Institute of Biological Anthropology, University of Oxford, Oxford, England
Correspondence: E-mail: nim21{at}cam.ac.uk.
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Abstract |
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Key Words: positive selection vomeronasal receptor V1R V1RL1 primate vomeronasal organ
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Introduction |
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During the evolution of higher primates the VNO decreased in size (reviewed in Martin 1990), and this has been interpreted as being due to a reduction in the importance of pheromone communication in these groups. In New World monkeys the VNO is small, whereas in Catarrhine primates (humans, apes, and Old World monkeys) it is vestigial, and probably nonfunctional. In spite of the apparent absence of a functional vomeronasal system in humans, interest in the role of pheromones in human biology is growing because of the increasing evidence for pheromonal effects in human mate choice (Wedekind et al. 1995; Jacob et al. 2002).
In humans, V1R diversity is low, and only five potentially functional vomeronasal receptor genes (V1R-like or V1RL genes) have been identified from human genome searches (V1RL1 to 5; Giorgi et al. 2000; Rodriguez et al. 2000; Kourso-Mehr et al. 2001; Rodriguez and Mombaerts 2002). The best characterized of these genes is V1RL1. Interestingly, V1RL1 mRNA is expressed not in the vestigial human VNO but in the main olfactory epithelium (Rodriguez et al. 2000). However, it is not known if a functional V1R1 receptor is produced.
There has been an acceleration in the number of examples of positive selection detected in diverse systems, which has been stimulated by the development of more powerful methods for its analysis (reviewed in Bielawski and Yang, 2002). As pheromones are species-specific and have important functions in social and reproductive behavior, pheromone receptors may be evolving rapidly, and we hypothesized that if V1RL genes code for functional pheromone receptors then they may be under positive Darwinian selection during primate evolution. To test this hypothesis we have sequenced the V1RL1 gene from anthropoid primates (monkeys and apes) and analyzed these data together with published human V1RL1 and recently described human V1RL2-5 gene sequences and marmoset V1R pseudogene sequences. The results provide evidence for the action of both positive and purifying selection on different sites in human and primate V1RLs. In addition, reconstruction of pseudogene formation of V1RL1 genes strongly suggests that it occurred independently in several lineages of great apes and New World monkeys.
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Materials and Methods |
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V1R genes contain a single coding exon of about 1 kb. External primer pairs designed to amplify a 735744-bp portion of this exon of the V1RL1 gene were VOMR1 (5'-GGAGTTGGGATCCTGGGAAA-3') and VOMR2 (5'-CTCATGATGAGGACAAAAGGGCT-3') (annealing temperature of 52°65°C), or VOMR5 (5'-TGGGAAATTCCTTTCTCCTTTG-3') and VOMR6 (5'-GGGCTGAGTGCTGGRAAAC-3') (annealing temperature of 55°60°C). Internal sequencing primers were VOMR3 (5'-AATCGCTTGGAATCCATTGAG-3') and VOMR4 (5'-TTTATGAGTTTGGGCTTCATGG-3').
Polymerase chain reactions (PCR) were prepared in a 25 µl total volume containing: 0.1 µl Taq polymerase (Thermoprime Plus DNA polymerase 5U/µl, ABGene, Surrey, U.K.). 2.5 µl 10x reaction buffer, 1.5 µl 25 mM magnesium chloride, 0.05 µl of each dNTP (25 mM), 1 µl of each primer (10 µM), and 25100 ng DNA. All PCRs were performed in a Techne Genius Cycler (Hybaid), with the following cycling parameters: 94°C for 2 min, 35x: (94°C for 30 s, annealing temperature for 45 s, 72°C for 90 s), 72°C for 5 min. The PCR products were directly sequenced on both strands by cycle sequencing using Big Dye Version 2 (PE Biosystems) under standard conditions, and run on an ABI 377 sequencer. Sequences were edited in Sequencher. The sequences have been deposited in GenBank (accession numbers AY314011AY314023).
Data Analysis
Sequences were aligned with ClustalX. A 765-bp alignment of all primate V1R sequences was used in PAUP*, and a 744-bp alignment of V1RL1-4 sequences was used for PAML analyses (this alignment was shorter because it was based on more closely related sequences). To infer evolutionary relationships among primate V1R genes, phylogenetic reconstructions were performed using the Neighbor-Joining method with HKY85 distances and maximum likelihood with the HKY85 model of substitution in PAUP* Version 4.0b10 (Swofford 1999). Branch support using Neighbor-Joining was estimated with 1,000 bootstrap replicates.
Estimation of dN/dS ratios was carried out by maximum likelihood using a codon-based substitution model in PAML Version 3.13 (Yang 1997). Two series of analyses were performed: (1) lineage-specific models in which all codon sites are assumed to be under the same selective pressure in a particular lineage but the selective pressure can vary among different lineages, and (2) site-specific models in which selective pressure varies among different sites but the site-specific pattern is identical across all lineages. Several different site-specific models were implemented: model M0 (null model with a single dN/dS ratio among all sites), M1 ("neutral" model, with two categories of site with fixed dN/dS ratios of 0 and 1), M2 ("selection" model: three categories of site, two with fixed dN/dS ratios of 0 and 1, and a third estimated dN/dS ratio), M3 ("discrete" model: three categories of site, with the dN/dS ratio free to vary for each site), M7 ("Beta model": eight categories of site, with eight dN/dS ratios in the range 01 taken from a discrete approximation of the beta distribution), and M8 ("Beta plus omega" model: eight categories of site from a beta distribution as in model M7 plus an additional category of site with a dN/dS ratio that is free to vary from 0 to greater than 1). PAML estimates the dN/dS ratios that are free to vary under these models, as well as the proportion of sites with each ratio. Likelihood ratio tests, to determine whether particular models provided a significantly better fit to the data than other nested models, were performed by comparing the likelihood ratio test statistic (2[LogLikelihood1 LogLikelihood2]) to critical values of the Chi square distribution with the appropriate degrees of freedom (Yang 1998). P values for sites potentially under positive selection were obtained using a Bayesian approach in PAML.
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Results and Discussion |
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The primate V1RL1 sequences contained an open reading frame (ORF) in the region sequenced in only six of the 14 species: human, gorilla, pygmy marmoset, and three species of howler monkey (fig. 1b). In the remaining eight species sequenced, all from different genera, the V1RL1 sequence was clearly a pseudogene, resulting from point substitutions creating stop codons (common marmoset), from a large deletion of 242 bp (saddle-backed tamarin), or from one or two small frameshifting deletions of 17 bp (chimpanzee, orangutan, night monkey, capuchin, lion tamarin, Goeldi's monkey). During the course of the study, full-length V1RL1 sequences were independently isolated from chimpanzee, gorilla, and orangutan (Giorgi and Rouquier 2002). These sequences differ from our V1RL1 sequences for these species at 2, 0, and 2 nucleotide sites, respectively, and they confirm the presence of a V1RL1 ORF in gorilla and pseudogenes in chimpanzee and orangutan. Reconstruction of V1RL1 pseudogene formation over the independently established primate phylogeny (fig. 1b) suggests that it occurred independently several times during anthropoid evolution, in terminal lineages.
Patterns of Selection in Primate V1R Gene Evolution
The relative rates of synonymous (dS) and nonsynonymous (dN) substitutions indicate the nature of the selective forces that have shaped the evolution of a coding sequence. Most codons are under purifying selection most of the time (dN/dS less than 1), because the majority of nonsynonymous mutations are deleterious and are removed by selection. Under neutral evolution, for example in pseudogenes, the expected dN/dS is one. Of particular interest are cases where dN/dS is greater than one (i.e., dN is greater than dS), because the rapid fixation of nonsynonymous substitutions must be due to the action of positive selection at the codons involved, and this therefore provides unequivocal evidence for adaptive evolution. Estimates of dN and dS during primate V1RL1-4 evolution were obtained by maximum likelihood using a codon-based substitution model in PAML (Yang 1997; tables 1 and 2). Human V1RL5 was excluded from this analysis as it is highly divergent and its ancestry with human V1RL1-4 predates the split between primates and rodents (Rodriguez and Mombaerts 2002).
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Given that there is a category of sites under positive selection, Bayesian inference can be used in PAML to identify specific codons that have a significant chance of being under positive selection. Results from all three models allowing positive selection (M2, M3, and M8) were compared to reduce the chance of false positives (Anisimova, Bielawski, and Yang 2001; Suzuki and Nei 2002). A single codon position (172, numbering from human V1RL1) has a greater than 95% probability of being under positive selection under all three models (fig 2). This residue is highly variable among the sequences studied (human and gorilla V1RL1s: serine; New World monkey V1RL1s: proline; human V1RL2: proline; human V1RL3: asparagine; human V1RL4: arginine). It is in the long third extracellular domain of the receptor, where it could potentially interact with odorant ligands, although it should be emphasized that the structure-function relationships of V1Rs in general are poorly understood.
The presence of positive selection on V1RL genes during human and non-human primate evolution is consistent with these genes having a role in species-specific pheromone detection in primates. The finding of V1RL1 polymorphism in humans (Rodriguez et al. 2000) suggests that diversification of V1RL1 function could be occurring within human populations, and it is intriguing that one of the two variable amino acid positions in humans (site 229alanine or glutamate in humans) has also undergone nonsynonymous substitution in gorillas (threonine).
The V1RL genes join the growing number of genes that have been shown to be under positive selection in one or more primate lineages, as inferred by dN/dS ratios greater than 1 (e.g., MHC class I: Hughes and Nei 1988; lysozyme: Messier and Stewart 1997; SRY: Pamilo and O'Neill 1997; ribonuclease: Zhang, Rosenberg, and Nei 1998; alanine glyoxylate aminotransferase (AGT): Holbrook et al. 2000; BRCA1: Huttley et al. 2000; Protamine-1, Protamine-2, Tnp-2: Wyckoff, Wang, and Wu 2000; morpheus: Johnson et al. 2001; glycophorin A: Baum, Ward, and Conway 2002). The majority of these genes have roles in the immune system (MHC genes), male reproduction (SRY, protamine, Tnp-2), or digestion/metabolism (lysozyme, ribonuclease, AGT). V1RLs are one of the first examples of genes with putative functions in the sensory system that have been under directional selection during primate evolution. Another example are the opsin genes involved in color vision, which underwent positive selection following duplication in Catarrhine primates, and are under balancing selection in most New World monkey lineages (Shyue et al. 1995; Surridge and Mundy 2002).
V1R Gene Family Evolution in Primates
Overall, data on V1R gene evolution in primates are still rather scarce, but some important conclusions are beginning to emerge. Pseudogene formation is not confined to the human lineage, but has occurred independently in different lineages and, in marmosets at least, in several genes (Giorgi and Rouquier 2002). This is reminiscent of the pattern of evolution in the large olfactory receptor gene family in non-human primates (Rouquier, Blancher, Giorgi 2000; Whinnett and Mundy 2003). The pattern of V1RL1 pseudogene formation among hominoids is particularly notablethis is the only V1R gene known to be expressed in humans, but it is a pseudogene in our closest relative, the chimpanzee, an ORF in gorillas, and a pseudogene in orangutans. It is seems likely that the repertoire of functional V1R genes is highly lineage specific in primates.
The V1Rs and the VNO are extremely important in mice, and in this lineage there has been a rapid evolutionary turnover of V1R genes, involving both duplication and pseudogene formation (Lane et al. 2002; Rodriguez and Mombaerts 2002). It remains to be seen whether lineages of non-human primates with a functional VNO, such as marmosets, have had V1R diversification in addition to the pseudogene formation documented here.
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Acknowledgements |
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Footnotes |
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Literature Cited |
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Anisimova, M., J. P. Bielawski, and Z. Yang. 2001. Accuracy and power of the likelihood ratio test in detecting adaptive molecular evolution. Mol. Biol. Evol. 19:950-958.[ISI]
Baum, J., R. H. Ward, and D. J. Conway. 2002. Natural selection on the erythrocyte surface. Mol. Biol. Evol. 19:223-229.
Bielawski, J. P., and Z. Yang. 2002. Statistical methods for detecting molecular adaptation. Trends Ecol. Evol. 15:496-503.
Dulac, C. 1999. Sensory coding of pheromone signals in mammals. Curr. Opin. Neurol. 10:511-518.
Dulac, C., and R. Axel. 1995. A novel family of genes encoding putative pheromone receptors in mammals. Cell 83:195-206.[ISI][Medline]
Giorgi, D., C. Friedman, B. J. Trask, and S. Rouquier. 2000. Characterization of nonfunctional V1R-like pheromone receptor sequences in human. Genome Res. 10:1979-1985.
Giorgi, D., and S. Rouquier. 2002. Identification of V1R-like putative pheromone receptor sequences in non-human primates. Characterization of V1R pseudogenes in marmoset, a primate species that possesses an intact vomeronasal organ. Chem. Senses 27:529-537.
Holbrook, J. D., G. M. Birdsey, Z. Yang, M. W. Bruford, and C. J. Danpure. 2000. Molecular adaptation of alanine: glyoxylate aminotransferase targeting in primates. Mol. Biol. Evol. 17:387-400.
Hughes, A. L., and M. Nei. 1988. Pattern of nucleotide substitution at MHC class I loci reveals overdominant selection. Nature 335:167-170.[CrossRef][ISI][Medline]
Huttley, G. A., et al. 2000. Adaptive evolution of the tumour suppressor BRCA1 in humans and chimpanzees. Nat. Genet. 25:410-413.[CrossRef][ISI][Medline]
Jacob, S., M. K. McClintock, B. Zelano, and C. Ober. 2002. Paternally inherited HLA alleles are associated with women's choice of male odor. Nat. Genet. 30:175-179.[CrossRef][ISI][Medline]
Johnson, M. E., L. Viggiano, J. A. Bailey, M. Abdul-Rauf, G. Goodwin, M. Rocchi, and E. E. Eichler. 2001. Positive selection of a gene family during the emergence of humans and African apes. Nature 413:514-519.[CrossRef][ISI][Medline]
Keverne, E. B. 1999. The vomeronasal organ. Science 286:716-719.
Kourso-Mehr, H., S. Pintchovski, J. Melnyk, Y. J. Chen, C. Frideman, B. Trask, and H. Shizuya. 2001. Identification of non-functional human VNO receptor genes provides evidence for vestigiality of the human VNO. Chem. Senses 26:1167-1174.
Lane, R. P., T. Cutforth, R. Axel, L. Hood, and B. J. Trask. 2002. Sequence analysis of mouse vomeronasal receptor gene clusters reveals common promoter motifs and a history of recent expansion. Proc. Natl. Acad. Sci. USA 99:291-296.
Martin, R. D. 1990. Primate origins and evolution. A phylogenetic reconstruction. Princeton, N.J.
Messier, W., and C.-B. Stewart. 1997. Episodic adaptive evolution of primate lysozymes. Nature 385:151-154.[CrossRef][ISI][Medline]
Pamilo, P., and R. J. W. O'Neill. 1997. Evolution of the Sry genes. Mol. Biol. Evol. 14:49-55.[Abstract]
Rodriguez, I., K. Del Punta, A. Rothman, T. Ishii, and P. Mombaerts. 2002. Multiple new and isolate families within the mouse superfamily of V1R vomeronasal receptors. Nat. Neurosci. 5:134-140.[CrossRef][ISI][Medline]
Rodriguez, I., C. A. Greer, Y. Mok, and P. Mombaerts. 2000. A putative pheromone receptor gene expressed in human olfactory mucosa. Nat. Genet. 26:18-19.[CrossRef][ISI][Medline]
Rodriguez, I., and P. Mombaerts. 2002. Novel human vomeronasal receptor-like genes reveal species-specific families. Curr. Biol. 12:R409-R411.[CrossRef][ISI][Medline]
Rouquier, S., A. Blancher, and D. Giorgi. 2000. The olfactory receptor gene repertoire in primates and mouse: evidence for reduction of the functional fraction in primates. Proc. Natl. Acad. Sci. USA 97:2870-2874.
Shyue, S. K., D. Hewett-Emmett, H. G. Sperling, D. M. Hunt, J. K. Bowmaker, J. D. Mollon, and W.-H. Li. 1995. Adaptive evolution of color vision genes in higher primates. Science 269:1265-1267.[ISI][Medline]
Surridge, A. K., and N. I. Mundy. 2002. Trans-specific evolution of opsin alleles and the maintenance of trichromatic colour vision in Callitrichine primates. Mol. Ecol. 11:2157-2169.[CrossRef][ISI][Medline]
Suzuki, Y., and M. Nei. 2002. Simulation study of the reliability and robustness of the statistical methods for detecting positive selection at single amino acid sites. Mol. Biol. Evol. 19:1865-1869.
Swofford, D. 1999. PAUP*: phylogenetic analysis using parsimony (and other methods). Sinauer Associates, Sunderland, Mass.
Wedekind, C., T. Seebeck, F. Bettens, and A. J. Papepke. 1995. MHC-dependent mate preferences in humans. Proc. R. Soc. Ser. B 260:245-249.[ISI][Medline]
Whinnett, A., and N. I. Mundy. 2003. Isolation of novel olfactory receptor genes in marmosets: insights into pseudogene formation and evidence for functional degeneracy in non-human primates. Gene 304:87-96.[CrossRef][ISI][Medline]
Wyckoff, G. J., W. Wang, and C.-I. Wu. 2000. Rapid evolution of male reproductive genes in the descent of man. Nature 403:304-309.[CrossRef][ISI][Medline]
Yang, Z. 1997. PAML: a program package for phylogenetic analysis by maximum likelihood. Comput. Appl. Biosci. 13:555-556.[Medline]
Yang, Z. 1998. Likelihood ratio tests for detecting positive selection and application to primate lysozyme evolution. Mol. Biol. Evol. 15:568-573.[Abstract]
Yang, Z., Nielsen, R., Goldman, N., and Pedersen, A.-M. K. 2000. Codon-substitution models for heterogeneous selection pressure at amino acid sites. Genetics 155:431-449.
Zhang, J., H. F. Rosenberg, and M. Nei. 1998. Positive Darwinian selection after gene duplication in primate ribonuclease genes. Proc. Natl. Acad. Sci. USA 95:3708-3713.