Evolutionary characteristics of the mitochondrial NADH dehydrogenase subunit 6 gene in some populations of four sympatric Mustela species (Mustelidae, Mammalia) from central Europe.

BACKGROUND: Selection on or reticulate evolution of mtDNA is documented in various mammalian taxa and could lead to misleading phylogenetic conclusions if not recognized. We sequenced the MT-ND6 gene of four sympatric Mustelid species of the genus Mustela from some central European populations. We hypothesised positive selection on MT-ND6, given its functional importance and the different body sizes and life histories of the species, even though climatic differences may be unimportant for adaptation in sympatry. METHODS AND RESULTS: MT-ND6 genes were sequenced in 187 sympatric specimens of weasels, Mustela nivalis, stoats, M. erminea, polecats, M. putorius, and steppe polecats, M. eversmannii, from eastern Austria and of fourteen allopatric polecats from eastern-central Germany. Median joining networks, neighbour joining and maximum likelihood analyses as well as Bayesian inference grouped all species according to earlier published phylogenetic models. However, polecats and steppe polecats, two very closely related species, shared the same two haplotypes. We found only negative selection within the Mustela sequences, including 131 downloaded ones covering thirteen species. Positive selection was observed on three MT-ND6 codons of other mustelid genera retrieved from GenBank. CONCLUSIONS: Negative selection for MT-ND6 within the genus Mustela suggests absence of both environmental and species-specific effects of cellular energy metabolism despite large species-specific differences in body size. The presently found shared polymorphism in European polecats and steppe polecats may result from ancestral polymorphism before speciation and historical or recent introgressive hybridization; it may indicate mtDNA capture of steppe polecats by M. putorius in Europe.

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.].

Purifying selection shaping the evolution of the Toll-like receptor 2 TIR domain in brown hares (Lepus europaeus) from Europe and the Middle East.

Toll-like receptors (TLRs) are transmembrane proteins of the innate immune system, composed of the ectodomain involved in pathogen recognition and the intracellular Toll/interleukin-1 receptor (TIR) domain important for downstream signal transduction. Here, we analyze the genetic variability of TIR nucleotide and amino-acid sequences of the TLR2 gene in 243 brown hares from Europe and the Middle East and tested for the presence of selection signals and spatial structuring. TLR2 TIR domain sequences were PCR amplified and sequenced, while genotyping was performed by phasing. Genetic diversity indices were calculated in DnaSP and Arlequin, while presence of selection signals was tested using MEGA and the Datamonkey web server. The presence of spatial patterns in TIR sequence distribution was tested by spatial Principal Component Analysis (sPCA) in adegenet. A total of 13 haplotypes were revealed with haplotype diversity of 0.424, and nucleotide diversity (π) of 0.00138. Two spatial clusters were revealed: "Anatolia/Middle East" and "Europe". In Anatolia the two most prevalent amino-acid variants, A and B (the latter being the most ancestral) were maintained at similar frequencies; but in Europe a shift in genotype frequencies was observed as well as a higher number of nonsynonymous substitutions giving rise to novel amino-acid protein variants originating from the evolutionarily younger protein variant. Molecular diversity (haplotype and nucleotide diversity) indices were significantly higher in the "Anatolia/Middle East" cluster. A signal of purifying selection was detected acting on the TIR sequences.

Islands and hybrid zones: combining the knowledge from "Natural Laboratories" to explain phylogeographic patterns of the European brown hare.

BACKGROUND: The aim of the study was to use hybrid populations as well as island populations of the European brown hare (Lepus europaeus) to explore the effect of evolutionary events, such as the post-deglaciation translocations, spontaneous and human-mediated, local adaptation and the genetic drift in the shaping of the phylogeographic patterns of the species. For this purpose, we used molecular markers, both nuclear and mitochondrial, that are indicative for local adaptation as well as neutral markers to elucidate the patterns of population differentiation based on geographic isolation and the clade of origin. To broaden our analysis, we included data from our previous studies concerning mainland populations, to explore the genetic differentiation in the base of the geographic origin (mainland/island) of the populations. RESULTS: Our results suggest that local adaptation shapes the differentiation in both genomes, favoring specific alleles in nuclear genes (e.g. DQA) or haplotypes in mtDNA (e.g. Control Region, CR). mtDNA variation was found to be in a higher level and was able to give a phylogeographic signal for the populations. Furthermore, the degree of variation was influenced not only by the geographic origin, but also by the clade of origin, since specific island populations of Anatolian origin showed a greater degree of variation compared to specific mainland populations of the European clade. Concerning the hybrid population, we confirmed the existence of both clades in the territory and we provided a possible explanation for the lack of introgression between the clades. CONCLUSION: Our results indicate that the Quaternary's climatic oscillations played a major role in the shaping of the phylogeographic patterns of the species, by isolating populations in the distinct refugia, where they adapted and differentiate in allopatry, leading to genome incompatibilities observed nowadays.

Selection on the mitochondrial ATP synthase 6 and the NADH dehydrogenase 2 genes in hares (Lepus capensis L., 1758) from a steep ecological gradient in North Africa.

BACKGROUND: Recent studies of selection on mitochondrial (mt) OXPHOS genes suggest adaptation due mainly to environmental variation. In this context, Tunisian hares that display several external phenotypes with phylogenetically rather homogenous gene pool and shallow population structure provide a good precondition to detect positive selection on mt genes related to environmental/climatic variation, specifically ambient temperature and precipitation. RESULTS: We used codon-based methods along with population genetic data to test for positive selection on ATPase synthase 6 (ATP6) and NADH dehydrogenase 2 (ND2) of cape hares (Lepus capensis) collected along a steep ecological gradient in Tunisia. We found significantly higher differentiation at the ATP6 locus across Tunisia, with sub-humid Mediterranean, semi-arid, and arid Sahara climate than for fourteen unlinked supposedly neutrally evolving nuclear microsatellites and mt control region sequences. This suggested positive selection on ATP6 sequences, which was confirmed by several codon-based tests for one sequence site that together with a second site translated into four different amino acids. Positive selection on ND2 sequences was also confirmed by several codon-based tests. The corresponding frequencies of the two most prevalent variants at each locus varied significantly across climate regions, and our logistic general linear models of occurrence of those proteins indicated significant effects of mean annual temperature for ATP6 and mean minimum temperature of the coldest month of the year for ND2, independent of geographical location, annual precipitation, and the respective co-occurring protein at the second locus. Moreover, presence of the ancestral ATP6 protein, as inferred from phylogenetic networks, was positively affected by the simultaneous presence of the derived ND2 protein and vice versa, independent of temperature, precipitation, or geographic location. Finally, we obtained a significant coevolution signal for the ancestral ATP6 and derived ND2 sequences and vice versa. CONCLUSIONS: positive selection was strongly suggested by the population genetic approach and the codon-based tests in both mtDNA genes. Moreover, the two most prevalent proteins at the ATP6 locus were distributed at significantly varying frequencies across the study area with a significant effect of mean annual temperature on the occurrence of the ATP6 proteins independent of geographical coordinates and the co-occuring ND2 protein variant. For ND2, occurrence of the two most frequent protein variants was significantly influenced by the mean minimum temperature of the coldest month, independent of the co-occurring ATP6 protein variant and geographical coordinates. This strongly suggests direct involvement of ambient temperature in the adaptation of the studied mtOXPHOS genes.

Variation of partial transferrin sequences and phylogenetic relationships among hares (Lepus capensis, Lagomorpha) from Tunisia.

North African hares are currently included in cape hares, Lepus capensis sensu lato, a taxon that may be considered a superspecies or a complex of closely related species. The existing molecular data, however, are not unequivocal, with mtDNA control region sequences suggesting a separate species status and nuclear loci (allozymes, microsatellites) revealing conspecificity of L. capensis and L. europaeus. Here, we study sequence variation in the intron 6 (468 bp) of the transferrin nuclear gene, of 105 hares with different coat colour from different regions in Tunisia with respect to genetic diversity and differentiation, as well as their phylogenetic status. Forty-six haplotypes (alleles) were revealed and compared phylogenetically to all available TF haplotypes of various Lepus species retrieved from GenBank. Maximum Likelihood, neighbor joining and median joining network analyses concordantly grouped all currently obtained haplotypes together with haplotypes belonging to six different Chinese hare species and the African scrub hare L. saxatilis. Moreover, two Tunisian haploypes were shared with L. capensis, L timidus, L. sinensis, L. yarkandensis, and L. hainanus from China. These results indicated the evolutionary complexity of the genus Lepus with the mixing of nuclear gene haplotypes resulting from introgressive hybridization or/and shared ancestral polymorphism. We report the presence of shared ancestral polymorphism between North African and Chinese hares. This has not been detected earlier in the mtDNA sequences of the same individuals. Genetic diversity of the TF sequences from the Tunisian populations was relatively high compared to other hare populations. However, genetic differentiation and gene flow analyses (AMOVA, FST, Nm) indicated little divergence with the absence of geographically meaningful phylogroups and lack of clustering with coat colour types. These results confirm the presence of a single hare species in Tunisia, but a sound inference on its phylogenetic position would require additional nuclear markers and numerous geographically meaningful samples from Africa and Eurasia.

Diversity of major histocompatibility complex (MHC) and natural killer cell receptor (NKR) genes and their interactions in domestic horses.

The immunogenome is the part of the genome that underlies immune mechanisms and evolves under various selective pressures. Two complex regions of the immunogenome, major histocompatibility complex (MHC) and natural killer cell receptor (NKR) genes, play an important role in the response to selective pressures of pathogens. Their importance is expressed by their genetic polymorphism at the molecular level, and their diversity associated with different types of diseases at the population level. Findings of associations between specific combinations of MHC/NKR haplotypes with different diseases in model species suggest that these gene complexes did not evolve independently. No such associations have been described in horses so far. The aim of the study was to detect associations between MHC and NKR gene/microsatellite haplotypes in three horse breed groups (Camargue, African, and Romanian) by statistical methods; chi-square test, Fisher's exact test, Pearson's goodness-of-fit test and logistic regression. Associations were detected for both MHC/NKR genes and microsatellites; the most significant associations were found between the most variable KLRA3 gene and the EQCA-1 or EQCA-2 genes. This finding supports the assumption that the KLRA3 is an important receptor for MHC I and that interactions of these molecules play important roles in the horse immunity and reproduction. Despite some limitations of the study such as low numbers of horses or lack of knowledge of the selected genes functions, the results were consistent across different statistical methods and remained significant even after overconservative Bonferroni corrections. We therefore consider them biologically plausible.

Prevalence of Eimeria spp. infections and major histocompatibility complex class II DRA diversity in Mongolian Bactrian camels (Camelus bactrianus).

Introduction: The two-humped Bactrian camel (Camelus bactrianus) is a large, even-toed ungulate native to the steppes of Central Asia. Domestic Bactrian camels are economically important in Mongolia and other Central Asian countries. These animals are used for transport, milk and meat production, and camel racing which is a great culture of nomads. Eimeriosis, also known as coccidiosis, is considered as an economically important parasitic diseases in Bactrian camels. There is still considerable lack of data concerning the spectrum of monoxenous Eimeria species, their epizootiology as well as their precise life cycles in Bactrian camels. This study was performed to determine the prevalence of Eimeria species in camelids from southern part of Mongolia. Methods: A total of 536 fresh camel fecal samples (n = 536) collected from herds located in five different Aimags (provinces) of Mongolia were examined. Eimeria spp. oocysts were isolated using the sugar flotation technique, and after sporulation, oocysts were identified by morphometric evaluation. Results: We identified the most common Eimeria species infecting Mongolian Bactrian camels: Eimeria cameli (22.3%), Eimeria rajasthani (37.3%) and Eimeria dromedarii (27.7%). Interestingly, mixed infections were detected in 24.8% (n = 133) of the samples, while 39.0% (n = 209) were negative for coccidian stages. To investigate the immunogenetic response of the Mongolian Bactrian camels to Eimeria spp. infection, we screened the genetic diversity in a functional important immune response gene of the major histocompatibility complex (MHC). We detected two polymorphic sites in the MHC class II DRA exon 2, which translated into one non-synonymous and one synonymous amino acid (aa) change. Discussion: The resulting aa alleles were not significantly associated with any of the three detected Eimeria species infections, nor could we show heterozygote advantage in non-infected Mongolian Bactrian camels. Further investigations on molecular epidemiology, in vitro culture, pathogenicity and host-parasite interactions will be necessary to better understand the impact of eimeriosis in Bactrian camels.

Insular giant leporid matured later than predicted by scaling.

The island syndrome describes morphological, behavioral, and life history traits that evolve in parallel in endemic insular organisms. A basic axiom of the island syndrome is that insular endemics slow down their pace of life. Although this is already confirmed for insular dwarfs, a slow life history in giants may not be adaptive, but merely a consequence of increasing body size. We tested this question in the fossil insular giant leporid Nuralagus rex. Using bone histology, we constructed both a continental extant taxon model derived from experimentally fluorochrome-labeled Lepus europaeus to calibrate life history events, and a growth model for the insular taxon. N. rex grew extremely slowly and delayed maturity well beyond predictions from continental phylogenetically corrected scaling models. Our results support the life history axiom of the island syndrome as generality for insular mammals, regardless of whether they have evolved into dwarfs or giants.

Sex-specific selection for MHC variability in Alpine chamois.

BACKGROUND: In mammals, males typically have shorter lives than females. This difference is thought to be due to behavioural traits which enhance competitive abilities, and hence male reproductive success, but impair survival. Furthermore, in many species males usually show higher parasite burden than females. Consequently, the intensity of selection for genetic factors which reduce susceptibility to pathogens may differ between sexes. High variability at the major histocompatibility complex (MHC) genes is believed to be advantageous for detecting and combating the range of infectious agents present in the environment. Increased heterozygosity at these immune genes is expected to be important for individual longevity. However, whether males in natural populations benefit more from MHC heterozygosity than females has rarely been investigated. We investigated this question in a long-term study of free-living Alpine chamois (Rupicapra rupicapra), a polygynous mountain ungulate. RESULTS: Here we show that male chamois survive significantly (P = 0.022) longer if heterozygous at the MHC class II DRB locus, whereas females do not. Improved survival of males was not a result of heterozygote advantage per se, as background heterozygosity (estimated across twelve microsatellite loci) did not change significantly with age. Furthermore, reproductively active males depleted their body fat reserves earlier than females leading to significantly impaired survival rates in this sex (P < 0.008). This sex-difference was even more pronounced in areas affected by scabies, a severe parasitosis, as reproductively active males were less likely to survive than females. However, we did not find evidence for a survival advantage associated with specific MHC alleles in areas affected by scabies. CONCLUSIONS: Increased MHC class II DRB heterozygosity with age in males, suggests that MHC heterozygous males survive longer than homozygotes. Reproductively active males appear to be less likely to survive than females most likely because of the energetic challenge of the winter rut, accompanied by earlier depletion of their body fat stores, and a generally higher parasite burden. This scenario renders the MHC-mediated immune response more important for males than for females, which implies a relatively stronger selection pressure on MHC genes in males than in females.

Pan-European phylogeography of the European roe deer (Capreolus capreolus).

To provide the most comprehensive picture of species phylogeny and phylogeography of European roe deer (Capreolus capreolus), we analyzed mtDNA control region (610 bp) of 1469 samples of roe deer from Central and Eastern Europe and included into the analyses additional 1541 mtDNA sequences from GenBank from other regions of the continent. We detected two mtDNA lineages of the species: European and Siberian (an introgression of C. pygargus mtDNA into C. capreolus). The Siberian lineage was most frequent in the eastern part of the continent and declined toward Central Europe. The European lineage contained three clades (Central, Eastern, and Western) composed of several haplogroups, many of which were separated in space. The Western clade appeared to have a discontinuous range from Portugal to Russia. Most of the haplogroups in the Central and the Eastern clades were under expansion during the Weichselian glacial period before the Last Glacial Maximum (LGM), while the expansion time of the Western clade overlapped with the Eemian interglacial. The high genetic diversity of extant roe deer is the result of their survival during the LGM probably in a large, contiguous range spanning from the Iberian Peninsula to the Caucasus Mts and in two northern refugia.

Evolutionary genetics of MHC class II beta genes in the brown hare, Lepus europaeus.

The genes of the major histocompatibility complex (MHC) are attractive candidates for investigating the link between adaptive variation and individual fitness. High levels of diversity at the MHC are thought to be the result of parasite-mediated selection and there is growing evidence to support this theory. Most studies, however, target just a single gene within the MHC and infer any evidence of selection to be representative of the entire gene region. Here we present data from three MHC class II beta genes (DPB, DQB, and DRB) for brown hares in two geographic regions and compare them against previous results from a class II alpha-chain gene (DQA). We report moderate levels of diversity and high levels of population differentiation in the DQB and DRB genes (Na = 11, D (est) = 0.071 and Na = 15, D (est) = 0.409, respectively), but not for the DPB gene (Na = 4, D (est) = 0.00). We also detected evidence of positive selection within the peptide binding region of the DQB and DRB genes (95% CI, ω > 1.0) but found no signature of selection for DPB. Mutation and recombination were both found to be important processes shaping the evolution of the class II genes. Our findings suggest that while diversifying selection is a significant contributor to the generally high levels of MHC diversity, it does not act in a uniform manner across the entire MHC class II region. The beta-chain genes that we have characterized provide a valuable set of MHC class II markers for future studies of the evolution of adaptive variation in Leporids.

Positive selection on two mitochondrial coding genes and adaptation signals in hares (genus Lepus) from China.

BACKGROUND: Animal mitochondria play a central role in energy production in the cells through the oxidative phosphorylation (OXPHOS) pathway. Recent studies of selection on different mitochondrial OXPHOS genes have revealed the adaptive implications of amino acid changes in these subunits. In hares, climatic variation and/or introgression were suggested to be at the origin of such adaptation. Here we looked for evidence of positive selection in three mitochondrial OXPHOS genes, using tests of selection, protein structure modelling and effects of amino acid substitutions on the protein function and stability. We also used statistical models to test for climate and introgression effects on sites under positive selection. RESULTS: Our results revealed seven sites under positive selection in ND4 and three sites in Cytb. However, no sites under positive selection were observed in the COX1 gene. All three subunits presented a high number of codons under negative selection. Sites under positive selection were mapped on the tridimensional structure of the predicted models for the respective mitochondrial subunit. Of the ten amino acid replacements inferred to have evolved under positive selection for both subunits, six were located in the transmembrane domain. On the other hand, three codons were identified as sites lining proton translocation channels. Furthermore, four codons were identified as destabilizing with a significant variation of Δ vibrational entropy energy between wild and mutant type. Moreover, our PROVEAN analysis suggested that among all positively selected sites two fixed amino acid replacements altered the protein functioning. Our statistical models indicated significant effects of climate on the presence of ND4 and Cytb protein variants, but no effect by trans-specific mitochondrial DNA introgression, which is not uncommon in a number of hare species. CONCLUSIONS: Positive selection was observed in several codons in two OXPHOS genes. We found that substitutions in the positively selected codons have structural and functional impacts on the encoded proteins. Our results are concordantly suggesting that adaptations have strongly affected the evolution of mtDNA of hare species with potential effects on the protein function. Environmental/climatic changes appear to be a major trigger of this adaptation, whereas trans-specific introgressive hybridization seems to play no major role for the occurrence of protein variants.

Positive selection on the MHC class II DLA-DQA1 gene in golden jackals (Canis aureus) from their recent expansion range in Europe and its effect on their body mass index.

BACKGROUND: In Europe, golden jackals (Canis aureus) have been expanding their range out of the southern and southeastern Balkans towards central Europe continually since the 1960s. Here, we investigated the level of functional diversity at the MHC class II DLA-DQA1 exon 2 in golden jackal populations from Bulgaria, Serbia, and Hungary. Specifically, we tested for positive selection on and geographic variation at that locus due to adaptation to supposedly regionally varying pathogenic landscapes. To test for potential fitness effects of different protein variants on individual body condition, we used linear modeling of individual body mass indexes (bmi) and accounted for possible age, sex, geographical, and climatic effects. The latter approach was performed, however, only on Serbian individuals with appropriate data. RESULTS: Only three different DLA-DQA1 alleles were detected, all coding for different amino-acid sequences. The neutrality tests revealed no significant but positive values; there was no signal of spatial structuring and no deviation from the Hardy-Weinberg equilibrium across the studied range of expansion. However, we found a signal of trans-species polymorphism and significant test results for positive selection on three codons. Our information-theory based linear modeling results indicated an effect of ambient temperature on the occurrence of individual DLA-DQA1 genotypes in individuals from across the studied expansion range, independent from geographical position. Our linear modeling results of individual bmi values indicated that yearlings homozygous for DLA-DQA1*03001 reached values typical for adults contrary to yearlings carrying other genotypes (protein combinations). This suggested better growth rates and thus a possible fitness advantage of yearlings homozygous for DLA-DQA1*03001. CONCLUSIONS: Our results indicate a demographic (stochastic) signal of reduced DLA-DQA1 exon 2 variation, in line with the documented historical demographic bottleneck. At the same time, however, allelic variation was also affected by positive selection and adaptation to varying ambient temperature, supposedly reflecting geographic variation in the pathogenic landscape. Moreover, an allele effect on body mass index values of yearlings suggested differential fitness associated with growth rates. Overall, a combination of a stochastic effect and positive selection has shaped and is still shaping the variation at the studied MHC locus.

Introducing mother's curse: low male fertility associated with an imported mtDNA haplotype in a captive colony of brown hares.

Mitochondrial DNA mutations create variation in the efficiency of the oxidative phosphorylation pathway and therefore cellular energy production. Mildly deleterious mutations may reduce the performance of sperm cells in particular, due to their high energy requirements and low number of mitochondria, yet have little or no effect on the viability of somatic cells or ova. Mutations will be maintained in the population, despite the fitness cost for males, because mtDNA is passed down the female line. We looked for this so-called mother's curse effect in our captive colony of European brown hares. Significantly reduced male reproductive success was detected for a divergent haplotype that could be traced back to hares imported from a remote population. Median reproductive success for these hares was 0.17 compared to 0.49 for the indigenous haplotypes (Wilcoxon rank-sum, P = 0.002). No difference was detected for female reproductive success, nor were we able to find a nuclear DNA component to variation in male fertility. Our data are strong evidence for a mother's curse effect persisting despite multiple crossings over seven generations. These data raise important issues relating to the reproductive fitness of small or intermixing populations and have particular implications for the management of populations for conservation.

Homozygosity at a class II MHC locus depresses female reproductive ability in European brown hares.

The link between adaptive genetic variation, individual fitness and wildlife population dynamics is fundamental to the study of ecology and evolutionary biology. In this study, a Bayesian modelling approach was employed to examine whether individual variability at two major histocompatibility complex (MHC) class II loci (DQA and DRB) and eight neutral microsatellite loci explained variation in female reproductive success for wild populations of European brown hare (Lepus europaeus). We examined two aspects of reproduction: the ability to reproduce (sterility) and the number of offspring produced (fecundity). Samples were collected from eastern Austria, experiencing a sub-continental climatic regime, and from Belgium with a more Atlantic-influenced climate. As expected, reproductive success (both sterility and fecundity) was significantly influenced by age regardless of sampling locality. For Belgium, there was also a significant effect of DQA heterozygosity in determining whether females were able to reproduce (95% highest posterior density interval of the regression parameter [-3.64, -0.52]), but no corresponding effect was found for Austria. In neither region was reproduction significantly associated with heterozygosity at the DRB locus. DQA heterozygotes from both regions also showed a clear tendency, but not significantly so, to produce a larger number of offspring. Predictive simulations showed that, in Belgium, sub-populations of homozygotes will have higher rates of sterile individuals and lower average offspring numbers than heterozygotes. No similar effect is predicted for Austria. The mechanism for the spatial MHC effect is likely to be connected to mate choice for increased heterozygosity or to the linkage of certain MHC alleles with lethal recessives at other loci.

Evolutionary history of an MHC gene in two leporid species: characterisation of Mhc-DQA in the European brown hare and comparison with the European rabbit.

We surveyed the genetic diversity of the expressed major histocompatibility complex class II DQA locus in natural populations of European brown hares, Lepus europaeus, from Austria and Belgium (267 individuals in total). Based on cDNA sequences, we designed hare-specific primers to amplify the highly variable second exon of the DQA gene. Using cloning-sequencing methodology and capillary electrophoresis single-strand conformation polymorphism, we found ten alleles of the DQA exon 2 locus across these two European regions, of which eight are described for the first time. To search for signals of selection and recombination in the evolution of the DQA gene within the leporids, we augmented our sample with orthologous DQA alleles from the European rabbit, Oryctolagus cuniculus, in order to carry out a species level, species pairwise comparison. We found evidence of recombination in the history of the DQA sequences in leporids with some recombinant alleles bridging the species divide. In both species, selection on peptide binding site codons can be detected, though stronger for the rabbit. This result suggests that there may be a differential selection pressure in the deeper evolutionary history of these two species due to differences in several demographic and ecological traits likely subjecting them to differential selection by parasites. Finally, evolutionary relationships show a widespread and statistically significant intermingling of alleles from the two species. The many macroparasites shared between hares and rabbits may explain this pattern of trans-species polymorphism.

Cytochrome b phylogeography of chamois (Rupicapra spp.). Population contractions, expansions and hybridizations governed the diversification of the genus.

The chamois provides an excellent model for exploring the effect of historical and evolutionary events on diversification. We investigate cytochrome b (cytb) sequences in the 10 recognized subspecies of Rupicapra classified within 2 species: Rupicapra pyrenaica, with the subspecies parva, pyrenaica, and ornata, and Rupicapra rupicapra, with cartusiana, rupicapra, tatrica, carpatica, balcanica, asiatica, and caucasica. A fragment of 349 bp of the cytb was sequenced in 189 individuals. We identified 3 cytb lineages: Clade West in Iberia and Western Alps; Clade Central in the Apennines and the Massif of Chartreuse; and Clade East present in populations to the east of the Alps. The 2 proposed species were polyphyletic; the clades West and Central are represented in both, whereas the Clade East is restricted to R. rupicapra. In contrast to the current systematic, cytb phylogenies suggest the classification of the 10 subspecies of chamois into a single species, R. rupicapra. Phylogeny and geographical distribution of the 3 lineages show the effects of limited latitudinal range expansions, contractions, and hybridizations among highly divergent lineages, along with a major role of the glacial ice sheets of the Alps and the Pyrenees as barriers to gene flow, on the diversification of extant taxa.

Positive selection and precipitation effects on the mitochondrial NADH dehydrogenase subunit 6 gene in brown hares (Lepus europaeus) under a phylogeographic perspective.

Previous studies in hares and jackrabbits have indicated that positive selection has shaped the genetic diversity of mitochondrial genes involved in oxidative phosphorylation, which may affect cellular energy production and cause regional adaptation to different environmental (climatic) pressures. In the present study, we sequenced the NADH dehydrogenase subunit 6 (MT-ND6) gene of 267 brown hares (L. europaeus) from Europe and Asia Minor and tested for positive selection and adaptations acting on amino acid sequences (protein variants). Molecular diversity indices and spatial clustering were assessed by DnaSP, Network, and Geneland, while the presence of selection signals was tested by codeml in PAML, and by using the Datamonkey Adaptive Evolution web server. The SPSS software was used to run multinomial regression models to test for possible effects of climate parameters on the currently obtained protein variants. Fifty-eight haplotypes were revealed with a haplotype diversity of 0.817, coding for 17 different protein variants. The MT-ND6 phylogeographic pattern as determined by the nucleotide sequences followed the earlier found model based on the neutrally evolving D-loop sequences, and reflected the earlier found phylogeographic Late Pleistocene scenario. Based on several selection tests, only one codon position consistently proved to be under positive selection. It did occur exclusively in the evolutionarily younger hares from Europe and it gave rise to several protein variants from the southeastern and south-central Balkans. The occurrence of several of those variants was significantly favored under certain precipitation conditions, as proved by our multinomial regression models. Possibly, the great altitudinal variation in the Balkans may have lead to bigger changes in precipitation across that region and this may have imposed an evolutionarily novel selective pressure on the protein variants and could have led to regional adaptation.

Recruitment of mitochondrial tRNA genes as auxiliary variability markers for both intra- and inter-species analysis: The paradigm of brown hare (Lepus europaeus).

We sequenced and analyzed the mitochondrial tRNA(Thr) and tRNA(Pro) genes from brown hare (Lepus europaeus) individuals of different geographic distribution and we investigated the role of various nucleotide substitutions that were detected. We compared these tRNAs with the respective available mitochondrial tRNA genes sequences within Lepus species and among mammals. The mutations that were detected represent specific and conserved polymorphisms that do not seem to affect the structural and functional features that are required for participation of tRNA molecules in mitochondrial protein synthesis. These changes however, possibly reflect on the evolutionary background of the species, which is based on the high intra-genomic variability and the evolutionary dynamic of the mitochondrial DNA. In an attempt to compare the phylogeny that is based on these specific tRNA genes with the phylogeny that is produced from sequencing data of the mitochondrial variable loop, we came up with results that indicate similar phylogeographic clusters. This observation implies that the tRNA mutations that were used for the present study have been well tolerated during evolution and they define an additional genetic and biochemical tag that can be used for such studies. Based on this notion and according to our results, we propose that mitochondrial tRNA genes can be used as valuable auxiliary molecular markers for contemporaneous and linked biochemical and genetic analyses.