Development of a high-density sub-species-specific targeted SNP assay for Rocky Mountain bighorn sheep (Ovis canadensis canadensis).

Due to their abundance and relative ease of genotyping, single nucleotide polymorphisms (SNPs) are a commonly used molecular marker for contemporary population genetic and genomic studies. A high-density and cost-effective way to type SNP loci is Allegro targeted genotyping (ATG), which is a form of targeted genotyping by sequencing developed and offered by Tecan genomics. One major drawback of this technology is the need for a reference genome and information on SNP loci when designing a SNP assay. However, for some non-model species genomic information from other closely related species can be used. Here we describe our process of developing an ATG assay to target 50,000 SNPs in Rocky Mountain bighorn sheep, using a reference genome from domestic sheep and SNP resources from prior bighorn sheep studies. We successfully developed a high accuracy, high-density, and relatively low-cost SNP assay for genotyping Rocky Mountain bighorn sheep that genotyped ~45,000 SNP loci. These loci were relatively evenly distributed throughout the genome. Furthermore, the assay produced genotypes at tens of thousands of SNP loci when tested on other mountain sheep species and subspecies.

Sexes in sync: phenotypic plasticity, sexual selection and phenological synchrony between the sexes in a wild hibernator.

Desynchrony of phenological responses to climate change is a major concern for ecological communities. Potential uncoupling between one of the most fundamental divisions within populations, males and females, has not been well studied. To address this gap, we examined sex-specific plasticity in hibernation phenology in two populations of Columbian ground squirrels (Urocitellus columbianus). We find that both sexes display similar phenological plasticity to spring snowmelt dates in their timing of torpor termination and behavioural emergence from hibernation. As a result of this plasticity, the degree of protandry (i.e. males' emergences from hibernation preceding those of females) did not change significantly over the 27-year study. Earlier male behavioural emergence, relative to females, improved the likelihood of securing a breeding territory and increased annual reproductive success. Sexual selection favouring earlier male emergence from hibernation may maintain protandry in this population, but did not contribute to further advances in male phenology. Together, our results provide evidence that the sexes should remain synchronized, at least in response to the weather variation investigated here, and further support the role of sexual selection in the evolution of protandry in sexually reproducing organisms.

Benefits of living closer to kin vary by genealogical relationship in a territorial mammal.

While cooperative interactions among kin are a key building block in the societies of group-living species, their importance for species with more variable social environments is unclear. North American red squirrels (Tamiasciurus hudsonicus) defend individual territories in dynamic neighbourhoods and are known to benefit from living among familiar conspecifics, but not relatives. However, kin-directed behaviours may be restricted to specific genealogical relationships or strongly mediated by geographical distance, masking their influence at broader scales. Using distance between territories as a proxy for the ability of individuals to interact, we estimated the influence of primary kin (parents, offspring, siblings) on the annual survival and reproductive success of red squirrels. This approach revealed associations between fitness and access to kin, but only for certain genealogical relationships and fitness components. For example, females had enhanced annual survival when living closer to their daughters, though the reverse was not true. Most surprising was the finding that males had higher annual reproductive success when living closer to their father, suggesting possible recognition and cooperation among fathers and sons. Together, these findings point to unexpected nuance in the fitness consequences of kinship dynamics for a species that is territorial and largely solitary.

Genetic variance in fitness indicates rapid contemporary adaptive evolution in wild animals.

The rate of adaptive evolution, the contribution of selection to genetic changes that increase mean fitness, is determined by the additive genetic variance in individual relative fitness. To date, there are few robust estimates of this parameter for natural populations, and it is therefore unclear whether adaptive evolution can play a meaningful role in short-term population dynamics. We developed and applied quantitative genetic methods to long-term datasets from 19 wild bird and mammal populations and found that, while estimates vary between populations, additive genetic variance in relative fitness is often substantial and, on average, twice that of previous estimates. We show that these rates of contemporary adaptive evolution can affect population dynamics and hence that natural selection has the potential to partly mitigate effects of current environmental change.

Ewe are what ewe wear: bigger horns, better ewes and the potential consequence of trophy hunting on female fitness in bighorn sheep.

In polygynous species, secondary sexual traits such as weapons or elaborate ornaments have evolved through intrasexual competition for mates. In some species, these traits are present in both sexes but are underdeveloped in the sex facing lower intrasexual competition for mates. It is often assumed that these underdeveloped sexually selected traits are a vestige of strong sexual selection on the other sex. Here, we challenge this assumption and investigate whether the expression of secondary sexual traits is associated with fitness in female bighorn sheep. Analyses of 45 years of data revealed that female horn length at 2 years, while accounting for mass and environmental variables, is associated with younger age at primiparity, younger age of first offspring weaned, greater reproductive lifespan and higher lifetime reproductive success. There was no association between horn length and fecundity. These findings highlight a potential conservation issue. In this population, trophy hunting selects against males with fast-growing horns. Intersexual genetic correlations imply that intense selective hunting of large-horned males before they can reproduce can decrease female horn size. Therefore, intense trophy hunting of males based on horn size could reduce female reproductive performance through the associations identified here, and ultimately reduce population growth and viability.

Ancient hybridization patterns between bighorn and thinhorn sheep.

Whole-genome sequencing has advanced the study of species evolution, including the detection of genealogical discordant events such as ancient hybridization and incomplete lineage sorting (ILS). The evolutionary history of bighorn (Ovis canadensis) and thinhorn (Ovis dalli) sheep present an ideal system to investigate evolutionary discordance due to their recent and rapid radiation and putative secondary contact between bighorn and thinhorn sheep subspecies, specifically the dark pelage Stone sheep (O. dalli stonei) and predominately white Dall sheep (O. dalli dalli), during the last ice age. Here, we used multiple genomes of bighorn and thinhorn sheep, together with snow (O. nivicola) and the domestic sheep (O. aries) as outgroups, to assess their phylogenomic history, potential introgression patterns and their adaptive consequences. Among the Pachyceriforms (snow, bighorn and thinhorn sheep) a consistent monophyletic species tree was retrieved; however, many genealogical discordance patterns were observed. Alternative phylogenies frequently placed Stone and bighorn as sister clades. This relationship occurred more often and was less divergent than that between Dall and bighorn. We also observed many blocks containing introgression signal between Stone and bighorn genomes in which coat colour genes were present. Introgression signals observed between Dall and bighorn were more random and less frequent, and therefore probably due to ILS or intermediary secondary contact. These results strongly suggest that Stone sheep originated from a complex series of events, characterized by multiple, ancient periods of secondary contact with bighorn sheep.

Whole-genome sequence analysis unveils different origins of European and Asiatic mouflon and domestication-related genes in sheep.

The domestication and subsequent development of sheep are crucial events in the history of human civilization and the agricultural revolution. However, the impact of interspecific introgression on the genomic regions under domestication and subsequent selection remains unclear. Here, we analyze the whole genomes of domestic sheep and their wild relative species. We found introgression from wild sheep such as the snow sheep and its American relatives (bighorn and thinhorn sheep) into urial, Asiatic and European mouflons. We observed independent events of adaptive introgression from wild sheep into the Asiatic and European mouflons, as well as shared introgressed regions from both snow sheep and argali into Asiatic mouflon before or during the domestication process. We revealed European mouflons might arise through hybridization events between a now extinct sheep in Europe and feral domesticated sheep around 6000-5000 years BP. We also unveiled later introgressions from wild sheep to their sympatric domestic sheep after domestication. Several of the introgression events contain loci with candidate domestication genes (e.g., PAPPA2, NR6A1, SH3GL3, RFX3 and CAMK4), associated with morphological, immune, reproduction or production traits (wool/meat/milk). We also detected introgression events that introduced genes related to nervous response (NEURL1), neurogenesis (PRUNE2), hearing ability (USH2A), and placental viability (PAG11 and PAG3) into domestic sheep and their ancestral wild species from other wild species.

Extent and direction of introgressive hybridization of mule and white-tailed deer in western Canada.

Hybridization of mule deer (Odocoileus hemionus) and white-tailed deer (O. virginianus) appears to be a semi-regular occurrence in western North America. Previous studies confirmed the presence of hybrids in a variety of sympatric habitats, but their developing molecular resources limited identification to the earliest, most admixed generations. For this reason, estimates of hybrid production in wild populations often rely on anecdotal reports. As well, white-tailed deer populations' continued encroachment into historically mule deer-occupied habitats due to changes in land use, habitat homogenization, and a warming climate may increase opportunities for interspecific encounters. We sought to quantify the prevalence and extent of hybrid deer in the prairies of western Canada using a SNP assay with enhanced discriminating power. By updating the available molecular resources, we sought to identify and characterize previously cryptic introgression. We also investigated the influence of various parameters on hybridity by way of logistic regression. We observed overall hybridization rates of ~1.0%, slightly lower than that reported by previous studies, and found white-tailed-like hybrids to be more common than their mule deer-like counterparts. Here, we build upon past studies of hybridization in North American deer by increasing hybrid detection power, expanding sample sizes, demonstrating a new molecular resource applicable to future research and observing asymmetrical directionality of introgression.

Determinants and long-term costs of early reproduction in males of a long-lived polygynous mammal.

In long-lived polygynous species, male reproductive success is often monopolized by a few mature dominant individuals. Young males are generally too small to be dominant and may employ alternative tactics; however, little is known about the determinants of reproductive success for young males. Understanding the causes and consequences of variability in early reproductive success may be crucial to assess the strength of sexual selection and possible long-term trade-offs among life-history traits. Selective pressures driven by fluctuating environmental conditions may depend on age class. We evaluated the determinants of reproduction in male bighorn sheep (Ovis canadensis) aged 2-4 years using 30 years of individual-level data. These young males cannot defend estrous ewes and use alternative mating tactics. We also investigated how the age of first detected reproduction was correlated to lifetime reproductive success and longevity. We found that reproductive success of males aged 3 years was positively correlated to body mass, to the proportion of males aged 2-4 years in the competitor pool, and to the number of females available per adult male. These results suggest that reproductive success depends on both competitive ability and population age-sex structure. None of these variables, however, had significant effects on the reproductive success of males aged 2 or 4 years. Known reproduction before the age of five increased lifetime reproductive success but decreased longevity, suggesting a long-term survival cost of early reproduction. Our analyses reveal that both individual-level phenotypic and population-level demographic variables influence reproductive success by young males and provide a rare assessment of fitness trade-offs in wild polygynous males.

Genetic association with boldness and maternal performance in a free-ranging population of grey seals (Halichoerus grypus).

Individual variation in quantitative traits clearly influence many ecological and evolutionary processes. Moderate to high heritability estimates of personality and life-history traits suggest some level of genetic control over these traits. Yet, we know very little of the underlying genetic architecture of phenotypic variation in the wild. In this study, we used a candidate gene approach to investigate the association of genetic variants with repeated measures of boldness and maternal performance traits (weaning mass and lactation duration) collected over an 11- and 28-year period, respectively, in a free-ranging population of grey seals on Sable Island National Park Reserve, Canada. We isolated and re-sequenced five genes: dopamine receptor D4 (DRD4), serotonin transporter (SERT), oxytocin receptor (OXTR), and melanocortin receptors 1 (MC1R) and 5 (MC5R). We discovered single nucleotide polymorphisms (SNPs) in each gene; and, after accounting for loci in linkage disequilibrium and filtering due to missing data, we were able to test for genotype-phenotype relationships at seven loci in three genes (DRD4, SERT, and MC1R). We tested for association between these loci and traits of 180 females having extreme shy-bold phenotypes using mixed-effects models. One locus within SERT was significantly associated with boldness (effect size = 0.189) and a second locus within DRD4 with weaning mass (effect size = 0.232). Altogether, genotypes explained 6.52-13.66% of total trait variation. Our study substantiates SERT and DRD4 as important determinants of behaviour, and provides unique insight into the molecular mechanisms underlying maternal performance variation in a marine predator.

Familiar Neighbors, but Not Relatives, Enhance Fitness in a Territorial Mammal.

One of the outstanding questions in evolutionary biology is the extent to which mutually beneficial interactions and kin selection can facilitate the evolution of cooperation by mitigating conflict between interacting organisms. The indirect fitness benefits gained from associating with kin are an important pathway to conflict resolution,1 but conflict can also be resolved if individuals gain direct benefits from cooperating with one another (e.g., mutualism or reciprocity).2 Because of the kin-structured nature of many animal societies, it has been difficult for previous research to assess the relative importance of these mechanisms.3-5 However, one area that might allow for the relative roles of kin selection and mutualistic benefits to be disentangled is in the resolution of conflict over territorial space.6 Although much research has focused on group-living species, the question of how cooperation can first be favored in solitary, territorial species remains a key question. Using 22 years of data from a population of North American red squirrels, we assessed how kinship and familiarity with neighbors affected fitness in a territorial mammal. Although living near kin did not enhance fitness, familiarity with neighbors increased survival and annual reproductive success. These fitness benefits were strong enough to compensate for the effects of aging later in life and have potential consequences for the evolution of senescence. We suggest that such substantial fitness benefits provide the opportunity for the evolution of cooperation between adversarial neighbors, offering insight into the role that mutually beneficial behaviors might play in facilitating and stabilizing social systems.

Historical Introgression from Wild Relatives Enhanced Climatic Adaptation and Resistance to Pneumonia in Sheep.

How animals, particularly livestock, adapt to various climates and environments over short evolutionary time is of fundamental biological interest. Further, understanding the genetic mechanisms of adaptation in indigenous livestock populations is important for designing appropriate breeding programs to cope with the impacts of changing climate. Here, we conducted a comprehensive genomic analysis of diversity, interspecies introgression, and climate-mediated selective signatures in a global sample of sheep and their wild relatives. By examining 600K and 50K genome-wide single nucleotide polymorphism data from 3,447 samples representing 111 domestic sheep populations and 403 samples from all their seven wild relatives (argali, Asiatic mouflon, European mouflon, urial, snow sheep, bighorn, and thinhorn sheep), coupled with 88 whole-genome sequences, we detected clear signals of common introgression from wild relatives into sympatric domestic populations, thereby increasing their genomic diversities. The introgressions provided beneficial genetic variants in native populations, which were significantly associated with local climatic adaptation. We observed common introgression signals of alleles in olfactory-related genes (e.g., ADCY3 and TRPV1) and the PADI gene family including in particular PADI2, which is associated with antibacterial innate immunity. Further analyses of whole-genome sequences showed that the introgressed alleles in a specific region of PADI2 (chr2: 248,302,667-248,306,614) correlate with resistance to pneumonia. We conclude that wild introgression enhanced climatic adaptation and resistance to pneumonia in sheep. This has enabled them to adapt to varying climatic and environmental conditions after domestication.

Whole-genome resequencing of wild and domestic sheep identifies genes associated with morphological and agronomic traits.

Understanding the genetic changes underlying phenotypic variation in sheep (Ovis aries) may facilitate our efforts towards further improvement. Here, we report the deep resequencing of 248 sheep including the wild ancestor (O. orientalis), landraces, and improved breeds. We explored the sheep variome and selection signatures. We detected genomic regions harboring genes associated with distinct morphological and agronomic traits, which may be past and potential future targets of domestication, breeding, and selection. Furthermore, we found non-synonymous mutations in a set of plausible candidate genes and significant differences in their allele frequency distributions across breeds. We identified PDGFD as a likely causal gene for fat deposition in the tails of sheep through transcriptome, RT-PCR, qPCR, and Western blot analyses. Our results provide insights into the demographic history of sheep and a valuable genomic resource for future genetic studies and improved genome-assisted breeding of sheep and other domestic animals.

The genetic basis of animal behavioural diversity in natural populations.

Individual differences in animal behaviour influence ecological and evolutionary processes. Much behavioural variation has a heritable component, suggesting that genetics may play a role in its development. Yet, the study of the mechanistic description linking genes to behaviour in nature remains in its infancy, and such research is considered a challenge in contemporary biology. Here, we performed a literature review and meta-analysis to assess trends in analytical approaches used to investigate the relationship between genes and behaviour in natural systems, specifically candidate gene approaches, quantitative trait locus (QTL) mapping, and genome-wide association studies (GWAS). We aimed to determine the efficacy and success of each approach, while also describing which behaviours and species were examined by researchers most often. We found that the majority of QTL mapping and GWAS results revealed a significant or suggestive effect (Zr = 0.3 [95% CI: 0.25:0.35] and Zr = 0.39 [0.33:0.46], respectively) between the trait of interest and genetic marker(s) tested, while over half of candidate gene accounts (Zr = 0.16 [0.11:0.21]) did not find a significant association. Approximately a third of all study estimates investigated animal personality traits; though, reproductive and migratory behaviours were also well-represented. Our findings show that despite widespread accessibility of molecular approaches given current sequencing technologies, efforts to elucidate the genetic basis of behaviour in free-ranging systems has been limited to relatively few species. We discuss challenges encountered by researchers, and recommend integration of novel genomic methods with longitudinal studies to usher in the next wave of behavioural genomic research.

Predicting the spread-risk potential of chronic wasting disease to sympatric ungulate species.

Wildlife disease incidence is increasing, resulting in negative impacts on the economy, biodiversity, and potentially human health. Chronic wasting disease (CWD) is a fatal, transmissible spongiform encephalopathy of cervids (wild and captive) which continues to spread geographically resulting in exposure to potential new host species. The disease agent (PrPCWD) is a misfolded conformer of the cellular prion protein (PrPC). In Canada, the disease is endemic in Alberta and Saskatchewan, affecting mule and white-tail deer, with lesser impact on elk and moose. As the disease continues to expand, additional wild ungulate species including bison, bighorn sheep, mountain goat, and pronghorn antelope may be exposed. To better understand the species-barrier, we reviewed the current literature on taxa naturally or experimentally exposed to CWD to identify susceptible and resistant species. We created a phylogeny of these taxa using cytochrome B and found that CWD susceptibility followed the species phylogeny. Using this phylogeny we estimated the probability of CWD susceptibility for wild ungulate species. We then compared PrPC amino acid polymorphisms among these species to identify which sites segregated between susceptible and resistant species. We identified sites that were significantly associated with susceptibility, but they were not fully discriminating. Finally, we sequenced Prnp from 578 wild ungulates to further evaluate their potential susceptibility. Together, these data suggest the host-range for CWD will potentially include pronghorn, mountain goat and bighorn sheep, but bison are likely to be more resistant. These findings highlight the need for monitoring potentially susceptible species as CWD continues to expand.

Linking genotype to phenotype to identify genetic variation relating to host susceptibility in the mountain pine beetle system.

Identifying genetic variants responsible for phenotypic variation under selective pressure has the potential to enable productive gains in natural resource conservation and management. Despite this potential, identifying adaptive candidate loci is not trivial, and linking genotype to phenotype is a major challenge in contemporary genetics. Many of the population genetic approaches commonly used to identify adaptive candidates will simultaneously detect false positives, particularly in nonmodel species, where experimental evidence is seldom provided for putative roles of the adaptive candidates identified by outlier approaches. In this study, we use outcomes from population genetics, phenotype association, and gene expression analyses as multiple lines of evidence to validate candidate genes. Using lodgepole and jack pine as our nonmodel study species, we analyzed 17 adaptive candidate loci together with 78 putatively neutral loci at 58 locations across Canada (N > 800) to determine whether relationships could be established between these candidate loci and phenotype related to mountain pine beetle susceptibility. We identified two candidate loci that were significant across all population genetic tests, and demonstrated significant changes in transcript abundance in trees subjected to wounding or inoculation with the mountain pine beetle fungal associate Grosmannia clavigera. Both candidates are involved in central physiological processes that are likely to be invoked in a trees response to stress. One of these two candidate loci showed a significant association with mountain pine beetle attack status in lodgepole pine. The spatial distribution of the attack-associated allele further coincides with other indicators of susceptibility in lodgepole pine. These analyses, in which population genetics was combined with laboratory and field experimental validation approaches, represent first steps toward linking genetic variation to the phenotype of mountain pine beetle susceptibility in lodgepole and jack pine, and provide a roadmap for more comprehensive analyses.

The new kid on the block: immigrant males win big whereas females pay fitness cost after dispersal.

Dispersal is nearly universal; yet, which sex tends to disperse more and their success thereafter depends on the fitness consequences of dispersal. We asked if lifetime fitness differed between residents and immigrants (successful between-population dispersers) and their offspring using 29 years of monitoring from North American red squirrels (Tamiasciurus hudsonicus) in Canada. Compared to residents, immigrant females had 23% lower lifetime breeding success (LBS), while immigrant males had 29% higher LBS. Male immigration and female residency were favoured. Offspring born to immigrants had 15-43% lower LBS than offspring born to residents. We conclude that immigration benefitted males, but not females, which appeared to be making the best of a bad lot. Our results are in line with male-biased dispersal being driven by local mate competition and local resource enhancement, while the intergenerational cost to immigration is a new complication in explaining the drivers of sex-biased dispersal.

Heritability of Horn Size in Thinhorn Sheep.

Understanding the genetic basis of fitness-related trait variation has long been of great interest to evolutionary biologists. Secondary sexual characteristics, such as horns in bovids, are particularly intriguing since they can be potentially affected by both natural and sexual selection. Until recently, however, the study of fitness-related quantitative trait variation in wild species has been hampered by a lack of genomic resources, pedigree, and/or phenotype data. Recent innovations in genomic technologies have enabled wildlife researchers to perform marker-based relatedness estimation and acquire adequate loci density, enabling both the "top-down" approach of quantitative genetics and the "bottom-up" approach of association studies to describe the genetic basis of fitness-related traits. Here we combine a cross species application of the OvineHD BeadChip and horn measurements (horn length, base circumference, and volume) from harvested thinhorn sheep to examine the heritability and to perform a genome-wide single-nucleotide polymorphism association study of horn size in the species. Thinhorn sheep are mountain ungulates that reside in the mountainous regions of northwestern North America. Thinhorn sheep males grow massive horns that determine the social rank and mating success. We found horn length, base circumference, and volume to be moderately heritable and two loci to be suggestively associated with horn length.