A Candidate Subspecies Discrimination System Involving
a Vomeronasal Receptor Gene with Different Alleles
Fixed in M. m. domesticus and M. m. musculus
Robert C. Karn1*, Janet M. Young2, Christina M. Laukaitis1
1Department of Medicine, College of Medicine, University of Arizona, Tucson, Arizona, United States of America, 2Division of Human Biology, Fred Hutchinson Cancer
Research Center, Seattle, Washington, United States of America
Assortative mating, a potentially efficient prezygotic reproductive barrier, may prevent loss of genetic potential by avoiding
the production of unfit hybrids (i.e., because of hybrid infertility or hybrid breakdown) that occur at regions of secondary
contact between incipient species. In the case of the mouse hybrid zone, where two subspecies of Mus musculus (M. m.
domesticus and M. m. musculus) meet and exchange genes to a limited extent, assortative mating requires a means of
subspecies recognition. We based the work reported here on the hypothesis that, if there is a pheromone sufficiently
diverged between M. m. domesticus and M. m. musculus to mediate subspecies recognition, then that process must also
require a specific receptor(s), also sufficiently diverged between the subspecies, to receive the signal and elicit an assortative
mating response. We studied the mouse V1R genes, which encode a large family of receptors in the vomeronasal organ
(VNO), by screening Perlegen SNP data and identified one, Vmn1r67, with 24 fixed SNP differences most of which (15/24) are
nonsynonymous nucleotide substitutions between M. m. domesticus and M. m. musculus. We observed substantial linkage
disequilibrium (LD) between Vmn1r67 and Abpa27, a mouse salivary androgen-binding protein gene that encodes a
proteinaceous pheromone (ABP) capable of mediating assortative mating, perhaps in conjunction with its bound small
lipophilic ligand. The LD we observed is likely a case of association rather than residual physical linkage from a very recent
selective sweep, because an intervening gene, Vmn1r71, shows significant intra(sub)specific polymorphism but no
inter(sub)specific divergence in its nucleotide sequence. We discuss alternative explanations of these observations, for
example that Abpa27 and Vmn1r67 are coevolving as signal and receptor to reinforce subspecies hybridization barriers or
that the unusually divergent Vmn1r67 allele was not a product of fast positive selection, but was derived from an
introgressed allele, possibly from Mus spretus.
Citation: Karn RC, Young JM, Laukaitis CM (2010) A Candidate Subspecies Discrimination System Involving a Vomeronasal Receptor Gene with Different Alleles
Fixed in M. m. domesticus and M. m. musculus. PLoS ONE 5(9): e12638. doi:10.1371/journal.pone.0012638
Editor: Sebastian D. Fugmann, National Institute on Aging, United States of America
Received June 3, 2010; Accepted August 8, 2010; Published September 9, 2010
Copyright: ? 2010 Karn et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: JMY was supported by National Institutes of Health grant DC004209 to Barbara Trask. CML was supported by career development funding from a
National Cancer Institute Specialized Program of Research Excellence in Gastrointestinal Cancer (P50 CA95060). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Pheromones, specific substances secreted to the exterior of an
organism, communicate information about sex, species and related
states to other members of the same species, whereupon the
pheromones elicit specific reactions such as behavior and/or
endocrine changes . Many pheromones in terrestrial animals
are volatile airborne molecules, however, large non-volatile
molecules such as peptides and proteins may also be utilized for
communication (reviewed in [2,3]). Olfactory cues represent the
primary means of communication in nocturnal animals such as the
house mouse [4,5] and two families of receptors in the
vomeronasal organ (VNO), the V1Rs and the V2Rs, are thought
to detect pheromonal signals . Studies of putative mouse
pheromones have proliferated over the past several decades,
however, it is one thing to propose a pheromonal function but
quite another to elucidate a mechanism, including identification of
the receptor by which the pheromone is recognized. In the case of
putative mouse pheromones, receptor identification has generally
relied on cell biology experiments which have indicated that the
VNO and the accessory olfactory bulb comprise the system
receiving and processing the information [7,8], but in those
experiments identification of the specific VNO receptors has been
elusive. In this report, we demonstrate a purely genetic and
evolutionary approach with which to identify specific receptors for
a particular pheromonal function, subspecies recognition [9–11],
thought to mediate assortative mating where two different
subspecies make secondary contact, e.g. the European house
mouse hybrid zone described below.
The house mouse, Mus musculus, comprises at least three
relatively distinct parapatric gene pools given subspecies status by
some and full species status by others (for reviews see [12,13]).
Two subspecies, Mus musculus domesticus and M. m. musculus, occupy
distinct geographic ranges in western and eastern Europe,
respectively. Where these make contact across southern Danish
Jutland and through Central Europe from the Baltic Sea to the
Black Sea coast, they form a narrow hybrid zone, which is a region
of limited gene exchange [12–14]. Two lines of indirect evidence
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suggest that selection is acting against hybrids: (1) hybrid male
sterility and partial female sterility have been described in different
crosses of laboratory or wild populations [15–23]; and (2) limited
introgression of sex chromosome markers as compared to
autosomes has been shown across four studied hybrid zone
transects [24–31]. The reduced fitness of hybrid animals within the
zone has been proposed to create a genetic sink, where genes
entering the zone are eliminated by selection .
Assortative mating is a potentially efficient prezygotic repro-
ductive barrier, which may prevent loss of genetic potential into
unfit hybrids [33–40]. When partial postzygotic isolation acts in
the presence of divergent specific mate recognition systems,
selection for increased mating specificity, the phenomenon of
reinforcement, may lead to complete speciation [34,41–43]. This
idea predicts that if hybrids are less fit, reinforcement should then
amplify homo(sub)specific preference most close to a contact zone,
a phenomenon called reproductive character displacement.
Reinforcement is best studied in closely related or recently
divergent taxa, such as the subspecies of house mice, where
limited hybridization still occurs and speciation may be incipient.
Here, selection may act to reinforce prezygotic isolation in regions
of secondary contact, e.g. the European mouse hybrid zone,
leading to avoidance of disadvantageous hetero(sub)specific
mating. A divergent subspecific mate recognition system, upon
which reinforcement in the mouse European hybrid zone is
predicated, requires some means by which members of a
subspecies can recognize their own subspecies from a foreign one.
Subspecies recognition was originally suggested by studies of
polymorphism of a mouse androgen-binding protein gene (Abpa,
now Abpa27)  in which different alleles were observed to be
fixed in different subspecies [45,46]. Those observations led to the
development of congenic strains that differ only in their Abpa27
alleles. Subsequent studies of mate recognition involving saliva
targets from the congenic strains showed that mice are capable of
recognizing their own subspecies from another and choose to mate
with their own more frequently than with a foreign subspecies
[9–11]. Thus one pheromonal function in house mice is
recognition of subspecies identity for the purpose of mediating
assortative mating and it has been proposed that the mouse VNO
is the tissue that recognizes such pheromones [10,11]. Since that
work, evidence has been obtained suggesting that mate preference
across the European mouse hybrid zone is a case of reproductive
character displacement . This has been observed as increased
interest in congenic saliva targets in populations offset from the
center of the hybrid zone  as predicted by the theory of
reinforcement [42,47,48]. Recently, Vos ˇlajerova ´ Bı ´mova ´ et al 
have tested mice across a transect of the mouse hybrid zone for
their preferences for saliva from the Abp congenic strains and have
shown that the incorporation of a reinforcement parameter into
the model for the behavioural data creates a significantly better fit
than other cline models.
We based the work reported here on the hypothesis that, if there
are pheromonal signals mediating subspecies recognition between
M. m. domesticus and M. m. musculus, then there must also be specific
receptors that are sufficiently diverged between the subspecies to
receive the signal and to elicit an assortative mating response. We
chose to study the V1R receptor genes because of their relatively
simple structure; they are intron-less genes less than 1 kb in length,
whereas V2R genes have many exons spread over ,20 kb of
DNA. We hypothesized that there should be at least one V1R
receptor gene exhibiting a high level of divergence, in the form of
fixed nonsynonymous differences between the two subspecies of
Mus musculus, arising by adaptive evolution to allow the receptor to
distinguish subspecies-specific signals. We screened a subset of the
most likely candidate V1R genes and found that one, Vmn1r67,
indeed shows a large number of fixed changes between the two
subspecies and an unusual pattern of evolution that may suggest its
involvement in subspecies recognition.
Screening for V1R genes significantly diverged between
M. m. domesticus and M. m. musculus
We began our search for a V1R gene(s) with different haplotypes
fixed in the two subspecies, M. m. domesticus and M. m. musculus, by
screening the 392 V1R genes for those with the most SNP
differences between the wild-derived inbred strains WSB and
PWD in the Perlegen SNP data . The microarrays used by
Perlegen for resequencing were designed from the mm6 version of
the mouse genome assembly, which is older and more incomplete
than the current build; thus, some regions of the genome simply
were not assayed with this technology.
We reasoned that positive selection would maximize the
number of fixed differences between the two haplotypes allowing
discrimination of signals communicating subspecies identification
(although there may be other explanations for large numbers of
fixed differences – see Discussion). We ranked the 392 V1R genes
according to how many SNPs were reported by Perlegen to have
different genotypes in WSB and PWD (Table S1). The screen
results showed that 165 V1Rs (42%) contain at least one SNP
where WSB and PWD are reported to have different genotypes,
while the rest (227) had no reported WSB-PWD differences.
Excluding pseudogenes, the ten V1R genes with the most
differences between the two strains were Vmn1r31 (9), Vmn1r75
(8), Vmn1r67 (6), Vmn1r39 (5), Vmn1r12 (5), Vmn1r64 (5), Vmn1r62
(5), Vmn1r175 (5), Vmn1r61 (5), and Vmn1r37 (4); the number of
SNPs where WSB and PWD have different alleles appears in
parentheses for each gene. To determine how many SNPs in these
candidate V1R genes might represent fixed nucleotide differences
between the two subspecies of interest, we produced DNA
sequences of the ten top-ranked genes from the Perlegen screen
in at least two wild-derived inbred strains of each subspecies
(except Vmn1r61, where only a single SNP of any kind was evident
between the single M. m. domesticus and M. m. musculus strains we
sequenced). Only Vmn1r67 (also known as V1RE10) had a
substantial number of fixed differences (24 differences) between
the sets of strains representing the two subspecies. We did not
pursue the other nine candidate genes because most had no fixed
differences between the subspecies (only Vmn1r12 had any, with
two in 906 bp; File S1). Therefore, most allelic differences
reported between WSB and PWD represent polymorphisms in
populations of both subspecies.
We sequenced the Vmn1r67 gene (915 bp) in three more wild-
derived inbred strains of each subspecies to bring the total number
of sequences to five each (File S2). For comparison, we also
sequenced Vmn1r71 (921 bp), also known as V1RE13 (File S3), one
of the V1R genes for which Perlegen did not report any differences
between WSB and PWD (Table S1). Figure 1 shows the SNPs
found in both V1R genes in these ten strains. Of 26 total variable
nucleotide sites in Vmn1r67, 24 are fixed differences representing
divergence in Vmn1r67 between the two subspecies, while only two
are polymorphisms, both of which are in the M. m. domesticus
subspecies. Of the 24 fixed sites in Vmn1r67, 15 are nonsynon-
ymous (i.e., result in amino acid substitutions) while nine are
synonymous. Of 35 total variable nucleotide sites in Vmn1r71, we
found no fixed differences (i.e., no divergent sites) between the two
subspecies; all differences were polymorphisms in one or both
strains. As expected, these extreme differences in divergence and
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polymorphism between the two V1R genes are reflected in much
higher (,ten-fold) nucleotide diversity (p) and nucleotide poly-
morphism (h) in Vmn1r71 compared to Vmn1r67 (Fig. 1).
Nucleotide diversity and nucleotide polymorphism had previously
been reported to be 0 for Abpa27 in five M. m. domesticus samples
from widely separated geographical locations .
Perlegen reported 19 SNPs for Vmn1r67, at only six of which did
they make a genotype assignment (G, A, T or C) for both WSB
and PWD. At the other thirteen sites, one strain genotype was
reported only as ‘‘N’’ (ambiguous site). By contrast, we discovered
26 SNPs by sequencing the DNA of WSB and PWD. Thus, we
confirmed polymorphisms at all of the sites reported by Perlegen,
and confirmed the genotypes called for each strain at 18 of 19 SNP
positions. Only one Perlegen genotype disagreed with our
sequencing data (Perlegen: G; our data: C; PWD genotype at
nucleotide 897 in Fig. 1). According to our sequencing, this is a
fixed SNP difference between the two subspecies, not a
polymorphic site. Thus the Perlegen data ascertained 73% of
the SNP sites but reported high quality genotypes that differ
between WSB and PWD at only 23% of those sites, instead
providing ambiguous calls at many sites. We discovered seven
additional sites that Perlegen did not report as SNPs.
Evolution of Vmn1r67
A possible interpretation of the high divergence between the
Vmn1r67 alleles fixed in M. m. domesticus and M. m. musculus,
combined with low/absent polymorphism is that positive selection
has acted on this gene in the house mouse (see Discussion for an
alternative explanation). In order to assess this, we used maximum
likelihood methods employed in the CODEML program of the
PAML package [52–54]. We obtained Vmn1r67 amplicons from
the third Mus musculus subspecies (M. m. castaneus) and four other
Figure 1. A comparison of polymorphism and divergence in two V1R genes. Reduced data sets summarizing SNP differences for Vmn1r67
(Panel A) and the closely linked gene Vmn1r71 (Panel B) in five wild-derived inbred strains each for M. m. domesticus and M. m. musculus. Strain
abbreviations are: LEW=LEWES; PER=PERA; TIR=TIRANO; ZAL=ZALENDE; CZ1=CZECHI; CZ2=CZECHII; and SKI=SKIVE. Fixed differences
(divergence) between M. m. domesticus and M. m. musculus are highlighted in green (there are 24 in Vmn1r67 and none in Vmn1r71) and
polymorphisms in both genes in the two subspecies are highlighted in gray to facilitate comparison. In addition, nonsynonymous sites in Vmn1r67
are indicated by yellow highlighting of the site numbers. Nucleotide diversity (p) and nucleotide polymorphism (h) were calculated for both
subspecies using DNAsp and are shown for each dataset. Full sequence data appear in Files S2 and S3.
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species of the genus Mus: M. spicilegus, M. spretus, M. caroli, M. pahari
with polymerase chain reaction (PCR) and sequenced them. Two
possible Rattus norvegicus Vmn1r67 orthologs were obtained from
. Orthology of all sequenced Vmn1r67 genes from taxa of the
genus Mus was confirmed by Blast searches against the mouse
genome . The phylogeny of Chevret et al.  was used for
the rodent species (Fig. 2A) for the PAML tests and the three
subspecies of M. musculus were treated as an unresolved polytomy.
See File S4 for the Vmn1r67 sequence data used in CODEML
analysis. Vmn1r67 showed significant signs of positive selection
within murid rodents when the species phylogeny was used as the
CODEML guide tree. However, we also noticed that the evolution
of the Vmn1r67 sequences appears not to follow the species tree
(Figure 2, File S4), and that the sequence data support an alternate
tree (Figure 2B) much better than the species tree. This
unexpected finding may be due to phenomena including one or
more introgression events, incomplete lineage sorting, and/or
homoplasies (recurrent mutations). Such introgression or homo-
plastic mutations might even have been fixed due to positive
PAML’s author, Ziheng Yang, has suggested that the gene tree
should be used when it differs substantially from the species
When the same taxa were analyzed with CODEML using the
Vmn1r67 gene phylogeny as the guide tree (Fig. 2B), we obtained
nonsignificant results (not shown), evidently because the Vmn1r67
gene phylogeny is widely incongruent with the murid rodent
species phylogeny. The branch-sites model  also failed to
yield significant evidence of positive selection using the gene tree.
We reconstructed ancestral sequences using CODEML and
Figure 2. A comparison of a murid rodent phylogeny and a V1R gene phylogeny. Panel A: A canonical phylogeny of murid rodents
(adapted from ); Panel B: A Vmn1r67 gene phylogeny for murid rodents using F84 nucleotide distances and neighbor-joining, with bootstrap
values expressed as percentages (see Methods). Full sequence data appear in File S4.
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parsed the rst output to look for evidence of homoplasy that could
explain the substantial incongruence between the two phyloge-
nies. Using the species tree, we encountered 19 apparent
homoplasies but none when we used the gene tree (File S5).
Most of the fixed nonsynonymous SNP differences (14/15) we
observed between the Vmn1r67 genes of the two M. musculus
subspecies are at sites exhibiting homoplasy if the species tree is
assumed. It is not clear whether these changes indeed represent
true recurrent mutations or whether this portion of the Mus
genome has evolved with a very different history than the
genomes in which it resides.
We used the Phyre threading program on one of the M. m.
domesticus sequences (PERA) to identify the most likely structural
model, d1ln6a (Family A G protein-coupled receptor-like;
rhodopsin-like) with 100% confidence mapping of the 305 amino
acids of Vmn1r67 onto 309 total positions of the model with an
identity of 10%. Fifteen Vmn1r67 amino acid sites were not
captured by the threading program: seven in the N-terminal
sequence, four between sites 115 and 120, two between sites 157
and 160, and two at the C-terminus of the model. The d1ln6a
model is a seven transmembrane domain structure consistent with
the general structure for V1R receptor proteins and we used it to
produce a diagram of the Vmn1r67 protein that reflects the details
of the transmembrane helix and coil structures.
Figure 3A is a diagram of the amino acid sequence of Vmn1r67
with the fixed amino acid differences between M. m. domesticus and
M. m. musculus represented with diamond shapes (amino acid
sequences appear in File S6). Amino acid residues affected by
nonsynonymous sites where apparent homoplasy was detected
assuming the species tree are mapped onto the sequence in red.
The figure also shows the same sites mapped in red onto three-
dimensional representations of the structure (Fig. 3B). The most
important conclusion from this comparison is that the majority of
amino acid residues detected with the test for apparent
homoplasies are also diverged between M. m. domesticus and M.
m. musculus Vmn1r67 receptors. Most of the amino acid residues
that differ between the two subspecies due to fixed SNPs appear in
either extracellular loops (four) or intracellular loops (six), although
four appear in transmembrane helices (see Discussion).
Linkage disequilibrium (LD) between Vmn1r67 and
In light of the data we present here, suggesting that Vmn1r67 has
alleles fixed in M. m. domesticus and M. m. musculus, and the
Figure 3. Amino acid differences in a V1R gene between M. m. domesticus and M. m. musculus. Diagrammatic representation of the amino
acid sequence of Vmn1r67 (Panel A) and the three-dimensional structure on which it was threaded (Panel B); amino acids in Panel A that were not
threaded on the d1ln6a model in Panel B are shaded gray. The amino acid positions affected by fifteen nonsynonymous differences fixed between M.
m. domesticus and M. m. musculus (File S6) are shown as diamonds. Fourteen nonsynonymous sites detected by a test for homoplasy (File S5) are
colored red. For orientation to the three-dimensional models in Panel B, the first helix is colored light orange. Panel B views are 1) front, 2) rear, 3) left
side, 4) right side, 5) top and 6) bottom.
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previously reported fixed alleles for Abpa27 in the three subspecies
of the house mouse [59,60], we evaluated linkage disequilibrium
(LD) between these two genes. In order to test whether LD might
be due to a very recent selective sweep covering the ,23 Mb
between them, we included Vmn1r71, which lies between the two
genes. Sequencing Abpa27 (1185 bp) in the same ten wild-derived
inbred strains in which we sequenced Vmn1r67 and Vmn1r71
[46,51,61,62]. Figure 4 illustrates LD for the reduced data set of
the three genes. Clearly there is strong LD between Vmn1r67 and
Abpa27 (red regions indicating maximum r2in the data) but not
between Vmn1r71 and either of those two. These observations
indicate a strong association between Vmn1r67 and Abpa27, and
the lack of LD with Vmn1r71 argues against residual physical
linkage resulting from a very recent selective sweep across the
entire Vmn1r67-Abpa27 region. This is not surprising given that the
distance between the Vmn1r67 and Abpa27 genes (23 Mb) is
substantially larger than that expected for strong linkage
disequilibrium (LD), which occurs over a distance that varies
depending on the genomic region, but is usually on the order of
tens of kilobases .
from previous studies
The origin of the prediction of vomeronasal receptors
differentiating the Mus musculus subspecies
We predicted that, if M. m. domesticus and M. m. musculus are each
capable of distinguishing members of their own subspecies from
members of another subspecies, there should be at least one
vomeronasal receptor that recognizes a distinct difference, i.e. a
signal or signals that advertise subspecies identity. In order to
accomplish this recognition function there should be at least one
V1R receptor encoded by a gene exhibiting a high level of
divergence, in the form of fixed nonsynonymous changes between
the two subspecies arising by adaptive evolution. Although our
initial hypothesis was neutral with regard to the pheromone that
might signal the subspecies difference, only one mouse pheromone
system, salivary androgen-binding protein (ABP) has been shown
to provide a recognizable cue of subspecies status between the
subspecies that form the mouse hybrid zone [9–11,49]. Even
though V1R receptors are not normally thought to recognize
proteinaceous pheromones, ABP appears at this time to be the best
candidate pheromone system for mediating prezygotic mating
Figure 4. Linkage disequilibrium (LD) between Vmn1r67 and Abpa27, with the gene Vmn1r71 mapping between them. The proximal
end of mouse chromosome 7 shown at the top of the figure represents the relative positions of the three genes which appear diagrammed as gray-
shaded blocks below the chromosome and over their portion of the LD diagram. A comparison of the degree of LD, represented as r2values, appears
in the triangle at the bottom of the figure and a shading scale for LD minimum to maximum appears under the LD diagram. The wild-derived inbred
mouse strains used in this study are numbered on the left side of the LD diagram with a strain legend immediately below. The genes Vmn1r67 and
Abpa27 show strong LD, while Vmn1r71 shows no significant LD with either of them.
PLoS ONE | www.plosone.org6September 2010 | Volume 5 | Issue 9 | e12638
isolation  leading to subspeciation, whether through the
protein structure itself or through the lipophilic ligand it binds.
The bioinformatic screen for subspecies-recognition
receptor gene candidates
Since the total collection of V1R receptor genes numbers 392,
we reasoned that we should begin our effort to find a highly
diverged V1R by selecting a subset of those with the highest
number of fixed differences between the wild-derived strains
representing the two subspecies in Perlegen SNP data. To that
end, we designed the screen we described above. Out of the top
ten V1R genes returned by our bioinformatic screen of Perlegen
SNP data, only one, Vmn1r67, had a significant number of fixed
SNP differences (24/26), revealed by DNA sequencing in five
wild-derived strains for each of the two subspecies M. m. domesticus
and M. m. musculus. In most other cases, WSB-PWD SNPs
represented polymorphisms that segregate within both subspecies,
rather than fixed differences. While this suggests that our screening
approach to identifying candidate subspecies-recognition receptor
genes is naı ¨ve and somewhat inefficient (,10%), it allowed us to
obtain one such gene rapidly and inexpensively. We note that
more than half (58%) of the 392 V1R genes had no WSB-PWD
differences reported by Perlegen. This clearly reflects the
conservative nature of Perlegen data because our DNA sequencing
revealed 35 SNP differences in Vmn1r71, a gene reported as having
no SNP differences. Therefore, our screen remains incomplete,
but provided one excellent candidate for a V1R gene involved in
Evolution of Vmn1r67
The number of fixed SNP differences in Vmn1r67 suggests that
this gene diverged dramatically between the two subspecies
accompanied by the acquisition of very little polymorphism (only
two sites in M. m. domesticus). By contrast, Vmn1r71 had 35 SNP
differences and all of those were polymorphisms within one or
both subspecies. Returning to Vmn1r67, nearly two-thirds of the
fixed SNP differences (15/24) were nonsynonymous substitutions.
While CODEML did not predict positive selection using the gene
phylogeny as the guide tree, our observations of low polymor-
phism and high divergence are suggestive of selection for
divergence in this region between the M. m. domesticus and M.
m. musculus subspecies. The extreme incongruence we observe
between the gene tree and species tree is intriguing, and may be
due to some combination of introgression (see below), incomplete
lineage sorting, and/or fixation of recurrent mutations, perhaps
under the influence of positive selection to fix divergent alleles.
We suggest that Vmn1r67’s unusual evolution in M. m domesticus
and M. m. musculus may have occurred in response to a parallel
evolution of unique Abpa27 signals in the two subspecies,
explaining the strong LD between Vmn1r67 and Abpa27. Thus,
it appears that the LD we report here is a case of association
between Vmn1r67 and Abpa27, rather than residual physical
linkage from a very recent selective sweep, because an intervening
gene, Vmn1r71, shows significant polymorphism within subspecies
but no fixed divergence between the two subspecies. In advancing
the view that the LD we have observed may be explained by
functional association, we also note that there may be many such
regions in the genome that are mutually fixed between the
subspecies. However, in conceiving our experimental approach,
we purposely narrowed the field substantially by examining a
group of vomeronasal receptors some of which could be
candidates for ABP receptor(s) and association between Vmn1r67
and Abpa27 is at least a plausible explanation. We also note that
among nine other V1Rs for which we obtained sequences, there
were only two nucleotide positions showing fixed differences
between the two subspecies (File S1), establishing Vmn1r67’s
pattern of divergence as being unusual.
The hypothesis that rapid positive selection produced the
unusual divergence of the Vmn1r67 alleles in M. m. domesticus and
M. m. musculus is not the only possible explanation of the
observations we report here. An alternative explanation is that
the M. m. domesticus allele was derived from an introgressed allele,
for example from M. spretus or M. macedonicus. In fact, M. m.
domesticus is broadly sympatric with M. spretus both on the southern
shore and the northern shore of the Mediterranean Sea, as well as
with M. macedonicus in the Middle East. Alternatively or in addition,
the M. m. musculus allele could have been derived from an allele
introgressed from another species of Mus because it is sympatric
with M. spicilegus in Central Europe (see  for a review of the
distributions of these taxa of the genus Mus). There is precedence
for such an introgression pattern  and so we explored this by
comparing the sequences of Vmn1r67 alleles from M. spretus, M.
macedonicus and M. spicilegus, as well as with the allele in the M. m.
castaneus subspecies (File S4). This comparison revealed that the
Vmn1r67 allele in each differs from those in the two subspecies of
M. musculus we studied here. Thus we conclude that, while we
cannot rule out the possibility that either M. m. domesticus or M. m.
musculus, or both acquired a Vmn1r67 allele from other Mus species,
we can at least rule out very recent acquisition from these taxa that
are known to be sympatric with one or the other of them.
Coevolution of Vmn1r67 and Abpa27
ABP is the only one of the three proteinaceous pheromone
families (ABPs, ESPs and MUPs) that has been shown to have a
different allele fixed in each of the three Mus musculus subspecies
(Abpa27ain M. m. domesticus, Abpa27bin M. m. musculus and Abpa27c
in M. m. castaneus; [45,46]; see  for revised nomenclature).
Because ESPs have been discovered only recently [66,67], it is not
yet known whether any of them show fixed differences between
rodent taxa. Fixed differences in MUPs between the Mus musculus
subspecies have been sought extensively by others, but not found.
Indeed, both ESPs and MUPs have been hypothesized to mediate
recognition of individual characteristics including gender ,
rather than recognition of subspecies. MUPs may even allow
detection of predators . Abpa27 is also the only proteinaceous
pheromone for which there is population genetic evidence that the
different fixed alleles came about by positive selection [51,61].
Moreover, Karn and Laukaitis  previously reported evidence
for substantial recent gene duplication and copy-number variation
in the Abp region, especially in mouse strains with all or part of the
M. m. musculus or M. m. castaneus Abp gene region. By contrast, they
did not find evidence for any particularly remarkable recent
duplication or copy-number variation in the other two pheromone
gene families (ESPs and MUPs). They concluded that there is a
striking similarity between the volatility for copy-number variation
of Abp genes and that reported by others for chemosensory
receptor genes, especially the V1R genes [71,72] and speculated
that some of the V1R genes encode vomeronasal receptors
recognizing ABP molecules, which are coevolving to provide a
recognition system to reinforce subspecies and species hybridiza-
tion barriers. At this time we can only suggest that the data we
report here are consistent with the idea that Abpa27 and Vmn1r67
are coevolving as signal and receptor, respectively. Nonetheless
this idea is entirely consistent with the evolution of a pheromonal
system resulting in incipient reinforcement acting on behavioural
isolation traits in the European mouse hybrid zone and our future
research will focus on directly testing it.
PLoS ONE | www.plosone.org7September 2010 | Volume 5 | Issue 9 | e12638
Most of the sites showing fixed differences representing
divergence at nonsynonymous nucleotides between the two
subspecies affect amino acid residues found in either extracellular
loops (four) or intracellular loops (seven), with only four in
transmembrane helices. These nonsynonymous sites affect eleven
amino acid residues found in either extracellular or intracellular
loops (Fig. 3). The sites in the extracellular loops are candidates for
interaction with the ABP dimer, which has all of its selected sites
on one exterior face . It is not entirely clear what the function
of the seven sites in intracellular loops and the four in the
transmembrane helices is. It may be that a clue to the function of
these residues lies in ABP’s ability to bind male sex steroids and/or
other ligands, possibly as a means of enhancing the signal
interaction with the receptor. While laboratory studies have shown
that ABP is capable of binding male sex steroid hormones [74,75],
the identity of the lipophilic ligand actually bound by ABP in
nature has not been identified, and its potential role in ABP’s
function is not understood. However, it has been shown that
mouse saliva contains approximately equal quantities of two
different ABP dimers, one composed of the alpha subunit (the
subunit encoded by Abpa27) disulfide-bridged to the beta (the
subunit encoded by Abpbg27 – see  for current nomenclature)
and the other of the alpha subunit disulfide-bridged to the gamma
subunit (the subunit encoded by Abpbg26; ). Expression studies
have shown that other Abpa and Abpbg genes are expressed in
various tissues but dimeric combinations involving them have yet
to be described . Karn and Clements  studied the two
dimers in saliva (see above) and showed that they bind testosterone
and dihydrotestosterone (DHT) with different affinities. If ligand
binding provides a mechanism for either dimer to impart
information about subspecies status or to enhance ABP’s ability
to communicate subspecies status (analogous to turning up the
volume of a radio), this may explain why we found fixed
nonsynonymous sites in transmembrane helices and in intracellu-
lar loops, where a lipophilic ligand might be found, as well as in
extracellular loops, where an environmental pheromonal protein
Physical vs. genetic evidence for pheromone receptors
A number of studies have identified the mouse VNO as the tissue
containing receptors for pheromones (reviewed in [7,8]). Here we
have used a genetic approach to look for a candidate receptor(s) for
a putative subspecies recognition pheromone, ABP. Admittedly the
approach is an indirect one but it has the efficacy of identifying
candidate receptor genes that canthen be subjected to other studies,
including experiments with mouse strains in which either the signal
gene (e.g., Abpa27) and/or the receptor gene (in this case, Vmn1r67)
has been knocked out. Additional or alternative experiments
involving mate-choice behavior, using subject mice with recombi-
nant Vmn1r67- Abpa27 haplotypes may not only reinforce the
conclusion that the Vmn1r67 receptor recognizes the ABP signal
but may also reveal more details about the interaction of the
receptor and the signal. In any event, future work should also
involve direct electrophysiological experiments that will corroborate
the genetic identification of a VNO receptor for the ABP molecules
species identity in other rodents of the genus Mus.
Materials and Methods
Genomic DNA from wild-derived inbred Mus musculus domesticus
strains WSB/EiJ, LEWES/EiJ, PERA/EiJ, TIRANO/EiJ, ZA-
LENDE/EiJ; wild-derived M. m. musculus strains CZECHI/EiJ,
CZECHII/EiJ, PWD/PhJ, PWK/PhJ and SKIVE/EiJ; and wild-
derived M. m. castaneus (CAST/EiJ), M. spicilegus (PANCEVO/EiJ),
M. spretus (SPRET/EiJ), M. caroli (CAROLI/EiJ) and M. pahari/EiJ
were obtained from Jackson Laboratory. The M. macedonicus
sample was trapped in Iran by Pavel Munclinger. PCR and DNA
sequencing primers were obtained from Bioneer; primer sequences
and conditions are available from the authors upon request.
Screening for maximum SNP differences between M. m.
musculus and M. m. domesticus
We downloaded Perlegen’s mouse SNP genotype data 
(release 4, where SNP coordinates are given relative to Build 37 of
the mouse genome assembly, mm9) from http://mouse.perlegen.
com. These data were obtained by using an array ‘‘re-sequencing’’
method for key inbred and wild-derived mouse strains. Other
strains’ genotypes were inferred based on homology blocks
determined by similarity of tag SNPs in those strains to the re-
sequenced strains. PWD and WSB were both directly re-
sequenced and their genotypes were not inferred. Perlegen data
is intended to be conservative and a false negative rate of 67% has
been estimated  and we note that, because the Perlegen data
underestimate both SNP numbers and number of allelic
differences between the two strains analyzed, our V1R screen
remains incomplete. Using a custom Perl script, we determined
whether PWD and WSB had different genotypes for each SNP
(conservatively not counting ‘‘N’’ genotypes as differences). The
same Perl script then determined for each V1R gene the total
number of SNPs with genotypes that differ between PWD and
WSB using the mm9 coordinates of the 392 V1Rs found in a
previous study . We selected the ten intact V1R genes with the
most reported WSB-PWD differences for further study.
The top ten V1R genes identified with the SNP screen described
above were amplified by PCR and sequenced. The PCR products
were evaluated on 1% agarose gels and diluted 1:4, and were
sequenced by the UAGC facility at the University of Arizona. The
V1R sequences obtained from other species were checked against
their known mouse genome coordinates with the BLAT tool on
the UCSC genome browser [56,79,80].
DNA sequence traces were edited with Chromas 2.3 (http://
www.technelysium.com.au). DNA sequence alignment, coding
region assembly, and in silico translation were done using the
DNAsis Max program 2.0 (Hitachi). Nucleotide diversity (p) and
nucleotide polymorphism (h) were calculated for both subspecies
using DnaSP 5.10 . Positive selection was assessed in the
program CODEML in the PAML 4.4 package [53,78]). The
phylogeny of Chevret et al.  was used for the mouse species for
initial PAML tests. The three subspecies of M. musculus were
treated as an unresolved polytomy. We also followed Yang’s advice
that the gene tree should be used rather than the species tree
if the two are incongruent (http://www.ucl.ac.uk/discussions/
viewtopic.php?t=7850), which required constructing a Vmn1r67
gene phylogeny. The dnadist program of the PHYLIP package 3.68
 was used to calculate nucleotide distances using the F84 model
and a tree was constructed from those distances using the neighbor-
joining method , as implemented in PHYLIP’s neighbor
program . PHYLIP’s seqboot was used to calculate bootstrap
values from 1000 replicate datasets, again using F84 distances with
neighbor-joining . We also constructed a maximum likelihood
PLoS ONE | www.plosone.org8 September 2010 | Volume 5 | Issue 9 | e12638
gene tree with PHYLIP’s dnaml  and obtained the same
Three different comparisons of neutral and selection models
were made (M1 vs. M2, M7 vs. M8, and M8A vs. M8 [54,85,86].
Model M1 (neutral) allows two classes of codons, one with dN/dS
over the interval (0,1) and the other with a dN/dS value of one.
Model M2 (selection) is similar to M1 except that it allows an
additional class of codons with a freely estimated dN/dS value.
Model M7 (neutral) estimates dN/dS with a beta-distribution over
the interval (0, 1), whereas model M8 (selection) adds parameters
to M7 for an additional class of codons with a freely estimated dN/
dS value. M8A (neutral) is a special case of M8 that fixes the
additional codon class at a dN/dS value of one. We also conducted
‘‘branch-sites’’ CODEML analysis  comparing Model A with
the corresponding neutral model. As well as sites under purifying
selection and neutrally evolving sites, Model A allows for a class of
amino acid sites that only experience positive selection on one or
more selected ‘‘foreground’’ branches of the tree, rather than over
the entire phylogeny under consideration. Tests were performed
specifying as foreground branches either the M. m. domesticus
terminal branch alone, the M. m. musculus terminal branch alone,
or both terminal branches. In order to detect apparent
homoplasies when the species tree is assumed, we used CODEML
to reconstruct ancestral sequences and list nucleotide changes
occurring along each branch (with settings model=1 to allow each
branch its own dN/dS value, and NSsites=0 to allow only one
category of site). We then used a custom Perl script to parse
CODEML’s rst output file to look for apparent homoplasies
(identical nucleotide changes happening on more than one branch
of the tree). No such homoplasies were detected if the gene tree is
Two possible rat orthologs of Vmn1r67 were obtained from
Young et al . Both are located on chromosome 1 of the Nov.
2004 rat genome assembly, at coordinates 63137741–63138658
and 63198769–63199686 (both on the forward strand). We used
the first of these two genes in our codeml analysis, and provide its
sequence in File S4. Unlike most V1Rs, there does not seem to
have been extensive post-rat-mouse-speciation duplication in this
gene . The three-dimensional structure of Vmn1r67 was
modeled using the PHYRE 0.2 threading program (http://www.
sbg.bio.ic.ac.uk/phyre/), and the resulting model was visualized
using PYMOL (open-source 1.2.8; http://www.pymol.org/). Sites
showing fixed domesticus-musculus changes in Vmn1r67 were mapped
onto structural models using PYMOL and transferred to a figure
of the amino sequence using PHOTOSHOP 10.1.
Linkage disequilibrium (LD) was assessed using the linkage
disequilibrium viewer of the genome variation server (http://gvs.
gs.washington.edu/GVS). The Vmn1r67, Vmn1r71 and Abpa27
sequences from five strains each of the M. m. domesticus and M.
m. musculus subspecies were concatenated and aligned using
BioEdit 188.8.131.52 (6/27/07) . Nucleotide sites varying between
the samples were combined into a single reduced data set,
manually reformatted into the recommended format and upload-
ed. The LD image output was saved and a figure constructed with
labels in PHOTOSHOP.
Perlegen SNP data on V1R genes ranked from most PWD-WSB
SNPs to least.
Found at: doi:10.1371/journal.pone.0012638.s001 (0.05 MB
Raw data from the Perlegen screen. Screen of the
genes selected in our screen of Perlegen SNPs. These were rejected
as having few or no fixed differences between PWD and WSB.
Found at: doi:10.1371/journal.pone.0012638.s002 (0.05 MB
The partial DNA sequences of nine of the top ten V1R
gene sequences that contributed to the reduced data set shown in
Found at: doi:10.1371/journal.pone.0012638.s003 (0.01 MB
The five M. m. domesticus and five M. m. musculus Vmn1r67
gene sequences that contributed to the reduced data set shown in
Found at: doi:10.1371/journal.pone.0012638.s004 (0.01 MB
The five M. m. domesticus and five M. m. musculus Vmn1r71
for CODEML analysis.
Found at: doi:10.1371/journal.pone.0012638.s005 (0.01 MB
Vmn1r67 gene sequences used to construct the gene tree
Synonymous and nonsynonymous sites where apparent homopla-
sy was discovered when the species tree was used instead of the
gene tree in CODEML analysis.
Found at: doi:10.1371/journal.pone.0012638.s006 (0.00 MB
Synonymous and nonsynonymous sites in Vmn1r67.
Found at: doi:10.1371/journal.pone.0012638.s007 (0.00 MB
Vmn1r67 amino acid sequences used in constructing
The authors wish to thank Corina Fuentes for technical assistance, Pavel
Munclinger for the M. macedonicus sample, and Willie Swanson, Gabrielle
Wlasiuk and Nathan Clark for helpful discussions.
Conceived and designed the experiments: RCK CML. Performed the
experiments: RCK JMY. Analyzed the data: RCK JMY CML.
Contributed reagents/materials/analysis tools: JMY. Wrote the paper:
RCK. Wrote computer scripts for sorting SNPs in V1R genes in Perlegen
data: JMY. Linkage disequilibrium analysis: CML.
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PLoS ONE | www.plosone.org 11September 2010 | Volume 5 | Issue 9 | e12638