Download Positive Selection During the Diversification of

Positive Selection During the Diversification of Class I Vomeronasal
Receptor-like (V1RL) Genes, Putative Pheromone Receptor Genes,
in Human and Primate Evolution
Nicholas I. Mundy and Shelley Cook
Institute of Biological Anthropology, University of Oxford, Oxford, England
Vomeronasal receptors are the major receptors for pheromones in vertebrates, and five putative type 1 vomeronasal
receptors (V1RL) have been identified in humans. The evolution of the V1RL1 gene in non-human primates, and
patterns of selection on V1RL genes, were investigated. The presumed ortholog of V1RL1 was sequenced from 13
species of nonhuman primate, and in eight of these species V1RL1 was a pseudogene. Phylogenetic reconstructions
reveal that V1RL1 pseudogene formation occurred independently in multiple primate lineages. Using maximum
likelihood estimates of dN/dS ratios in PAML, we show that V1RL genes have evolved under neutral evolution in
lineages in which they became a pseudogene. In contrast, among lineages in which V1RL genes contain an open reading
frame, the majority of sites are under purifying selection and a minority are under significant positive selection. These
results provide an interesting case where all three categories of selection can be teased apart in the same data set using
maximum likelihood methods. The finding of positive selection on V1RL genes during primate evolution provides
indirect support for the hypothesis that V1RL genes have a function in species-specific pheromone detection in primates.
Introduction
The vomeronasal system is a phylogenetically ancient
chemosensory system of vertebrates that is anatomically
and functionally distinct from the main olfactory system
(reviewed in Keverne 1999). This system has a major role
in the detection of pheromones, which are odorant
molecules used in species-specific communication. Primary detection of odorants in the vomeronasal system occurs
in the vomeronasal organ (VNO). At the molecular level,
detection in the VNO is via vomeronasal receptors, which
are part of the large superfamily of 7-transmembrane G
protein–coupled receptors (Dulac and Axel 1995; reviewed in Dulac 1999). There are two classes of
vomeronasal receptor, V1Rs and V2Rs (Type 1 and type
2 vomeronasal receptors), that have distinctive structures,
G protein–coupling systems, and expression patterns in the
VNO (Keverne 1999). Pheromone detection is important
in most mammalian groups including rodents, and in mice
there is a well-developed VNO and a high diversity of
vomeronasal receptors, with at least 137 V1Rs and around
100 V2Rs expressed from separate genomic loci (Rodriguez et al. 2002).
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
Key words: positive selection, vomeronasal receptor, V1R, V1RL1,
primate, vomeronasal organ.
E-mail: [email protected].
Mol. Biol. Evol. 20(11):1805–1810. 2003
DOI: 10.1093/molbev/msg192
Molecular Biology and Evolution, Vol. 20, No. 11,
Ó Society for Molecular Biology and Evolution 2003; all rights reserved.
potentially functional vomeronasal receptor genes (V1Rlike 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.
Materials and Methods
Laboratory Work
Genomic DNA samples had previously been extracted from tissue samples of Anthropoid primates using
Qiagen kits. The following samples were used (lab ID is
given in parentheses): Apes: common chimpanzee, Pan
troglodytes (Ptr4); western lowland gorilla: Gorilla gorilla
gorilla (Ggo6); orangutan: Pongo pygmaeus (Ppy2); lar
gibbon, Hylobates lar, (Hla1). Old World monkeys: olive
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1806 Mundy and Cook
baboon, Papio anubis (Pan1); ebony langur, Trachypithecus auratus (Tau1); De Brazza’s monkey, Cercopithecus
neglectus (Cne1) New World monkeys: common marmoset: Callithrix jacchus (Cja13); pygmy marmoset: Cebuella pygmaea (Cpy690); Goeldi’s monkey: Callimico
goeldii (Cgo20); night monkey: Aotus sp (Aot1); goldenheaded lion tamarin: Leontopithecus chrysomelas (Lch2);
saddle-backed tamarin: Saguinus fuscicollis (Sfu1); whitefronted capuchin: Cebus albifrons (Cal1); mantled howler:
Alouatta palliata mexicana (Apm2); Bolivian red howler:
Alouatta sara (Asa3); red howler: Alouatta seniculus
(Ase2).
V1R genes contain a single coding exon of about 1 kb.
External primer pairs designed to amplify a 735–744-bp
portion of this exon of the V1RL1 gene were VOMR1
(59-GGAGTTGGGATCCTGGGAAA-39) and VOMR2
(59-CTCATGATGAGGACAAAAGGGCT-39) (annealing
temperature of 528658C), or VOMR5 (59-TGGGAAATTCCTTTCTCCTTTG-39) and VOMR6 (59-GGGCTGAGTGCTGGRAAAC-39) (annealing temperature of
558608C). Internal sequencing primers were VOMR3
(59-AATCGCTTGGAATCCATTGAG-39) and VOMR4
(59-TTTATGAGTTTGGGCTTCATGG-39).
Polymerase chain reactions (PCR) were prepared in
a 25 ll total volume containing: 0.1 ll Taq polymerase
(Thermoprime Plus DNA polymerase 5U/ll, ABGene,
Surrey, U.K.). 2.5 ll 103 reaction buffer, 1.5 ll 25 mM
magnesium chloride, 0.05 ll of each dNTP (25 mM), 1 ll
of each primer (10 lM), and 25–100 ng DNA. All PCRs
were performed in a Techne Genius Cycler (Hybaid), with
the following cycling parameters: 948C for 2 min, 353:
(948C for 30 s, annealing temperature for 45 s, 728C for
90 s), 728C 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 AY314011–AY314023).
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) sitespecific 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 0–1 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.
Results and Discussion
V1RL1 Genes in Non-human Primates
V1RL1 sequences were obtained for 13 species of
non-human primates including great apes and several New
World monkeys such as marmosets, tamarins, and howler
monkeys (see fig. 1b for a full list of species). The region
sequenced comprises about 80% of the predicted V1R
protein, extending from the first transmembrane domain to
the seventh transmembrane domain (fig 2). No PCR
products within 300 bp of the expected size were amplified
from any of the Old World monkey and gibbon species
with three pairs of non-overlapping conserved forward and
reverse primers, or combinations of these primers. As 1-kb
amplicons from other loci (e.g., melanocortin-1 receptor
gene) were easily obtained from these samples, we
consider it most likely that V1RL1 is absent or highly
modified in these species.
The V1RL1 sequences were aligned with all other
available primate V1R sequences. Phylogenetic reconstructions using Neighbor-Joining and maximum likelihood methods produced similar topologies in which
V1RL1 sequences from humans and all other primates
formed a well-supported monophyletic clade, which was
the sister group to a clade comprising human V1RL2,
V1RL3, V1RL4 genes and the marmoset V1R pseudogene
MsM11 (fig 1a). The V1RL1 gene phylogeny is generally
consistent with the accepted primate species phylogeny.
Together, these results strongly suggest that the V1R
sequences obtained from non-human primates are orthologous to human V1RL1. Previous work has shown that
human V1RL1-4 are distantly related to the 12 mouse V1R
subfamilies and that a clade comprising these human
V1RL1-4 is monophyletic with respect to the 137 mouse
V1Rs (Rodriguez and Mombaerts 2002). We confirmed
Primate V1RL Evolution 1807
that the new primate V1RL1 sequences had no close
matches among other V1R genes in GenBank. The clade
containing all primate V1RL1 to V1RL4 genes has strong
bootstrap support in our analysis, and we used this clade to
investigate patterns of selection described below.
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 1–7 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).
First, we were interested in whether it was possible to
detect changes in selection pressure on V1RL genes before
and after pseudogene formation. All of the sequences in
figure 1b were used for this analysis. All codon sites were
treated equivalently, and lineages in which V1RL1 became
a pseudogene (‘‘pseudogene lineages’’—see fig 1b) were
considered as a separate category from lineages in which
V1RL1s and V1RL2-4 were reconstructed as potentially
functional (‘‘ORF lineages’’; table 1). The overall estimated
dN/dS ratio (known as x in PAML) among pseudogene
lineages (1.02) was close to 1, as expected for neutral
evolution. In contrast, the estimated dN/dS ratio over all
FIG. 1.—Phylogenetic analysis of V1RL sequences and reconstruction of V1RL1 pseudogene evolution in primates. Pseudogene sequences
are labeled ‘‘w.’’ a. Maximum likelihood tree of all primate V1R
sequences. Numbers on branches represent percentage bootstrap support
using Neighbor-Joining. b. Reconstruction of V1RL1 pseudogene
evolution over the known primate phylogeny obtained from independent
data sets. These sequences and phylogeny were used in the first part of the
PAML analyses. Bars across lineages indicate pseudogene formation.
Lineages with a bar were defined as ‘‘pseudogene lineages’’ for the PAML
analysis, with all other lineages being defined as ‘‘ORF lineages.’’ The
second part of the PAML analyses using V1RL ORFs alone excluded the
pseudogenes. GenBank accession numbers for published human and
marmoset V1RL pseudogene sequences are as follows: Human V1RL1:
AF255342; Human V1RL2: AF370359; Human V1RL3: AF336873;
Human V1RL4: AY114733; Human V1RL5: AY114735; Human PH2:
AF253312; Human PHB4C5: AF253314; Human PH2D1: AF253315;
Human PhH8: U73853; Human PHRET: AF253316; Human PhH5:
U73852; Human PHA4: AF253313; Marmoset MsM11: AF397897;
Marmoset MsM4: AF397898; Marmoset MsM5: AF397899; Marmoset
MsM6: AF397900; Marmoset MsM7: AF397901.
1808 Mundy and Cook
FIG. 2.—Alignment of V1RL ORF protein sequences. Numbering follows human V1RL1, and only the region sequenced in this study is shown.
The position marked by an asterisk (172) is inferred to be under positive selection (see text for details). Species abbreviations are as follows:
Hsa, human; Ggo, gorilla; Cpy, pygmy marmoset; Asa, Bolivian red howler; Ase, red howler; Apm, mantled howler.
V1RL ‘‘ORF’’ lineages was 0.64. This value was significantly less than 1 in a likelihood ratio test (test I in table 3),
providing evidence for purifying selection among primate
V1RLs when all sites are treated equally. This lends support
to the reconstruction of pseudogene evolution shown in
figure 1b, and similar significant results were obtained using
PAML analyses on V1RL1 sequences alone (not shown).
This provides an example of the use of PAML to detect
differences in selection pressure on functional genes and
pseudogenes.
The finding of purifying selection among V1R
‘‘ORF’’ lineages when dN/dS ratios are averaged over all
sites is not surprising, because even if certain sites are
under positive selection this would be masked if purifying
selection is occurring at the majority of sites. To investigate the nature of selection in the V1RL ORF lineages
further, we carried out a separate series of analyses on the
ORF lineages alone (see fig. 1b) in which the type of
selection varies among different categories of codon site
(Yang et al. 2000). Several different site-specific models of
increasing complexity were implemented (see Materials
and Methods for details). Three of these models (M2, M3,
and M8) permit a proportion of sites to have a dN/dS ratio
greater than 1, (i.e., they accommodate the possibility of
Table 1
Maximum Likelihood Estimates of dN/dS Ratios (x) Using
PAML: Lineage-Specific Models Over All Sites
(18 ORF + Pseudogene Lineages)
positive selection). When applied to the V1R data, these
three models all led to a category of sites in the V1R data
with an estimated dN/dS ratio greater than 1 (2.22–4.75;
table 1), suggesting that a proportion of codons (estimated
to be 0.05–0.16; table 1) are under positive selection
during V1R evolution in primates. Statistical support for
this was obtained in likelihood ratio tests with nested
simpler models (tests II to IV in table 2). Thus positive
selection is present in primate V1RL gene evolution from
the duplication events that gave rise to V1RL1 to V1RL4
through to diversification of V1RL1 in anthropoid
primates. Analyses performed separately on V1RL1
sequences alone also gave a proportion of sites under
positive selection, but in this case likelihood ratio tests for
positive selection were not significant at the 5% level (not
shown).
Table 2
Maximum Likelihood Estimates of dN/dS Ratios (x) Using
PAML: Site-Specific Models Over All Lineages
(9 ORF Lineages)
x1
Model
M0 (one ratio)
M1 (neutral)
M2 (selection)
M3 (discrete)
x2
0.00
0.56
Model
x1
x2
Log Likelihood
A: One ratio
B: 2 ratios
xORF lineages,
xpseudogene
0.69
—
4384.35
M7 (Beta)
4382.17
M8 (Beta þ x) p ¼ 0.89 q ¼ 0.66 p0 ¼ 0.91
x ¼ 2.73c p1 ¼ 0.09
lineages
C: 2 ratios
xORF lineages ¼ 1,
xpseudogene lineages
a
0.64
1.02
Values in parentheses are fixed, not estimated.
4393.02
3571.10
3556.39
3551.06
3543.21
pd ¼ 0.60 q ¼ 0.35
3548.57
3543.60
Values in parentheses are fixed, not estimated.
‘‘p’’ indicates proportion of sites with that value of x.
c
Values in bold indicate positive selection.
d
‘‘p’’ and ‘‘q’’ under models M7 and M8 indicate parameters defining the
beta function.
b
1.03
2.22c
Log Likelihood
p1 ¼ 0.11 p2 ¼ 0.72 p3 ¼ 0.16
a
(1.0)a
x3
0.61
—
—
(0.00)a
(1.00)a —
p1b ¼ 0.17 p2 ¼ 0.83
(0.00)a
(1.00)a 4.75c
p1 ¼ 0.16 p2 ¼ 0.78 p3 ¼ 0.05
Primate V1RL Evolution 1809
Table 3
Likelihood Ratio Tests Using Models Presented in
Tables 1 and 2
Hypothesis Tested
xORF lineages
x . 1 for a
of sites
III x . 1 for a
of sites
IV x . 1 for a
of sites
I
II
Models
Compared
Likelihood Ratio
Statistic: 2(L1L2) df
,1
B versus C
proportion M1 versus M2
21.70**
10.66**
1
2
proportion M1 versus M3
26.36**
4
proportion M7 versus M8
9.94**
2
** Significant at the 1% level.
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 speciesspecific 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 229—alanine 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; Protamine1, 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 notable—this 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.
Acknowledgments
We thank Andrew Kitchener, Liliana Cortés-Ortiz,
Alcides Pissinatti, Jim Dietz, and Mike Bruford for
providing samples. Brian Golding and two anonymous
reviewers made helpful comments that greatly improved
the manuscript. This work was funded by the Biotechnology and Biological Sciences Research Council.
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Brian Golding, Associate Editor
Accepted June 6, 2003