De novo transcriptome assembly facilitates characterisation of fast-evolving gene families, MHC class I in the bank vole (Myodes glareolus).

The major histocompatibility complex (MHC) plays a central role in the adaptive immune response and is the most polymorphic gene family in vertebrates. Although high-throughput sequencing has increasingly been used for genotyping families of co-amplifying MHC genes, its potential to facilitate early steps in the characterisation of MHC variation in nonmodel organism has not been fully explored. In this study we evaluated the usefulness of de novo transcriptome assembly in characterisation of MHC sequence diversity. We found that although de novo transcriptome assembly of MHC I genes does not reconstruct sequences of individual alleles, it does allow the identification of conserved regions for PCR primer design. Using the newly designed primers, we characterised MHC I sequences in the bank vole. Phylogenetic analysis of the partial MHC I coding sequence (2-4 exons) of the bank vole revealed a lack of orthology to MHC I of other Cricetidae, consistent with the high gene turnover of this region. The diversity of expressed alleles was characterised using ultra-deep sequencing of the third exon that codes for the peptide-binding region of the MHC molecule. High allelic diversity was demonstrated, with 72 alleles found in 29 individuals. Interindividual variation in the number of expressed loci was found, with the number of alleles per individual ranging from 5 to 14. Strong signatures of positive selection were found for 8 amino acid sites, most of which are inferred to bind antigens in human MHC, indicating conservation of structure despite rapid sequence evolution.

Relaxed functional constraints on triplicate α-globin gene in the bank vole suggest a different evolutionary history from other rodents.

Gene duplication plays an important role in the origin of evolutionary novelties, but the mechanisms responsible for the retention and functional divergence of the duplicated copy are not fully understood. The α-globin genes provide an example of a gene family with different numbers of gene duplicates among rodents. Whereas Rattus and Peromyscus each have three adult α-globin genes (HBA-T1, HBA-T2 and HBA-T3), Mus has only two copies. High rates of amino acid evolution in the independently derived HBA-T3 genes of Peromyscus and Rattus have been attributed to positive selection. Using RACE PCR, reverse transcription-PCR (RT-PCR) and RNA-seq, we show that another rodent, the bank vole Clethrionomys glareolus, possesses three transcriptionally active α-globin genes. The bank vole HBA-T3 gene is distinguished from each HBA-T1 and HBA-T2 by 20 amino acids and is transcribed 23- and 4-fold lower than HBA-T1 and HBA-T2, respectively. Polypeptides corresponding to all three genes are detected by electrophoresis, demonstrating that the translated products of HBA-T3 are present in adult erythrocytes. Patterns of codon substitution and the presence of low-frequency null alleles suggest a postduplication relaxation of purifying selection on bank vole HBA-T3.

PCR-RFLP assays to distinguish the Western and Eastern phylogroups in wild and cultured tench Tinca tinca.

The tench Tinca tinca is a valued table fish native to Europe and Asia, but which is now widely distributed in many temperate freshwater regions of the world as the result of human-mediated translocations. Fish are currently being transplanted between watersheds without concern for genetic similarity to wild populations or local adaptation, and efficient phylogeographic markers are therefore urgently needed to rapidly distinguish genetically distinct geographical populations and to assess their contribution to the hatchery breeds and to the stocked wild populations. Here, we present a new method to distinguish recently discovered and morphologically undistinguishable Western and Eastern phylogroups of the tench. The method relies on PCR-RFLP assays of two independent nuclear-encoded exon-primed intron-crossing (EPIC) markers and of one mitochondrial DNA (mDNA) marker and allows the rapid identification of the Western and Eastern phylogroup and also of three geographical mtDNA clades within the Eastern phylogroup. Our method will enable researchers and fishery practitioners to rapidly distinguish genetically divergent geographical populations of the tench and will be useful for monitoring the introduction and human-mediated spread of the phylogroups in wild populations, for characterization of cultured strains and in breeding experiments.

Rare and asymmetrical hybridization of the endemic Barbus carpathicus with its widespread congener Barbus barbus.

In endemic species that co-occur with widespread congeners, hybridization can lead to an influx of novel and beneficial genetic variation, but high rates of introgression may cause genetic swamping of the endemic species and have detrimental effects on its survival potential. This study examines hybridization between sympatric populations of the Carpathian barbel Barbus carpathicus, a recently discovered cryptic species with a restricted range, and the widespread common barbel Barbus barbus. Based on six diagnostic allozyme loci, a microsatellite locus and mtDNA, hybrids were found to be present at multiple localities within the Vistula River drainage (Baltic Sea) as well as in the Tisza River system of the Danube River drainage (Black Sea). However, the numbers of hybrids were very low; four individuals of 230 fish sampled from the Vistula drainage. Bayesian assessment of their nuclear genotypes suggested that two hybrids in the Vistula drainage and nine in the Tisza system were F1 generation, and one in the Vistula drainage and one in the Tisza system were backcrosses (BC) to B. barbus, while no F2 or BC to B. carpathicus were detected. No hybrid carried B. carpathicus mtDNA and cytonuclear linkage disequilibria showed significant positive associations between hybrid genotypes and B. barbus mtDNA, suggesting unidirectionality in the interspecific mating with a disproportionate contribution of B. barbus mothers. Despite geographically broad occurrence of hybrids, these data provide evidence of strong constraints on hybridization in the native breeding habitats and the lack of introgression towards B. carpathicus.

Ice age cloning--comparison of the Quaternary evolutionary histories of sexual and clonal forms of spiny loaches (Cobitis; Teleostei) using the analysis of mitochondrial DNA variation.

Recent advances in population history reconstruction offered a powerful tool for comparisons of the abilities of sexual and clonal forms to respond to Quaternary climatic oscillations, ultimately leading to inferences about the advantages and disadvantages of a given mode of reproduction. We reconstructed the Quaternary historical biogeography of the sexual parental species and clonal hybrid lineages within the Europe-wide hybrid complex of Cobitis spiny loaches. Cobitis elongatoides and Cobitis taenia recolonizing Europe from separated refuges met in central Europe and the Pontic region giving rise to hybrid lineages during the Holocene. Cobitis elongatoides due to its long-term reproductive contact with the remaining parental species of the complex--C. tanaitica and C. spec.--gave rise to two clonal hybrid lineages probably during the last interglacial or even earlier, which survived the Würmian glaciation with C. elongatoides. These lineages followed C. elongatoides postglacial expansion and probably decreased its dispersal rate. Our data indicate the frequent origins of asexuality irrespective of the parental populations involved and the comparable dispersal potential of diploid and triploid lineages.

Beyond the Mediterranean peninsulas: evidence of central European glacial refugia for a temperate forest mammal species, the bank vole (Clethrionomys glareolus).

This study details the phylogeographic pattern of the bank vole, Clethrionomys glareolus, a European rodent species strongly associated with forest habitat. We used sequences of 1011 base pairs of the mitochondrial DNA cytochrome b gene from 207 bank voles collected in 62 localities spread throughout its distribution area. Our results reveal the presence of three Mediterranean (Spanish, Italian and Balkan) and three continental (western, eastern and 'Ural') phylogroups. The endemic Mediterranean phylogroups did not contribute to the post-glacial recolonization of much of the Palaearctic range of species. Instead, the major part of this region was apparently recolonized by bank voles that survived in glacial refugia in central Europe. Moreover, our phylogeographic analyses also reveal differentiated populations of bank voles in the Ural mountains and elsewhere, which carry the mitochondrial DNA of another related vole species, the ruddy vole (Clethrionomys rutilus). In conclusion, this study demonstrates a complex phylogeographic history for a forest species in Europe which is sufficiently adaptable that, facing climate change, survives in relict southern and northern habitats. The high level of genetic diversity characterizing vole populations from parts of central Europe also highlights the importance of such regions as a source of intraspecific genetic biodiversity.

Circum Black Sea phylogeography of Barbus freshwater fishes: divergence in the Pontic glacial refugium.

We used DNA sequence variation at the mitochondrial cytochrome b gene (1141 bp) to assess the phylogeography of Barbus fishes in the Black Sea region. Our aim was to test whether the recent ( approximately 22 000-7500 years ago) freshwater phase of the Black Sea was a conduit for gene flow among freshwater fishes that today are found in streams entering the saltwater Black Sea. Deep phylogeographical breaks suggestive of allopatric divergence were observed between four regional groups of populations. Coalescent simulation used to distinguish between this and an alternative scenario that the phylogeographical structure was due to random lineage sorting showed that the contemporary populations were unlikely (P < 0.001) to have been founded by a single ancestral population. Divergences between the lineages (0.86-2.54%) were dated to the Middle to Late Pleistocene using distances and a molecular clock corrected for superimposed substitutions. Taken together, this evidence suggests that multiple refugial populations survived over several later glaciations in the vicinity of the Black Sea. This Pontic refugium served as the primary source for the postglacial expansion throughout Europe as far as the Atlantic basin. However, only one of the phylogeographical lineages contributed to this dispersion, whereas the others remain restricted to the Black Sea region and followed independent evolutionary trajectories.

Evolutionary history of asexual hybrid loaches (Cobitis: Teleostei) inferred from phylogenetic analysis of mitochondrial DNA variation.

Reconstruction of the evolutionary history of asexual lineages undermines their suitability as models for the studies of evolutionary consequences of sexual reproduction. Using molecular tools we addressed the origin, age and maternal ancestry of diploid and triploid asexual lineages arisen through the hybridization between spiny loaches Cobitis elongatoides, C. taenia and C. tanaitica. Reconstructions of the phylogenetic relationships among mitochondrial DNA (mtDNA) haplotypes, revealed by sequence analyses, suggest that both hybrid complexes (C. elongatoides-taenia and C. elongatoides-tanaitica) contained several asexual lineages of independent origin. Cobitis elongatoides was the exclusive maternal ancestor of all the C. elongatoides-tanaitica hybrids, whereas within the C. elongatoides-taenia complex, hybridization was reciprocal. In both complexes the low haplotype divergences were consistent with a recent origin of asexual lineages. Combined mtDNA and allozyme data suggest that the triploids arose through the incorporation of a haploid sperm genome into unreduced ova produced by diploid hybrids.

Phylogeography of the barbel (Barbus barbus) assessed by mitochondrial DNA variation.

Using the phylogeographic framework, we assessed the DNA sequence variation at the mitochondrial cytochrome b gene across the distribution range of the barbel Barbus barbus, a widely distributed European cyprinid. Reciprocal monophyly of non-Mediterranean European and Balkan/Anatolian populations is taken as evidence for a long-term barrier to gene flow, and interpreted as a consequence of survival of the species in two separate refugia during several later glacial cycles. Lack of profound genealogical divergence across Europe from western France to the northwestern Black Sea basin is consistent with recent colonization of this area from a single glacial refuge, which was probably located in the Danube River basin. This may have occurred in two steps: into the Western European river basins during the last interglacial, and throughout the Central European river basins after the last glacial. The populations from the Balkans and Anatolia apparently did not contribute mitochondrial DNA to the post-Pleistocene colonization of non-Mediterranean Europe. Lack of detectable variation within the Balkans/Anatolia is attributed mainly to recent expansion throughout these regions, facilitated by the freshwater conditions and seashore regression in the Black Sea during the Late Pleistocene and Early Holocene.