Owing to the high levels of DNA sequence divergence among the primate species in our sample, orthologous OR genes could not be amplified by primers designed based on human sequences (Gilad et al. 2003
). Instead, we used two sets of degenerate primer pairs, constructed to amplify OR genes from all of the species studied (see Materials and Methods
). We then cloned the PCR products and determined the sequences of clones until we had identified 100 distinct OR genes from each species. A danger of this approach is that degenerate primers may bind preferentially to certain OR genes, thereby resulting in a biased representation of the OR repertoire. To safeguard against this, we tested the degenerate primers on human and mouse, for which the entire OR gene repertoire is known, by using them to amplify 100 OR genes from the two species. The sample thus obtained faithfully represented the composition of the full OR gene repertoire in human and mouse with respect to the 17 OR gene families (). Moreover, the sample estimates of the fractions of pseudogenes were accurate (see Materials and Methods
; ). This pilot study demonstrates that the degenerate primers yield an unbiased representation of the OR gene repertoire, as measured by the family composition and pseudogene content of the human and mouse samples. Since the primers performed well both in human and a distantly related species, the mouse, there was no reason to assume that they would not do so in nonhuman primate species.
Results of the Pilot Study in Human and Mouse
The Proportion of OR Pseudogenes in 20 Species
We therefore proceeded to sequence 100 genes from 18 nonhuman primates using these primer pairs. Since the genome sequence is not available for these species, we were not able to compare the familial composition of our samples of OR genes to that of the full OR repertoires. However, with the exception of OR families 3, 11, 12, and 55 (whose absence in a sample of 100 genes is not unlikely, as they represent less than 1.8% of human OR genes), we identified OR genes from all families in all species (). Moreover, the representation of the three largest OR gene families in the sample varied across species, again suggesting that there is no strong bias towards the amplification of specific families.
Distribution of OR Genes in Families across Species
We then tabulated the proportion of OR pseudogenes in each species (). Consistent with previous results based on direct sequencing of full-length OR orthologs (Gilad et al. 2003
), we found that the proportion of OR pseudogene in the great apes and rhesus macaque is approximately 30% (). Together, these findings confirm the validity of this degenerate primer approach.
We further found that the proportion of OR pseudogenes in OWMs (29.3% ± 2.4%) is very similar to that of nonhuman apes (33.0% ± 0.8%), but notably higher than that of NWMs (18.4% ± 5.6%). One NWM species, the howler monkey, was a conspicuous exception, with an elevated proportion of OR pseudogenes, similar to that of OWMs and apes (31.0%) () and significantly higher than any other NWM (one-tailed p < 0.02 for the difference between the howler monkey and the NWM with the second highest proportion of pseudogenes, the Wooly monkey, as assessed by a Fisher's exact test [FET]). Thus, it appears that a deterioration of the olfactory repertoire occurred in all apes and OWMs as well as, independently, in the howler monkey lineage.
Strikingly, a second phenotype is shared only by the howler monkey, OWMs, and apes: full (or “routine”) trichromatic color vision. In primates, trichromatic color vision is accomplished by three opsin genes whose products are pigments sensitive to short, medium, or long wavelength ranges of visible light (Nathans et al. 1986
). In OWMs and apes, the short-wavelength opsin gene is found on an autosome, while two distinct X-linked loci for medium and long wavelengths underlie full trichromatic color vision (and so are present in both males and females). In contrast, most NWM species carry an autosomal gene and only one X-linked gene, where different alleles encode for photopigment opsins that respond to medium or long wavelengths. Heterozygous females can therefore possess trichromatic vision, but males are dichromatic (Jacobs 1996
; Boissinot et al. 1998
; Hunt et al. 1998
). The sole exception among NWMs is the howler monkey (Jacobs et al. 1996
; Jacobs and Deegan 2001
; Surridge et al. 2003
), which has a duplication of the opsin genes on the X chromosome (Goodman et al. 1998
; Jacobs and Deegan 2001
) (). Thus, full trichromatic vision arose twice in primates, once in the common ancestor of OWMs and apes and once in the howler monkey lineage.
While OWMs, apes, and the howler monkey carry 32.5% ± 6.3% OR pseudogenes in their OR gene repertoire, species without full trichromatic vision have 16.7% ± 1.0%, significantly fewer (p < 10−4, or, excluding humans from the full trichromatic group, p < 10−3, as assessed by a Mann–Whitney U test). This p value is only indicative since the species lineages are not all independent. However, if significance is instead assessed by a FET for all pairwise comparisons of species with full trichromatic color vision and without, the difference is again striking: 94 out of 96 comparisons are significant at the 5% level. Thus, the evolution of full trichromatic vision coincided with an increase in the fraction of OR pseudogenes, indicative of a deterioration of the sense of smell.
Apes and OWMs acquired trichromatic color vision approximately 23 million years ago (Yokoyama and Yokoyama 1989
), while the duplication of the opsin genes in the howler monkey occurred approximately 7–16 million years ago (Jacobs 1996
; Cortes-Ortiz et al. 2003
). In spite of this difference in timing, the proportion of OR pseudogenes in species from both lineages is very similar. We estimated the rate of fixation of neutral gene disruptions for OR genes to be approximately 0.12 per gene per million years (Y. Gilad, S. Pääbo, and G. Glusman, unpublished data). This estimate implies that both apes, OWMs and the howler monkey could have a much higher proportion of OR pseudogenes than observed (data not shown), indicating that the process of functional OR gene loss has decreased or stopped in these species. A plausible explanation for the similar proportion of OR pseudogenes in the different lineages is that while full trichromatic vision relaxed the need for a sensitive sense of smell, it did not render olfaction unnecessary. Accordingly, while some OR genes can accumulate coding region disruptions, others are still evolving under evolutionary constraint. This model predicts that the possession of full trichromatic color vision alone allows for the loss of some but not all OR genes. A natural next step would then be to identify which OR genes or families were lost after the acquisition of full trichromatic vision. The answer to this question awaits sequence from a large number of orthologous OR genes.
In this respect, it is interesting to note that the TRP2
gene, a major component of the vomeronasal pheromone transduction pathway, was found to be intact in several NWM species, but is a pseudogene in OWMs and apes (Liman and Innan 2003
; Zhang and Webb 2003
). The authors raised the possibility of a connection between the acquisition of full trichromatic color vision and decreased pheromone perception, based on the difference between OWMs and apes on the one hand and NWMs on the other (Liman and Innan 2003
; Zhang and Webb 2003
). However, since many traits can potentially be mapped to the lineage that leads to OWMs and apes, the connection between full trichromatic vision and pheromone perception was tenuous. Furthermore, Liman and Innan (2003
) did not find a coding region disruption in four exons of TRP2
in the howler monkey. An intact TRP2
gene in the howler monkey would be inconsistent with the hypothesis that the enhancement of color vision replaced pheromone signaling in primates.
In contrast, in the present study, we find that the deterioration of the olfactory repertoire occurred concomitant with the evolution of full trichromatic vision in two separate primate lineages. Thus, although at this point we are unable to demonstrate that the decline in the sense of smell is a direct result of the evolution of color vision, our results strongly suggest an exchange in the importance of these two senses in primate evolution. Future studies of the sensory cues involved in detection and selection of food (e.g., Smith et al. 2003
), or the choice of a mate, may test this association directly.