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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.

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.

Major histocompatibility complex (MHC) class I molecules display peptides on cell surfaces for subsequent T-cell recognition and are involved in the immune response against intracellular pathogens. In this study, a BAC library was created from a single three-spined stickleback and screened for clones containing MHC class I genes. In a 163.2-kb genomic sequence segment of a single clone, we identified three MHC class I genes in the same transcriptional orientation. Two class I genes are potentially expressed and functional. In one class I gene, the transmembrane region is missing and could therefore present a pseudogene. Alternatively, it presents a functional gene that encodes a soluble MHC class Ib molecule. Despite genomic similarities to the MHC class II region, which is characterized by interlocus recombination, we did not find any evidence for this kind of recombination in the class I genes. It thus seems that interlocus recombination may play a rather minor role in generating class I diversity in stickleback and that the class I region displays a higher genomic stability (i.e., lower local recombination rate). In addition, two non-MHC genes (Oct-2 beta and Na(+),K(+)-ATPasealpha3) have been identified in the analyzed class I region. The Oct-2 beta gene is a transcription factor that is expressed primarily in B lymphocytes, in activated T-cells, and in neuronal cells. The Na(+),K(+)-ATPasealpha3 gene is primarily expressed in the brain and heart and mediates catalytic activities. Both genes are located on the same linkage group together with the MHC class I genes in the zebra fish. In humans, however, homologues of Oct-2 beta and ATPasealpha3 lie outside the MHC region, which indicates that the concentration of immune genes found in mammalian genomes is a derived state.

We examined the evolutionary processes contributing to genetic diversity at the major histocompatibility complex (MHC) class II DRB locus in chamois (Rupicapra spp., subfamily Caprinae). We characterised the pattern of intragenic recombination (or homologous gene conversion) and quantified the amount of recombination in the genealogical history of the two chamois species, Pyrenean chamois (Rupicapra pyrenaica) and Alpine chamois (Rupicapra rupicapra). We found evidence for intragenic recombination, and the estimated amount of population recombination suggests that recombination has been a significant process in generating DRB allelic diversity in the genealogical history of the genus Rupicapra. Moreover, positive selection appears to act on the same peptide-binding residues in both analysed chamois species, but not in identical intensity. Recombination coupled with positive selection drives the rapid evolution at the peptide-binding sites in the MHC class II DRB gene. Many chamois MHC class II DRB alleles are thus much younger than previously assumed.

Using a bacterial artificial chromosome (BAC) library, we analysed a 99.5 kb genomic segment containing the major histocompatibility class II genes of a teleost, the three-spined stickleback Gasterosteus aculeatus. Experiments with G. aculeatus have provided direct evidence for balancing selection by pathogens and mate choice driving MH class II beta polymorphism. Two sets of paralogous class II alpha genes and beta genes in a tandem arrangement were identified, designated Gaac-DAA/DAB and Gaac-DBA/DBB. Expression analysis of the beta genes using single-strand conformation polymorphism revealed that both gene copies are expressed. Based on an analysis of pairwise nucleotide polymorphisms, we estimate that the duplication into two paralogous class II loci occurred only 1.2-2.4 million years ago, 1-2 orders of magnitude more recently than in other fish, bird or mammalian species. At the 3'-direction of the classical MH loci, we identified another seven genes or gene fragments, two of which (small inducible cytokine, complement regulatory factor) are related to immune function in other vertebrates. None of these genes were associated with MH class II genes in zebrafish, suggesting a markedly different organisation of the MH class II region in sticklebacks, and presumably, across bony fishes. When the nucleotide substitution pattern of the novel class II beta genes was analysed together with a representative sequence sample isolated from fish in northern Germany (n=27), we found that the peptide binding region of the Gaac-DAB and Gaac-DBB loci had undergone an inter-locus gene conversion (P=0.007). In accordance, we found a 10- to 20-fold higher frequency of CpG-islands on the MH class II segment compared to other species, a feature that may be conducive for inter-locus recombination.