Low MHC DRB class II diversity in the mountain goat: past bottlenecks and possible role of pathogens and parasites

Major histocompatibility complex (MHC) genes are the most polymorphic in vertebrates and code for molecules playing a central role in pathogen resistance. We studied levels of MHC DRB class II diversity in a long-term study population of mountain goats (Oreamnos americanus) at Caw Ridge, Alberta, and two other populations from British Columbia, Canada. Only two alleles were found among the three populations sampled. The Caw Ridge population was fixed for one of the two MHC DRB alleles, but this lack of variation did not appear to have affected it negatively because the population doubled over two decades and had no history of any apparent infectious diseases. Past population bottlenecks during Pleistocene glaciations are thought to have been the main factor contributing to the low levels of MHC diversity in mountain goats, a hypothesis supported by our previous work reporting low polymorphism at neutral loci. Additionally, the limited MHC variability in mountain goats may be related to its northern distribution as we found that allelic diversity at MHC DRB class II in wild ungulates decreases with increasing latitude, possibly as a result of low parasite diversity at high latitudes. The low MHC variation in mountain goats and other northern ungulates such as muskoxen (Ovibos moschatus) may expose these species to population outbreaks that could be generated by introduced pathogens or northward shifts in the distribution of pathogens with global climate warming.     

Characterization of major histocompatibility complex class I and class II genes from the Tasmanian devil (<Emphasis Type="Italic">Sarcophilus harrisii</Emphasis>)

The Tasmanian devil (Sarcophilus harrisii) is currently threatened by an emerging wildlife disease, devil facial tumour disease. The disease is decreasing devil numbers dramatically and may lead to the extinction of the species. At present, nothing is known about the immune genes or basic immunology of the devil. In this study, we report the construction of the first genetic library for the Tasmanian devil, a spleen cDNA library, and the isolation of full-length MHC Class I and Class II genes. We describe six unique Class II beta chain sequences from at least three loci, which belong to the marsupial Class II DA gene family. We have isolated 13 unique devil Class I sequences, representing at least seven Class I loci, two of which are most likely non-classical genes. The MHC Class I sequences from the devil have little heterogeneity, indicating recent divergence. The MHC genes described here are most likely involved in antigen presentation and are an important first step for studying MHC diversity and immune response in the devil.     

Lack of genetic diversity across diverse immune genes in an endangered mammal,the Tasmanian devil (Sarcophilus harrisii)

The Tasmanian devil (Sarcophilus harrisii) is threatened with extinction due to the spread of devil facial tumour disease. Polymorphisms in immune genes can provide adaptive potential to resist diseases. Previous studies in diversity at immune loci in wild species have almost exclusively focused on genes of the major histocompatibility complex (MHC); however, these genes only account for a fraction of immune gene diversity. Devils lack diversity at functionally important immunity loci, including MHC and Toll‐like receptor genes. Whether there are polymorphisms at devil immune genes outside these two families is unknown. Here, we identify polymorphisms in a wide range of key immune genes, and develop assays to type single nucleotide polymorphisms (SNPs) within a subset of these genes. A total of 167 immune genes were examined, including cytokines, chemokines and natural killer cell receptors. Using genome‐level data from ten devils, SNPs within coding regions, introns and 10 kb flanking genes of interest were identified. We found low polymorphism across 167 immune genes examined bioinformatically using whole‐genome data. From this data, we developed long amplicon assays to target nine genes. These amplicons were sequenced in 29–220 devils and found to contain 78 SNPs, including eight SNPS within exons. Despite the extreme paucity of genetic diversity within these genes, signatures of balancing selection were exhibited by one chemokine gene, suggesting that remaining diversity may hold adaptive potential. The low functional diversity may leave devils highly vulnerable to infectious disease, and therefore, monitoring and preserving remaining diversity will be critical for the long‐term management of this species. Examining genetic variation in diverse immune genes should be a priority for threatened wildlife species. This study can act as a model for broad‐scale immunogenetic diversity analysis in threatened species.     

Unraveling the Effects of Selection and Demography on Immune Gene Variation in Free-Ranging Plains Zebra (Equus quagga) Populations

Demography, migration and natural selection are predominant processes affecting the distribution of genetic variation among natural populations. Many studies use neutral genetic markers to make inferences about population history. However, the investigation of functional coding loci, which directly reflect fitness, is critical to our understanding of species'' ecology and evolution. Immune genes, such as those of the Major Histocompatibility Complex (MHC), play an important role in pathogen recognition and provide a potent model system for studying selection. We contrasted diversity patterns of neutral data with MHC loci, ELA-DRA and -DQA, in two southern African plains zebra (Equus quagga) populations: Etosha National Park, Namibia, and Kruger National Park, South Africa. Results from neutrality tests, along with observations of elevated diversity and low differentiation across populations, supported previous genus-level evidence for balancing selection at these loci. Despite being low, MHC divergence across populations was significant and may be attributed to drift effects typical of geographically separated populations experiencing little to no gene flow, or alternatively to shifting allele frequency distributions driven by spatially variable and fluctuating pathogen communities. At the DRA, zebra exhibited geographic differentiation concordant with microsatellites and reduced levels of diversity in Etosha due to highly skewed allele frequencies that could not be explained by demography, suggestive of spatially heterogeneous selection and local adaptation. This study highlights the complexity in which selection affects immune gene diversity and warrants the need for further research on the ecological mechanisms shaping patterns of adaptive variation among natural populations.     

Retracted: MHC diversity and differential exposure to pathogens in kestrels (Aves: Falconidae)

Pathogen diversity is thought to drive major histocompatibility complex (MHC) polymorphism given that host’s immune repertories are dependent on antigen recognition capabilities. Here, we surveyed an extensive community of pathogens (n = 35 taxa) and MHC diversity in mainland versus island subspecies of the Eurasian kestrel Falco tinnunculus and in a sympatric mainland population of the phylogenetically related lesser kestrel Falco naumanni. Insular subspecies are commonly exposed to impoverished pathogen communities whilst different species’ ecologies and contrasting life‐history traits may lead to different levels of pathogen exposure. Although specific host traits may explain differential particular infections, overall pathogen diversity, richness and prevalence were higher in the truly cosmopolitan, euriphagous and long‐distance disperser Eurasian kestrel than in the estenophagous, steppe‐specialist, philopatric but long‐distance migratory lesser kestrel. Accordingly, the continental population of Eurasian kestrels displayed a higher number (64 vs. 49) as well as more divergent alleles at both MHC class I and class II loci. Detailed analyses of amino acid diversity revealed that significant differences between both species were exclusive to those functionally important codons comprising the antigen binding sites. The lowest pathogen burdens and the smallest but still quite divergent set of MHC alleles (n = 16) were found in island Eurasian kestrels, where the rates of allele fixation at MHC loci seem to have occurred faster than at neutral markers. The results presented in this study would therefore support the role of pathogen diversity and abundance in shaping patterns of genetic variation at evolutionary relevant MHC genes.     

Isolation and characterization of 10 MHC Class I-associated microsatellite loci in tammar wallaby (Macropus eugenii)

The major histocompatibility complex (MHC) contain genes which play a key role in immune response and mate choice, and are therefore of functional importance to molecular ecologists. Here we describe the design of 10 MHC Class I-associated microsatellite loci from the tammar wallaby. All 10 loci are highly polymorphic, with the expected heterozygosity ranging from 0.547 to 0.919. Six loci successfully cross-amplify in other macropodid species. These microsatellites will serve as useful tools for studying the level of MHC diversity, the impact of selection on genetic variation and the unique structure of the tammar wallaby MHC.     

Genetic structure in insular and mainland populations of house sparrows (Passer domesticus) and their hemosporidian parasites

Small and isolated populations usually exhibit low levels of genetic variability, and thus, they are expected to have a lower capacity to adapt to changes in environmental conditions, such as exposure to pathogens and parasites. Comparing the genetic variability of selectively neutral versus functional loci allows one to assess the evolutionary history of populations and their future evolutionary potential. The genes of the major histocompatibility complex (MHC) control immune recognition of parasites, and their unusually high diversity is genes which is likely driven by parasite‐mediated balancing selection. Here, we examined diversity and differentiation of neutral microsatellite loci and functional MHC class I genes in house sparrows (Passer domesticus), living in six insular and six mainland populations, and we aimed to determine whether their diversity or differentiation correlates with the diversity and the prevalence of infection of hemosporidian parasites. We found that island bird populations tended to have lower neutral genetic variability, whereas MHC variability gene was similar between island and mainland populations. Similarly, island populations tended to show greater genetic differentiation than mainland populations, especially at microsatellite markers. The maintenance of MHC genetic diversity and its less marked structure in the island populations could be attributed to balancing‐selection. The greater MHC differentiation among populations was negatively correlated with similarity in blood parasites (prevalence and diversity of parasite strains) between populations. Even at low prevalence and small geographical scale, haemosporidian parasites might contribute to structure the variability of immune genes among populations of hosts.     

Evidence for selection at cytokine loci in a natural population of field voles (Microtus agrestis)

Individuals in natural populations are frequently exposed to a wide range of pathogens. Given the diverse profile of gene products involved in responses to different types of pathogen, this potentially results in complex pathogen-specific selection pressures acting on a broad spectrum of immune system genes in wild animals. Thus far, studies into the evolution of immune genes in natural populations have focused almost exclusively on the Major Histocompatibility Complex (MHC). However, the MHC represents only a fraction of the immune system and there is a need to broaden research in wild species to include other immune genes. Here, we examine the evidence for natural selection in a range of non-MHC genes in a natural population of field voles (Microtus agrestis). We concentrate primarily on genes encoding cytokines, signalling molecules critical in eliciting and mediating immune responses and identify signatures of natural selection acting on several of these genes. In particular, genetic diversity within Interleukin 1 beta and Interleukin 2 appears to have been maintained through balancing selection. Taken together with previous findings that polymorphism within these genes is associated with variation in resistance to multiple pathogens, this suggests that pathogen-mediated selection may be an important force driving genetic diversity at cytokine loci in voles and other natural populations. These results also suggest that, along with the MHC, preservation of genetic variation within cytokine genes should be a priority for the conservation genetics of threatened wildlife populations.     

Is promiscuity associated with enhanced selection on MHC-DQα in mice (genus Peromyscus)?

Reproductive behavior may play an important role in shaping selection on Major Histocompatibility Complex (MHC) genes. For example, the number of sexual partners that an individual has may affect exposure to sexually transmitted pathogens, with more partners leading to greater exposure and, hence, potentially greater selection for variation at MHC loci. To explore this hypothesis, we examined the strength of selection on exon 2 of the MHC-DQα locus in two species of Peromyscus. While the California mouse (P. californicus) is characterized by lifetime social and genetic monogamy, the deer mouse (P. maniculatus) is socially and genetically promiscuous; consistent with these differences in mating behavior, the diversity of bacteria present within the reproductive tracts of females is significantly greater for P. maniculatus. To test the prediction that more reproductive partners and exposure to a greater range of sexually transmitted pathogens are associated with enhanced diversifying selection on genes responsible for immune function, we compared patterns and levels of diversity at the Class II MHC-DQα locus in sympatric populations of P. maniculatus and P. californicus. Using likelihood based analyses, we show that selection is enhanced in the promiscuous P. maniculatus. This study is the first to compare the strength of selection in wild sympatric rodents with known differences in pathogen milieu.     

Parasite-mediated selection drives an immunogenetic trade-off in plains zebras (Equus quagga)

Pathogen evasion of the host immune system is a key force driving extreme polymorphism in genes of the major histocompatibility complex (MHC). Although this gene family is well characterized in structure and function, there is still much debate surrounding the mechanisms by which MHC diversity is selectively maintained. Many studies have investigated relationships between MHC variation and specific pathogens, and have found mixed support for and against the hypotheses of heterozygote advantage, frequency-dependent or fluctuating selection. Few, however, have focused on the selective effects of multiple parasite types on host immunogenetic patterns. Here, we examined relationships between variation in the equine MHC gene, ELA-DRA, and both gastrointestinal (GI) and ectoparasitism in plains zebras (Equus quagga). Specific alleles present at opposing population frequencies had antagonistic effects, with rare alleles associated with increased GI parasitism and common alleles with increased tick burdens. These results support a frequency-dependent mechanism, but are also consistent with fluctuating selection. Maladaptive GI parasite ‘susceptibility alleles’ were reduced in frequency, suggesting that these parasites may play a greater selective role at this locus. Heterozygote advantage, in terms of allele mutational divergence, also predicted decreased GI parasite burden in genotypes with a common allele. We conclude that an immunogenetic trade-off affects resistance/susceptibility to parasites in this system. Because GI and ectoparasites do not directly interact within hosts, our results uniquely show that antagonistic parasite interactions can be indirectly modulated through the host immune system. This study highlights the importance of investigating the role of multiple parasites in shaping patterns of host immunogenetic variation.     

Variation in MHC genotypes in two populations of house sparrow (Passer domesticus) with different population histories

Small populations are likely to have a low genetic ability for disease resistance due to loss of genetic variation through inbreeding and genetic drift. In vertebrates, the highest genetic diversity of the immune system is located at genes within the major histocompatibility complex (MHC). Interestingly, parasite‐mediated selection is thought to potentially maintain variation at MHC loci even in populations that are monomorphic at other loci. Therefore, general loss of genetic variation in the genome may not necessarily be associated with low variation at MHC loci. We evaluated inter‐ and intrapopulation variation in MHC genotypes between an inbred (Aldra) and a relatively outbred population (Hestmannøy) of house sparrows (Passer domesticus) in a metapopulation at Helgeland, Norway. Genomic (gDNA) and transcribed (cDNA) alleles of functional MHC class I and IIB loci, along with neutral noncoding microsatellite markers, were analyzed to obtain relevant estimates of genetic variation. We found lower allelic richness in microsatellites in the inbred population, but high genetic variation in MHC class I and IIB loci in both populations. This suggests that also the inbred population could be under balancing selection to maintain genetic variation for pathogen resistance.     

Coloniality and migration are related to selection on MHC genes in birds

The major histocompatibility complex (MHC) plays a key role in pathogen recognition as a part of the vertebrate adaptive immune system. The great diversity of MHC genes in natural populations is maintained by different forms of balancing selection and its strength should correlate with the diversity of pathogens to which a population is exposed and the rate of exposure. Despite this prediction, little is known about how life‐history characteristics affect selection at the MHC. Here, we examined whether the strength of balancing selection on MHC class II genes in birds (as measured with nonsynonymous nucleotide substitutions, dN) was related to their social or migratory behavior, two life‐history characteristics correlated with pathogen exposure. Our comparative analysis indicated that the rate of nonsynonymous substitutions was higher in colonial and migratory species than solitary and resident species, suggesting that the strength of balancing selection increases with coloniality and migratory status. These patterns could be attributed to: (1) elevated transmission rates of pathogens in species that breed in dense aggregations, or (2) exposure to a more diverse fauna of pathogens and parasites in migratory species. Our study suggests that differences in social structure and basic ecological traits influence MHC diversity in natural vertebrate populations.     

Contrasting patterns of selection acting on MHC class I and class II DRB genes in the Alpine marmot (Marmota marmota)

The major histocompatibility complex (MHC) genes code for proteins that play a critical role in the immune system response. The MHC genes are among the most polymorphic genes in vertebrates, presumably due to balancing selection. The two MHC classes appear to differ in the rate of evolution, but the reasons for this variation are not well understood. Here, we investigate the level of polymorphism and the evolution of sequences that code for the peptide-binding regions of MHC class I and class II DRB genes in the Alpine marmot (Marmota marmota). We found evidence for four expressed MHC class I loci and two expressed MHC class II loci. MHC genes in marmots were characterized by low polymorphism, as one to eight alleles per putative locus were detected in 38 individuals from three French Alps populations. The generally limited degree of polymorphism, which was more pronounced in class I genes, is likely due to bottleneck the populations undergone. Additionally, gene duplication within each class might have compensated for the loss of polymorphism at particular loci. The two gene classes showed different patterns of evolution. The most polymorphic of the putative loci, Mama-DRB1, showed clear evidence of historical positive selection for amino acid replacements. However, no signal of positive selection was evident in the MHC class I genes. These contrasting patterns of sequence evolution may reflect differences in selection pressures acting on class I and class II genes.     

Genetic drift outweighs balancing selection in shaping post-bottleneck major histocompatibility complex variation in New Zealand robins (Petroicidae)

The Chatham Island black robin, Petroica traversi, is a highly inbred, endangered passerine with extremely low levels of variation at hypervariable neutral DNA markers. In this study we investigated variation in major histocompatibility complex (MHC) class II genes in both the black robin and its nonendangered relative, the South Island robin Petroica australis australis. Previous studies have shown that Petroica have at least four expressed class II B MHC genes. In this study, the sequences of introns flanking exon 2 of these loci were characterized to design primers for peptide-binding region (PBR) sequence analysis. Intron sequences were comprised of varying numbers of repeated units, with highly conserved regions immediately flanking exon 2. Polymerase chain reaction primers designed to this region amplified three or four sequences per black robin individual, and eight to 14 sequences per South Island robin individual. MHC genes are fitness-related genes thought to be under balancing selection, so they may be more likely to retain variation in bottlenecked populations. To test this, we compared MHC variation in the black robin with artificially bottlenecked populations of South Island robin, and with their respective source populations, using restriction fragment length polymorphism analyses and DNA sequencing of the PBR. Our results indicate that the black robin is monomorphic at class II B MHC loci, while both source and bottlenecked populations of South Island robin have retained moderate levels of variation. Comparison of MHC variation with minisatellite DNA variation indicates that genetic drift outweighs balancing selection in determining MHC diversity in the bottlenecked populations. However, balancing selection appears to influence MHC diversity over evolutionary timescales, and the effects of gene conversion are evident.     

Two patterns of variation among MHC class I loci in Tuatara (Sphenodon punctatus)

The genes of the major histocompatibility complex (MHC) are a central component of the immune system in vertebrates and have become important markers of functional, fitness-related genetic variation. We have investigated the evolutionary processes that generate diversity at MHC class I genes in a large population of an archaic reptile species, the tuatara (Sphenodon punctatus), found on Stephens Island, Cook Strait, New Zealand. We identified at least 2 highly polymorphic (UA type) loci and one locus (UZ) exhibiting low polymorphism. The UZ locus is characterized by low nucleotide diversity and weak balancing selection and may be either a nonclassical class I gene or a pseudogene. In contrast, the UA-type alleles have high nucleotide diversity and show evidence of balancing selection at putative peptide-binding sites. Twenty-one different UA-type genotypes were identified among 26 individuals, suggesting that the Stephens Island population has high levels of MHC class I variation. UA-type allelic diversity is generated by a mixture of point mutation and gene conversion. As has been found in birds and fish, gene conversion obscures the genealogical relationships among alleles and prevents the assignment of alleles to loci. Our results suggest that the molecular mechanisms that underpin MHC evolution in nonmammals make locus-specific amplification impossible in some species.     

High variability in the MHC class II DA beta chain of the brushtail possum (<Emphasis Type="Italic">Trichosurus vulpecula</Emphasis>)

The diversity of class II major histocompatibility complex (MHC) loci was investigated in the brushtail possum, an important marsupial pest species in New Zealand. Immunocontraception, a form of fertility control that generates an autoimmune response, is being developed as a population control method for the possum. Because the immune response is partly under genetic control, an understanding of immunogenetics in possum will be crucial to the development of immunocontraceptive vaccines. MHC molecules are critical in the vertebrate immune response. Class II MHC molecules bind and present exogenously derived peptides to T lymphocytes and may be important in the presentation of immunocontraceptives. We used polymerase chain reaction primers designed to amplify the peptide binding region of possum class II MHC genes to isolate sequences from 49 animals. We have previously described 19 novel alleles from the DAB locus in the possum (Holland et al., Immunogenetics 60:449–460, 2008). Here, we report on another 11 novel alleles isolated from possum DAB, making this the most diverse marsupial locus described so far. This high level of diversity indicates that DAB is an important MHC locus in the possum and will need to be taken into account in the design of immunocontraceptive vaccines.     

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.     

Maintenance of MHC Class IIB diversity in a recently established songbird population

We examined variation at MHC Class IIB genes in a recently established population of dark-eyed juncos (Junco hyemalis) in a coastal urban environment in southern California, USA relative to an ancestral-range population from a nearby species-typical montane environment. The founding population is estimated to have been quite small, but we predicted that variation at the major histocompatibility complex (MHC) among the founders would nevertheless be preserved owing to the high functional significance of MHC. Previous studies of MHC in songbirds have had varying degrees of success in isolating loci, as passerines show extensive MHC gene duplication. In order to compare diversity in the two populations, we employed two published approaches to sequencing MHC Class II exon 2: direct sequencing with exon-based primers, and traditional cloning and sequencing with intron-based primers. Results from both methods show that the colonist population has maintained high levels of variation. Our results also indicate varying numbers of alleles across individuals, corroborating evidence for gene duplication in songbird MHC. While future studies in songbirds may need to take a genomic approach to fully understand the structure of MHC in this lineage, our results show that it is possible to use traditional methods to reveal functional variation across populations.