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 
). Olfactory cues represent the primary means of communication in nocturnal animals such as the house mouse 
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 
, 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 
, 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 
). 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 
. Two lines of indirect evidence 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 
; and (2) limited introgression of sex chromosome markers as compared to autosomes has been shown across four studied hybrid zone transects 
. 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 reproductive barrier, which may prevent loss of genetic potential into unfit hybrids 
. 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 
. 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 
. 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 
. 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 
. 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 
. Recently, Vošlajerová Bímová 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.