Home-loving boreal hare mitochondria survived several invasions in Iberia: the relative roles of recurrent hybridisation and allele surfing.

Genetic introgression from a resident species into an invading close relative can result from repeated hybridisation along the invasion front and/or allele surfing on the expansion wave. Cases where the phenomenon is massive and systematic, such as for hares (genus Lepus) in Iberia, would be best explained by recurrent hybridisation but this is difficult to prove because the donor populations are generally extinct. In the Pyrenean foothills, Lepus europaeus presumably replaced Lepus granatensis recently and the present species border is parallel to the direction of invasion, so that populations of L. granatensis in the contact zone represent proxies of existing variation before the invasion. Among three pairs of populations sampled across this border, we find less differentiation of mitochondrial DNA (mtDNA) across than along it, as predicted under recurrent hybridisation at the invasion front. Using autosomal microsatellite loci and X- and Y-linked diagnostic loci, we show that admixture across the border is quasi-absent, making it unlikely that lack of interspecific mtDNA differentiation results from ongoing gene flow. Furthermore, we find that the local species ranges are climatically contrasted, making it also unlikely that ongoing ecology-driven movement of the contact account for mtDNA introgression. The lack of mtDNA differentiation across the boundary is mostly due to sharing of mtDNA from a boreal species currently extinct in Iberia (Lepus timidus) whose mitochondria have thus remained in place since the last deglaciation despite successive invasions by two other species. Home-loving mitochondria thus witness past species distribution rather than ongoing exchanges across stabilised contact zones.

Recurrent introgression of mitochondrial DNA among hares (Lepus spp.) revealed by species-tree inference and coalescent simulations.

Understanding recent speciation history requires merging phylogenetic and population genetics approaches, taking into account the persistence of ancestral polymorphism and possible introgression. The emergence of a clear phylogeny of hares (genus Lepus) has been hampered by poor genomic sampling and possible occurrence of mitochondrial DNA (mtDNA) introgression from the arctic/boreal Lepus timidus into several European temperate and possibly American boreal species. However, no formal test of introgression, taking also incomplete lineage sorting into account, has been done. Here, to clarify the yet poorly resolved species phylogeny of hares and test hypotheses of mtDNA introgression, we sequenced 14 nuclear DNA and 2 mtDNA fragments (8205 and 1113 bp, respectively) in 50 specimens from 11 hare species from Eurasia, North America, and Africa. By applying an isolation-with-migration model to the nuclear data on subsets of species, we find evidence for very limited gene flow from L. timidus into most temperate European species, and not into the American boreal ones. Using a multilocus coalescent-based method, we infer the species phylogeny, which we find highly incongruent with mtDNA phylogeny using parametric bootstrap. Simulations of mtDNA evolution under the speciation history inferred from nuclear genes did not support the hypothesis of mtDNA introgression from L. timidus into the American L. townsendii but did suggest introgression from L. timidus into 4 temperate European species. One such event likely resulted in the complete replacement of the aboriginal mtDNA of L. castroviejoi and of its sister species L. corsicanus. It is remarkable that mtDNA introgression in hares is frequent, extensive, and always from the same donor arctic species. We discuss possible explanations for the phenomenon in relation to the dynamics of range expansions and species replacements during the climatic oscillations of the Pleistocene.

The genomic legacy from the extinct Lepus timidus to the three hare species of Iberia: contrast between mtDNA, sex chromosomes and autosomes.

Extensive interspecific genetic introgression is often reported, and appraising its genomic impact can serve to determine whether it results from selection on specific loci or from demographic processes affecting the whole genome. The three species of hares present in the Iberian Peninsula harbour high frequencies of mitochondrial DNA (mtDNA) from Lepus timidus, an arctic/boreal species now extinct in the region. This could result from the invasive replacement of L. timidus by the temperate species during deglaciation but should then have left traces in the nuclear genome. We typed single nucleotide polymorphisms (SNPs) discovered by sequencing 10 autosomal loci, two X-linked and one Y-linked in species-wide samples of the four taxa. Based on lineage-diagnostic SNPs, we detected no trace of L. timidus sex chromosomes in Iberia. From the frequencies of inferred haplotypes, autosomal introgression into L. granatensis appeared mostly sporadic but always widespread instead of restricted to the north as mtDNA. Autosomal introgression into Iberian L. europaeus, inhabiting the Pyrenean foothills, was hardly detectable, despite quasi-fixation of L. timidus mtDNA. L. castroviejoi, endemic to the Cantabrian Mountains and fixed for L. timidus mtDNA, showed little traces of autosomal introgression. The absence of sex-chromosome introgression presumably resulted from X-linked hybrid male unfitness. The contrasting patterns between the autosomes and mtDNA could reflect general gender asymmetric processes such as frequency-dependent female assortative mating, lower mtDNA migration and higher male dispersal, but adaptive mtDNA introgression cannot be dismissed. Additionally, we document reciprocal introgression between L. europaeus and both L. granatensis in Iberia and L. timidus outside Iberia.

The evolutionary fate of recently duplicated retrogenes in mice.

Inferences about the evolutionary impact of gene duplications often rely on the analysis of their long-term outcome. The fate of the majority of them must, however, be decided shortly after duplication. Here we analysed the evolutionary pattern of 10 mouse genes very recently duplicated by retrotransposition, by sequencing the retroposed copy in five to 10 closely related mouse species. In all cases the retroposed copy experienced accelerated nonsynonymous evolution whereas the divergence pattern of the source copy appeared unaffected by the duplication, consistent with the neofunctionalization model. The analysis further revealed that most retrogenes, including pseudogenes, did not experience a period of relaxed neutral evolution, but have been submitted to purifying selection ever since their retroposition. We propose that these duplicates play a biochemical role but are not indispensable. Purifying selection prevents them from acquiring a negative role until they are lost or silenced. This period of unnecessary redundancy could in rare cases give the time for new functions to evolve.

The rise and fall of the mountain hare (Lepus timidus) during Pleistocene glaciations: expansion and retreat with hybridization in the Iberian Peninsula.

The climatic fluctuations during glaciations have affected differently arctic and temperate species. In the northern hemisphere, cooling periods induced the expansion of many arctic species to the south, while temperate species were forced to retract in southern refugia. Consequently, in some areas the alternation of these species set the conditions for competition and eventually hybridization. Hares in the Iberian Peninsula appear to illustrate this phenomenon. Populations of Iberian hare (Lepus granatensis), brown hare (Lepus europaeus) and broom hare (Lepus castroviejoi) in Northern Iberia harbour mitochondrial haplotypes from the mountain hare (Lepus timidus), a mainly boreal and arctic species presently absent from the peninsula. To understand the history of this past introgression we analysed sequence variation and geographical distribution of mitochondrial control region and cytochrome b haplotypes of L. timidus origin found in 378 specimens of these four species. Among 124 L. timidus from the Northern Palaearctic and the Alps we found substantial nucleotide diversity (2.3%) but little differentiation between populations. Based on the mismatch distribution of the L. timidus sequences, this could result from an expansion at a time of temperature decrease favourable to this arctic species. The nucleotide diversity of L. timidus mtDNA found in Iberian L. granatensis, L. europaeus and L. castroviejoi (183, 70 and 1 specimens, respectively) was of the same order as that in L. timidus over its range (1.9%), suggesting repeated introgression of multiple lineages. The structure of the coalescent of L. granatensis sequences indicates that hybridization with L. timidus was followed by expansion of the introgressed haplotypes, as expected during a replacement with competition, and occurred when temperatures started to rise, favouring the temperate species. Whether a similar scenario explains the introgression into Iberian L. europaeus remains unclear but it is possible that it hybridized with already introgressed L. granatensis.

Invasion from the cold past: extensive introgression of mountain hare (Lepus timidus) mitochondrial DNA into three other hare species in northern Iberia.

Mitochondrial DNA introgression from Lepus timidus into Lepus granatensis and Lepus europaeus was recently reported in Iberia, although L. timidus presumably retreated from this region at the end of the last ice age. Here we assess the extent of this ancient mtDNA introgression by RFLP analysis of 695 specimens representing the three hare species present in Iberia. The introgressed L. timidus lineage was found in 23 of the 37 populations sampled. It is almost fixed in L. europaeus across its Iberian range in the Pyrenean foothills, and in L. granatensis, which occupies the rest of the peninsula, it is predominant in the north and gradually disappears further south. We also found it in Lepus castroviejoi, a species endemic to Cantabria. Multiple hybridizations and, potentially, a selective advantage for the L. timidus lineage can explain the remarkable taxonomic and geographical range of this mitochondrial introgression.

Mouse biodiversity in the genomic era.

Comparative genomics has developed by comparison of distantly related genomes, for which the link between the reported evolutionary changes and species development/physiology/ecology is not obvious. It is argued that the mouse (genus Mus) is an optimal model for microevolutionary genomics in vertebrates. This is because the mouse genome sequence, physical and genetic map have been completed, because mouse genetics, morpho-anatomy, pathology, behavior and ecology are well-studied, and because the Mus genus is a diverse, well- documented taxon, allowing comparative studies at the level of individual, population, subspecies, and species. The potential of the interaction between mouse genome and mouse biodiversity is illustrated by recent studies of speciation in the house mouse Mus musculus, and studies about the evolution of isochores, the peculiar pattern of GC-content variation across mammalian genomes.

DetSel 1.0: a computer program to detect markers responding to selection.

Estimating population parameters from polymorphism frequency data requires neutral genetic markers. Any departure from neutrality may invalidate the inferences drawn from such analyses. We recently discussed the possibility of identifying markers that show deviation from neutral expectations in pairwise comparisons of diverging populations. We are now releasing a user-friendly software package that implements all the necessary steps to identify the signature of selection among molecular markers in a set of polymorphism data. This software can be downloaded free of charge at http://www.univ-montp2.fr/~genetix/detsel/detsel.html.

Nuclear ribosomal DNA monophyly versus mitochondrial DNA polyphyly in two closely related mite species: the influence of life history and molecular drive.

In two very closely related but reproductively isolated mite species, Tetranychus urticae and T. turkestani, we found nucleotide diversity to be extensive for mitochondrial DNA (mtDNA) cytochrome oxidase 1 (COI) (3-4%) but extremely reduced for nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS2) (less than 0.5%). By contrast, ITS2 was shown to evolve much faster than COI between species of this genus. Furthermore, we found that these two species are polyphyletic for mtDNA but monophyletic for rDNA. Thus it appears that despite its biparental transmission and multiplicity of copies in the genome, nuclear rDNA has a smaller effective population size than mtDNA in these species. The conjunction of efficient concerted evolution and/or gene conversion in the rDNA cluster, the haplodiploidy of these species and their female-biased sex ratio could account for this apparent contradiction.

Interpretation of variation across marker loci as evidence of selection.

Population structure and history have similar effects on the genetic diversity at all neutral loci. However, some marker loci may also have been strongly influenced by natural selection. Selection shapes genetic diversity in a locus-specific manner. If we could identify those loci that have responded to selection during the divergence of populations, then we may obtain better estimates of the parameters of population history by excluding these loci. Previous attempts were made to identify outlier loci from the distribution of sample statistics under neutral models of population structure and history. Unfortunately these methods depend on assumptions about population structure and history that usually cannot be verified. In this article, we define new population-specific parameters of population divergence and construct sample statistics that are estimators of these parameters. We then use the joint distribution of these estimators to identify outlier loci that may be subject to selection. We found that outlier loci are easier to recognize when this joint distribution is conditioned on the total number of allelic states represented in the pooled sample at each locus. This is so because the conditional distribution is less sensitive to the values of nuisance parameters.

Comparison of ribosomal ITS regions among Androctonus spp. sorpions (Scorpionida: Buthidae) from Tunisia.

Three species of scorpions have been reported from Tunisia: Androctonus amoreuxi Audoin, A. aeneas Koch, and A. australis L. The latest has been divided in two subspecies: A. australis garzonii Goyffon & Lamy and A. australis hector Koch. Despite the public health importance of these animals, which cause several cases of poisoning each year, nothing is know about the genetic diversity of the group. To gain a better understanding of the group, we studied the variation of rDNA sequences of the ITS1, 5.8S, and ITS2 region of 14 representatives of the four taxa in Tunisia. The main result is the high polymorphism of the ITS regions. In some instances in both intra- and interspecific comparisons, it was difficult to unambiguously align the sequences. However, some representatives of different species were relatively similar, so that it appeared difficult to recognize the species on the basis of these sequences. A phylogenetic analysis was conducted that inquires the validity of the subspecies status of A. australis garzonii and A. australis hector. Moreover, the taxonomic status of A. aeneas is also questioned. Our studies highlighted the need of a revision of the taxonomy of the scorpions in Tunisia; however, the use of other genetic markers will be necessary to solve this question.

A new method for locating changes in a tree reveals distinct nucleotide polymorphism vs. divergence patterns in mouse mitochondrial control region.

A new, model-based method was devised to locate nucleotide changes in a given phylogenetic tree. For each site, the posterior probability of any possible change in each branch of the tree is computed. This probabilistic method is a valuable alternative to the maximum parsimony method when base composition is skewed (i.e., different from 25% A, 25% C, 25% G, 25% T): computer simulations showed that parsimony misses more rare --> common than common --> rare changes, resulting in biased inferred change matrices, whereas the new method appeared unbiased. The probabilistic method was applied to the analysis of the mutation and substitution processes in the mitochondrial control region of mouse. Distinct change patterns were found at the polymorphism (within species) and divergence (between species) levels, rejecting the hypothesis of a neutral evolution of base composition in mitochondrial DNA.

Species-wide homogeneity of nuclear ribosomal ITS2 sequences in the spider mite Tetranychus urticae contrasts with extensive mitochondrial COI polymorphism.

We compared patterns of intraspecific polymorphism of two markers with contrasted modes of evolution, nuclear ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA), in the phytophagous mite Tetranychus urticae Koch. The second internal transcribed spacer (ITS2) of rDNA and a fragment in the mtDNA gene coding for Cytochrome Oxidase I (COI), were PCR-amplified and sequenced in samples of various geographical origins distributed worldwide. The 15 COI haplotypes found fell into two major phylogenetic lineages differing by an average of 5% nucleotide divergence. Samples from the Mediterranean basin were represented in both lineages, and showed no phylogeographical structure. The other samples, from temperate regions of the northern hemisphere, were clustered in one of the lineages and displayed little variation, indicating a recent colonization of this region. In contrast, no variation at all was found at the ITS2 in this species. We sequenced both COI and ITS2 in four other species of the genus Tetranychus and found that, despite the absence of intraspecific polymorphism, ITS appears to evolve 2.5 times faster than COI. We argue that rDNA homogeneity over the species range of T. urticae results from the high colonization potential of this species, preventing long-term differentiation. Preliminary data on two other mite species (Amphitetranychus viennensis Zacher and Mononychellus progresivus Doreste) with stricter ecological requirements and more restricted colonization potential revealed substantial and concordant geographical differentiation for both ITS2 and COI.

Population genetic structure in a Robertsonian race of house mice: evidence from microsatellite polymorphism.

Genetic evidence was assessed for inbreeding and population subdivision in a Robertsonian fusion (Rb) race of the western European form of house mouse, Mus musculus domesticus, in central Belgium. Inbreeding, and the factors responsible for subdivision (genetic drift and extinction-recolonization) can theoretically influence the fixation of underdominant Rb variants. The data consisted of allele frequencies of eight microsatellite loci and of the Rb(4.12) and Rb(5.10) chromosomes. Six populations were sampled once, and a seventh was sampled successively over 3 years. No evidence for inbreeding within populations was found. Levels of between-population subdivision were high (theta = 0.15-0.39), and showed no association with either karyotype or geographical distance over 8-60 km. In addition, low values of effective size were found in the successively sampled population (Ne = 5-20). Cases of significant two-locus disequilibria were associated with the most closely linked pair of microsatellite loci (r = 0.15): also consistent with small effective sizes. These results suggest that both the lack of inbreeding, and the combined effects of genetic drift and extinction-recolonization, may promote Rb polymorphism in M. m. domesticus.

Social structure of the mound-building mouse Mus spicilegus revealed by genetic analysis with microsatellites.

The Mound-building mouse Mus spicilegus possesses a unique behaviour amongst mice. It constructs large earthen mounds and associated nesting chambers which serve to store food for immature individuals during the winter nesting period. We have used genetic analysis of four autosomal and four X-linked microsatellite loci to determine relationships between individuals inhabiting 40 mounds in Bulgaria. We show that, in almost all cases, individuals in a mound are the product of multiple parentage. We estimate the minimum number of males and female parents contributing offspring to each mound and demonstrate that at least two male and two female parents contribute offspring to a minimum of seven mounds. Analyses of relatedness coefficients and allele sharing values demonstrate that parents of different sibships within mounds are more related than if they had been chosen at random from the population and suggest that it is the female parents that contribute this excess relatedness. These results suggest that the mechanism by which individuals congregate to build mounds is kin-based and that the evolution of mound building and communal nesting in M. spicilegus is due in part to kin selection. This study represents a novel approach to the study of mammalian behavioural ecology. We have used a genetic dataset to construct an outline of social structure in the absence of behavioural data. These inferences can now be used to direct further work on this species.

Discordant phylogeographic patterns between the Y chromosome and mitochondrial DNA in the house mouse: selection on the Y chromosome?

We have compared patterns of geographic variation and molecular divergence of mitochondrial DNA (mtDNA) and Y chromosome over the range of the different subspecies of Mus musculus. MtDNA was typed for 305 nucleotides in the control region, the Y chromosome for 834 base pairs (bp) in Zfy introns and 242 bp in Sry, a Zfy2 18-bp deletion, and two microsatellites. Apparent discrepancies exist between the distributions of the lineages of mtDNA and of the two major Y-chromosome lineages thus defined: some subspecies share the same mtDNA lineage but have different Y-chromosome lineages or vice versa. One microsatellite reveals a geographically clustered variation inside the distribution of each Y-chromosome lineage, showing that new Y-chromosome variants can rapidly spread locally. The two major Y-chromosome lineages have a divergence time only about one fourth of that between mtDNA lineages. Although this recent coalescence of the Y chromosomes between subspecies could partly be due to a lower ancestral polymorphism of the Y chromosome, it suggests that secondary introgression after the radiation of the subspecies might have occurred. There is evidence that the differentiation of the Y-chromosome lineages contributes to partial reproductive isolation between subspecies, and patterns of molecular evolution suggest that selection has played a role in the rapid spread across subspecies.

Mitochondrial COI sequences in mites: evidence for variations in base composition.

Studies of mitochondrial DNA sequences in a variety of animals have shown important differences between phyla, including differences in the genetic codes used, and varying constraints on base composition. In that respect, little is known of mites, an important and diversified group. We sequenced a portion (340 nt) of the cytochrome oxidase subunit I (COI) encoding gene in twenty species of phytophagous mites belonging to nine genera of the two families Tetranychidae and Tenuipalpidae. The mitochondrial genetic code used in mites appeared to be the same as in insects. As is generally also the case in insects, the mite sequences were very rich in A + T (75% on average), especially at the third codon position (94%). However, important variations of base composition were observed among mite species, one of them showing as little as 69% A + T. Variations of base composition occur mostly through synonymous transitions, and do not have detectable effects on polypeptide evolution in this group.

[Polytypic species Mus musculus in Transcaucasia]. / L'espèce polytypique Mus musculus en Transcaucasie.

It has been suggested that the house mouse, Mus musculus, is a polytypic species that originated in the northern part of the Indian sub-continent. Its subspecies have established secondary contact zones in east-centre China and in western Europe. However, the exact colonization routes taken by these subtaxa and their regions of primary differentiation have not yet been identified. We analyzed 89 mice from Transcaucasia at 35 enzyme loci and for polymorphism of the mitochondrial control region and a deletion of the Y chromosome. The various samples analyzed are a mosaic of populations intermediates between M. m. domesticus and M. m. musculus. Trancaucasia appears thus as a broad secondary contact zone, a fact which reinforces the idea that the species has retained large possibilities of remixing.