The evolutionary pathways for local adaptation in mountain hares.

Understanding the evolution of local adaptations is a central aim of evolutionary biology and key for the identification of unique populations and lineages of conservation relevance. By combining RAD sequencing and whole-genome sequencing, we identify genetic signatures of local adaptation in mountain hares (Lepus timidus) from isolated and distinctive habitats of its wide distribution: Ireland, the Alps and Fennoscandia. Demographic modelling suggested that the split of these mountain hares occurred around 20 thousand years ago, providing the opportunity to study adaptive evolution over a short timescale. Using genome-wide scans, we identified signatures of extreme differentiation among hares from distinct geographic areas that overlap with area-specific selective sweeps, suggesting targets for local adaptation. Several identified candidate genes are associated with traits related to the uniqueness of the different environments inhabited by the three groups of mountain hares, including coat colour, ability to live at high altitudes and variation in body size. In Irish mountain hares, a variant of ASIP, a gene previously implicated in introgression-driven winter coat colour variation in mountain and snowshoe hares (L. americanus), may underlie brown winter coats, reinforcing the repeated nature of evolution at ASIP moulding adaptive seasonal colouration. Comparative genomic analyses across several hare species suggested that mountain hares' adaptive variants appear predominantly species-specific. However, using coalescent simulations, we also show instances where the candidate adaptive variants have been introduced via introgressive hybridization. Our study shows that standing adaptive variation, including that introgressed from other species, was a crucial component of the post-glacial dynamics of species.

Androgen-Binding Protein (Abp) Evolutionary History: Has Positive Selection Caused Fixation of Different Paralogs in Different Taxa of the Genus Mus?

Comparison of the androgen-binding protein (Abp) gene regions of six Mus genomes provides insights into the evolutionary history of this large murid rodent gene family. We identified 206 unique Abp sequences and mapped their physical relationships. At least 48 are duplicated and thus present in more than two identical copies. All six taxa have substantially elevated LINE1 densities in Abp regions compared with flanking regions, similar to levels in mouse and rat genomes, although nonallelic homologous recombination seems to have only occurred in Mus musculus domesticus. Phylogenetic and structural relationships support the hypothesis that the extensive Abp expansion began in an ancestor of the genus Mus. We also found duplicated Abpa27's in two taxa, suggesting that previously reported selection on a27 alleles may have actually detected selection on haplotypes wherein different paralogs were lost in each. Other studies reported that a27 gene and species trees were incongruent, likely because of homoplasy. However, L1MC3 phylogenies, supposed to be homoplasy-free compared with coding regions, support our paralog hypothesis because the L1MC3 phylogeny was congruent with the a27 topology. This paralog hypothesis provides an alternative explanation for the origin of the a27 gene that is suggested to be fixed in the three different subspecies of Mus musculus and to mediate sexual selection and incipient reinforcement between at least two of them. Finally, we ask why there are so many Abp genes, especially given the high frequency of pseudogenes and suggest that relaxed selection operates over a large part of the gene clusters.

The Legacy of Recurrent Introgression during the Radiation of Hares.

Hybridization may often be an important source of adaptive variation, but the extent and long-term impacts of introgression have seldom been evaluated in the phylogenetic context of a radiation. Hares (Lepus) represent a widespread mammalian radiation of 32 extant species characterized by striking ecological adaptations and recurrent admixture. To understand the relevance of introgressive hybridization during the diversification of Lepus, we analyzed whole exome sequences (61.7 Mb) from 15 species of hares (1-4 individuals per species), spanning the global distribution of the genus, and two outgroups. We used a coalescent framework to infer species relationships and divergence times, despite extensive genealogical discordance. We found high levels of allele sharing among species and show that this reflects extensive incomplete lineage sorting and temporally layered hybridization. Our results revealed recurrent introgression at all stages along the Lepus radiation, including recent gene flow between extant species since the last glacial maximum but also pervasive ancient introgression occurring since near the origin of the hare lineages. We show that ancient hybridization between northern hemisphere species has resulted in shared variation of potential adaptive relevance to highly seasonal environments, including genes involved in circadian rhythm regulation, pigmentation, and thermoregulation. Our results illustrate how the genetic legacy of ancestral hybridization may persist across a radiation, leaving a long-lasting signature of shared genetic variation that may contribute to adaptation. [Adaptation; ancient introgression; hybridization; Lepus; phylogenomics.].

An Annotated Draft Genome of the Mountain Hare (Lepus timidus).

Hares (genus Lepus) provide clear examples of repeated and often massive introgressive hybridization and striking local adaptations. Genomic studies on this group have so far relied on comparisons to the European rabbit (Oryctolagus cuniculus) reference genome. Here, we report the first de novo draft reference genome for a hare species, the mountain hare (Lepus timidus), and evaluate the efficacy of whole-genome re-sequencing analyses using the new reference versus using the rabbit reference genome. The genome was assembled using the ALLPATHS-LG protocol with a combination of overlapping pair and mate-pair Illumina sequencing (77x coverage). The assembly contained 32,294 scaffolds with a total length of 2.7 Gb and a scaffold N50 of 3.4 Mb. Re-scaffolding based on the rabbit reference reduced the total number of scaffolds to 4,205 with a scaffold N50 of 194 Mb. A correspondence was found between 22 of these hare scaffolds and the rabbit chromosomes, based on gene content and direct alignment. We annotated 24,578 protein coding genes by combining ab-initio predictions, homology search, and transcriptome data, of which 683 were solely derived from hare-specific transcriptome data. The hare reference genome is therefore a new resource to discover and investigate hare-specific variation. Similar estimates of heterozygosity and inferred demographic history profiles were obtained when mapping hare whole-genome re-sequencing data to the new hare draft genome or to alternative references based on the rabbit genome. Our results validate previous reference-based strategies and suggest that the chromosome-scale hare draft genome should enable chromosome-wide analyses and genome scans on hares.

The genomic impact of historical hybridization with massive mitochondrial DNA introgression.

BACKGROUND: The extent to which selection determines interspecific patterns of genetic exchange enlightens the role of adaptation in evolution and speciation. Often reported extensive interspecific introgression could be selection-driven, but also result from demographic processes, especially in cases of invasive species replacements, which can promote introgression at their invasion front. Because invasion and selective sweeps similarly mold variation, population genetics evidence for selection can only be gathered in an explicit demographic framework. The Iberian hare, Lepus granatensis, displays in its northern range extensive mitochondrial DNA introgression from L. timidus, an arctic/boreal species that it replaced locally after the last glacial maximum. We use whole-genome sequencing to infer geographic and genomic patterns of nuclear introgression and fit a neutral model of species replacement with hybridization, allowing us to evaluate how selection influenced introgression genome-wide, including for mtDNA. RESULTS: Although the average nuclear and mtDNA introgression patterns contrast strongly, they fit a single demographic model of post-glacial invasive replacement of timidus by granatensis. Outliers of elevated introgression include several genes related to immunity, spermatogenesis, and mitochondrial metabolism. Introgression is reduced on the X chromosome and in low recombining regions. CONCLUSIONS: General nuclear and mtDNA patterns of introgression can be explained by purely demographic processes. Hybrid incompatibilities and interplay between selection and recombination locally modulate levels of nuclear introgression. Selection promoted introgression of some genes involved in conflicts, either interspecific (parasites) or possibly cytonuclear. In the latter case, nuclear introgression could mitigate the potential negative effects of alien mtDNA on mitochondrial metabolism and male-specific traits.

Whole exome sequencing of wild-derived inbred strains of mice improves power to link phenotype and genotype.

The house mouse is a powerful model to dissect the genetic basis of phenotypic variation, and serves as a model to study human diseases. Despite a wealth of discoveries, most classical laboratory strains have captured only a small fraction of genetic variation known to segregate in their wild progenitors, and existing strains are often related to each other in complex ways. Inbred strains of mice independently derived from natural populations have the potential to increase power in genetic studies with the addition of novel genetic variation. Here, we perform exome-enrichment and high-throughput sequencing (~8× coverage) of 26 wild-derived strains known in the mouse research community as the "Montpellier strains." We identified 1.46 million SNPs in our dataset, approximately 19% of which have not been detected from other inbred strains. This novel genetic variation is expected to contribute to phenotypic variation, as they include 18,496 nonsynonymous variants and 262 early stop codons. Simulations demonstrate that the higher density of genetic variation in the Montpellier strains provides increased power for quantitative genetic studies. Inasmuch as the power to connect genotype to phenotype depends on genetic variation, it is important to incorporate these additional genetic strains into future research programs.

Do changes in gene expression contribute to sexual isolation and reinforcement in the house mouse?

Expression divergence, rather than sequence divergence, has been shown to be important in speciation, particularly in the early stages of divergence of traits involved in reproductive isolation. In the two European subspecies of house mice, Mus musculus musculus and Mus musculus domesticus, earlier studies have demonstrated olfactory-based assortative mate preference in populations close to their hybrid zone. It has been suggested that this behaviour evolved following the recent secondary contact between the two taxa (~3,000 years ago) in response to selection against hybridization. To test for a role of changes in gene expression in the observed behavioural shift, we conducted a RNA sequencing experiment on mouse vomeronasal organs. Key candidate genes for pheromone-based subspecies recognition, the vomeronasal receptors, are expressed in these organs. Overall patterns of gene expression varied significantly between samples from the two subspecies, with a large number of differentially expressed genes between the two taxa. In contrast, only ~200 genes were found repeatedly differentially expressed between populations within M. m. musculus that did or did not display assortative mate preferences (close to or more distant from the hybrid zone, respectively), with an overrepresentation of genes belonging to vomeronasal receptor family 2. These receptors are known to play a key role in recognition of chemical cues that handle information about genetic identity. Interestingly, four of five of these differentially expressed receptors belong to the same phylogenetic cluster, suggesting specialization of a group of closely related receptors in the recognition of odorant signals that may allow subspecies recognition and assortative mating.

Range expansion underlies historical introgressive hybridization in the Iberian hare.

Introgressive hybridization is an important and widespread evolutionary process, but the relative roles of neutral demography and natural selection in promoting massive introgression are difficult to assess and an important matter of debate. Hares from the Iberian Peninsula provide an appropriate system to study this question. In its northern range, the Iberian hare, Lepus granatensis, shows a northwards gradient of increasing mitochondrial DNA (mtDNA) introgression from the arctic/boreal L. timidus, which it presumably replaced after the last glacial maximum. Here, we asked whether a south-north expansion wave of L. granatensis into L. timidus territory could underlie mtDNA introgression, and whether nuclear genes interacting with mitochondria ("mitonuc" genes) were affected. We extended previous RNA-sequencing and produced a comprehensive annotated transcriptome assembly for L. granatensis. We then genotyped 100 discovered nuclear SNPs in 317 specimens spanning the species range. The distribution of allele frequencies across populations suggests a northwards range expansion, particularly in the region of mtDNA introgression. We found no correlation between variants at 39 mitonuc genes and mtDNA introgression frequency. Whether the nuclear and mitochondrial genomes coevolved will need a thorough investigation of the hundreds of mitonuc genes, but range expansion and species replacement likely promoted massive mtDNA introgression.

Seeking signatures of reinforcement at the genetic level: a hitchhiking mapping and candidate gene approach in the house mouse.

Reinforcement is the process by which prezygotic isolation is strengthened as a response to selection against hybridization. Most empirical support for reinforcement comes from the observation of its possible phenotypic signature: an accentuated degree of prezygotic isolation in the hybrid zone as compared to allopatry. Here, we implemented a novel approach to this question by seeking for the signature of reinforcement at the genetic level. In the house mouse, selection against hybrids and enhanced olfactory-based assortative mate preferences are observed in a hybrid zone between the two European subspecies Mus musculus musculus and M. m. domesticus, suggesting a possible recent reinforcement event. To test for the genetic signature of reinforcing selection and identify genes involved in sexual isolation, we adopted a hitchhiking mapping approach targeting genomic regions containing candidate genes for assortative mating in mice. We densely scanned these genomic regions in hybrid zone and allopatric samples using a large number of fast evolving microsatellite loci that allow the detection of recent selection events. We found a handful of loci showing the expected pattern of significant reduction in variability in populations close to the hybrid zone, showing assortative odour preference in mate choice experiments as compared to populations further away and displaying no such preference. These loci lie close to genes that we pinpoint as testable candidates for further investigation.

The elusive nature of adaptive mitochondrial DNA evolution of an arctic lineage prone to frequent introgression.

Mitochondria play a fundamental role in cellular metabolism, being responsible for most of the energy production of the cell in the oxidative phosphorylation (OXPHOS) pathway. Mitochondrial DNA (mtDNA) encodes for key components of this process, but its direct role in adaptation remains far from understood. Hares (Lepus spp.) are privileged models to study the impact of natural selection on mitogenomic evolution because 1) species are adapted to contrasting environments, including arctic, with different metabolic pressures, and 2) mtDNA introgression from arctic into temperate species is widespread. Here, we analyzed the sequences of 11 complete mitogenomes (ten newly obtained) of hares of temperate and arctic origins (including two of arctic origin introgressed into temperate species). The analysis of patterns of codon substitutions along the reconstructed phylogeny showed evidence for positive selection in several codons in genes of the OXPHOS complexes, most notably affecting the arctic lineage. However, using theoretical models, no predictable effect of these differences was found on the structure and physicochemical properties of the encoded proteins, suggesting that the focus of selection may lie on complex interactions with nuclear encoded peptides. Also, a cloverleaf structure was detected in the control region only from the arctic mtDNA lineage, which may influence mtDNA replication and transcription. These results suggest that adaptation impacted the evolution of hare mtDNA and may have influenced the occurrence and consequences of the many reported cases of massive mtDNA introgression. However, the origin of adaptation remains elusive.

Diversity of Prdm9 zinc finger array in wild mice unravels new facets of the evolutionary turnover of this coding minisatellite.

In humans and mice, meiotic recombination events cluster into narrow hotspots whose genomic positions are defined by the PRDM9 protein via its DNA binding domain constituted of an array of zinc fingers (ZnFs). High polymorphism and rapid divergence of the Prdm9 gene ZnF domain appear to involve positive selection at DNA-recognition amino-acid positions, but the nature of the underlying evolutionary pressures remains a puzzle. Here we explore the variability of the Prdm9 ZnF array in wild mice, and uncovered a high allelic diversity of both ZnF copy number and identity with the caracterization of 113 alleles. We analyze features of the diversity of ZnF identity which is mostly due to non-synonymous changes at codons -1, 3 and 6 of each ZnF, corresponding to amino-acids involved in DNA binding. Using methods adapted to the minisatellite structure of the ZnF array, we infer a phylogenetic tree of these alleles. We find the sister species Mus spicilegus and M. macedonicus as well as the three house mouse (Mus musculus) subspecies to be polyphyletic. However some sublineages have expanded independently in Mus musculus musculus and M. m. domesticus, the latter further showing phylogeographic substructure. Compared to random genomic regions and non-coding minisatellites, none of these patterns appears exceptional. In silico prediction of DNA binding sites for each allele, overlap of their alignments to the genome and relative coverage of the different families of interspersed repeated elements suggest a large diversity between PRDM9 variants with a potential for highly divergent distributions of recombination events in the genome with little correlation to evolutionary distance. By compiling PRDM9 ZnF protein sequences in Primates, Muridae and Equids, we find different diversity patterns among the three amino-acids most critical for the DNA-recognition function, suggesting different diversification timescales.

Sexual selection against natural hybrids may contribute to reinforcement in a house mouse hybrid zone.

Sexual selection may hinder gene flow across contact zones when hybrid recognition signals are discriminated against. We tested this hypothesis in a unimodal hybrid zone between Mus musculus musculus and Mus musculus domesticus where a pattern of reinforcement was described and lower hybrid fitness documented. We presented mice from the border of the hybrid zone with a choice between opposite sex urine from the same subspecies versus hybrids sampled in different locations across the zone. While no preference was evidenced in domesticus mice, musculus males discriminated in favour of musculus signals and against hybrid signals. Remarkably, the pattern of hybrid unattractiveness did not vary across the hybrid zone. Moreover, allopatric populations tested in the same conditions did not discriminate against hybrid signals, indicating character displacement for signal perception or preference. Finally, habituation-discrimination tests assessing similarities between signals pointed out that hybrid signals differed from the parental ones. Overall, our results suggest that perception of hybrids as unattractive has evolved in border populations of musculus after the secondary contact with domesticus. We discuss the mechanisms involved in hybrid unattractiveness, and the potential impact of asymmetric sexual selection on the hybrid zone dynamics and gene flow between the two subspecies.

Adaptive evolution and effective population size in wild house mice.

Estimates of the proportion of amino acid substitutions that have been fixed by selection (α) vary widely among taxa, ranging from zero in humans to over 50% in Drosophila. This wide range may reflect differences in the efficacy of selection due to differences in the effective population size (N(e)). However, most comparisons have been made among distantly related organisms that differ not only in N(e) but also in many other aspects of their biology. Here, we estimate α in three closely related lineages of house mice that have a similar ecology but differ widely in N(e): Mus musculus musculus (N(e) ∼ 25,000-120,000), M. m. domesticus (N(e) ∼ 58,000-200,000), and M. m. castaneus (N(e) ∼ 200,000-733,000). Mice were genotyped using a high-density single nucleotide polymorphism array, and the proportions of replacement and silent mutations within subspecies were compared with those fixed between each subspecies and an outgroup, Mus spretus. There was significant evidence of positive selection in M. m. castaneus, the lineage with the largest N(e), with α estimated to be approximately 40%. In contrast, estimates of α for M. m. domesticus (α = 13%) and for M. m. musculus (α = 12 %) were much smaller. Interestingly, the higher estimate of α for M. m. castaneus appears to reflect not only more adaptive fixations but also more effective purifying selection. These results support the hypothesis that differences in N(e) contribute to differences among species in the efficacy of selection.

Isolation and gene flow: inferring the speciation history of European house mice.

Inferring the history of isolation and gene flow during species differentiation can inform us on the processes underlying their formation. Following their recent expansion in Europe, two subspecies of the house mouse (Mus musculus domesticus and Mus musculus musculus) have formed a hybrid zone maintained by hybrid incompatibilities and possibly behavioural reinforcement, offering a good model of incipient speciation. We reconstruct the history of their divergence using an approximate Bayesian computation framework and sequence variation at 57 autosomal loci. We find support for a long isolation period preceding the advent of gene flow around 200,000 generations ago, much before the formation of the European hybrid zone a few thousand years ago. The duration of the allopatric episode appears long enough (74% of divergence time) to explain the accumulation of many post-zygotic incompatibilities expressed in the present hybrid zone. The ancient contact inferred could have played a role in mating behaviour divergence and laid the ground for further reinforcement. We suggest that both subspecies originally colonized the Middle East from the northern Indian subcontinent, domesticus settling on the shores of the Persian Gulf and musculus on those of the Caspian Sea. Range expansions during interglacials would have induced secondary contacts, presumably in Iran, where they must have also interacted with Mus musculus castaneus. Future studies should incorporate this possibility, and we point to Iran and its surroundings as a hot spot for house mouse diversity and speciation studies.

Interspecific X-chromosome and mitochondrial DNA introgression in the Iberian hare: selection or allele surfing?

Introgression from a resident species into an invading one is predicted to occur through the demographic process of "allele surfing," and to particularly affect genomic regions transmitted by the lower migrating sex, such as mtDNA. This could explain that northern Iberian populations of Lepus granatensis harbor high frequencies of mtDNA from L. timidus, an arctic hare it replaced there after deglaciation. We report that variation of introgressed timidus-like mtDNA reflects several predicted effects of this process: increasing frequency and diversity in the direction of expansion, strong perpendicular phylogeographic structure and signs of postglacial demographic growth. However, demographic inferences for the granatensis and timidus-like mtDNA lineages suggest the latter may have outcompeted the former in northern Iberia. Autosomal introgression occurs at low frequencies and species-wide rather than only in the north. If this difference with mtDNA resulted from sex-biased migration, an intermediate pattern should prevail for the X-chromosome, but we report species-wide and high-frequency introgression of an X-fragment. Either selection favored this ubiquitous X-introgression, or more complex postglacial expansion patterns prevailed, with different consequences depending on the genomic and geographic region. This illustrates the difficulty of distinguishing demographic and selective effects and the need for genome and species-wide based demographic models.

Subspecific origin and haplotype diversity in the laboratory mouse.

Here we provide a genome-wide, high-resolution map of the phylogenetic origin of the genome of most extant laboratory mouse inbred strains. Our analysis is based on the genotypes of wild-caught mice from three subspecies of Mus musculus. We show that classical laboratory strains are derived from a few fancy mice with limited haplotype diversity. Their genomes are overwhelmingly Mus musculus domesticus in origin, and the remainder is mostly of Japanese origin. We generated genome-wide haplotype maps based on identity by descent from fancy mice and show that classical inbred strains have limited and non-randomly distributed genetic diversity. In contrast, wild-derived laboratory strains represent a broad sampling of diversity within M. musculus. Intersubspecific introgression is pervasive in these strains, and contamination by laboratory stocks has played a role in this process. The subspecific origin, haplotype diversity and identity by descent maps can be visualized using the Mouse Phylogeny Viewer (see URLs).

Genetic differentiation of the house mouse around the Mediterranean basin: matrilineal footprints of early and late colonization.

The molecular signatures of the recent expansion of the western house mouse, Mus musculus domesticus, around the Mediterranean basin are investigated through the study of mitochondrial D-loop polymorphism on a 1313 individual dataset. When reducing the complexity of the matrilineal network to a series of haplogroups (HGs), our main results indicate that: (i) several HGs are recognized which seem to have almost simultaneously diverged from each other, confirming a recent expansion for the whole subspecies; (ii) some HGs are geographically delimited while others are widespread, indicative of multiple introductions or secondary exchanges; (iii) mice from the western and the eastern coasts of Africa harbour largely different sets of HGs; and (iv) HGs from the two shores of the Mediterranean are more similar in the west than in the east. This pattern is in keeping with the two-step westward expansion proposed by zooarchaeological data, an early one coincident with the Neolithic progression and limited to the eastern Mediterranean and a later one, particularly evident in the western Mediterranean, related to the generalization of maritime trade during the first millennium BC and onwards. The dispersal of mice along with humans, which continues until today, has for instance left complex footprints on the long ago colonized Cyprus or more simple ones on the much more recently populated Canary Islands.

The ubiquitous mountain hare mitochondria: multiple introgressive hybridization in hares, genus Lepus.

Climatic oscillations during the glaciations forced dramatic changes in species distributions, such that some presently temperate regions were alternately occupied by temperate and arctic species. These species could have met and hybridized during climatic transitions. This phenomenon happened for three hare species present in Iberia (Lepus granatensis, Lepus europaeus and Lepus castroviejoi), which display high frequencies of mitochondrial DNA (mtDNA) from Lepus timidus, an arctic/boreal species presently extinct in Iberia. Here, we extend our previous geographical survey to determine whether the distribution of this mtDNA lineage extends beyond the northern half of the Iberian Peninsula, where it is found at high frequencies. We also review the taxonomy, distribution and molecular phylogeny of the genus Lepus. The phylogenetic inference reveals the presence of L. timidus-like mtDNA in several other hare species in Asia and North America, suggesting that the mitochondrial introgression observed in Iberia might be generalized. Comparison with the available nuclear gene phylogenies suggests that introgression could have happened repeatedly, possibly during different climatic transitions. We discuss demographic and adaptive scenarios that could account for the repetition in time and space of this spectacular phenomenon and suggest ways to improve our understanding of its determinants and consequences. Such high levels of introgressive hybridization should discourage attempts to revise hare taxonomy based solely on mtDNA.