Pheromones are secreted chemicals recognized by members of the same species (conspecifics) that convey information about individual members. The vomeronasal organ (VNO) of terrestrial vertebrates is responsible for pheromonal responses that evoke social and reproductive behaviors, including male territorial aggression, sexual preference, and sexual maturity (reviewed in [1
]). These responses are thought to be mediated by at least two unrelated gene families, referred to as V1Rs [2
] and V2Rs [3
], encoding G protein-coupled receptors (GPCRs) that are expressed on surfaces of sensory neurons in the VNO.
In addition to being the principle chemoreceptors in the olfactory and gustatory sensory systems, the large GPCR superfamily has prominent roles in transducing signals for diverse ligands, including ions, hormones, neurotransmitters, nucleotides, and photons [6
]. The GPCR repertoire has a common seven transmembrane structure, and mammalian GPCR proteins are classified into six major families: the peptide-binding secretin types, the adhesion types that contain N-terminal domains with motifs implicated in cell adhesion functions (e.g., EGF
-like repeats), the glutamate types (including TAS1
taste receptors and the V2Rs expressed in the VNO), the frizzled
types, the Taste2 types (TAS2
taste receptors), and the large set of rhodopsin types (including the olfactory receptors) [6
]. V1Rs share a distant relationship with the Taste2- and rhodopsin-types of GPCRs that bind ligands within transmembrane cavities (and that lack the large N-terminal binding domains characteristic of other GPCRs).
V1R gene repertoires vary significantly among species. In mouse and rat, there are a total of approximately 150 V1R genes from 12 phylogenetically distinct subfamilies [7
]; members of each subfamily tend to be clustered at one or two chromosomal locations, reflective of a recent history of expansion by tandem duplications. In human and chimpanzee, the V1R repertoire consists almost entirely of non-functional pseudogenes, consistent with the loss of VNO function possibly concurrent with the advent of trichromatic color vision during primate evolution [8
]. Surprisingly, the dog genome encodes only five intact V1R genes [8
], the cow and opossum genomes encode less than a third the number of V1Rs as mouse [9
], and several other mammalian species (such as pig [10
], sheep [11
], and ferret [12
]) seem to similarly exhibit reduced VNO function as compared to rodents. Therefore, rodents appear to be exceptional with respect to their reliance on the VNO and V1R gene repertoires for mediating pheromone responses.
The mouse and rat V1R repertoires have modulated significantly since these two species diverged. In general, orthologous relationships between genes and syntenic relationships between clusters are difficult to map as a result of lineage-specific duplications and deletions. Two striking examples of delineation between the two species are the complete deletion of two subfamilies (V1Rh
) in rat that represent the largest gene cluster in mouse, and the independent generation of two subfamilies (V1Ra
) in both mouse and rat from a small number of ancestral genes in the presumptive ancestor [7
]. Moreover, the V1Ra/V1Rb
duplications in both species appear to have occurred over a very short period of evolution just following mouse-rat speciation, probably driven in part by a wave of LINE repeat integration within the locus [13
]. These observations suggest that adaptive modulation of V1R repertoires were important in the reinforcement of interspecies communication barriers in the period immediately following speciation.
Unlike the odorant receptors of the main olfactory system, which exhibit ligand promiscuity as part of a combinatorial system of odor recognition (reviewed in [14
]), the V1Rs of the VNO might respond to a very narrow range of ligands, if not exclusively to one ligand [15
]. Moreover, while odorant receptor sensory neurons of the main olfactory system exhibit responsiveness in a concentration-dependent manner, the subset of vomeronasal sensory neurons responding to a particular stimulus do not change with the concentration of that stimulus. These observations suggest that different members of a V1R subfamily detect highly specific ligands, implying that modulation of subfamily repertoires could modulate physiologically distinct functions.
Little is currently known about V1R function. A recent knockout of the entire V1Ra-V1Rb
cluster in mouse perturbed normal patterns of female aggression and caused male mice to show reduced sexual behavior towards females [16
]. Similarly broad effects in sexual and social behaviors are evident in mice genetically deficient in Trp2
, a protein required for vomeronasal neuronal signalling [17
]. The low resolution of these phenotypes and the apparent phenotypic overlap between mice deficient in a subset of V1Rs and those fully deficient in V1R response suggest that individual V1Rs make combinatorial or additive contributions to broad behavioural patterns, as opposed to each V1R directing a distinct behaviour. To our knowledge, the hypothesis that V1Rs contribute to the establishment of mating barriers between species has not yet been investigated.
It is presumed that one function of pheromones is the establishment of prezygotic mating barriers so that unproductive mating is not attempted (reviewed in [18
]). Prezygotic mating barriers can quickly arise to prevent non-productive interbreeding attempts between subpopulations otherwise capable of mating [19
], and therefore, modulation of pheromones and their receptors might be especially important for selective breeding within sympatric populations. For example, the Mediterranean short-tailed mouse, Mus spretus, is sympatric with certain subspecies of Mus musculus in parts of southern Europe and North Africa. No hybrids of these two species have been observed in nature, suggesting that they will not interbreed (presumably due to a selective disadvantage in hybrid offspring), even though they can produce viable offspring in the laboratory [20
]. These two species diverged approximately 1.1 million years ago [21
]; with an estimated neutral substitution rate of ~1% per million years [23
], we expect that typical orthologs will be ~98% identical between the two species.
In this study, we compare V1Ra and V1Rb gene repertoires in Mus spretus and Mus musculus in order to investigate whether, even over these very short evolutionary periods and in a background of very high sequence identity, these V1R repertoires exhibit the dynamic functional modulation observed between mouse and rat. Our results indicate that functional modulation of rodent V1R repertoires has occurred, albeit by diverse evolutionary paths, supporting the hypothesis that adaptive changes in these V1R repertoires have contributed to the delineation of even very closely related species.