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 (Figure 1). Moreover, the sample estimates of the fractions of pseudogenes were accurate (see Materials and Methods; Figure 2).
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.
Figure 1 Results of the Pilot Study in Human and Mouse.The
percentage of OR genes from each family is given for the entire
repertoire (filled bars) and a sample of 100 genes amplified using PC1
and PC2 degenerate primers (open bars). (A) OR genes in human. (B) OR
genes in mouse. None of the differences between the full repertoires
and the samples are significant at the 5% level. Only full-length OR
genes (larger than 850 bp) were considered.
Figure 2 The Proportion of OR Pseudogenes in 20 Species Primate
species are color-coded according to family. The arrow points to the
howler monkey. Datapoints (from left to right) are for apes (green):
human (Homo sapiens), chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus), gibbon (Hylobates syndactylus); for OWMs (blue): Guinea baboon (Papio papio), rhesus macaque (Macaca mulatta), silver langur (Trachypithecus auratus), mona (Cercopithecus mona), agile mangabey (Cercocebus agilis), black-and-white colobus (Colobus guereza); for NWMs (red): brown capuchin monkey (Cebus apella), southern owl monkey (Aotus azarai), spider monkey (Ateles fusciceps), black howler monkey (Alouatta caraya), squirrel monkey (Saimiri sciureus), wooly monkey (Lagothrix lagotricha), common marmoset (Callithrix jacchus); for one prosimian primate (brown): crowed lemur (Eulemur mongoz); and for the mouse (Mus musculus) (grey).
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 (Table 1).
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.
Table 1. Distribution of OR Genes in Families across Species
We then tabulated the proportion of OR pseudogenes in each species (Figure 2
). 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% (Figure 2
). Together, these findings confirm the validity of this degenerate primer approach.
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%) (Figure 2) 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.
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) (Figure 3).
Thus, full trichromatic vision arose twice in primates, once in the
common ancestor of OWMs and apes and once in the howler monkey lineage.
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
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.