Population fragmentation and major histocompatibility complex variation in the spotted suslik,Spermophilus suslicus

The fragmentation of populations typically enhances depletion of genetic variation, but highly polymorphic major histocompatibility complex (MHC) genes are thought to be under balancing selection and therefore retain polymorphism despite population bottlenecks. In this study, we investigate MHC DRB (class II) exon 2 variation in 14 spotted suslik populations from two regions differing in their degree of habitat fragmentation and gene flow. We found 16 alleles that segregated in a sample of 248 individuals. The alleles were highly divergent and revealed the hallmark signs of positive selection acting on them in the past, showing a significant excess of nonsynonymous substitutions. This excess was concentrated in putative antigen‐binding sites, which suggests that past selection was driven by pathogens. MHC diversity was significantly lower in fragmented western populations than in the eastern populations, characterized by significant gene flow. In contrast to neutral variation, amova did not reveal genetic differentiation between the two regions. This may indicate similar selective pressures shaping MHC variation in both regions until the recent past. However, MHC allelic richness within a population was correlated with that for microsatellites. F_ST outlier analyses have shown that population differentiation at DRB was neither higher nor lower than expected under neutrality. The results suggest that selection on MHC is not strong enough to counteract drift that results from recent fragmentation of spotted suslik populations.     

Sequence diversity of the MHC DRB gene in the Eurasian beaver (Castor fiber)

Major histocompatibility complex (MHC) genes, coding molecules which play an important role in immune response, are the most polymorphic genes known in vertebrates. However, MHC polymorphism in some species is limited. MHC monomorphism at several MHC class I and II loci was previously reported for two neighbouring northern European populations of the Eurasian beaver (Castor fiber) and reduced selection for polymorphism has been hypothesized. Here, we analysed a partial sequence of the second exon of the MHC II DRB locus from seven relict European and Asian beaver populations. We detected 10 unique alleles among 76 beavers analysed. Only a western Siberian population was polymorphic, with four alleles detected in 10 individuals. Each of the remaining populations was fixed for a different allele. Sequences showed considerable divergence, suggesting the long persistence of allelic lineages. A significant excess of nonsynonymous substitutions was detected at the antigen binding sites, indicating that sequence evolution of beaver DRB was driven by positive selection. Current MHC monomorphism in the majority of populations may be the result of the superimposition of the recent bottleneck on pre-existing genetic structure resulting from population subdivision and differential pathogen pressure.     

Major histocompatibility complex class II genetic variation in forest musk deer (Moschus berezovskii) in China

The major histocompatibility complex (MHC) plays an important role in the immune system of vertebrates. We used the second exon of four MHC class II genes (DRA, DQA1, DQA2 and DRB3) to assess the overall MHC variation in forest musk deer (Moschus berezovskii). We also compared the MHC variation in captive and wild populations. We observed 22 alleles at four loci (four at DRA, four at DQA1, four at DQA2 and 10 at DRB3), 15 of which were newly identified alleles. Results suggest that forest musk deer maintain relatively high MHC variation, which may result from balancing selection. Moreover, considerable diversity was observed at the DRA locus. We found a high frequency of Mobe‐DRA*02, Mobe‐DQA1*01 and Mobe‐DQA2*05 alleles, which may be important for pathogen resistance. A Ewens–Watterson test showed that the DRB3 locus in the wild population had experienced recent balancing selection. We detected a small divergence at the DRA locus, suggesting the effect of weak positive selection on the DRA gene. Alternatively, this locus may be young and not yet adapted a wide spectrum of alleles for pathogen resistance. The significant heterozygosity deficit observed at the DQA1 and DRB3 loci in the captive population and at all four loci in the wild population may be the result of a population bottleneck. Additionally, MHC genetic diversity was higher in the wild population than in the captive, suggesting that the wild population may have the ability to respond to a wider range of pathogens.     

Impact of historical founder effects and a recent bottleneck on MHC variability in Commander Arctic foxes (Vulpes lagopus)

Populations of Arctic foxes (Vulpes lagopus) have been isolated on two of the Commander Islands (Bering and Mednyi) from the circumpolar distributed mainland population since the Pleistocene. In 1970-1980, an epizootic outbreak of mange caused a severe population decline on Mednyi Island. Genes of the major histocompatibility complex (MHC) play a primary role in infectious disease resistance. The main objectives of our study were to compare contemporary variation of MHC class II in mainland and island Arctic foxes, and to document the effects of the isolation and the recent bottleneck on MHC polymorphism by analyzing samples from historical and contemporary Arctic foxes. In 184 individuals, we found 25 unique MHC class II DRB and DQB alleles, and identified evidence of balancing selection maintaining allelic lineages over time at both loci. Twenty different MHC alleles were observed in mainland foxes and eight in Bering Island foxes. The historical Mednyi population contained five alleles and all contemporary individuals were monomorphic at both DRB and DQB. Our data indicate that despite positive and diversifying selection leading to elevated rates of amino acid replacement in functionally important antigen-binding sites, below a certain population size, balancing selection may not be strong enough to maintain genetic diversity in functionally important genes. This may have important fitness consequences and might explain the high pathogen susceptibility in some island populations. This is the first study that compares MHC diversity before and after a bottleneck in a wild canid population using DNA from museum samples.     

Density-related changes in selection pattern for major histocompatibility complex genes in fluctuating populations of voles

Host-pathogen interactions are of particular interest in studies of the interplay between population dynamics and natural selection. The major histocompatibility complex (MHC) genes of demographically fluctuating species are highly suitable markers for such studies, because they are involved in initiating the immune response against pathogens and display a high level of adaptive genetic variation. We investigated whether two MHC class II genes (DQA1, DRB) were subjected to contemporary selection during increases in the density of fossorial water vole (Arvicola terrestris) populations, by comparing the neutral genetic structure of seven populations with that estimated from MHC genes. Tests for heterozygosity excess indicated that DQA1 was subject to intense balancing selection. No such selection operated on neutral markers. This pattern of selection became more marked with increasing abundance. In the low-abundance phase, when populations were geographically isolated, both overall differentiation and isolation-by-distance were more marked for MHC genes than for neutral markers. Model-based simulations identified DQA1 as an outlier (i.e. under selection) in a single population, suggesting the action of local selection in fragmented populations. The differences between MHC and neutral markers gradually disappeared with increasing effective migration between sites. In the high-abundance year, DQA1 displayed significantly lower levels of overall differentiation than the neutral markers. This gene therefore displayed stronger homogenization than observed under drift and migration alone. The observed signs of selection were much weaker for DRB. Spatial and temporal fluctuations in parasite pressure and locus-specific selection are probably the most plausible mechanisms underlying the observed changes in selection pattern during the demographic cycle.     

A temporal analysis shows major histocompatibility complex loci in the Scandinavian wolf population are consistent with neutral evolution

The major histocompatibility complex (MHC) has an integral role in the immune system, and hence diversity at its genes may be of particular importance for the health of populations. In large populations, balancing selection maintains diversity in MHC genes, but theoretical expectations indicate that this form of selection is absent or inefficient in small populations. We examine the level of diversity at three MHC class II loci in the wolf population of Scandinavia, a population naturally recolonized with a genetic contribution from as few as three founders, and in four neighbouring wolf populations. In the Scandinavian wolf population, two alleles were found for each locus and the distribution of alleles is compatible with their linkage into two haplotypes. Changes in the level of heterozygosity over time since recolonization demonstrate the effects of the proposed arrival of an immigrant wolf. The maintenance of diversity is shown to be compatible with a neutral, random allocation of alleles, in conjunction with crossing between packs. A total of 15 DRB1, seven DQA and 10 DQB1 alleles are found in four neighbouring wolf populations, with substantial sharing across populations. Even in these larger populations, bottlenecks and fragmentation with consequent genetic drift are likely to have resulted in few indicators for balancing selection and significant differentiation of populations.     

MHC-DRB exon 2 allele polymorphism in Indian river buffalo (Bubalus bubalis)

The polymorphism of the major histocompatibility complex (MHC) class II DRB gene of riverine buffalo (Bubalus bubalis) was studied. Second exon sequences from the buffalo DRB locus, homologous to the cattle DRB3 gene, were amplified and characterized. A combination of single strand conformation polymorphism (SSCP) and heteroduplex analysis (HA) in a non-denaturing gel was used to identify new DRB second exon sequences. SSCP, HA and finally sequencing allowed the identification of 22 MHC-DRB exon 2 alleles from 25 unrelated Indian river buffalo. These are the first river buffalo DRB second exon sequences reported. A high degree of polymorphism in the sequences encoding the peptide binding regions was observed and some amino acid substitutions were found unique to the river buffalo.     

Duplication and population dynamics shape historic patterns of selection and genetic variation at the major histocompatibility complex in rodents

Genetic variation at the major histocompatibility complex (MHC) is vitally important for wildlife populations to respond to pathogen threats. As natural populations can fluctuate greatly in size, a key issue concerns how population cycles and bottlenecks that could reduce genetic diversity will influence MHC genes. Using 454 sequencing, we characterized genetic diversity at the DRB Class II locus in montane voles (Microtus montanus), a North American rodent that regularly undergoes high‐amplitude fluctuations in population size. We tested for evidence of historic balancing selection, recombination, and gene duplication to identify mechanisms maintaining allelic diversity. Counter to our expectations, we found strong evidence of purifying selection acting on the DRB locus in montane voles. We speculate that the interplay between population fluctuations and gene duplication might be responsible for the weak evidence of historic balancing selection and strong evidence of purifying selection detected. To further explore this idea, we conducted a phylogenetically controlled comparative analysis across 16 rodent species with varying demographic histories and MHC duplication events (based on the maximum number of alleles detected per individual). On the basis of phylogenetic generalized linear model‐averaging, we found evidence that the estimated number of duplicated loci was positively related to allelic diversity and, surprisingly, to the strength of purifying selection at the DRB locus. Our analyses also revealed that species that had undergone population bottlenecks had lower allelic richness than stable species. This study highlights the need to consider demographic history and genetic structure alongside patterns of natural selection to understand resulting patterns of genetic variation at the MHC.     

Selection and recombination drive the evolution of MHC class II DRB diversity in ungulates

Major histocompatibility complex (MHC) antigen-presenting genes are the most variable loci in vertebrate genomes. Host-parasite co-evolution is assumed to maintain the excessive polymorphism in the MHC loci. However, the molecular mechanisms underlying the striking diversity in the MHC remain contentious. The extent to which recombination contributes to the diversity at MHC loci in natural populations is still controversial, and there have been only few comparative studies that make quantitative estimates of recombination rates. In this study, we performed a comparative analysis for 15 different ungulates species to estimate the population recombination rate, and to quantify levels of selection. As expected for all species, we observed signatures of strong positive selection, and identified individual residues experiencing selection that were congruent with those constituting the peptide-binding region of the human DRB gene. However, in addition for each species, we also observed recombination rates that were significantly different from zero on the basis of likelihood-permutation tests, and in other non-quantitative analyses. Patterns of synonymous and non-synonymous sequence diversity were consistent with differing demographic histories between species, but recent simulation studies by other authors suggest inference of selection and recombination is likely to be robust to such deviations from standard models. If high rates of recombination are common in MHC genes of other taxa, re-evaluation of many inference-based phylogenetic analyses of MHC loci, such as estimates of the divergence time of alleles and trans-specific polymorphism, may be required.     

MHC‐disassortative mate choice and inbreeding avoidance in a solitary primate

Sexual selection theory suggests that choice for partners carrying dissimilar genes at the major histocompatibility complex (MHC) may play a role in maintaining genetic variation in animal populations by limiting inbreeding or improving the immunity of future offspring. However, it is often difficult to establish whether the observed MHC dissimilarity among mates drives mate choice or represents a by‐product of inbreeding avoidance based on MHC‐independent cues. Here, we used 454‐sequencing and a 10‐year study of wild grey mouse lemurs (Microcebus murinus), small, solitary primates from western Madagascar, to compare the relative importance on the mate choice of two MHC class II genes, DRB and DQB, that are equally variable but display contrasting patterns of selection at the molecular level, with DRB under stronger diversifying selection. We further assessed the effect of the genetic relatedness and of the spatial distance among candidate mates on the detection of MHC‐dependent mate choice. Our results reveal inbreeding avoidance, along with disassortative mate choice at DRB, but not at DQB. DRB‐disassortative mate choice remains detectable after excluding all related dyads (characterized by a relatedness coefficient r > 0), but varies slightly with the spatial distance among candidate mates. These findings suggest that the observed deviations from random mate choice at MHC are driven by functionally important MHC genes (like DRB) rather than passively resulting from inbreeding avoidance and further emphasize the need for taking into account the spatial and genetic structure of the population in correlative tests of MHC‐dependent mate choice.     

Diversification of porcine MHC class II genes: evidence for selective advantage

The major histocompatibility complex (MHC) is an immunological gene-dense region of high diversity in mammalian species. Sus scrofa was domesticated by at least six independent events over Eurasia during the Holocene period. It has been hypothesized that the level and distribution of MHC variation in pig populations reflect genetic selection and environmental influences. In an effort to define the complexity of MHC polymorphisms and the role of selection in the generation of class II gene diversity (DQB, DRB1, and pseudogene ΨDRB3), DNA from globally distributed unrelated domestic pigs of European and Asian origins and a Suidae out-group was analyzed. The number of pseudogene alleles identified (ΨDRB3 33) was greater than those found in the expressed genes (DQB 20 and DRB1 23) but the level of observed heterozygosity (ΨDRB3 0.452, DQB 0.732, and DRB1 0.767) and sequence diversity (ΨDRB3 0.029, DQB 0.062, and DRB1 0.074) were significantly lower in the pseudogene, respectively. The substitution ratios reflected an excess of d _N (DQB 1.476, DRB1 1.724, and ΨDRB3 0.508) and the persistence of expressed gene alleles suggesting the influence of balancing selection, while the pseudogene was undergoing purifying selection. The lack of a clear MHC phylogeographic tree, coupled with close genetic distances observed between the European and Asian populations (DQB 0.047 and DRB1 0.063) suggested that unlike observations using mtDNA, the MHC diversity lacks phylogeographic structure and appears to be globally uniform. Taken together, these results suggest that, despite regional differences in selective breeding and environments, no skewing of MHC diversity has occurred. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Substantial functional diversity accompanies limited major histocompatibility complex class II variability in golden jackal (Canis aureus): A comparison between two wild Canis species in Croatia

The golden jackal (Canis aureus) is one of the less studied carnivores and research on its major histocompatibility complex (MHC) variability is just at its early stages. MHC genes encode cell-surface receptors that serve to bind and present antigens to T cells, which is essential to initiating specific immunological responses in vertebrates. In this paper we present for the first time patterns of genetic diversity and natural selection on MHC class II DLA-DRB1, DQA1 and DQB1 loci in the golden jackal using samples from two geographically distinct regions in Croatia and further compare them to the values found in its congener grey wolf (Canis lupus). Diversity of golden jackals at all three loci was markedly lower than that of grey wolves (allelic richness values were 4, 2 and 3 in jackal versus 11.9, 6.6 and 10.2 in wolves for DRB1, DQA1 and DQB1, respectively) and can be attributed to a genetic drift rather than to the lack of historical positive selection. The finding of high evolutionary distances (16.3% for DRB1 and 8.5% for DQB1) and a substantial number of codons predicted to be under the influence of positive selection (11 for DRB1 and 9 for DQB1) suggests that the investigated golden jackal population still contains considerable functional diversity necessary for the presentation of varied foreign peptides. In contrast to neutral genetic variation, our results suggest that the Dalmatian population has a higher MHC diversity than the Slavonian population, casting doubt on its supposed isolation and calling for a more extensive investigation of the MHC variability of southern Balkan jackal populations.     

Polymorphisms in MHC-DRA and -DRB alleles of water buffalo (Bubalus bubalis) reveal different features from cattle DR alleles

Seventy-five individuals of Bubalus bubalis belonging to four different breeds, three of river buffalo and one of swamp buffalo, were studied for polymorphism in MHC DRB (Bubu-DRB) and DRA (Bubu-DRA) loci. Eight alleles of Bubu-DRB were found, and all alleles in the swamp type were shared with the three river breeds. All alleles sampled from the breed of European origin (Mediterranean) were present in breeds sampled in Brazil, thus variability of this locus may have been preserved to a great extent in the more recently founded Brazilian population. Bubu-DRB alleles contained higher proportions of synonymous vs. non-synonymous substitutions in the non-peptide-binding sites (PBS) region, in contrast to the pattern of variation found in BoLA-DRB3, the orthologous locus in cattle. This indicated that either the first domain exon (exon 2) of Bubu-DRB has not undergone as much recombination and/or gene conversion as in cattle alleles, or Bubu-DRB may be more ancient than BoLA-DRB3 alleles. Phylogenetic analysis of DRB alleles from Bubalus, Syncerus c. caffer, the Cape buffalo, and domestic cattle demonstrated transspecies polymorphism. Water buffalo contained two alleles of DRA that differed from each other in two amino acid positions, including one in the PBS (alpha22) that was also shared with Anoa depressicornis, the anoa. Discovery of variation in DRA was surprising as the first domain of DRA is a highly conserved polypeptide in mammals in general and especially in ruminants, where no other substitution in PBS was seen.     

Selective pressures on MHC class II genes in the guppy (Poecilia reticulata) as inferred by hierarchical analysis of population structure

Genes of the major histocompatibility complex, which are the most polymorphic of all vertebrate genes, are a pre‐eminent system for the study of selective pressures that arise from host–pathogen interactions. Balancing selection capable of maintaining high polymorphism should lead to the homogenization of MHC allele frequencies among populations, but there is some evidence to suggest that diversifying selection also operates on the MHC. However, the pattern of population structure observed at MHC loci is likely to depend on the spatial and/or temporal scale examined. Here, we investigated selection acting on MHC genes at different geographic scales using Venezuelan guppy populations inhabiting four regions. We found a significant correlation between MHC and microsatellite allelic richness across populations, which suggests the role of genetic drift in shaping MHC diversity. However, compared to microsatellites, more MHC variation was explained by differences between populations within larger geographic regions and less by the differences between the regions. Furthermore, among proximate populations, variation in MHC allele frequencies was significantly higher compared to microsatellites, indicating that selection acting on MHC may increase population structure at small spatial scales. However, in populations that have significantly diverged at neutral markers, the population‐genetic signature of diversifying selection may be eradicated in the long term by that of balancing selection, which acts to preserve rare alleles and thus maintain a common pool of MHC alleles.     

MHC Variability of a Small Lemur in the Littoral Forest Fragments of Southeastern Madagascar

Habitat fragmentation inhibits gene flow between populations often resulting in a loss of genetic diversity with possible negative effects on fitness parameters. In vertebrates, growing evidence suggests that such genetic diversity is particularly important at the level of the major histocompatibility complex (MHC) because its gene products play an important role in immune functions. Diversity in the MHC is assumed to improve population viability. Here, we investigated the impact of forest fragmentation on the genetic variability of one of the functionally important parts of the MHC, DRB exon 2, of the endemic mouse lemur Microcebus murinus by comparing populations inhabiting two littoral forest fragments of different size in southeastern Madagascar. Twelve different alleles of DRB exon 2 were found in 145 individuals of M. murinus with high levels of sequence divergence between alleles. In both subpopulations, levels of genetic diversity were high, and the genetic analyses revealed only limited effects of fragmentation. Significantly more non-synonymous than synonymous substitutions were found in the functionally important antigen recognition and binding sites indicating selection processes maintaining MHC polymorphism. This is the first study on MHC variation in a free-ranging Malagasy lemur population.     

Are there Ubiquitous Parasite-driven Major Histocompatibility Complex Selection Mechanisms in Gray Mouse Lemurs?

A major goal of evolutionary biology is to understand how selection drives local adaptation. For example, the major histocompatibility complex (MHC) plays an important role in the immune system, and high levels of MHC variation are thought to be a form of adaptation in natural populations. Individual MHC composition may influence parasite resistance via advantages associated with 1) heterozygosity, because heterozygotes recognize a broader range of different antigens than homozygotes (heterozygote advantage); 2) highly variable amino acid sequences in MHC alleles, allowing individuals to bind a broader spectrum of parasite-derived peptides (divergent-alleles advantage, a mechanistic variant of the heterozygote advantage model); or 3) specific MHC alleles (rare allele advantage or frequency dependent selection). We investigated relationships between gastrointestinal nematode burden and both adaptive immune gene variability (MHC class II DRB) and neutral microsatellites in free-living gray mouse lemurs (Microcebus murinus) native to a dry deciduous forest population in western Madagascar to test these hypotheses. The individual MHC composition was related to parasite infestation. Specific MHC alleles were involved in parasite resistance and the presence of common alleles negatively influenced infestation intensity. We found no support for the heterozygote advantage hypothesis, but we did find support for the divergent-MHC allele advantage hypothesis: Individuals with very divergent MHC alleles carried fewer and less intense nematode infestations than individuals with more similar alleles in the more variable dry deciduous forest population. These results indicate that intestinal parasites are important selection pressures under natural conditions and suggest that different selection mechanisms are not mutually exclusive. In contrast, we detected no association between neutral overall individual genetic diversity (measured via 17 microsatellites) and parasite load. Finally, we investigated the ubiquity of parasite-driven selection mechanisms by comparing our results with a previous study of a mouse lemur population from the climatically different littoral forest in southeastern Madagascar, ca. 500 km away. This revealed that different specific MHC alleles were involved in parasite resistance in the 2 habitats, showing that gene-parasite associations are not consistent between populations.     

MHC heterozygosity and survival in red junglefowl

Genes of the major histocompatibility complex (MHC) form a vital part of the vertebrate immune system and play a major role in pathogen resistance. The extremely high levels of polymorphism observed at the MHC are hypothesised to be driven by pathogen‐mediated selection. Although the exact nature of selection remains unclear, three main hypotheses have been put forward; heterozygote advantage, negative frequency‐dependence and fluctuating selection. Here, we report the effects of MHC genotype on survival in a cohort of semi‐natural red junglefowl (Gallus gallus) that suffered severe mortality as a result of an outbreak of the disease coccidiosis. The cohort was followed from hatching until 250 days of age, approximately the age of sexual maturity in this species, during which time over 80% of the birds died. We show that on average birds with MHC heterozygote genotypes survived infection longer than homozygotes and that this effect was independent of genome‐wide heterozygosity, estimated across microsatellite loci. This MHC effect appeared to be caused by a single susceptible haplotype (CD_c) the effect of which was masked in all heterozygote genotypes by other dominant haplotypes. The CD_c homozygous genotype had lower survival than all other genotypes, but CD_c heterozygous genotypes had survival probabilities equal to the most resistant homozygote genotype. Importantly, no heterozygotes conferred greater resistance than the most resistant homozygote genotype, indicating that the observed survival advantage of MHC heterozygotes was the product of dominant, rather than overdominant processes. This pattern and effect of MHC diversity in our population could reflect the processes ongoing in similarly small, fragmented natural populations.     

Heterozygosity excess at the cattle DRB locus revealed by large scale genotyping of two closely linked microsatellites

A method for MHC DRB typing in cattle based on two closely linked and highly polymorphic microsatellites is described. The two microsatellites DRBP1ms and DRB3ms are located in intron 2 of the corresponding DRB gene. The very strong linkage disequilibrium between the two loci made it possible to establish DRB microsatellite haplotypes. The typing results with this method on reference samples followed closely that obtained with RFLP and direct sequence analysis of DRB3 exon 2. The method is well suited for large scale genotyping and was successfully applied for typing more than 600 unrelated animals representing 23 breeds. The data were used to test whether the observed DRB allele frequency distributions were consistent with that expected for selectively neutral alleles in populations at mutation-drift equilibrium. A significant heterozygosity excess was detected and there was an obvious trend across breeds towards a more even allele frequency distribution than expected. The deviation may be due to balancing selection acting on the DRB locus or by recent population bottlenecks.     

Major histocompatibility complex (MHC) heterozygote superiority to natural multi-parasite infections in the water vole (Arvicola terrestris)

The fundamental role of the major histocompatibility complex (MHC) in immune recognition has led to a general consensus that the characteristically high levels of functional polymorphism at MHC genes is maintained by balancing selection operating through host–parasite coevolution. However, the actual mechanism by which selection operates is unclear. Two hypotheses have been proposed: overdominance (or heterozygote superiority) and negative frequency-dependent selection. Evidence for these hypotheses was evaluated by examining MHC–parasite relationships in an island population of water voles (Arvicola terrestris). Generalized linear mixed models were used to examine whether individual variation at an MHC class II DRB locus explained variation in the individual burdens of five different parasites. MHC genotype explained a significant amount of variation in the burden of gamasid mites, fleas (Megabothris walkeri) and nymphs of sheep ticks (Ixodes ricinus). Additionally, MHC heterozygotes were simultaneously co-infected by fewer parasite types than homozygotes. In each case where an MHC-dependent effect on parasite burden was resolved, the heterozygote genotype was associated with fewer parasites, and the heterozygote outperformed each homozygote in two of three cases, suggesting an overall superiority against parasitism for MHC heterozygote genotypes. This is the first demonstration of MHC heterozygote superiority against multiple parasites in a natural population, a mechanism that could help maintain high levels of functional MHC genetic diversity in natural populations.