44 (fugu) to 102 (zebrafish), and from 12 (spotted green puffer fish Tetraodon nigroviridis) to 46 (zebrafish), respectively (Niimura and Nei 2005; Hashiguchi and Nishida 2006). In addition, we found at least 93 putatively functional OR genes in the stickleback genomic sequence (Hashiguchi Y and Nishida M, unpublished data). Thus, the number of intact TAAR genes in zebrafish and stickleback is approximately equal to the number of ORs. If almost all intact TAARs in these fishes are used as chemosensory receptors, the relative contribution of TAARs to fish olfaction is substantially large.
In contrast, only a few TAAR genes were found in the frog and chicken genomes (table 1). This suggests that TAARs are less important chemosensory receptors in amphibians and avians. In mammals, particularly in the mouse and opossum, the number of subfamily I TAAR genes slightly increased (fig. 2A). In the mouse, these TAAR genes perceive volatile amines contained in the urine that function as sex pheromones (Liberles and Buck 2006). Male opossums prefer the odor of nonestrus female urine (Zuri et al. 2003). In the opossum, subfamily I TAARs might also be used to discriminate some volatile amines contained in urine. For humans, the number of subfamily I TAAR genes was relatively small (3 genes). This may reflect the relatively low importance of pheromones in humans (Brennan and Zufall 2006). Further studies are needed to elucidate the functions of human TAARs.
It is interesting to note that the number of TAAR genes highly diverse in some fishes. However, in tetrapods, the number of TAAR genes does not increase except in the mouse and opossum. In contrast, in the OR gene family, the number of OR genes is highly expanded in the tetrapod lineage; this is thought to reflect adaptation to diverse volatile chemicals in terrestrial environments (Freitag et al. 1995; Niimura and Nei 2005). However, the diversification of TAARs in fishes cannot be explained by this hypothesis. Why has the number of TAAR genes increased specifically in fishes? The reason is unclear, but it is possible that biogenic amines are more important odorants for fishes than tetrapods. It has been reported that the olfactory sensory neurons in zebrafish and goldfish respond to several polyamines (Michel et al. 2003; Rolen et al. 2003). In addition, the goldfish is also known to have olfactory sensitivity to catecholamines (Hubbard et al. 2003). Interestingly, Michel et al. (2003) reported that the signaling pathway of polyamine stimuli is different from that of amino acids. In fishes, amino acid stimuli are detected by olfactory cells expressing ORs and V2Rs (Hansen et al. 2003, 2004). Thus, in fishes, polyamines might be detected specifically by olfactory cells expressing TAARs.
Evolution of Vertebrate TAAR Genes
Our phylogenetic analysis revealed that jawed vertebrate TAAR genes are subdivided into 5 distinct subfamilies originating before the divergence of teleost fishes and tetrapods (fig. 1). Subfamily I, the largest subfamily of TAARs, was expanded in fishes, particularly in zebrafish and stickleback. Most subfamily I TAAR genes in these fishes formed species-specific monophyletic clades (fig. 1), indicating that rapid turnover and lineage-specific expansion of TAAR genes occurred in these fishes. In addition, many subfamily I TAAR genes in zebrafish and stickleback were expressed in the olfactory organ (fig. 1, indicated by blue arrows). Also, in mammals, most TAAR genes belonged to subfamily I (figs. 1 and 2A
). All mouse TAARs in subfamily I function as chemosensory receptors (Liberles and Buck 2006). Thus, in fishes and mammals, subfamily I TAARs are considered to be used mainly as chemosensory receptors. However, no subfamily I TAAR genes were found in the frog genome. In all tetrapod species examined, virtually all TAAR genes (except subfamily V) were encoded within a specific region as a gene cluster (fig. 2A). In the frog, we could not find any subfamily I TAAR genes in the gene cluster. Thus, the loss of subfamily I TAAR genes in frogs is likely true. Of course, we cannot exclude the possibility of subfamily I TAARs in the missing portion of the frog genome assembly. It was recently reported that the western clawed frog has numerous V2R-type vomeronasal receptor genes, with 249 intact V2R genes identified in its genome sequence (Shi and Zhang 2007). The increase of V2Rs in frogs might have reduced the relative importance of TAARs as chemosensory receptors and caused the loss of subfamily I TAAR genes. Interestingly, frogs also lack T1R-type taste receptors (Shi and Zhang 2006).
In subfamily II, several genes and pseudogenes were identified in tetrapods and zebrafish, but not in stickleback, medaka, or fugu (fig. 1). Two subfamily II TAARs in the mouse are expressed in the olfactory epithelium and function as receptors for volatile amines (Liberles and Buck 2006). In addition, 1 subfamily II TAAR gene in zebrafish was found in its olfactory epithelium EST sequences (fig. 1). These findings suggest that the TAARs in this subfamily are also used as chemosensory receptors.
Subfamily III TAAR genes were found in all teleost fishes examined (fig. 1). Four zebrafish and 1 stickleback TAAR genes in subfamily III were suggested to be expressed in the olfactory organ (figs. 1 and 3). In fishes, subfamily III TAARs likely function as chemosensory receptors in the olfactory epithelium. No subfamily IV TAARs were expressed in the nose. One mouse TAAR in this subfamily, TAAR1, respond to trace amines localized in the central nervous system (Borowsky et al. 2001; Bunzow et al. 2001), and mouse TAAR1 is thought to be expressed in the brain and various tissues (Borowsky et al. 2001). In addition, in all tetrapods examined, subfamily IV TAARs were encoded by a single-copy gene (fig. 1). The subfamily IV TAAR (ortholog of the mouse TAAR1 gene) may have specific functions, such as a neurotransmitter receptor.
Our phylogenetic analysis showed that subfamily V TAAR genes were the most ancestral group of TAAR genes in jawed vertebrates. This relationship may also be supported by the observation that the genomic location of subfamily V genes differs from the others in 4 fishes and the frog (fig. 2). In zebrafish and stickleback, we failed to detect expression of subfamily V genes in any tissue examined (fig. 3). Genes in this subfamily might have some specialized functions in a tissue not examined, or they could be expressed within a limited life stage.
We now consider the long-term evolutionary scenario for the TAAR gene family. Figure 4 shows a hypothetical evolutionary trajectory for the vertebrate TAAR gene family. The most recent common ancestor (MRCA) of fishes and mammals had at least 5 ancestral TAARs corresponding to the 5 subfamilies. The complicated genomic distributions of fish TAAR genes (fig. 2B) can be explained by a whole-genome duplication thought to have occurred in the MRCA of ray-finned fishes (Crollius and Weissenbach 2005 and references therein) and subsequent lineage-specific gene diversifications and losses. Gloriam et al. (2005) attempted to explain the genomic distribution pattern of zebrafish TAAR genes by considering the whole-genome duplication and subsequent local duplications. In this study, we refined their hypothesis by comparing the repertoires and genomic distributions of TAAR genes in multiple vertebrate species. The tetrapod lineage retains the basic structure of the ancestral TAAR gene cluster. The TAAR gene family repertoires in the frog, chicken, and mammals can be explained by considering the loss of subfamily III genes in the MRCA of tetrapods, the loss of subfamily I in the amphibian lineage, and the loss of subfamily V in the MRCA of avian and mammalian lineages.
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We have identified the near-complete repertoire of TAAR genes in 10 vertebrate species and revealed the evolutionary dynamics of the TAAR gene family by phylogenetic and comparative genomic analyses. Further studies of TAARs as chemosensory receptors may give profound insights into the mechanisms and evolution of vertebrate chemoreception.
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Supplementary Material |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Supplementary figures 1–3, tables 1 and 2, and data sets are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
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Acknowledgements |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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The zebrafish, fugu, stickleback, and medaka sequence data were produced by the Sanger Institute (SI), International Fugu Genome Consortium, Broad Institute (BI), and the National Institute of Genetics, respectively. The frog sequence data were produced by the Joint Genome Institute. The chicken and sea lamprey sequence data were produced by the GSC at Washington University, St Louis, MO. The human sequence data were produced by the International Human Genome Sequencing Consortium. The mouse data were produced by a joint project between the Whitehead Institute/MIT Center for Genome Research, the Washington University GSC, SI, and European Molecular Biology Laboratory-European Bioinformatics Institute. The opossum genome sequences were produced by the BI. We thank associate editor Laura Katz and 3 anonymous reviewers for comments that improved the manuscript substantially. This work was partially supported by Grants-in-Aid from the Japan Society for the Promotion of Science.
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Footnotes |
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Laura Katz, Associate Editor
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References |
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