Diversity and evolutionary history of the MHC DQA gene in leporids

The European rabbit (Oryctolagus cuniculus) is used as a model for many human diseases, yet comparatively little is known of its genetics, particularly at important loci such as the major histocompatibility complex (MHC). This study investigated genetic diversity and evolutionary history of the DQA gene in a range of leporid species by analysing coding sequence diversity of exon 2 and intron 2 in 53 individuals of 16 different species. Fifty leporid DQA alleles were detected, including 13 novel European rabbit alleles. In the rabbit, the highest levels of diversity were observed in wild rabbits from Portugal, with wild rabbits from England and domestic rabbits showing less diversity. Within the sample, several recombination events were detected and trans-specific evolution of alleles was evidenced, both being general characteristics of mammalian MHC genes. Positive selection is implicated as operating on six codons within exon 2, which are also subject to positive selection in other mammals. Some of these positions are putative antigen recognition sites and underline the importance of pathogen-driven selection on these MHC genes.     

Evolution of MHC-<Emphasis Type="Italic">DRB</Emphasis> class II polymorphism in the genus <Emphasis Type="Italic">Apodemus</Emphasis> and a comparison of <Emphasis Type="Italic">DRB</Emphasis> sequences within the family Muridae (Mammalia: Rodentia)

Allelic diversity at major histocompatibility complex (MHC) genes is thought to be maintained by balancing selection over long periods of time, even across multiple speciation events. Trans-species sharing of MHC alleles among genera has been supported by many studies on mammals and fish, but in rodents, the results are ambiguous. We investigated natural levels of MHC-DRB variability and evolutionary processes in the wood mouse (Apodemus sylvaticus) and the yellow-necked mouse (Apodemus flavicollis), which are common, sympatric murid rodents in European forests. Using single-strand conformation polymorphism analysis and DNA sequencing, 38 DRB exon 2 alleles were detected among 162 A. sylvaticus from nine different locations in Germany and Switzerland, and 15 DRB exon 2 alleles were detected among 60 A. flavicollis from three different locations in northern Germany. There was evidence for balancing selection in both species. Phylogenetic analysis, including additional murid taxa, showed that the DRB exon 2 sequences did not separate according to species, consistent with trans-species evolution of the MHC in these taxa.     

Local Patterns of Nucleotide Polymorphism Are Highly Variable in the Selfing Species Arabidopsis thaliana

Neighboring genes predictably share similar evolutionary histories to an extent delineated by recombination. This correlation should extend across multiple linked genes in a selfing species such as Arabidopsis thaliana due to its low effective recombination rate. To test this prediction, we performed a molecular population genetics analysis of nucleotide polymorphism and divergence in chromosomal regions surrounding four low-diversity loci. Three of these loci, At1g67140, At3g03700, and TERMINAL FLOWER1 (TFL1), have been previously implicated as targets of selection and we would predict stronger correlations in polymorphism between neighboring loci due to genetic hitchhiking around these loci. The remaining locus, At1g04300, was identified in a study of linkage disequilibrium surrounding the CRYPTOCHROME2 (CRY2) locus. Although we found broad valleys of reduced nucleotide variation around two of our focal genes, At1g67140 and At3g03700, all chromosomal regions exhibited extreme variation in the patterns of polymorphism and evolution between neighboring loci. Although three of our four regions contained potential targets of selection, application of the composite-likelihood-ratio test of selection in conjunction with a goodness-of-fit test supports the selection hypothesis only for the region containing At3g03700. The degree of discordance in evolutionary histories between linked loci within each region generally correlated with estimates of recombination and linkage disequilibrium for that region, with the exception of the region containing At1g04300. We discuss the implications of these data for future population genetics analyses and genomics studies in A. thaliana. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Allelic diversity at the Mhc-DQA locus of woodmouse populations (Apodemus sylvaticus) present in the islands and mainland of the northern Mediterranean

Aim Polymorphism at neutral markers and at MHC loci in rodent populations living on islands is generally low. The main genetic factors that may contribute to a reduced level of genetic variability are genetic drift, reduced gene flow and founder events. We investigated the pattern of polymorphism at the second exon of the Mhc‐DQA gene in island populations of Apodemus sylvaticus and in their mainland counterparts to investigate the pattern of MHC polymorphism in a phylogeographical context and to assess the impact of insularity on diversity at this locus. Location Eight north Mediterranean populations of Apodemus sylvaticus were studied, including five island populations (Majorca, Minorca, Porquerolles, Port‐Cros and Sicily) and three mainland populations. Methods cDNA sequencing and nucleotide sequences analyses. Synonymous and non‐synonymous substitutions were examined at the PBR and non‐PBR sites. The DQA allelic distribution in populations was compared with the woodmouse phylogeography. Results This study presents novel DQA alleles. High polymorphism of the DQA locus is recorded in natural populations of A. sylvaticus with 13 alleles being widely distributed irrespective of the geographical origin and palaeoclimatic history of populations. The DQA locus does not show the expected pattern for non‐synonymous substitutions at the PBR sites. However, island populations show a weak loss of polymorphism in comparison with their mainland counterparts. Main conclusions The DQA locus in the woodmouse seems to be subject to weak selection and does not allow resolution of phylogeographical relationships among European woodmouse populations. The presence of at least three alleles in island populations and the maintenance of five alleles between the two European lineages over 1.5 Myr suggest that balancing selection may act within populations, and more precisely within island populations, to maintain genetic variability. This study shows that phylogeographical studies are a prerequisite for any genetic investigation of selected genes in natural populations.     

Extremely high MHC class I variation in a population of a long-distance migrant,the Scarlet Rosefinch (<Emphasis Type="Italic">Carpodacus erythrinus</Emphasis>)

Although the number of studies focusing on the major histocompatibility complex (MHC) in non-model vertebrates is increasing, results are often contradictory, and the structure of MHC is still poorly understood in wild species. Here, we describe the structure and diversity of exon 3 of MHC class I in a passerine bird, the Scarlet Rosefinch (Carpodacus erythrinus). Using capillary electrophoresis single-strand conformation polymorphism, we identified 82 different MHC class I variants in one Rosefinch population nesting at one site in the Czech Republic. Thus far, this is the highest intra-populational MHC class I variation observed in birds. We have not found support for ‘minimal essential’ MHC in this species since individuals exhibited between three and nine different exon 3 sequences, indicating that there may be at least five amplified MHC class I genes. By cloning, we obtained and analysed 29 exon sequences and found that all of them could be translated into potentially functional proteins. We also show that strong positive selection appears to be acting mainly, but not only, on previously described antigen-binding sites in MHC class I genes. Furthermore, our results indicate that recombination has played an important role in generating genetic diversity of these genes in the Scarlet Rosefinch; we discuss the significance of this extremely high genetic diversity in light of the life history traits of this species, such as long-distance migration. The sequence data described in this paper are accessible in GenBank data library under accession numbers FJ392762–FJ392790.     

Trans-species polymorphism and evidence of selection on class II MHC loci in tuco-tucos (Rodentia: Ctenomyidae)

Balancing selection acting over the evolutionary history of a lineage can result in the retention of alleles among species for longer than expected under neutral evolution. The associated pattern of trans-species polymorphism, in which similar or even identical alleles are shared among species, is often used to infer that balancing selection has occurred. The genes of the major histocompatibility complex (MHC) are thought to be subject to balancing selection that maintains alleles associated with response to specific pathogens. To explore the role of balancing selection in shaping MHC diversity in ctenomyid rodents, we examined allelic variability at the class II DRB and DQA loci in 18 species in the genus Ctenomys. Previous studies of four of these species had revealed significant within-population evidence of positive selection on MHC loci. The current study expands upon these analyses to (1) evaluate among-species evidence of positive selection and (2) explore the potential for balancing selection on MHC genes. Interspecific nucleotide sequence variation revealed significant evidence of positive selection on the DRB and DQA loci. At the same time, comparisons of phylogenetic trees for these MHC loci with a putative species tree based on mitochondrial sequence data revealed multiple examples of trans-specific polymorphism, including sharing of identical DRB and DQA alleles among distantly related species of Ctenomys. These findings suggest that MHC genes in these animals have historically been subject to balancing selection and yield new insights into the complex suite of forces shaping MHC diversity in free-living vertebrates.     

Gene duplication,allelic diversity,selection processes and adaptive value of MHC class II <Emphasis Type="Italic">DRB</Emphasis> genes of the bank vole, <Emphasis Type="Italic">Clethrionomys glareolus</Emphasis>

The generation and maintenance of allelic polymorphism in genes of the major histocompatibility complex (MHC) is a central issue in evolutionary genetics. Recently, the focus has changed from ex situ to in situ populations to understand the mechanisms that determine adaptive MHC polymorphism under natural selection. Birth-and-death evolution and gene conversion events are considered to generate sequence diversity in MHC genes, which subsequently is maintained by balancing selection through parasites. The ongoing arms race between the host and parasites leads to an adaptive selection pressure upon the MHC, evident in high rates of non-synonymous vs synonymous substitution rates. We characterised the MHC class II DRB exon 2 of free living bank voles, Clethrionomys glareolus by single-strand conformation polymorphism and direct sequencing. Unlike other arvicolid species, the DRB locus of the bank vole is at least quadruplicated. No evidence for gene conversion events in the Clgl-DRB sequences was observed. We found not only high allelic polymorphism with 26 alleles in 36 individuals but also high rates of silent polymorphism. Exceptional for MHC class II genes is a purifying selection pressure upon the majority of MHC-DRB sequences. Further, we analysed the association between certain DRB alleles and the parasite burden with gastrointestinal trichostrongyle nematodes Heligmosomum mixtum and Heligmosomoides glareoli and found significant quality differences between specific alleles with respect to infection intensity. Our findings suggest a snapshot in an evolutionary process of ongoing birth-and-death evolution. One allele cluster has lost its function and is already silenced, another is loosing its adaptive value in terms of gastrointestinal nematode resistance, while a third group of alleles indicates all signs of classical functional MHC alleles.     

Polymorphic microsatellites identified by cross-species amplifications in the European Coot Fulica atra

We tested the cross-amplification of 26 microsatellites developed for passerines and an additional three developed for Gallinula species in eight European Coots from two populations. Sixteen microsatellite markers successfully amplified, of which nine were polymorphic with 2–6 alleles (mean 3.7 alleles) and an expected heterozygosity (H _e) ranging from 0.375 to 0.805 (mean H _e = 0.589). On average, we found 2.22 alleles/locus and a mean H _e of 0.440 in one nest, and 2.56 alleles/locus and a mean H _e of 0.494 in the other one. These nine polymorphic markers could be of potential use in studies of genetic variability, population structure and reproductive strategy of European Coots.     

The Flow of Antimicrobial Peptide Genes Through a Genetic Barrier Between <Emphasis Type="Italic">Mytilus edulis</Emphasis> and <Emphasis Type="Italic">M. galloprovincialis</Emphasis>

We studied the population genetics of two antimicrobial peptide (AMP) loci, called Mytilin B and Mytilus galloprovincialis defensin 2 (MGD2), in the secondary contact mosaic hybrid zone between Mytilus edulis and M. galloprovincialis. The isolation period between the two species was estimated to be ~1 million years (range, 0.5 million to 2 million years) long. During this period, coevolution between microbes and the immune system has likely occurred. The secondary contact, which would date back to ~25,000 (0–200,000) years, recently allowed these coadaptations to be rearranged through hybridization. Distinctive polymorphisms were uncovered in coding sequences of the two AMP loci such as insertion/deletion of codons or bisubstituted codons. Very low levels of differentiation were observed between populations of the two species at both loci, while other nuclear loci often showed marked structure among the same samples. The absence of population differentiation proved to be the consequence of secondary introgression of highly divergent alleles. While only a few recombinants were observed at the Mytilin B locus, the MGD2 locus showed a high intragenic recombination rate, which increased in the exon coding for the mature peptide. In addition, standard neutrality tests revealed significant deviations from the mutation-drift equilibrium at both loci. These results suggest that either balancing or directional selection is likely to play a role in the evolution of the two AMPs and introgression would be adaptive. However, evidence accumulated at the Mytilin B locus allows neither for identification of the direction of selection nor for any conclusions on whether selection acted directly on the antimicrobial peptide itself. At the MGD2 locus, a spatial variation of polymorphism patterns along the sequence suggests that selection was direct, although the precise nature of the selection (directional vs. balancing) remains unclear. This study concurs with previous reports of an effect of slight selection on AMP genes evolution in other invertebrates, although selection does not necessarily act on the mature peptides. E. Boon and M. F. Faure contributed equally to this work.     

Major histocompatibility complex variation and evolution at a single, expressed DQA locus in two genera of elephants

Genes of the vertebrate major histocompatibility complex (MHC) are crucial to defense against infectious disease, provide an important measure of functional genetic diversity, and have been implicated in mate choice and kin recognition. As a result, MHC loci have been characterized for a number of vertebrate species, especially mammals; however, elephants are a notable exception. Our study is the first to characterize patterns of genetic diversity and natural selection in the elephant MHC. We did so using DNA sequences from a single, expressed DQA locus in elephants. We characterized six alleles in 30 African elephants (Loxodonta africana) and four alleles in three Asian elephants (Elephas maximus). In addition, for two of the African alleles and three of the Asian alleles, we characterized complete coding sequences (exons 1–5) and nearly complete non-coding sequences (introns 2–4) for the class II DQA loci. Compared to DQA in other wild mammals, we found moderate polymorphism and allelic diversity and similar patterns of selection; patterns of non-synonymous and synonymous substitutions were consistent with balancing selection acting on the peptides involved in antigen binding in the second exon. In addition, balancing selection has led to strong trans-species allelism that has maintained multiple allelic lineages across both genera of extant elephants for at least 6 million years. We discuss our results in the context of MHC diversity in other mammals and patterns of evolution in elephants.     

MHC class II genes in European wolves: a comparison with dogs

The genome of the grey wolf, one of the most widely distributed land mammal species, has been subjected to both stochastic factors, including biogeographical subdivision and population fragmentation, and strong selection during the domestication of the dog. To explore the effects of drift and selection on the partitioning of MHC variation in the diversification of species, we present nine DQA, 10 DQB, and 17 DRB1 sequences of the second exon for European wolves and compare them with sequences of North American wolves and dogs. The relatively large number of class II alleles present in both European and North American wolves attests to their large historical population sizes, yet there are few alleles shared between these regions at DQB and DRB1. Similarly, the dog has an extensive array of class II MHC alleles, a consequence of a genetically diverse origin, but allelic overlap with wolves only at DQA. Although we might expect a progression from shared alleles to shared allelic lineages during differentiation, the partitioning of diversity between wolves and dogs at DQB and DRB1 differs from that at DQA. Furthermore, an extensive region of nucleotide sequence shared between DRB1 and DQB alleles and a shared motif suggests intergenic recombination may have contributed to MHC diversity in the Canidae.     

Isolation and characterization of microsatellite loci in the Amami rabbit (Pentalagus furnessi)

We report on the isolation and characterization of eight polymorphic and five monomorphic microsatellites in the Amami rabbit (Pentalagus furnessi). Microsatellite polymorphism was determined using 25 individuals. There were 2–11 alleles for each polymorphic locus with heterozygosity ranging between 0.08 and 0.76. Linkage disequilibrium was not suggested between any pairs among the eight polymorphic loci. We suggest that these primers be used in future studies to monitor population size, determine dispersal patterns, and genetic diversity within and between populations of this and related species.     

Postglacial-period and Recent Invasions Shape the Population Genetics of Botryllid Ascidians along European Atlantic Coasts

The colonial urochordate Botryllus schlosseri is a sedentary species of Mediterranean origin that became cosmopolitan, probably because of postglacial-period dispersal and human-mediated invasions of colonies attached to ship hulls. Here we studied microsatellite allele diversity of Atlantic coast populations from an area ranging from European regions south of the last glacial front to regions that had been permanently ice-covered. Gene diversity levels varied dramatically among populations residing in areas subject to different glacial conditions. Five populations from the Iberian Peninsula, in an area south of the last glacial front, as well as two populations from presumed refugia in Brittany, expressed high gene diversity values (expected heterozygosity [He]: 0.76–0.80; average number of alleles per locus [A]: 7.25–8.75). Two populations inhabiting areas that experienced permafrost conditions (Helgoland Island, Germany, and Plymouth, England) had intermediate values (He: 0.40–0.42; A: 3.0–4.0), whereas the Auchenmalg, Scotland, population, from an area previously covered by ice, showed a remarkably low value (He: 0.17; A: 1.75). Therefore, most European populations of B. schlosseri mirrored the movement of the ice front in the last ice age. A second population from the area that was covered by permanent ice (Lossiemouth, Scotland), however, had a high He of 0.61 and an intermediate A of 3.67. Results were compared with recent invasions (populations less than 200 years old) in the United States and New Zealand that had a higher degree of genetic variation than the European native populations established thousands of years ago. Given the overall dearth of studies on this subject, we suggest that in contemporary established Botryllus populations, gene diversity is affected by ecological factors, some of which can be traced directly to the last ice age. Other parameters of gene diversity are influenced by selection pressure, which might be more intense in northern regions.     

Genetic diversity within Anatolian brown hares (Lepus europaeus Pallas, 1778) and differentiation among Anatolian and European populations

Genetic variability of Anatolian hares and relationships between Anatolian and European populations were assessed by a multilocus allozyme approach to infer evolutionary relationships between hares from Asia Minor and Europe. Of the 48 loci assayed, 19 (39.6%) were polymorphic with two to four alleles in the Anatolian hares. Among all Anatolian alleles, 14 were so far not found in the compared 717 brown hares from Europe. Overall, genetic diversity was highest in Anatolian hares, intermediate in brown hares from the southern and southeastern Balkans and lowest in central European populations. The rich genetic diversity in Anatolian hares might be a consequence of Anatolias biogeographic position with the chance of multiple gene flow from neighbouring regions, and the likelihood of long-term presence of hares during the last ice age, when large parts of more northern latitudes did not provide suitable habitats.However, among 28 loci used for the comparison between European and Anatolian populations, most common alleles of European brown hares were also common in Anatolian populations and no alternately fixed alleles were found for Anatolian and European populations. This together with only little or moderately varying allele frequencies produced low genetic divergence between Anatolian and European populations. Genetic differentiation among Anatolian populations was also low. Even between the two forms with different coat colour (brownish and yellowish) in Anatolian hares, there was little genetic differentiation. Altogether, all Anatolian hares studied presently are closely related to European brown hare populations, and only some distantly spaced population pairs revealed increased genetic divergence.

Zusammenfassung

Genetische Diversität anatolischer Feldhasen (Lepus europaeus Pallas, 1778) und Differenzierung zwischen anatolischen und europäischen PopulationenZur Beurteilung der phylogenetischen Beziehungen zwischen anatolischen Hasen und europäischen Feldhasenpopulationen wurde die allelische Variabilität anatolischer Hasen mittels horizontaler Stärkegelelektrophorese erfaßt und gemeinsam mit unmittelbar vergleichbaren Daten griechischer, bulgarischer und österreichischer Populationen aus früheren Studien populationsgenetischen Analysen unterzogen. Neunzehn der 48 untersuchten Loci der anatolischen Hasen zeigten allelische Variabilität. Unter den anatolischen Allelen kamen 14 bisher in den europäischen Polulationen nicht vor. Insgesamt zeigten anatolische Hasen die höchste und österreichische Populationen die niedrigste genetische Diversität; die jeweiligen Werte der griechischen und bulgarischen Populationen lagen dazwischen. Dies entspricht unserer Hypothese hoher genetischer Diversität in Anatolien, auf Grund der biogeografischen Position und der klimatischen bzw. Lebensraumbedingungen während des Pleistozäns, die, im Gegensatz zu Mitteleuropa, kontinuierliche Hasenpopulation in Anatolien wahrscheinlich erscheinen lassen. Kontinuierliche Populationen und Genflüsse aus verschiedenen Nachbarregionen könnten bei langfristig relative ungestörten Populationen zur Anreicherung genetischer Varianten in Anatolien geführt haben, während mitteleuropäische Feldhasenpopulationen im Zuge ihrer postglazialen Einwanderung aus Refugial-gebieten an genetischer Vielfalt eingebüßt haben. Allerdings waren die häufigen Allele der anatolischen Hasen ebenfalls häufig bei den europäischen Feldhasen vertreten; somit ergab sich insgesamt nur eine geringe genetische Differenzierung zwischen anatolischen und europäischen Feldhasen. Die zwei in Anatolien gefundenen Fellfärbungstypen (brauner vs. gelber Grundton) zeigten ebenfalls keine besondere genetische Differenzierung.     

Polymorphism and signatures of selection in the multimammate rat DQB gene

In order to test if DQB is a good candidate marker to investigate the relationship between major histocompatibility complex genes and pathogens in natural populations of Mastomys natalensis, we assessed the polymorphism and evolutionary history of this gene. Twenty-four individuals were genotyped for exon 2 of DQB using capillary electrophoresis single-strand conformation polymorphism, cloning, and sequencing. We found 21 different alleles. Four individuals show three alleles implying a duplication event in the history of this gene. Each distinct sequence translates to give a distinct amino acid sequence and there are strong signals of positive selection on peptide binding sites. Signals of recombination were found in the sequences suggesting that recombination has played a role in generating allelic diversity. Although trans-taxon polymorphism is present at the interspecific level in DQB exon 2 sequences of Mus species, we did not find any evidence of allele sharing among Muridae genera. This indicates a temporal limit of DQB allele sharing in Muridae of less than 8 Mya. In conclusion, although DQB seems to be a good marker to investigate pathogen-driven selection, the polymorphism of gene copy number may restrict its utility in natural populations.     

Effect of natural selection on the duplicated lysyl oxidase gene in Atlantic salmon

We examined the polymorphism of the lysyl oxidase (LOX) locus, involved in the initiation of muscle collagen cross-linking, in three populations of Atlantic salmon with different life histories and growth rates and compared it with a closely related species (rainbow trout). Up to four alleles were observed per individual, probably as a consequence of the tetraploid origin of the salmonid genome. We found high polymorphism in the LOX locus (16 alleles expressed in total and several low frequency private alleles) in two natural Atlantic salmon populations and extremely reduced diversity in a farmed population (3 alleles) with low density of collagen crosslinks. We also assessed the relative role of selection in maintaining LOX genetic variability in Atlantic salmon. Results from several neutrality tests suggest that selection is playing a role in shaping diversity at the LOX locus. Positive selection was inferred by three different likelihood phylogeny-based methods and one selected site, identified by all three different methods (PAML, FEL and REL) was located within the “copper-talon” characteristic of LOX proteins. We suggest that the retention of four alleles in the salmon LOX locus could be related to its multiple functions.     

Mitochondrial DNA evolution in lagomorphs: Origin of systematic heteroplasmy and organization of diversity in European rabbits

Summary A characterization was conducted on mitochondrial DNA (mtDNA) molecules extracted separately from 107 European rabbits (Oryctolagus cuniculus) both wild and domestic, 13 European hares (Lepus capensis), and 1 eastern cottontail (Sylvilagus floridanus). Experimentally this study took into account restriction site polymorphism, overall length variation of the noncoding region, and numbers of repeated sequences. Nucleotide divergences indicate that the mtDNAs from the three species derived from a common ancestor some 6–8 million years (Myr) ago. Every animal appeared heteroplasmic for a set of molecules with various lengths of the noncoding region and variable numbers of repeated sequences that contribute to them. This systematic heteroplasmy, most probably generated by a rate of localized mtDNA rearrangements high enough to counterbalance the cellular segregation of rearranged molecules, is a shared derived character of leporids.The geographic distribution of mtDNA polymorphism among wild rabbit populations over the western European basin shows that two molecular lineages are represented, one in southern Spain, the second over northern Spain, France, and Tunisia. These two lineages derived from a common ancestor some 2 Myr ago. Their present geographical distribution may be correlated to the separation of rabbits into two stocks at the time of Mindel glaciation.Finally the distribution of mtDNA diversity exhibits a mosaic pattern both at inter- and intrapopulation levels.     

Major histocompatibility complex variation at class II DQA locus in the brown hare (Lepus europaeus)

The major histocompatability complex (MHC) is a multigene family of receptors that bind and present antigenic peptides to T-cells. Genes of the MHC are characterized by an outstanding genetic polymorphism, which is considered to be maintained by positive selection. Sites involved in peptide binding form binding pockets (P) that are collectively termed the peptide-binding region (PBR). In this study, we examined the level of MHC genetic diversity within and among natural populations of brown hare ( Lepus europaeus ) from Europe and Anatolia choosing for analysis of the second exon of the DQA locus, one of the most polymorphic class II loci. We aimed at an integrated population genetic analysis of L. europeaus by (i) correlating MHC polymorphism to genetic variability and phylogenetic status estimated previously from maternally (mtDNA) and biparentally (allozymes, microsatellites) inherited loci; and (ii) comparing full-length exon amino acid polymorphism with functional polymorphism in the PBR and the binding pockets P1, P6 and P9. A substantial level of DQA exon 2 polymorphism was detected with two completely different set of alleles between the Anatolian and European populations. However, the phylogeny of full-length exon 2 Leeu-DQA alleles did not show a strong phylogeographic signal. The presence of balancing selection was supported by a statistically significant excess of nonsynonymous substitutions over synonymous in the PBR and a trans-species pattern of evolution detected after phylogenetic reconstruction. The differentiating patterns detected between genetic and functional polymorphism, i.e. the number and the distribution of pocket variants within and among populations, indicated a hierarchical action of selection pressures.