Island demographics and trait associations in white-tailed deer.

When a population is isolated and composed of few individuals, genetic drift is the paramount evolutionary force and results in the loss of genetic diversity. Inbreeding might also occur, resulting in genomic regions that are identical by descent, manifesting as runs of homozygosity (ROHs) and the expression of recessive traits. Likewise, the genes underlying traits of interest can be revealed by comparing fixed SNPs and divergent haplotypes between affected and unaffected individuals. Populations of white-tailed deer (Odocoileus virginianus) on islands of Saint Pierre and Miquelon (SPM, France) have high incidences of leucism and malocclusions, both considered genetic defects; on the Florida Keys islands (USA) deer exhibit smaller body sizes, a polygenic trait. Here we aimed to reconstruct island demography and identify the genes associated with these traits in a pseudo case-control design. The two island populations showed reduced levels of genomic diversity and a build-up of deleterious mutations compared to mainland deer; there was also significant genome-wide divergence in Key deer. Key deer showed higher inbreeding levels, but not longer ROHs, consistent with long-term isolation. We identified multiple trait-related genes in ROHs including LAMTOR2 which has links to pigmentation changes, and NPVF which is linked to craniofacial abnormalities. Our mixed approach of linking ROHs, fixed SNPs and haplotypes matched a high number (~50) of a-priori body size candidate genes in Key deer. This suite of biomarkers and candidate genes should prove useful for population monitoring, noting all three phenotypes show patterns consistent with a complex trait and non-Mendelian inheritance.

Epigenetic clocks, sex markers and age-class diagnostics in three harvested large mammals.

The development of epigenetic clocks, or the DNA methylation-based inference of age, is an emerging tool for ageing in free ranging populations. In this study, we developed epigenetic clocks for three species of large mammals that are the focus of extensive management throughout their range in North America: white-tailed deer, black bear and mountain goat. We quantified differential DNA methylation patterns at over 30,000 cytosine-guanine sites (CpGs) from tissue samples of all three species (black bear n = 49; white-tailed deer n = 47; mountain goat n = 45). We used a penalized regression model (elastic net) to build explanatory (black bear r = .95; white-tailed deer r = .99; mountain goat r = .97) and robust (black bear Median Absolute Error or MAE = 1.33; white-tailed deer MAE = 0.29; mountain goat MAE = 0.61) models of age or clocks. We also characterized individual CpG sites within each species that demonstrated clear differences in methylation levels between age classes and sex, which can be used to develop a suite of accessible diagnostic markers. This tool has the potential to contribute to wildlife monitoring by providing easily obtainable representations of age structure in managed populations.

Genomic Analyses Capture the Human-Induced Demographic Collapse and Recovery in a Wide-Ranging Cervid.

The glacial cycles of the Quaternary heavily impacted species through successions of population contractions and expansions. Similarly, populations have been intensely shaped by human pressures such as unregulated hunting and land use changes. White-tailed and mule deer survived in different refugia through the Last Glacial Maximum, and their populations were severely reduced after the European colonization. Here, we analyzed 73 resequenced deer genomes from across their North American range to understand the consequences of climatic and anthropogenic pressures on deer demographic and adaptive history. We found strong signals of climate-induced vicariance and demographic decline; notably, multiple sequentially Markovian coalescent recovers a severe decline in mainland white-tailed deer effective population size (Ne) at the end of the Last Glacial Maximum. We found robust evidence for colonial overharvest in the form of a recent and dramatic drop in Ne in all analyzed populations. Historical census size and restocking data show a clear parallel to historical Ne estimates, and temporal Ne/Nc ratio shows patterns of conservation concern for mule deer. Signatures of selection highlight genes related to temperature, including a cold receptor previously highlighted in woolly mammoth. We also detected immune genes that we surmise reflect the changing land use patterns in North America. Our study provides a detailed picture of anthropogenic and climatic-induced decline in deer diversity and clues to understanding the conservation concerns of mule deer and the successful demographic recovery of white-tailed deer.

Contrasting whole-genome and reduced representation sequencing for population demographic and adaptive inference: an alpine mammal case study.

Genomes capture the adaptive and demographic history of a species, but the choice of sequencing strategy and sample size can impact such inferences. We compared whole genome and reduced representation sequencing approaches to study the population demographic and adaptive signals of the North American mountain goat (Oreamnos americanus). We applied the restriction site-associated DNA sequencing (RADseq) approach to 254 individuals and whole genome resequencing (WGS) approach to 35 individuals across the species range at mid-level coverage (9X) and to 5 individuals at high coverage (30X). We used ANGSD to estimate the genotype likelihoods and estimated the effective population size (Ne), population structure, and explicitly modelled the demographic history with δaδi and MSMC2. The data sets were overall concordant in supporting a glacial induced vicariance and extremely low Ne in mountain goats. We evaluated a set of climatic variables and geographic location as predictors of genetic diversity using redundancy analysis. A moderate proportion of total variance (36% for WGS and 21% for RADseq data sets) was explained by geography and climate variables; both data sets support a large impact of drift and some degree of local adaptation. The empirical similarities of WGS and RADseq presented herein reassuringly suggest that both approaches will recover large demographic and adaptive signals in a population; however, WGS offers several advantages over RADseq, such as inferring adaptive processes and calculating runs-of-homozygosity estimates. Considering the predicted climate-induced changes in alpine environments and the genetically depauperate mountain goat, the long-term adaptive capabilities of this enigmatic species are questionable.

Genomic health is dependent on long-term population demographic history.

Current genetic methods of population assessment in conservation biology have been challenged by genome-scale analyses due to their quantitatively novel insights. These analyses include assessments of runs-of-homozygosity (ROH), genomic evolutionary rate profiling (GERP), and mutational load. Here, we aim to elucidate the relationships between these measures using three divergent ungulates: white-tailed deer, caribou, and mountain goat. The white-tailed deer is currently expanding, while caribou are in the midst of a significant decline. Mountain goats remain stable, having suffered a large historical bottleneck. We assessed genome-wide signatures of inbreeding using the inbreeding coefficient F and %ROH (FROH ) and identified evolutionarily constrained regions with GERP. Mutational load was estimated by identifying mutations in highly constrained elements (CEs) and sorting intolerant from tolerant (SIFT) mutations. Our results showed that F and FROH are higher in mountain goats than in caribou and white-tailed deer. Given the extended bottleneck and low Ne of the mountain goat, this supports the idea that the genome-wide effects of demographic change take time to accrue. Similarly, we found that mountain goats possess more highly constrained CEs and the lowest dN/dS values, both of which are indicative of greater purifying selection; this is also reflected by fewer mutations in CEs and deleterious mutations identified by SIFT. In contrast, white-tailed deer presented the highest mutational load with both metrics, in addition to dN/dS, while caribou were intermediate. Our results demonstrate that extended bottlenecks may lead to reduced diversity and increased FROH in ungulates, but not necessarily an increase in mutational load, probably due to the purging of deleterious alleles in small populations.

Epigenetics and island-mainland divergence in an insectivorous small mammal.

Geographically isolated populations, specifically island-mainland counterparts, tend to exhibit phenotypic variation in many species. The so-called island syndrome occurs when different environmental pressures lead to insular divergence from mainland populations. This phenomenon can be seen in an island population of Nova Scotia masked shrews (Sorex cinereus), which have developed a specialized feeding habit and digestive enzyme compared to their mainland counterparts. Epigenetic modifications, such as DNA methylation (DNAm), can impact phenotypes by altering gene expression without changing the DNA sequence. Here, we used a de novo masked shrew genome assembly and a mammalian methylation array profiling 37 thousand conserved CpGs to investigate morphological and DNA methylation patterns between island and mainland populations. Island shrews were morphologically and epigenetically different than their mainland counterparts, exhibiting a smaller body size. A gene ontology enrichment analyses of differentially methylated CpGs implicated developmental and digestive system related pathways. Based on our shrew epigenetic clock, island shrews might also be aging faster than their mainland counterparts. This study provides novel insight on phenotypic and epigenetic divergence in island-mainland mammal populations and suggests an underlying role of methylation in island-mainland divergence.

Characterizing nutritional phenotypes using experimental nutrigenomics: Is there nutrient-specificity to different types of dietary stress?

The ability to directly measure and monitor poor nutrition in individual animals and ecological communities is hampered by methodological limitations. In this study, we use nutrigenomics to identify nutritional biomarkers in a freshwater zooplankter, Daphnia pulex, a ubiquitous primary consumer in lakes and a sentinel of environmental change. We grew animals in six ecologically relevant nutritional treatments: nutrient replete, low carbon (food), low phosphorus, low nitrogen, low calcium and high Cyanobacteria. We extracted RNA for transcriptome sequencing to identify genes that were nutrient responsive and capable of predicting nutritional status with a high degree of accuracy. We selected a list of 125 candidate genes, which were subsequently pruned to 13 predictive potential biomarkers. Using a nearest-neighbour classification algorithm, we demonstrate that these potential biomarkers are capable of classifying our samples into the correct nutritional group with 100% accuracy. The functional annotation of the selected biomarkers revealed some specific nutritional pathways and supported our hypothesis that animal responses to poor nutrition are nutrient specific and not simply different presentations of slow growth or energy limitation. This is a key step in uncovering the causes and consequences of nutritional limitation in animal consumers and their responses to small- and large-scale changes in biogeochemical cycles.

Speciation without gene-flow in hybridizing deer.

Under the ecological speciation model, divergent selection acts on ecological differences between populations, gradually creating barriers to gene flow and ultimately leading to reproductive isolation. Hybridisation is part of this continuum and can both promote and inhibit the speciation process. Here, we used white-tailed (Odocoileus virginianus) and mule deer (O. hemionus) to investigate patterns of speciation in hybridizing sister species. We quantified genome-wide historical introgression and performed genome scans to look for signatures of four different selection scenarios. Despite ample modern evidence of hybridisation, we found negligible patterns of ancestral introgression and no signatures of divergence with gene flow, rather localized patterns of allopatric and balancing selection were detected across the genome. Genes under balancing selection were related to immunity, MHC and sensory perception of smell, the latter of which is consistent with deer biology. The deficiency of historical gene-flow suggests that white-tailed and mule deer were spatially separated during the glaciation cycles of the Pleistocene and genome wide differentiation accrued via genetic drift. Dobzhansky-Muller incompatibilities and selection against hybrids are hypothesised to be acting, and diversity correlations to recombination rates suggests these sister species are far along the speciation continuum.

Genomic correlates for migratory direction in a free-ranging cervid.

Animal migrations are some of the most ubiquitous and one of the most threatened ecological processes globally. A wide range of migratory behaviours occur in nature, and this behaviour is not uniform among and within species, where even individuals in the same population can exhibit differences. While the environment largely drives migratory behaviour, it is necessary to understand the genetic mechanisms influencing migration to elucidate the potential of migratory species to cope with novel conditions and adapt to environmental change. In this study, we identified genes associated with a migratory trait by undertaking pooled genome-wide scans on a natural population of migrating mule deer. We identified genomic regions associated with variation in migratory direction, including FITM1, a gene linked to the formation of lipids, and DPPA3, a gene linked to epigenetic modifications of the maternal line. Such a genetic basis for a migratory trait contributes to the adaptive potential of the species and might affect the flexibility of individuals to change their behaviour in the face of changes in their environment.

Demographic history and conservation genomics of caribou (Rangifer tarandus) in Québec.

The loss of genetic diversity is a challenge many species are facing, with genomics being a potential tool to inform and prioritize decision-making. Most caribou (Rangifer tarandus) populations have experienced significant recent declines throughout Québec, Canada, and are considered of concern, threatened or endangered. Here, we calculated the ancestral and contemporary patterns of genomic diversity of five representative caribou populations and applied a comparative population genomics framework to assess the interplay between demographic events and genomic diversity. We first calculated a caribou specific mutation rate, µ, by extracting orthologous genes from related ungulates and estimating the rate of synonymous mutations. Whole genome re-sequencing was then completed on 67 caribou: from these data we calculated nucleotide diversity, θ π and estimated the coalescent or ancestral effective population size (N e), which ranged from 12,030 to 15,513. When compared to the census size, N C, the endangered Gaspésie Mountain caribou population had the highest ancestral N e:N C ratio which is consistent with recent work suggesting high ancestral N e:N C is of conservation concern. In contrast, values of contemporary N e, estimated from linkage-disequilibrium, ranged from 11 to 162, with Gaspésie having among the highest contemporary N e:N C ratio. Importantly, classic conservation genetics theory would predict this population to be of less concern based on this ratio. Interestingly, F varied only slightly between populations, and despite evidence of bottlenecks across the province, runs of homozygosity were not abundant in the genome. Tajima's D estimates mirrored the demographic models and current conservation status. Our study highlights how genomic patterns are nuanced and potentially misleading if viewed only through a contemporary lens; we argue a holistic conservation genomics view should integrate ancestral N e and Tajima's D into management decisions.

Using total RNA quality metrics for time since deposition estimates in degrading bloodstains.

The physicochemical changes occurring in biomolecules in degrading bloodstains can be used to approximate the time since deposition (TSD) of bloodstains. This would provide forensic scientists with critical information regarding the timeline of the events involving bloodshed. Our study aims to quantify the timewise degradation trends and temperature dependence found in total RNA from bloodstains without the use of amplification, expanding the scope of the RNA TSD research which has traditionally targeted mRNA and miRNA. Bovine blood with ACD-A anticoagulant was deposited and stored in plastic microcentrifuge tubes at 21 or 4°C and tested over different timepoints spanning 1 week. Total RNA was extracted from each sample and analyzed using automated high sensitivity gel electrophoresis. Nine RNA metrics were visually assessed and quantified using linear and mixed models. The RNA Integrity Number equivalent (RINe) and DV200 were not influenced by the addition of anticoagulant and demonstrated strong negative trends over time. The RINe model fit was high (R2  = 0.60), and while including the biological replicate as a random effect increased the fit for all RNA metrics, no significant differences were found between biological replicates stored at the same temperature for the RINe and DV200. This suggests that these standardized metrics can be directly compared between scenarios and individuals, with DV200 having an inflection point at approximately 28 h. This study provides a novel approach for blood TSD research, revealing metrics that are not affected by inter-individual variation, and improving our understanding of the rapid RNA degradation occurring in bloodstains.

Genomic diversity and demographic history of the Dromiciops genus (Marsupialia: Microbiotheriidae).

Three orders represent the South American fauna of marsupials. Of these, Microbiotheria was until recently known as a monotypic genus with the only surviving species Dromiciops gliroides (monito del monte). The recent proposal of a new Dromiciops species (Dromiciops bozinovici), together with new information on the origin and diversification of living microbioterians has changed the prevailing paradigm around the evolutionary history of these emblematic marsupials. Here, we used a RADseq approach to test for evidence of admixture and past or current gene flow among both species of Dromiciops and evaluate the genetic structure within D. gliroides. We analyzed 127 samples of Dromiciops distributed across the known distribution range of both species. We also inferred the joint demographic history of these lineages, thus corroborating the status of D. bozinovici as a distinct species. Demographic history reconstruction indicated that D. bozinovici diverged from D. gliroides around 4my ago and has remained isolated and demographically stable ever since. In contrast, D. gliroides is subdivided into three subclades that experienced recent expansions and moderate gene flow among them (mostly from north to south). Furthermore, genetic distances among populations within D. gliroides were significantly correlated with geographic distances. These results suggest that some of the D. gliroides populations would have survived in glacial refuges, with posterior expansions after ice retreat. Our results have important implications for the systematics of the genus and have profound conservation consequences for the new species, especially considering the fragmentation level of the temperate rainforest.

Genome assembly, annotation, and comparative analysis of the cattail Typha latifolia.

Cattails (Typha species) comprise a genus of emergent wetland plants with a global distribution. Typha latifolia and Typha angustifolia are two of the most widespread species, and in areas of sympatry can interbreed to produce the hybrid Typha × glauca. In some regions, the relatively high fitness of Typha × glauca allows it to outcompete and displace both parent species, while simultaneously reducing plant and invertebrate biodiversity, and modifying nutrient and water cycling. We generated a high-quality whole-genome assembly of T. latifolia using PacBio long-read and high coverage Illumina sequences that will facilitate evolutionary and ecological studies in this hybrid zone. Genome size was 287 Mb and consisted of 1158 scaffolds, with an N50 of 8.71 Mb; 43.84% of the genome were identified as repetitive elements. The assembly has a BUSCO score of 96.03%, and 27,432 genes and 2700 RNA sequences were putatively identified. Comparative analysis detected over 9000 shared orthologs with related taxa and phylogenomic analysis supporting T. latifolia as a divergent lineage within Poales. This high-quality scaffold-level reference genome will provide a useful resource for future population genomic analyses and improve our understanding of Typha hybrid dynamics.

Characterizing the demographic history and prion protein variation to infer susceptibility to chronic wasting disease in a naïve population of white-tailed deer (Odocoileus virginianus).

Assessments of the adaptive potential in natural populations are essential for understanding and predicting responses to environmental stressors like climate change and infectious disease. Species face a range of stressors in human-dominated landscapes, often with contrasting effects. White-tailed deer (Odocoileus virginianus; deer) are expanding in the northern part of their range following decreasing winter severity and increasing forage availability. Chronic wasting disease (CWD), a prion disease affecting deer, is likewise expanding and represents a major threat to deer and other cervids. We obtained tissue samples from free-ranging deer across their native range in Ontario, Canada, which has yet to detect CWD in wild populations. We used high-throughput sequencing to assess neutral genomic variation and variation in the prion protein gene (PRNP) that is partly responsible for the protein misfolding when deer contract CWD. Neutral variation revealed a high number of rare alleles and no population structure, and demographic models suggested a rapid historical population expansion. Allele frequencies of PRNP variants associated with CWD susceptibility and disease progression were evenly distributed across the landscape and consistent with deer populations not infected with CWD. We estimated the selection coefficient of CWD, with simulations showing an observable and rapid shift in PRNP allele frequencies that coincides with the start of a novel CWD outbreak. Sustained surveillance of genomic and PRNP variation can be a useful tool for guiding management practices, which is especially important for CWD-free regions where deer are managed for ecological and economic benefits.

The biogeography of Dromiciops in southern South America: Middle Miocene transgressions, speciation and associations with Nothofagus.

The current distribution of the flora and fauna of southern South America is the result of drastic geological events that occurred during the last 20 million years, including marine transgressions, glaciations and active vulcanism. All these have been associated with fragmentation, isolation and subsequent expansion of the biota, south of 35°S, such as the temperate rainforest. This forest is mostly dominated by Nothofagus trees and is the habitat of the relict marsupial monito del monte, genus Dromiciops, sole survivor of the order Microbiotheria. Preliminary analyses using mtDNA proposed the existence of three main Dromiciops lineages, distributed latitudinally, whose divergence was initially attributed to recent Pleistocene glaciations. Using fossil-calibrated dating on nuclear and mitochondrial genes, here we reevaluate this hypothesis and report an older (Miocene) biogeographic history for the genus. We performed phylogenetic reconstructions using sequences from two mitochondrial DNA and four nuclear DNA genes in 159 specimens from 31 sites across Chile and Argentina. Our phylogenetic analysis resolved three main clades with discrete geographic distributions. The oldest and most differentiated clade corresponds to that of the northern distribution (35.2°S to 39.3°S), which should be considered a distinct species (D. bozinovici, sensu D'Elía et al. 2016). According to our estimations, this species shared a common ancestor with D. gliroides (southern clades) about ~13 million years ago. Divergence time estimates for the southern clades (39.6°S to 42.0°S) ranged from 9.57 to 6.5 Mya. A strong genetic structure was also detected within and between clades. Demographic analyses suggest population size stability for the northern clade (D. bozinovici), and recent demographic expansions for the central and southern clades. All together, our results suggest that the diversification of Dromiciops were initiated by the Middle Miocene transgression (MMT), the massive marine flooding that covered several lowlands of the western face of Los Andes between 37 and 48°S. The MMT resulted from an increase in global sea levels at the Miocene climatic optimum, which shaped the biogeographic origin of several species, including Nothofagus forests, the habitat of Dromiciops.

Whole bovine blood use in forensic research: Sample preparation and storage considerations.

Mammalian whole blood sources are often used for forensic research and training when human samples cannot be sourced. While porcine, ovine and equine blood have been shown to be viable alternatives to whole human blood for forensic purposes, procurement can still pose a problem, especially for smaller and remote institutions. This work explores the use of whole bovine blood for basic bloodstain simulation. Sample preparation through the addition of ACD-A anticoagulant was optimized and storability was explored. Viscosity, surface tension, density, and packed cell volume, four fluid properties relevant to bloodstain pattern analysis, were monitored over four days and in two temperature conditions. Linear mixed models accounting for variation in the donor demonstrated that these fluid properties of the bovine blood changed predictably over time and with temperature. Whole bovine blood with 12.5% v/v ACD-A was found to be viable for use in basic bloodstain simulation at ambient and physiological temperature.

Gut microbiome composition predicts summer core range size in two divergent ungulates.

The gut microbiome of animals vary by age, diet, and habitat, and directly influences an individual's health. Similarly, variation in home ranges is linked to feeding strategies and fitness. Ungulates (hooved mammals) exhibit species-specific microbiomes and habitat use patterns. We combined gut microbiome and movement data to assess relationships between space use and the gut microbiome in a specialist and a generalist ungulate. We GPS radiocollared 24 mountain goats (Oreamnos americanus) and 34 white-tailed deer (Odocoileus virginianus), collected fecal samples, and conducted high-throughput sequencing of the 16S rRNA gene. We generated gut diversity metrics and key bacterial ratios. Our research question centred around the idea that larger Firmicutes to Bacteroidetes ratios confer body size or fat advantages that allow for larger home ranges, and relationships of disproportionate habitat use are stronger in the habitat specialist mountain goat. Firmicutes to Bacteroidetes ratios were positively correlated with core range area in both species. Mountain goats exhibited a negative relationship between gut diversity and proportional use of treed areas and escape terrain, and no relationships were detected in the habitat generalist white-tailed deer. This is the first study to relate range size to the gut microbiome in wild ungulates and is an important proof of concept that advances the information that can be gleaned from non-invasive sampling.

Quantifying visible absorbance changes and DNA degradation in aging bloodstains under extreme temperatures.

Physicochemical property changes observed in a degrading bloodstain can be used to estimate its time since deposition (TSD) and provide a timestamp to the sample's age. Many of the time-dependent processes that occur as a bloodstain degrades, such as DNA fragmentation and changes in hemoglobin structure, also exhibit temperature-dependent behaviours. Previous studies have demonstrated that pairing high-resolution automated gel electrophoresis and visible absorbance spectroscopy could be used to quantify the rate of degradation of a bloodstain in relation to time and storage substrate. Our study investigates such trends with an added factor, extreme temperatures. Passive drip stains were stored in either microcentrifuge tubes or on FTA cards at either -20°C, 21°C or 40°C and tested over 11 time points spanning 15 days. For both storage substrates, the wavelength at maximum absorbance for the Soret band and the maximum absorbance of the Alpha band showed a negative trend over time suggesting that spectral shifts are informative for TSD estimates. The ratio of the maximum peak height for DNA fragments lengths of 500-1000 base pairs to 1000-5000 base pairs was the most informative DNA variable in relation to time for both substrates. Cross-validation suggested the appropriate fit of the models with the data and reasonable predictive ability. We integrated both DNA concentration and hemoglobin visible absorbance metrics using principal component analysis (PCA) into a single model. Adding the random effect of the donor to the PCA model accounted for a large portion of the variation as did storage method and temperature. Additionally, canonical correspondence showed that temperature corresponded differently to the response variables for FTA card and microcentrifuge tube samples, suggesting a substrate specific effect. This study confirms that pairing DNA concentration and hemoglobin's visible absorbance can provide insight on the effect of different environmental and storage conditions on bloodstain degradation. While the level of uncertainty surrounding TSD estimates still precludes its use in the field, this study provides a valuable framework that improves our understanding of variation surrounding TSD estimates, which will be critical to any eventual application.