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Pathogens are recognized as major drivers of local adaptation in wildlife systems. By determining which gene variants are favored in local interactions among populations with and without disease, spatially explicit adaptive responses to pathogens can be elucidated. Much of our current understanding of host responses to disease comes from a small number of genes associated with an immune response. High-throughput sequencing ( HTS) technologies, such as genotype-by-sequencing ( GBS), facilitate expanded explorations of genomic variation among populations. Hybridization-based GBS techniques can be leveraged in systems not well characterized for specific variants associated with disease outcome to 'capture' specific genes and regulatory regions known to influence expression and disease outcome. We developed a multiplexed, sequence capture assay for red foxes to simultaneously assess ~300-kbp of genomic sequence from 116 adaptive, intrinsic, and innate immunity genes of predicted adaptive significance and their putative upstream regulatory regions along with 23 neutral microsatellite regions to control for demographic effects. The assay was applied to 45 fox DNA samples from Alaska, where three arctic rabies strains are geographically restricted and endemic to coastal tundra regions, yet absent from the boreal interior. The assay provided 61.5% on-target enrichment with relatively even sequence coverage across all targeted loci and samples (mean = 50×), which allowed us to elucidate genetic variation across introns, exons, and potential regulatory regions (4,819 SNPs). Challenges remained in accurately describing microsatellite variation using this technique; however, longer-read HTS technologies should overcome these issues. We used these data to conduct preliminary analyses and detected genetic structure in a subset of red fox immune-related genes between regions with and without endemic arctic rabies. This assay provides a template to assess immunogenetic variation in wildlife disease systems. [ABSTRACT FROM AUTHOR]

Interglacial-glacial cycles of the Quaternary are widely recognized in shaping phylogeographic structure. Patterns from cold adapted species can be especially informative - in particular, uncovering additional glacial refugia, identifying likely recolonization patterns, and increasing our understanding of species’ responses to climate change. We investigated phylogenetic structure of the wolverine, a wide-ranging cold adapted carnivore, using a 318 bp of the mitochondrial DNA control region for 983 wolverines (n = 209 this study, n = 774 from GenBank) from across their full Holarctic distribution. Bayesian phylogenetic tree reconstruction and the distribution of observed pairwise haplotype differences (mismatch distribution) provided evidence of a single rapid population expansion across the wolverine’s Holarctic range. Even though molecular evidence corroborated a single refugium, significant subdivisions of population genetic structure (0.01< ΦST <0.99, P<0.05) were detected. Pairwise ΦST estimates separated Scandinavia from Russia and Mongolia, and identified five main divisions within North America - the Central Arctic, a western region, an eastern region consisting of Ontario and Quebec/Labrador, Manitoba, and California. These data are in contrast to the nearly panmictic structure observed in northwestern North America using nuclear microsatellites, but largely support the nuclear DNA separation of contemporary Manitoba and Ontario wolverines from northern populations. Historic samples (c. 1900) from the functionally extirpated eastern population of Quebec/Labrador displayed genetic similarities to contemporary Ontario wolverines. To understand these divergence patterns, four hypotheses were tested using Approximate Bayesian Computation (ABC). The most supported hypothesis was a single Beringia incursion during the last glacial maximum that established the northwestern population, followed by a west-to-east colonization during the Holocene. This pattern is suggestive of colonization occurring in accordance with glacial retreat, and supports expansion from a single refugium. These data are significant relative to current discussions on the conservation status of this species across its range. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The correlation of landscape features with genetic discontinuities reveals barriers to dispersal that can contribute to understanding present and future spread of wildlife diseases. This knowledge can then be used for targeting control efforts. The impact of natural barriers on raccoon dispersal was assessed through genetic analysis of samples from two regions, Niagara ( N = 666) and St. Lawrence ( N = 802). These areas are transected by major rivers and are at the northern front of a raccoon rabies epizootic. Genetic clusters were identified in each region using Bayesian clustering algorithms. In the Niagara region, two clusters were identified corresponding to either side of the Niagara River. For the St. Lawrence region, spatially congruent clusters were not identified, despite the presence of the intervening St. Lawrence River. These genetic data are consistent with raccoon rabies incidence data where rabies has been detected across the St. Lawrence River in Ontario while no cases have been detected in Ontario across the Niagara River. This is despite expectations of rabies incidence in Niagara before the St. Lawrence based on the progression of rabies from New York. The results from the two regions suggest different permeabilities to raccoons between New York and Ontario that may be attributed to the rivers. However, other factors have also been explored that could contribute to this difference between these study sites including the shape of the landscape and resource distribution. [ABSTRACT FROM AUTHOR]

Frog virus 3 (FV3) and FV3-like ranaviruses can infect a variety of cold-blooded aquatic species and present a primary threat to amphibians across the globe. Previous studies of FV3-like viruses have largely investigated higher-level phylogenetic distinctions of these pathogens via portions of the conserved major capsid protein (MCP), and the putative virulence gene vIF-2α. Few studies, however, have investigated the spatial distribution of FV3 variants at the population level3-data that can be used to further understand the spatial epidemiology of this disease. In this study, we sequenced the MCP and vIF-2α of 127 FV3-positive amphibians sampled from Canadian water bodies in Ontario, northeastern Alberta, and southern Northwest Territories to explore whether intraspecific genetic variation exists within FV3. There was a lack of variation at the 2 markers across these regions, suggesting that there is a lack of FV3 sequence diversity in Canada, which may hint at a single source of infection that has spread. However, an undocumented variant termed Wood Buffalo ranavirus (WBRV) was detected in samples from 3 sites in Alberta and Northwest Territories that clustered within the FV3-like lineage with 99.3% sequence homology for MCP. For vIF-2α, all sequences were the expected truncated variant except for 6 samples in Ontario. These latter sequences were suggestive of recombination with common midwife toad virus (CMTV). The lack of variation suggests that higher-resolution genome analyses will be required to further explore the spatial spread and intraspecific variation of the disease.