Discussion
This study represents an effort to comprehensively evaluate multiple major hypotheses concerning the maintenance of MHC diversity, using observational data from 26 stickleback populations. Despite the large scale of sampling, we did not find strong support for any of the three popular parasite-mediated selection hypotheses, namely heterozygote advantage, frequency-dependent selection, and fluctuating selection. In contrast, neutral processes seem to best explain MHC diversity in this system, both in terms of allelic richness and between-population divergence.
Consistent with previous studies in other vertebrates (Kaufman, 2018), MHC IIβ genes are highly polymorphic in the surveyed stickleback metapopulation. We found 1115 unique MHC IIβ alleles from 1277 individuals, with a majority of alleles only present in one population. However, although on average fish from lake habitats are infected with more diverse parasite species than the ones from river and estuary habitats (Bolnick et al., 2020), lake stickleback have less MHC allelic diversity than others. The extremely high level of polymorphism in MHC genes is often attributed to parasite-mediated selection; however, the discrepancy between parasite richness and MHC allelic diversity among habitat types suggests that parasite-mediated selection alone is unlikely to explain the high level of MHC polymorphism in the studied stickleback populations. This finding contradicts previous results from studies of stickleback inhabiting different habitats in Germany (K. M. Wegner, Reusch, & Kalbe, 2003), where lake fish had higher parasite diversity (as in this study), and higher MHC diversity (unlike this study).
The above finding is further supported through testing specific predictions derived from heterozygote advantage model. The heterozygote advantage hypothesis predicts that the number of MHC alleles in the host genome would be maximized to recognize a wide range of parasites. But previous studies (K. Mathias Wegner et al., 2003) in stickleback found support for the theoretical model that the number of MHC alleles should be optimized at an intermediate level, rather thanmaximized . The argument is that too many MHC alleles would reduce the T cell portfolio due to negative selection of self-recognition T cell receptors. In this study, we applied similar analytical approaches as Wegner et al. (2003) to a larger sample size and many more populations. Contrary to those previous results, we did not find support for either the maximizing or optimizing hypothesis.
Negative frequency-dependent selection suggests that the frequency of MHC alleles might experience a cyclical pattern through time, with different low frequency alleles playing the role of conferring resistance at different time points. For negative frequency dependence to be effective, on average, it must be true that currently rare alleles exhibit an advantage over common ones. In this study, we found that allele prevalence in general is not associated with whether an allele is effective in parasite resistance. We further examined whether alleles have consistent effects across sites. We found inconsistent allele effects in some cases, but we also found consistent effects in a larger fraction of cases. So negative frequency-dependent selection might be able to explain a limited number of allele-parasite dynamics in our study. But, in most cases MHC alleles confer equivalent protection in multiple isolated populations, inconsistent with cyclic changes due to coevolution. Ideally, to demonstrate negative frequency-dependent selection, one would need to carry out a longitudinal study that samples parasite genotypes and host genotypes through time. However, this type of study is very challenging to implement in a field setting, especially for annually reproducing animals.
Finally, fluctuating selection, either temporally or spatially, is frequently invoked to explain the maintenance of genetic diversity across a landscape (Charbonnel & Pemberton, 2005; Schemske & Bierzychudek, 2007). Our sampling sites on Vancouver Island differ in many physical parameters, such as water area, depth, temperature, flow speed, etc . It is also documented that parasite communities differ significantly among different sites (Bolnick et al., 2020). This spatial heterogeneity in parasite communities is due to abiotic and biotic variables (e.g., lake size, fish diet; Bolnick et al. (2019)), and could generate spatially fluctuating selection that maintains MHC diversity. However, we did not find significant correlation between the parasite distance matrix and the MHC distance matrix, suggesting that the spatial heterogeneity in parasite community could not explain the observed spatial variation in MHC diversity.
Interestingly, we found that neutral processes could contribute to MHC diversity in two different analyses: (a). populations with higher genomic heterozygosity also have higher MHC allelic richness, and (b). less divergent populations (low genomic Fst) have similar MHC genotypes and frequencies. Similar to analysis (a), a previous study in stickleback (K. M. Wegner et al., 2003) also found a positive, albeit weak, association between neutral genetic diversity and population MHC allelic diversity. The role of neutral processes in the maintenance of MHC diversity has often been studied in the context of conservation, which is usually concerned with small and isolated populations (Miller & Lambert, 2004; Seddon & Ellegren, 2004). Our result suggested that at larger scales, neutral processes are also important factors in explaining MHC diversity within- and between-population, even when there is no specific evidence for past bottlenecks.
Parasite-mediated selection is believed to have an important role in maintaining MHC polymorphism. Consistent with this, we found some associations between certain parasites and individual MHC allele. However, we did not find strong support for parasite-mediated selection hypotheses in the scale of metapopulations. There are several possible explanations. First, in this study, we examined the three hypotheses separately, but in reality, the mechanisms are not mutually exclusive. For example, low frequency alleles are more likely to be in a heterozygous state in a population. Therefore, if an allele were effective in resistance at low frequency, it would be consistent with both negative frequency-dependent selection and heterozygote advantage. Furthermore, if these mechanisms work in combination in different MHC-parasite pairs or in different populations, we may not detect a clear signal of each mechanism when combining data from multiple MHC-parasite pairs and many populations. Second, parasites are not the only selective agents that act upon MHC loci. MHC genes are also involved in interactions with other species, such as symbionts comprising the gut microbiome (Bolnick et al., 2014), and within species interactions, such as mate choice (Milinski et al., 2005). It is unknown which MHC genes are relevant to which selective forces. If other selective forces are also at work, the influences of parasite-mediated selection on MHC diversity could be obscured. Third, we only measured parasite richness and MHC allelic richness in this study to represent parasite diversity and MHC diversity, respectively, so the detailed differences among individual parasite and individual MHC alleles were not considered. In the future, it is worthwhile to test the parasite-mediated selection hypotheses with diversity measurements that take into account of nuances such as the phylogenetic relationship of parasite taxa and the sequence divergence between distinct MHC alleles. Finally, our survey does not span multiple seasons, so we cannot evaluate how MHC variation is shaped across time by temporally fluctuating selection.
To summarize, we used a large-scale field survey to evaluate the three popular parasite-mediated selection hypotheses on MHC diversity, namely, heterozygote advantage, negative frequency-dependent selection, and fluctuating selection. We found that neutral processes best explain MHC diversity (allelic richness and population divergence), instead of those parasite-mediated selection mechanisms. Because MHC diversity can be influenced by many selective forces, and the outcome of this selection could be further shaped by spatial and temporal heterogeneity, our study suggests it may not be possible to parse out how each selective force influences MHC diversity at large scales directly from observational data. We propose that it is worthwhile to instead investigate how each selective force acts upon MHC diversity with experimental approaches (e.g. controlling other selective forces) at smaller scales. Combining insights from these small-scale controlled studies in a step-wise manner can be a fertile future direction to understand the complex process of MHC evolution and diversification.