Balancing selection and genetic drift create unusual patterns of MHCIIβ variation in Galápagos mockingbirds

The extracellular subunit of the major histocompatibility complex MHCIIβ plays an important role in the recognition of pathogens and the initiation of the adaptive immune response of vertebrates. It is widely accepted that pathogen‐mediated selection in combination with neutral micro‐evolutionary forces (e.g. genetic drift) shape the diversity of MHCIIβ, but it has proved difficult to determine the relative effects of these forces. We evaluated the effect of genetic drift and balancing selection on MHCIIβ diversity in 12 small populations of Galápagos mockingbirds belonging to four different species, and one larger population of the Northern mockingbird from the continental USA. After genotyping MHCIIβ loci by high‐throughput sequencing, we applied a correlational approach to explore the relationships between MHCIIβ diversity and population size by proxy of island size. As expected when drift predominates, we found a positive effect of population size on the number of MHCIIβ alleles present in a population. However, the number of MHCIIβ alleles per individual and number of supertypes were not correlated with population size. This discrepancy points to an interesting feature of MHCIIβ diversity dynamics: some levels of diversity might be shaped by genetic drift while others are independent and possibly maintained by balancing selection.     

Ultra‐deep Illumina sequencing accurately identifies MHC class IIb alleles and provides evidence for copy number variation in the guppy (Poecilia reticulata)

We address the bioinformatic issue of accurately separating amplified genes of the major histocompatibility complex (MHC) from artefacts generated during high‐throughput sequencing workflows. We fit observed ultra‐deep sequencing depths (hundreds to thousands of sequences per amplicon) of allelic variants to expectations from genetic models of copy number variation (CNV). We provide a simple, accurate and repeatable method for genotyping multigene families, evaluating our method via analyses of 209 b of MHC class IIb exon 2 in guppies (Poecilia reticulata). Genotype repeatability for resequenced individuals (N = 49) was high (100%) within the same sequencing run. However, repeatability dropped to 83.7% between independent runs, either because of lower mean amplicon sequencing depth in the initial run or random PCR effects. This highlights the importance of fully independent replicates. Significant improvements in genotyping accuracy were made by greatly reducing type I genotyping error (i.e. accepting an artefact as a true allele), which may occur when using low‐depth allele validation thresholds used by previous methods. Only a small amount (4.9%) of type II error (i.e. rejecting a genuine allele as an artefact) was detected through fully independent sequencing runs. We observed 1–6 alleles per individual, and evidence of sharing of alleles across loci. Variation in the total number of MHC class II loci among individuals, both among and within populations was also observed, and some genotypes appeared to be partially hemizygous; total allelic dosage added up to an odd number of allelic copies. Collectively, observations provide evidence of MHC CNV and its complex basis in natural populations.     

Extra‐pair mating in a passerine bird with highly duplicated major histocompatibility complex class II: Preference for the golden mean

Genes of the major histocompatibility complex (MHC) are essential in vertebrate adaptive immunity, and they are highly diverse and duplicated in many lineages. While it is widely established that pathogen‐mediated selection maintains MHC diversity through balancing selection, the role of mate choice in shaping MHC diversity is debated. Here, we investigate female mating preferences for MHC class II (MHCII) in the bluethroat (Luscinia svecica), a passerine bird with high levels of extra‐pair paternity and extremely duplicated MHCII. We genotyped family samples with mixed brood paternity and categorized their MHCII alleles according to their functional properties in peptide binding. Our results strongly indicate that females select extra‐pair males in a nonrandom, self‐matching manner that provides offspring with an allelic repertoire size closer to the population mean, as compared to offspring sired by the social male. This is consistent with a compatible genes model for extra‐pair mate choice where the optimal allelic diversity is intermediate, not maximal. This golden mean presumably reflects a trade‐off between maximizing pathogen recognition benefits and minimizing autoimmunity costs. Our study exemplifies how mate choice can reduce the population variance in individual MHC diversity and exert strong stabilizing selection on the trait. It also supports the hypothesis that extra‐pair mating is adaptive through altered genetic constitution in offspring.     

Testing genotyping strategies for ultra‐deep sequencing of a co‐amplifying gene family: MHC class I in a passerine bird

Characterization of highly duplicated genes, such as genes of the major histocompatibility complex (MHC), where multiple loci often co‐amplify, has until recently been hindered by insufficient read depths per amplicon. Here, we used ultra‐deep Illumina sequencing to resolve genotypes at exon 3 of MHC class I genes in the sedge warbler (Acrocephalus schoenobaenus). We sequenced 24 individuals in two replicates and used this data, as well as a simulated data set, to test the effect of amplicon coverage (range: 500–20 000 reads per amplicon) on the repeatability of genotyping using four different genotyping approaches. A third replicate employed unique barcoding to assess the extent of tag jumping, that is swapping of individual tag identifiers, which may confound genotyping. The reliability of MHC genotyping increased with coverage and approached or exceeded 90% within‐method repeatability of allele calling at coverages of >5000 reads per amplicon. We found generally high agreement between genotyping methods, especially at high coverages. High reliability of the tested genotyping approaches was further supported by our analysis of the simulated data set, although the genotyping approach relying primarily on replication of variants in independent amplicons proved sensitive to repeatable errors. According to the most repeatable genotyping method, the number of co‐amplifying variants per individual ranged from 19 to 42. Tag jumping was detectable, but at such low frequencies that it did not affect the reliability of genotyping. We thus demonstrate that gene families with many co‐amplifying genes can be reliably genotyped using HTS, provided that there is sufficient per amplicon coverage.     

Gene duplication and divergence produce divergent MHC genotypes without disassortative mating

Genes of the major histocompatibility complex (MHC) exhibit heterozygote advantage in immune defence, which in turn can select for MHC‐disassortative mate choice. However, many species lack this expected pattern of MHC‐disassortative mating. A possible explanation lies in evolutionary processes following gene duplication: if two duplicated MHC genes become functionally diverged from each other, offspring will inherit diverse multilocus genotypes even under random mating. We used locus‐specific primers for high‐throughput sequencing of two expressed MHC Class II B genes in Leach's storm‐petrels, Oceanodroma leucorhoa, and found that exon 2 alleles fall into two gene‐specific monophyletic clades. We tested for disassortative vs. random mating at these two functionally diverged Class II B genes, using multiple metrics and different subsets of exon 2 sequence data. With good statistical power, we consistently found random assortment of mates at MHC. Despite random mating, birds had MHC genotypes with functionally diverged alleles, averaging 13 amino acid differences in pairwise comparisons of exon 2 alleles within individuals. To test whether this high MHC diversity in individuals is driven by evolutionary divergence of the two duplicated genes, we built a phylogenetic permutation model. The model showed that genotypic diversity was strongly impacted by sequence divergence between the most common allele of each gene, with a smaller additional impact of monophyly of the two genes. Divergence of allele sequences between genes may have reduced the benefits of actively seeking MHC‐dissimilar mates, in which case the evolutionary history of duplicated genes is shaping the adaptive landscape of sexual selection.     

Nonparallelism in MHCIIβ diversity accompanies nonparallelism in pathogen infection of lake whitefish (Coregonus clupeaformis) species pairs as revealed by next‐generation sequencing

Major histocompatibility (MHC) immune system genes may evolve in response to pathogens in the environment. Because they also may affect mate choice, they are candidates for having great importance in ecological speciation. Here, we use next‐generation sequencing to test the general hypothesis of parallelism in patterns of MHCIIβ diversity and bacterial infections among five dwarf and normal whitefish sympatric pairs. A second objective was to assess the functional relationships between specific MHCIIβ alleles and pathogens in natural conditions. Each individual had between one and four alleles, indicating two paralogous loci. In Cliff Lake, the dwarf ecotype was monomorphic for the most common allele. In Webster Lake, the skew in the allelic distribution was towards the same allele but in the normal ecotype, underscoring the nonparallel divergence among lakes. Our signal of balancing selection matched putative peptide binding region residues in some cases, but not in others, supporting other recent findings of substantial functional differences in fish MHCIIβ compared with mammals. Individuals with fewer alleles were less likely to be infected; thus, we found no evidence for the heterozygote advantage hypothesis. MHCIIβ alleles and pathogenic bacteria formed distinct clusters in multivariate analyses, and clusters of certain alleles were associated with clusters of pathogens, or sometimes the absence of pathogens, indicating functional relationships at the individual level. Given that patterns of MHCIIβ and bacteria were nonparallel among dwarf and normal whitefish pairs, we conclude that pathogens driving MHCIIβ evolution did not play a direct role in their parallel phenotypic evolution.     

Family‐assisted inference of the genetic architecture of major histocompatibility complex variation

With their direct link to individual fitness, genes of the major histocompatibility complex (MHC) are a popular system to study the evolution of adaptive genetic diversity. However, owing to the highly dynamic evolution of the MHC region, the isolation, characterization and genotyping of MHC genes remain a major challenge. While high‐throughput sequencing technologies now provide unprecedented resolution of the high allelic diversity observed at the MHC, in many species, it remains unclear (i) how alleles are distributed among MHC loci, (ii) whether MHC loci are linked or segregate independently and (iii) how much copy number variation (CNV) can be observed for MHC genes in natural populations. Here, we show that the study of allele segregation patterns within families can provide significant insights in this context. We sequenced two MHC class I (MHC‐I) loci in 1267 European barn owls (Tyto alba), including 590 offspring from 130 families using Illumina MiSeq technology. Coupled with a high per‐individual sequencing coverage (~3000×), the study of allele segregation patterns within families provided information on three aspects of the architecture of MHC‐I variation in barn owls: (i) extensive sharing of alleles among loci, (ii) strong linkage of MHC‐I loci indicating tandem architecture and (iii) the presence of CNV in the barn owl MHC‐I. We conclude that the additional information that can be gained from high‐coverage amplicon sequencing by investigating allele segregation patterns in families not only helps improving the accuracy of MHC genotyping, but also contributes towards enhanced analyses in the context of MHC evolutionary ecology.     

Microsatellite and major histocompatibility complex variation in an endangered rattlesnake,the Eastern Massasauga (Sistrurus catenatus)

Genetic diversity is fundamental to maintaining the long‐term viability of populations, yet reduced genetic variation is often associated with small, isolated populations. To examine the relationship between demography and genetic variation, variation at hypervariable loci (e.g., microsatellite DNA loci) is often measured. However, these loci are selectively neutral (or near neutral) and may not accurately reflect genomewide variation. Variation at functional trait loci, such as the major histocompatibility complex (MHC), can provide a better assessment of adaptive genetic variation in fragmented populations. We compared patterns of microsatellite and MHC variation across three Eastern Massasauga (Sistrurus catenatus) populations representing a gradient of demographic histories to assess the relative roles of natural selection and genetic drift. Using 454 deep amplicon sequencing, we identified 24 putatively functional MHC IIB exon 2 alleles belonging to a minimum of six loci. Analysis of synonymous and nonsynonymous substitution rates provided evidence of historical positive selection at the nucleotide level, and Tajima's D provided support for balancing selection in each population. As predicted, estimates of microsatellite allelic richness, observed, heterozygosity, and expected heterozygosity varied among populations in a pattern qualitatively consistent with demographic history and abundance. While MHC allelic richness at the population and individual levels revealed similar trends, MHC nucleotide diversity was unexpectedly high in the smallest population. Overall, these results suggest that genetic variation in the Eastern Massasauga populations in Illinois has been shaped by multiple evolutionary mechanisms. Thus, conservation efforts should consider both neutral and functional genetic variation when managing captive and wild Eastern Massasauga populations.     

Evaluation of two approaches to genotyping major histocompatibility complex class I in a passerine-CE-SSCP and 454 pyrosequencing

Genes of the highly dynamic major histocompatibility complex (MHC) are directly linked to individual fitness and are of high interest in evolutionary ecology and conservation genetics. Gene duplication and positive selection usually lead to high levels of polymorphism in the MHC region, making genotyping of MHC a challenging task. Here, we compare the performance of two methods for MHC class I genotyping in a passerine with highly duplicated MHC class I genes: capillary electrophoresis-single-strand conformation polymorphism (CE-SSCP) analysis and 454 GS FLX Titanium pyrosequencing. According to our findings, the number of MHC variants (called alleles for simplicity) detected by CE-SSCP is significantly lower than detected by 454. To resolve discrepancies between the two methods, we cloned and Sanger sequenced a MHC class I amplicon for an individual with high number of alleles. We found a perfect congruence between cloning/Sanger sequencing results and 454. Thus, in case of multi-locus amplification, CE-SSCP considerably underestimates individual MHC diversity. However, numbers of alleles detected by both methods are significantly correlated, although the correlation is weak (r = 0.32). Thus, in systems with highly duplicated MHC, 454 provides more reliable information on individual diversity than CE-SSCP.     

Comparison of 454 pyrosequencing methods for characterizing the major histocompatibility complex of nonmodel species and the advantages of ultra deep coverage

Characterization and population genetic analysis of multilocus genes, such as those found in the major histocompatibility complex (MHC) is challenging in nonmodel vertebrates. The traditional method of extensive cloning and Sanger sequencing is costly and time‐intensive and indirect methods of assessment often underestimate total variation. Here, we explored the suitability of 454 pyrosequencing for characterizing multilocus genes for use in population genetic studies. We compared two sample tagging protocols and two bioinformatic procedures for 454 sequencing through characterization of a 185‐bp fragment of MHC DRB exon 2 in wolverines (Gulo gulo) and further compared the results with those from cloning and Sanger sequencing. We found 10 putative DRB alleles in the 88 individuals screened with between two and four alleles per individual, suggesting amplification of a duplicated DRB gene. In addition to the putative alleles, all individuals possessed an easily identifiable pseudogene. In our system, sequence variants with a frequency below 6% in an individual sample were usually artefacts. However, we found that sample preparation and data processing procedures can greatly affect variant frequencies in addition to the complexity of the multilocus system. Therefore, we recommend determining a per‐amplicon‐variant frequency threshold for each unique system. The extremely deep coverage obtained in our study (approximately 5000×) coupled with the semi‐quantitative nature of pyrosequencing enabled us to assign all putative alleles to the two DRB loci, which is generally not possible using traditional methods. Our method of obtaining locus‐specific MHC genotypes will enhance population genetic analyses and studies on disease susceptibility in nonmodel wildlife species.     

Sequence-based genotyping of the sheep MHC class II DRB1 locus

The immunopolymorphism database (IPD) provides a single nomenclature for alleles at the major histocompatibility complex (MHC) loci for a range of different species. The minimum requirements for inclusion of a sheep class II DRB1 sequence is a submission that includes all polymorphic sites within the second exon from at least two independent polymerase chain reactions (PCR). In order to meet these requirements, we have developed a DNA-based genotyping method for the rapid analysis of allelic diversity at the DRB1 locus in domestic sheep, Ovis aries. Using a series of primers located within introns flanking exon 2 and genomic DNA from a cohort of 214 sheep representing 15 different breeds and crossbreeds, the complete exon 2 sequences of 38 Ovar-DRB1 alleles were obtained. This sequence resource allowed the development of a generic set of locus-specific primers which amplify a fragment that includes all polymorphic sites within the second exon. Bidirectional sequence analysis of the PCR product provides a composite sequence where each polymorphic site is represented by the corresponding International Union of Biochemistry nucleotide code. A Basic Local Alignment Search Tool search of alleles held within the IPD or National Center for Biotechnology Information databases allows individual allele sequences to be identified. Low levels of homozygosity (7.48%) within the cohort and verification of previously genotyped samples confirmed the broad allelic specificity of this method. It improves on currently available methods and is broadly applicable to the analysis of MHC diversity in studies investigating linkages with resistance or susceptibility to disease.     

Major histocompatibility complex variation is similar in little brown bats before and after white‐nose syndrome outbreak

White‐nose syndrome (WNS), caused by the fungal pathogen Pseudogymnoascus destructans (Pd), has driven alarming declines in North American hibernating bats, such as little brown bat (Myotis lucifugus). During hibernation, infected little brown bats are able to initiate anti‐Pd immune responses, indicating pathogen‐mediated selection on the major histocompatibility complex (MHC) genes. However, such immune responses may not be protective as they interrupt torpor, elevate energy costs, and potentially lead to higher mortality rates. To assess whether WNS drives selection on MHC genes, we compared the MHC DRB gene in little brown bats pre‐ (Wisconsin) and post‐ (Michigan, New York, Vermont, and Pennsylvania) WNS (detection spanning 2014–2015). We genotyped 131 individuals and found 45 nucleotide alleles (27 amino acid alleles) indicating a maximum of 3 loci (1–5 alleles per individual). We observed high allelic admixture and a lack of genetic differentiation both among sampling sites and between pre‐ and post‐WNS populations, indicating no signal of selection on MHC genes. However, post‐WNS populations exhibited decreased allelic richness, reflecting effects from bottleneck and drift following rapid population declines. We propose that mechanisms other than adaptive immunity are more likely driving current persistence of little brown bats in affected regions.     

High MHC gene copy number maintains diversity despite homozygosity in a Critically Endangered single‐island endemic bird,but no evidence of MHC‐based mate choice

Small population sizes can, over time, put species at risk due to the loss of genetic variation and the deleterious effects of inbreeding. Losing diversity in the major histocompatibility complex (MHC) could be particularly harmful, given its key role in the immune system. Here, we assess MHC class I (MHC‐I) diversity and its effects on mate choice and survival in the Critically Endangered Raso lark Alauda razae, a species restricted to the 7 km² islet of Raso, Cape Verde, since ~1460, whose population size has dropped as low as 20 pairs. Exhaustively genotyping 122 individuals, we find no effect of MHC‐I genotype/diversity on mate choice or survival. However, we demonstrate that MHC‐I diversity has been maintained through extreme bottlenecks by retention of a high number of gene copies (at least 14), aided by cosegregation of multiple haplotypes comprising 2–8 linked MHC‐I loci. Within‐locus homozygosity is high, contributing to low population‐wide diversity. Conversely, each individual had comparably many alleles, 6–16 (average 11), and the large and divergent haplotypes occur at high frequency in the population, resulting in high within‐individual MHC‐I diversity. This functional immune gene diversity will be of critical importance for this highly threatened species’ adaptive potential.     

Evolution of MHC class II SLA‐DRB1 locus in the Croatian wild boar (Sus scrofa) implies duplication and weak signals of positive selection

The wild boar is an ancestor of the domestic pig and an important game species with the widest geographical range of all ungulates. Although a large amount of data are available on major histocompatibility complex (MHC) variability in domestic pigs, only a few studies have been performed on wild boars. Due to their crucial role in appropriate immune responses and extreme polymorphism, MHC genes represent some of the best candidates for studying the processes of adaptive evolution. Here, we present the results on the variability and evolution of the entire MHC class II SLA‐DRB1 locus exon 2 in 133 wild boars from Croatia. Using direct sequencing and cloning methods, we identified 20 SLA‐DRB1 alleles, including eight new variants, with notable divergence. In some individuals, we documented functional locus duplication, and SLA‐DRB1*04:10 was identified as the allele involved in the duplication. The expression of a duplicated locus was confirmed by cloning and sequencing cDNA‐derived amplicons. Based on individual genotypes, we were able to assume that alleles SLA‐DRB1*04:10 and SLA‐DRB1*06:07 are linked as an allelic combination that co‐evolves as a two‐locus haplotype. Our investigation of evolutionary processes at the SLA‐DRB1 locus confirmed the role of intralocus recombination in generating allelic variability, whereas tests of positive selection based on the dN/dS (non‐synonymous/synonymous substitution rate ratio) test revealed atypically weak and ambiguous signals.     

Stepwise Threshold Clustering: A New Method for Genotyping MHC Loci Using Next-Generation Sequencing Technology

Genes of the vertebrate major histocompatibility complex (MHC) are of great interest to biologists because of their important role in immunity and disease, and their extremely high levels of genetic diversity. Next generation sequencing (NGS) technologies are quickly becoming the method of choice for high-throughput genotyping of multi-locus templates like MHC in non-model organisms.Previous approaches to genotyping MHC genes using NGS technologies suffer from two problems:1) a “gray zone” where low frequency alleles and high frequency artifacts can be difficult to disentangle and 2) a similar sequence problem, where very similar alleles can be difficult to distinguish as two distinct alleles. Here were present a new method for genotyping MHC loci – Stepwise Threshold Clustering (STC) – that addresses these problems by taking full advantage of the increase in sequence data provided by NGS technologies. Unlike previous approaches for genotyping MHC with NGS data that attempt to classify individual sequences as alleles or artifacts, STC uses a quasi-Dirichlet clustering algorithm to cluster similar sequences at increasing levels of sequence similarity. By applying frequency and similarity based criteria to clusters rather than individual sequences, STC is able to successfully identify clusters of sequences that correspond to individual or similar alleles present in the genomes of individual samples. Furthermore, STC does not require duplicate runs of all samples, increasing the number of samples that can be genotyped in a given project. We show how the STC method works using a single sample library. We then apply STC to 295 threespine stickleback (Gasterosteus aculeatus) samples from four populations and show that neighboring populations differ significantly in MHC allele pools. We show that STC is a reliable, accurate, efficient, and flexible method for genotyping MHC that will be of use to biologists interested in a variety of downstream applications.     

Shared evolutionary origin of major histocompatibility complex polymorphism in sympatric lemurs

Genes of the major histocompatibility complex (MHC) play a central role in adaptive immune responses of vertebrates. They exhibit remarkable polymorphism, often crossing species boundaries with similar alleles or allelic motifs shared across species. This pattern may reflect parallel parasite‐mediated selective pressures, either favouring the long maintenance of ancestral MHC allelic lineages across successive speciation events by balancing selection (“trans‐species polymorphism”), or alternatively favouring the independent emergence of functionally similar alleles post‐speciation via convergent evolution. Here, we investigate the origins of MHC similarity across several species of dwarf and mouse lemurs (Cheirogaleidae). We examined MHC class II variation in two highly polymorphic loci (DRB, DQB) and evaluated the overlap of gut–parasite communities in four sympatric lemurs. We tested for parasite‐MHC associations across species to determine whether similar parasite pressures may select for similar MHC alleles in different species. Next, we integrated our MHC data with those previously obtained from other Cheirogaleidae to investigate the relative contribution of convergent evolution and co‐ancestry to shared MHC polymorphism by contrasting patterns of codon usage at functional vs. neutral sites. Our results indicate that parasites shared across species may select for functionally similar MHC alleles, implying that the dynamics of MHC‐parasite co‐evolution should be envisaged at the community level. We further show that balancing selection maintaining trans‐species polymorphism, rather than convergent evolution, is the primary mechanism explaining shared MHC sequence motifs between species that diverged up to 30 million years ago.     

Estimation of genotyping error rate from repeat genotyping,unintentional recaptures and known parent–offspring comparisons in 16 microsatellite loci for brown rockfish (Sebastes auriculatus)

Genotyping errors are present in almost all genetic data and can affect biological conclusions of a study, particularly for studies based on individual identification and parentage. Many statistical approaches can incorporate genotyping errors, but usually need accurate estimates of error rates. Here, we used a new microsatellite data set developed for brown rockfish (Sebastes auriculatus) to estimate genotyping error using three approaches: (i) repeat genotyping 5% of samples, (ii) comparing unintentionally recaptured individuals and (iii) Mendelian inheritance error checking for known parent–offspring pairs. In each data set, we quantified genotyping error rate per allele due to allele drop‐out and false alleles. Genotyping error rate per locus revealed an average overall genotyping error rate by direct count of 0.3%, 1.5% and 1.7% (0.002, 0.007 and 0.008 per allele error rate) from replicate genotypes, known parent–offspring pairs and unintentionally recaptured individuals, respectively. By direct‐count error estimates, the recapture and known parent–offspring data sets revealed an error rate four times greater than estimated using repeat genotypes. There was no evidence of correlation between error rates and locus variability for all three data sets, and errors appeared to occur randomly over loci in the repeat genotypes, but not in recaptures and parent–offspring comparisons. Furthermore, there was no correlation in locus‐specific error rates between any two of the three data sets. Our data suggest that repeat genotyping may underestimate true error rates and may not estimate locus‐specific error rates accurately. We therefore suggest using methods for error estimation that correspond to the overall aim of the study (e.g. known parent–offspring comparisons in parentage studies).     

MHC class I variation in a natural blue tit population (Cyanistes caeruleus)

The major histocompatibility complex (MHC) is central to the vertebrate immune system and its highly polymorphic genes are considered to influence several life-history traits of individuals. To characterize the MHC in a natural population of blue tits (Cyanistes caeruleus) we investigated the class I exon 3 diversity of more than 900 individuals. We designed two pairs of motif-specific primers that reliably amplify independent subsets of MHC alleles. Applying denaturing gradient gel electrophoresis (DGGE) we obtained 48 independently inherited units of unique band patterns (DGGE-haplogroups), which were validated in a segregation analysis within 105 families. In a second approach, we extensively sequenced 6 unrelated individuals to confirm that DGGE-haplogroup composition reflects individual allelic variation. The highest number of different DGGE-haplogroups in a single individual corresponded in 19 MHC exon 3 sequences, suggesting a minimum of 10 amplified MHC class I loci in the blue tit. In total, we identified 50 unique functional and 3 non-functional sequences. Functional sequences showed high levels of recombination and strong positive selection in the antigen binding region, whereas nucleotide diversity was comparatively low in the range of all passerine species. Finally, in a phylogenetic comparison of passerine MHC class I exon 3 sequences we discuss conflicting evolutionary signals possibly due to recent gene duplication, recombination events and concerted evolution. Our results indicate that the described method is suitable to effectively explore the MHC diversity and its ecological impacts in blue tits in future studies.