Dynamic evolution of V1R putative pheromone receptors between Mus musculus and Mus spretus

Abstract

Background: The mammalian vomeronasal organ (VNO) expresses two G-protein coupled receptor gene families that mediate pheromone responses, the V1R and V2R receptor genes. In rodents, there are ~150 V1R genes comprising 12 subfamilies organized in gene clusters at multiple chromosomal locations. Previously, we showed that several of these subfamilies had been extensively modulated by gene duplications, deletions, and gene conversions around the time of the evolutionary split of the mouse and rat lineages, consistent with the hypothesis that V1R repertoires might be involved in reinforcing speciation events. Here, we generated genome sequence for one large cluster containing two V1R subfamilies in Mus spretus, a closely related and sympatric species to Mus musculus, and investigated evolutionary change in these repertoires along the two mouse lineages.

Results: We describe a comparison of spretus and musculus with respect to genome organization and synteny, as well as V1R gene content and phylogeny, with reference to previous observations made between mouse and rat. Unlike the mouse-rat comparisons, synteny seems to be largely conserved between the two mouse species. Disruption of local synteny is generally associated with differences in repeat content, although these differences appear to arise more from deletion than new integrations. Even though unambiguous V1R orthology is evident, we observe dynamic modulation of the functional repertoires, with two of seven V1Rb and one of eleven V1Ra genes lost in spretus, two V1Ra genes becoming pseudogenes in musculus, two additional orthologous pairs apparently subject to strong adaptive selection, and another divergent orthologous pair that apparently was subjected to gene conversion.

Conclusion: Therefore, eight of the 18 (~44%) presumptive V1Ra/V1Rb genes in the musculus-spretus ancestor appear to have undergone functional modulation since these two species diverged. As compared to the rat-mouse split, where modulation is evident by independent expansions of these two V1R subfamilies, divergence between musculus and spretus has arisen more by mutations within coding sequences. These results support the hypothesis that adaptive changes in functional V1R repertoires contribute to the delineation of very closely related species.

Figure 1

Synteny map for Mus spretus versus Mus musculus. A. Map of the 690.4-kb Mus musculus V1Ra/V1Rb gene cluster on chromosome 6 (89617451–90307871 in July, 2007 assembly, UCSC Genome Browser) showing flanking non-V1R genes (Txnrd3, Uroc1) and the 16 intact V1R genes in the cluster. LINE repeats (black stripes) are shown below gene annotations. The locus assembly in Mus spretus is shown above the musculus map, with the ordered contigs indicated by rectangles (contig numbers and orientations above/below rectangles). The total spretus assembly is ~716.5 kb. Dotted boxed regions indicate the unresolved spretus assembly corresponding to the V1ra3-V1ra4 region in musculus and the presumed deleted segment in spretus corresponding to the V1ra8-V1rb3 region in musculus. The contiguity of the spretus-musculus assemblies are illustrated by the diagonal in the dot matrix in panel B.

Figure 2

Unexpected low orthology in the vicinity of the V1rb7 gene. PipMaker plot with chained alignments illustrating the contiguous high level of orthology (97–98%) evident upstream and downstream of the musculus V1rb7 gene. Hash marks indicate 1-kb intervals. The lower level of more disrupted synteny is confined to the region encompassing the musculus V1rb7/spretus YUA.5 orthologs, whose synonymous substitution rate (dS) is ~17%, or approximately 8.5-fold more diverged than expected. This gene pair might have been subject to gene conversion in one or both lineages.

Figure 3

Comparison of LINE ages at the three rodent V1Ra/V1Rb clusters to the mouse genome at large. The percentage of total LINE repeat content likely to be lineage-specific with respect to the musculus-spretus split (annotated in RepeatMasker with <2% substitution) or with respect to the mouse-rat split (annotated in RepeatMasker with <10% substitution), as well as older LINE content (>20% substitution), is compared between Mus spretus (sp.V1R, light gray), Mus musculus (mm.V1R, dark gray), and rat (rn.V1R, black), as well as to the Mus musculus genome at large (wider, open rectangles). The assumption that LINE repeats integrating since the mouse-rat split would exhibit <10% substitution is based on an estimate of neutral substitution levels of <20% among orthologous sequences (20% substitution between orthologs = 10% substitution along both lineages since the ancestral node). These data indicate that the majority of dense populations of LINE repeats at these rodent V1R loci integrated around the time of the mouse-rat split (but not since the musculus-spretus split) and that rodent V1R loci have younger LINE repeat content as compared to the genome at large.

Figure 4

V1R gene tree. Distance tree produced using 56 codon-aligned V1R sequences (906 nucleotides length) from mouse (musculus V1Rs denoted with "MUS" prefix; spretus V1Rs denoted with "YU" prefix) and rat ("RAT" prefix; dotted boxed clades). Pseudogenes are denoted by "pg" in their names. Musculus-spretus orthologous pairs in which one species encodes an intact V1R and the other species encodes an apparent pseudogene are shaded. Deleted V1Rs in one species but not the other are also shaded. Brackets with asterisks denote the two orthologous gene pairs apparently subject to adaptive selection (dS/dN < 1; see text), and the bracket without asterisk denotes the one orthologous gene pair apparently subject to gene conversion (Fig. 2). From this phylogeny, we infer that the rodent ancestor had five V1R genes: a homolog to rat A16, a homolog to mouse A1, a homolog to mouse A9, a homolog to mouse A7, and a B-like homolog. Bootstrap values (1000 replicas) for nodes <95% are indicated. Dashed lines with arrows show different locations of the MUS.A7, RAT.A2, and RAT.B9 branches in a parsimony bootstrap tree (1000 replicas) (not shown).