Lewis G. Spurgin

How pathogens drive genetic diversity: MHC, mechanisms and misunderstandings

Major histocompatibility complex (MHC) genes have been put forward as a model for studying how genetic diversity is maintained in wild populations. Pathogen-mediated selection (PMS) is believed to generate the extraordinary levels of MHC diversity observed. However, establishing the relative importance of the three proposed mechanisms of PMS (heterozygote advantage, rare-allele advantage and fluctuating selection) has proved extremely difficult. Studies have attempted to differentiate between mechanisms of PMS using two approaches: (i) comparing MHC diversity with that expected under neutrality and (ii) relating MHC diversity to pathogen regime. Here, we show that in many cases the same predictions arise from the different mechanisms under these approaches, and that most studies that have inferred one mechanism of selection have not fully considered the alternative explanations. We argue that, while it may be possible to demonstrate that particular mechanisms of PMS are occurring, resolving their relative importance within a system is probably impossible. A more realistic target is to continue to demonstrate when and where the different mechanisms of PMS occur, with the aim of determining their relative importance across systems. We put forward what we believe to be the most promising approaches that will allow us to progress towards achieving this.

New methods to identify conserved microsatellite loci and develop primer sets of high cross-species utility - as demonstrated for birds

We have developed a new approach to create microsatellite primer sets that have high utility across a wide range of species. The success of this method was demonstrated using birds. We selected 35 avian EST microsatellite loci that had a high degree of sequence homology between the zebra finch Taeniopygia guttata and the chicken Gallus gallus and designed primer sets in which the primer bind sites were identical in both species. For 33 conserved primer sets, on average, 100% of loci amplified in each of 17 passerine species and 99% of loci in five non‐passerine species. The genotyping of four individuals per species revealed that 24–76% (mean 48%) of loci were polymorphic in the passerines and 18–26% (mean 21%) in the non‐passerines. When at least 17 individuals were genotyped per species for four Fringillidae finch species, 71–85% of loci were polymorphic, observed heterozygosity was above 0.50 for most loci and no locus deviated significantly from Hardy–Weinberg proportions.

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.

Gene conversion rapidly generates major histocompatibility complex diversity in recently founded bird populations

Population bottlenecks can restrict variation at functional genes, reducing the ability of populations to adapt to new and changing environments. Understanding how populations generate adaptive genetic variation following bottlenecks is therefore central to evolutionary biology. Genes of the major histocompatibility complex (MHC) are ideal models for studying adaptive genetic variation due to their central role in pathogen recognition. While de novo MHC sequence variation is generated by point mutation, gene conversion can generate new haplotypes by transferring sections of DNA within and across duplicated MHC loci. However, the extent to which gene conversion generates new MHC haplotypes in wild populations is poorly understood. We developed a 454 sequencing protocol to screen MHC class I exon 3 variation across all 13 island populations of Berthelot’s pipit (Anthus berthelotii). We reveal that just 11–15 MHC haplotypes were retained when the Berthelot’s pipit dispersed across its island range in the North Atlantic ca. 75 000 years ago. Since then, at least 26 new haplotypes have been generated in situ across populations. We show that most of these haplotypes were generated by gene conversion across divergent lineages, and that the rate of gene conversion exceeded that of point mutation by an order of magnitude. Gene conversion resulted in significantly more changes at nucleotide sites directly involved with pathogen recognition, indicating selection for functional variants. We suggest that the creation of new variants by gene conversion is the predominant mechanism generating MHC variation in genetically depauperate populations, thus allowing them to respond to pathogenic challenges.

Sexual selection protects against extinction

Reproduction through sex carries substantial costs, mainly because only half of sexual adults produce offspring. It has been theorized that these costs could be countered if sex allows sexual selection to clear the universal fitness constraint of mutation load. Under sexual selection, competition between (usually) males and mate choice by (usually) females create important intraspecific filters for reproductive success, so that only a subset of males gains paternity. If reproductive success under sexual selection is dependent on individual condition, which is contingent to mutation load, then sexually selected filtering through ‘genic capture’ could offset the costs of sex because it provides genetic benefits to populations. Here we test this theory experimentally by comparing whether populations with histories of strong versus weak sexual selection purge mutation load and resist extinction differently. After evolving replicate populations of the flour beetle Tribolium castaneum for 6 to 7 years under conditions that differed solely in the strengths of sexual selection, we revealed mutation load using inbreeding. Lineages from populations that had previously experienced strong sexual selection were resilient to extinction and maintained fitness under inbreeding, with some families continuing to survive after 20 generations of sib × sib mating. By contrast, lineages derived from populations that experienced weak or non-existent sexual selection showed rapid fitness declines under inbreeding, and all were extinct after generation 10. Multiple mutations across the genome with individually small effects can be difficult to clear, yet sum to a significant fitness load; our findings reveal that sexual selection reduces this load, improving population viability in the face of genetic stress.

High-utility conserved avian microsatellite markers enable parentage and population studies across a wide range of species

BackgroundMicrosatellites are widely used for many genetic studies. In contrast to single nucleotide polymorphism (SNP) and genotyping-by-sequencing methods, they are readily typed in samples of low DNA quality/concentration (e.g. museum/non-invasive samples), and enable the quick, cheap identification of species, hybrids, clones and ploidy. Microsatellites also have the highest cross-species utility of all types of markers used for genotyping, but, despite this, when isolated from a single species, only a relatively small proportion will be of utility. Marker development of any type requires skill and time. The availability of sufficient “off-the-shelf” markers that are suitable for genotyping a wide range of species would not only save resources but also uniquely enable new comparisons of diversity among taxa at the same set of loci. No other marker types are capable of enabling this. We therefore developed a set of avian microsatellite markers with enhanced cross-species utility.ResultsWe selected highly-conserved sequences with a high number of repeat units in both of two genetically distant species. Twenty-four primer sets were designed from homologous sequences that possessed at least eight repeat units in both the zebra finch (Taeniopygia guttata) and chicken (Gallus gallus). Each primer sequence was a complete match to zebra finch and, after accounting for degenerate bases, at least 86% similar to chicken. We assessed primer-set utility by genotyping individuals belonging to eight passerine and four non-passerine species. The majority of the new Conserved Avian Microsatellite (CAM) markers amplified in all 12 species tested (on average, 94% in passerines and 95% in non-passerines). This new marker set is of especially high utility in passerines, with a mean 68% of loci polymorphic per species, compared with 42% in non-passerine species.ConclusionsWhen combined with previously described conserved loci, this new set of conserved markers will not only reduce the necessity and expense of microsatellite isolation for a wide range of genetic studies, including avian parentage and population analyses, but will also now enable comparisons of genetic diversity among different species (and populations) at the same set of loci, with no or reduced bias. Finally, the approach used here can be applied to other taxa in which appropriate genome sequences are available.

Genetic and phenotypic divergence in an island bird : Isolation by distance, by colonization or by adaptation?

Discerning the relative roles of adaptive and nonadaptive processes in generating differences among populations and species, as well as how these processes interact, is a fundamental aim in biology. Both genetic and phenotypic divergence across populations can be the product of limited dispersal and gradual genetic drift across populations (isolation by distance), of colonization history and founder effects (isolation by colonization) or of adaptation to different environments preventing migration between populations (isolation by adaptation). Here, we attempt to differentiate between these processes using island populations of Berthelots pipit (Anthus berthelotii), a passerine bird endemic to three Atlantic archipelagos. Using microsatellite markers and approximate Bayesian computation, we reveal that the northward colonization of this species ca. 8500 years ago resulted in genetic bottlenecks in the colonized archipelagos. We then show that high levels of genetic structure exist across archipelagos and that these are consistent with a pattern of isolation by colonization, but not with isolation by distance or adaptation. Finally, we show that substantial morphological divergence also exists and that this is strongly concordant with patterns of genetic structure and bottleneck history, but not with environmental differences or geographic distance. Overall, our data suggest that founder effects are responsible for both genetic and phenotypic changes across archipelagos. Our findings provide a rare example of how founder effects can persist over evolutionary timescales and suggest that they may play an important role in the early stages of speciation.

Microsatellite resources for Passeridae species: a predicted microsatellite map of the house sparrow Passer domesticus

We identified microsatellite sequences of potential utility in the house sparrow (Passer domesticus) and assigned their predicted genome locations. These sequences included newly isolated house sparrow loci, which we fully characterized. Many of the newly isolated loci were polymorphic in two other species of Passeridae: Berthelot’s pipit Anthus berthelotii and zebra finch Taeniopygia guttata. In total, we identified 179 microsatellite markers that were either isolated directly from, or are of known utility in, the house sparrow. Sixty‐seven of these markers were designed from unique sequences that we isolated from a house sparrow genomic library. These new markers were combined with 36 house sparrow markers isolated by other studies and 76 markers isolated from other passerine species but known to be polymorphic in the house sparrow. We utilized sequence homology to assign chromosomal locations for these loci in the assembled zebra finch genome. One hundred and thirty‐four loci were assigned to 25 different autosomes and eight loci to the Z chromosome. Examination of the genotypes of known‐sex house sparrows for 37 of the new loci revealed a W‐linked locus and an additional Z‐linked locus. Locus Pdoμ2, previously reported as autosomal, was found to be Z‐linked. These loci enable the creation of powerful and cost‐effective house sparrow multiplex primer sets for population and parentage studies. They can be used to create a house sparrow linkage map and will aid the identification of quantitative trait loci in passerine species.

Biogeographical patterns and co-occurrence of pathogenic infection across island populations of Berthelot's pipit (Anthus berthelotii)

Pathogens can exert strong selective forces upon host populations. However, before we can make any predictions about the consequences of pathogen-mediated selection, we first need to determine whether patterns of pathogen distribution are consistent over spatiotemporal scales. We used molecular techniques to screen for a variety of blood pathogens (avian malaria, pox and trypanosomes) over a three-year time period across 13 island populations of the Berthelot’s pipit (Anthus berthelotii). This species has only recently dispersed across its range in the North Atlantic, with little subsequent migration, providing an ideal opportunity to examine the causes and effects of pathogenic infection in populations in the early stages of differentiation. We screened 832 individuals, and identified two strains of Plasmodium, four strains of Leucocytozoon, and one pox strain. We found strong differences in pathogen prevalence across populations, ranging from 0 to 65%, and while some fluctuations in prevalence occurred, these differences were largely stable over the time period studied. Smaller, more isolated islands harboured fewer pathogen strains than larger, less isolated islands, indicating that at the population level, colonization and extinction play an important role in determining pathogen distribution. Individual-level analyses confirmed the island effect, and also revealed a positive association between Plasmodium and pox infection, which could have arisen due to dual transmission of the pathogens by the same vectors, or because one pathogen lowers resistance to the other. Our findings, combined with an effect of infection on host body condition, suggest that Berthelot’s pipits are subject to different levels of pathogen-mediated selection both across and within populations, and that these selective pressures are consistent over time.

Museum DNA reveals the demographic history of the endangered Seychelles warbler

The importance of evolutionary conservation – how understanding evolutionary forces can help guide conservation decisions – is widely recognized. However, the historical demography of many endangered species is unknown, despite the fact that this can have important implications for contemporary ecological processes and for extinction risk. Here, we reconstruct the population history of the Seychelles warbler (Acrocephalus sechellensis) – an ecological model species. By the 1960s, this species was on the brink of extinction, but its previous history is unknown. We used DNA samples from contemporary and museum specimens spanning 140 years to reconstruct bottleneck history. We found a 25% reduction in genetic diversity between museum and contemporary populations, and strong genetic structure. Simulations indicate that the Seychelles warbler was bottlenecked from a large population, with an ancestral Ne of several thousands falling to <50 within the last century. Such a rapid decline, due to anthropogenic factors, has important implications for extinction risk in the Seychelles warbler, and our results will inform conservation practices. Reconstructing the population history of this species also allows us to better understand patterns of genetic diversity, inbreeding and promiscuity in the contemporary populations. Our approaches can be applied across species to test ecological hypotheses and inform conservation.