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.

Assessing shared respiratory pathogens between domestic (Ovis aries) and bighorn (Ovis canadensis) sheep; methods for multiplex PCR, amplicon sequencing, and bioinformatics to characterize respiratory flora.

Respiratory disease is responsible for dramatic population declines in bighorn sheep (Ovis canadensis), and respiratory pathogen diagnostics contribute to the management of bighorn populations. To create a comprehensive and consistent approach to bighorn sheep respiratory diagnostics, we created a culture-independent assay to detect and strain type Mannheimia haemolytica, Bibersteinia trehalosi, Pasteurella multocida, and Mycoplasma ovipneumoniae. The assay also detects and characterizes the Pasteurellaceae leukotoxin A gene, and broadly assesses the bacterial composition of each sample based on 16S rRNA sequences. The assay is based on a three-step approach: 1) Multiplex PCR to amplify targets including eight loci for each bacterial species, the Pasteurellaceae lktA gene, and the 16S rRNA gene 2) Library preparation, barcoding, and short-read Illumina sequencing to determine the genetic sequences of each target, and 3) Bioinformatics in the form of automated software to analyze genetic sequences. The assay was designed to assess shared pathogens between domestic and bighorn sheep, but could be useful for many applications in bighorn sheep respiratory disease research and management.

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.

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.

A bioinformatic pipeline for identifying informative SNP panels for parentage assignment from RADseq data.

The development of high-throughput sequencing technologies is dramatically increasing the use of single nucleotide polymorphisms (SNPs) across the field of genetics, but most parentage studies of wild populations still rely on microsatellites. We developed a bioinformatic pipeline for identifying SNP panels that are informative for parentage analysis from restriction site-associated DNA sequencing (RADseq) data. This pipeline includes options for analysis with or without a reference genome, and provides methods to maximize genotyping accuracy and select sets of unlinked loci that have high statistical power. We test this pipeline on small populations of Mexican gray wolf and bighorn sheep, for which parentage analyses are expected to be challenging due to low genetic diversity and the presence of many closely related individuals. We compare the results of parentage analysis across SNP panels generated with or without the use of a reference genome, and between SNPs and microsatellites. For Mexican gray wolf, we conducted parentage analyses for 30 pups from a single cohort where samples were available from 64% of possible mothers and 53% of possible fathers, and the accuracy of parentage assignments could be estimated because true identities of parents were known a priori based on field data. For bighorn sheep, we conducted maternity analyses for 39 lambs from five cohorts where 77% of possible mothers were sampled, but true identities of parents were unknown. Analyses with and without a reference genome produced SNP panels with ≥95% parentage assignment accuracy for Mexican gray wolf, outperforming microsatellites at 78% accuracy. Maternity assignments were completely consistent across all SNP panels for the bighorn sheep, and were 74.4% consistent with assignments from microsatellites. Accuracy and consistency of parentage analysis were not reduced when using as few as 284 SNPs for Mexican gray wolf and 142 SNPs for bighorn sheep, indicating our pipeline can be used to develop SNP genotyping assays for parentage analysis with relatively small numbers of loci.

Simulating the spread of selection-driven genotypes using landscape resistance models for desert bighorn sheep.

Landscape genetic studies based on neutral genetic markers have contributed to our understanding of the influence of landscape composition and configuration on gene flow and genetic variation. However, the potential for species to adapt to changing landscapes will depend on how natural selection influences adaptive genetic variation. We demonstrate how landscape resistance models can be combined with genetic simulations incorporating natural selection to explore how the spread of adaptive variation is affected by landscape characteristics, using desert bighorn sheep (Ovis canadensis nelsoni) in three differing regions of the southwestern United States as an example. We conducted genetic sampling and least-cost path modeling to optimize landscape resistance models independently for each region, and then simulated the spread of an adaptive allele favored by selection across each region. Optimized landscape resistance models differed between regions with respect to landscape variables included and their relationships to resistance, but the slope of terrain and the presence of water barriers and major roads had the greatest impacts on gene flow. Genetic simulations showed that differences among landscapes strongly influenced spread of adaptive genetic variation, with faster spread (1) in landscapes with more continuously distributed habitat and (2) when a pre-existing allele (i.e., standing genetic variation) rather than a novel allele (i.e., mutation) served as the source of adaptive genetic variation. The combination of landscape resistance models and genetic simulations has broad conservation applications and can facilitate comparisons of adaptive potential within and between landscapes.

Towards robust evolutionary inference with integral projection models.

Integral projection models (IPMs) are extremely flexible tools for ecological and evolutionary inference. IPMs track the distribution of phenotype in populations through time, using functions describing phenotype-dependent development, inheritance, survival and fecundity. For evolutionary inference, two important features of any model are the ability to (i) characterize relationships among traits (including values of the same traits across ages) within individuals, and (ii) characterize similarity between individuals and their descendants. In IPM analyses, the former depends on regressions of observed trait values at each age on values at the previous age (development functions), and the latter on regressions of offspring values at birth on parent values as adults (inheritance functions). We show analytically that development functions, characterized this way, will typically underestimate covariances of trait values across ages, due to compounding of regression to the mean across projection steps. Similarly, we show that inheritance, characterized this way, is inconsistent with a modern understanding of inheritance, and underestimates the degree to which relatives are phenotypically similar. Additionally, we show that the use of a constant biometric inheritance function, particularly with a constant intercept, is incompatible with evolution. Consequently, current implementations of IPMs will predict little or no phenotypic evolution, purely as artefacts of their construction. We present alternative approaches to constructing development and inheritance functions, based on a quantitative genetic approach, and show analytically and through an empirical example on a population of bighorn sheep how they can potentially recover patterns that are critical to evolutionary inference.

Paternal reproductive success drives sex allocation in a wild mammal.

Parents should bias sex allocation toward offspring of the sex most likely to provide higher fitness returns. Trivers and Willard proposed that for polygynous mammals, females should adjust sex-ratio at conception or bias allocation of resources toward the most profitable sex, according to their own body condition. However, the possibility that mammalian fathers may influence sex allocation has seldom been considered. Here, we show that the probability of having a son increased from 0.31 to 0.60 with sire reproductive success in wild bighorn sheep (Ovis canadensis). Furthermore, our results suggest that females fertilized by relatively unsuccessful sires allocated more energy during lactation to daughters than to sons, while the opposite occurred for females fertilized by successful sires. The pattern of sex-biased offspring production appears adaptive because paternal reproductive success reduced the fitness of daughters and increased the average annual weaning success of sons, independently of maternal allocation to the offspring. Our results illustrate that sex allocation can be driven by paternal phenotype, with profound influences on the strength of sexual selection and on conflicts of interest between parents.

Phenotypic and molecular characterization of intrauterine fetal growth restriction in interspecies sheep pregnancy.

Interspecies pregnancies between closely related species are usually performed in livestock to obtain improved and enriched offspring. Indeed, different hybrids have been obtained for research purposes since many years ago, and the maternal-fetal interactions have been studied as a possible strategy for species preservation. The aim of this study was to characterize by physiological and molecular approaches the interspecies pregnancy between bighorn sheep () and domestic sheep (). Hybrids were obtained by artificial insemination; the blood pressure and protein urine levels were measured during the last two-thirds of gestation. After parturition, offspring and placentas were weighed and measured and cotyledons were counted and weighed and their surface area determined. Plasma samples were obtained between wk 8 and 21 of gestation to assess progesterone (P4), vascular endothelial growth factor (VEGF), and placental growth factor (PlGF) levels and cell-free RNA was isolated during the same period to assess hypoxia-inducible factor-1 α (α) gene expression. Hybrid and normal pregnancies were analyzed using physiological and molecular parameters during the last two-thirds of gestation (wk 8-21). The results show that during the measurement period, ewes with a hybrid pregnancy presented normal blood pressure and no alteration in urinary protein content. However, compared with sheep with a normal pregnancy, those with a hybrid pregnancy had a decrease in fetal and placental growth as well as in the cotyledonary surface area. Furthermore, in the hybrid group, there was placental insufficiency, characterized by a decrease in P4 production, as well as indications of endothelial dysfunction, characterized an increase in plasma levels of VEGF and PlGF as well as in plasma gene expression of α. Overall, the results indicate that hybrids of and presented intrauterine growth restriction, essentially due to altered endothelial function and chronic placental insufficiency. Further studies are necessary to overcome this primary placental dysfunction and thus obtain improved offspring for future molecular and genomic evaluations.

Whole-genome resequencing uncovers molecular signatures of natural and sexual selection in wild bighorn sheep.

The identification of genes influencing fitness is central to our understanding of the genetic basis of adaptation and how it shapes phenotypic variation in wild populations. Here, we used whole-genome resequencing of wild Rocky Mountain bighorn sheep (Ovis canadensis) to >50-fold coverage to identify 2.8 million single nucleotide polymorphisms (SNPs) and genomic regions bearing signatures of directional selection (i.e. selective sweeps). A comparison of SNP diversity between the X chromosome and the autosomes indicated that bighorn males had a dramatically reduced long-term effective population size compared to females. This probably reflects a long history of intense sexual selection mediated by male-male competition for mates. Selective sweep scans based on heterozygosity and nucleotide diversity revealed evidence for a selective sweep shared across multiple populations at RXFP2, a gene that strongly affects horn size in domestic ungulates. The massive horns carried by bighorn rams appear to have evolved in part via strong positive selection at RXFP2. We identified evidence for selection within individual populations at genes affecting early body growth and cellular response to hypoxia; however, these must be interpreted more cautiously as genetic drift is strong within local populations and may have caused false positives. These results represent a rare example of strong genomic signatures of selection identified at genes with known function in wild populations of a nonmodel species. Our results also showcase the value of reference genome assemblies from agricultural or model species for studies of the genomic basis of adaptation in closely related wild taxa.

Sex-based differences in the adaptive value of social behavior contrasted against morphology and environment.

The adaptive nature of sociality has long been a central question in ecology and evolution. However, the relative importance of social behavior for fitness, compared to morphology and environment, remains largely unknown. We assessed the importance of sociality for fitness (lamb production and survival) in a population of mark6d bighorn sheep (Ovis canadensis) over 16 years (n = 1022 sheep-years). We constructed social networks from observations (n = 38,350) of group membership (n = 3150 groups). We then tested whether consistent individual differences in social behavior (centrality) exist and evaluated their relative importance compared to factors known to affect fitness: mass, age, parental effects, and population density. Sheep exhibited consistent individual differences in social centrality. Controlling for maternal carryover effects and age, the positive effect of centrality in a social network on adult female lamb production and survival was equal or greater than the effect of body mass or population density. Social centrality had less effect on male survival and no effect on adult male lamb production or lamb survival. Through its effect on lamb production and survival, sociality in fission-fusion animal societies may ultimately influence population dynamics equally or more than morphological or environmental effects.

Harnessing cross-species alignment to discover SNPs and generate a draft genome sequence of a bighorn sheep (Ovis canadensis).

BACKGROUND: Whole genome sequences (WGS) have proliferated as sequencing technology continues to improve and costs decline. While many WGS of model or domestic organisms have been produced, a growing number of non-model species are also being sequenced. In the absence of a reference, construction of a genome sequence necessitates de novo assembly which may be beyond the ability of many labs due to the large volumes of raw sequence data and extensive bioinformatics required. In contrast, the presence of a reference WGS allows for alignment which is more tractable than assembly. Recent work has highlighted that the reference need not come from the same species, potentially enabling a wide array of species WGS to be constructed using cross-species alignment. Here we report on the creation a draft WGS from a single bighorn sheep (Ovis canadensis) using alignment to the closely related domestic sheep (Ovis aries). RESULTS: Two sequencing libraries on SOLiD platforms yielded over 865 million reads, and combined alignment to the domestic sheep reference resulted in a nearly complete sequence (95% coverage of the reference) at an average of 12x read depth (104 SD). From this we discovered over 15 million variants and annotated them relative to the domestic sheep reference. We then conducted an enrichment analysis of those SNPs showing fixed differences between the reference and sequenced individual and found significant differences in a number of gene ontology (GO) terms, including those associated with reproduction, muscle properties, and bone deposition. CONCLUSION: Our results demonstrate that cross-species alignment enables the creation of novel WGS for non-model organisms. The bighorn sheep WGS will provide a resource for future resequencing studies or comparative genomics.

Demography, not inheritance, drives phenotypic change in hunted bighorn sheep.

Selective harvest, such as trophy hunting, can shift the distribution of a quantitative character such as body size. If the targeted character is heritable, then there will be an evolutionary response to selection, and where the trait is not, then any response will be plastic or demographic. Identifying the relative contributions of these different mechanisms is a major challenge in wildlife conservation. New mathematical approaches can provide insight not previously available. Here we develop a size- and age-based two-sex integral projection model based on individual-based data from a long-term study of hunted bighorn sheep (Ovis canadensis) at Ram Mountain, Canada. We simulate the effect of trophy hunting on body size and find that the inheritance of body mass is weak and that any perceived decline in body mass of the bighorn population is largely attributable to demographic change and environmental factors. To our knowledge, this work provides the first use of two-sex integral projection models to investigate the potential eco-evolutionary consequences of selective harvest.

Local extinction and unintentional rewilding of bighorn sheep (Ovis canadensis) on a desert island.

Bighorn sheep (Ovis canadensis) were not known to live on Tiburón Island, the largest island in the Gulf of California and Mexico, prior to the surprisingly successful introduction of 20 individuals as a conservation measure in 1975. Today, a stable island population of ∼500 sheep supports limited big game hunting and restocking of depleted areas on the Mexican mainland. We discovered fossil dung morphologically similar to that of bighorn sheep in a dung mat deposit from Mojet Cave, in the mountains of Tiburón Island. To determine the origin of this cave deposit we compared pellet shape to fecal pellets of other large mammals, and extracted DNA to sequence mitochondrial DNA fragments at the 12S ribosomal RNA and control regions. The fossil dung was 14C-dated to 1476-1632 calendar years before present and was confirmed as bighorn sheep by morphological and ancient DNA (aDNA) analysis. 12S sequences closely or exactly matched known bighorn sheep sequences; control region sequences exactly matched a haplotype described in desert bighorn sheep populations in southwest Arizona and southern California and showed subtle differentiation from the extant Tiburón population. Native desert bighorn sheep previously colonized this land-bridge island, most likely during the Pleistocene, when lower sea levels connected Tiburón to the mainland. They were extirpated sometime in the last ∼1500 years, probably due to inherent dynamics of isolated populations, prolonged drought, and (or) human overkill. The reintroduced population is vulnerable to similar extinction risks. The discovery presented here refutes conventional wisdom that bighorn sheep are not native to Tiburón Island, and establishes its recent introduction as an example of unintentional rewilding, defined here as the introduction of a species without knowledge that it was once native and has since gone locally extinct.

Sexually antagonistic association between paternal phenotype and offspring viability reinforces total selection on a sexually selected trait.

The evolution of conspicuous sexually selected traits, such as horns or antlers, has fascinated biologists for more than a century. Elaborate traits can only evolve if they substantially increase reproduction, because they probably incur survival costs to the bearer. Total selection on these traits, however, includes sexual selection on sires and viability selection on offspring and can be influenced by changes in each of these components. Non-random associations between paternal phenotype and offspring viability may thus affect total selection on sexually selected traits. Long-term data on wild bighorn sheep (Ovis canadensis) provide the first evidence in nature that association between paternal phenotype and lamb viability strengthens total selection on horn size of adult rams, a sexually selected trait. The association of paternal horn length and offspring viability was sexually antagonistic: long-horned males sired sons with high viability but daughters of low viability. These results shed new light on the evolutionary dynamics of an iconic sexually selected trait and have important implications for sustainable wildlife management.

Estimating genome-wide heterozygosity: effects of demographic history and marker type.

Heterozygosity-fitness correlations (HFCs) are often used to link individual genetic variation to differences in fitness. However, most studies examining HFCs find weak or no correlations. Here, we derive broad theoretical predictions about how many loci are needed to adequately measure genomic heterozygosity assuming different levels of identity disequilibrium (ID), a proxy for inbreeding. We then evaluate the expected ability to detect HFCs using an empirical data set of 200 microsatellites and 412 single nucleotide polymorphisms (SNPs) genotyped in two populations of bighorn sheep (Ovis canadensis), with different demographic histories. In both populations, heterozygosity was significantly correlated across marker types, although the strength of the correlation was weaker in a native population compared with one founded via translocation and later supplemented with additional individuals. Despite being bi-allelic, SNPs had similar correlations to genome-wide heterozygosity as microsatellites in both populations. For both marker types, this association became stronger and less variable as more markers were considered. Both populations had significant levels of ID; however, estimates were an order of magnitude lower in the native population. As with heterozygosity, SNPs performed similarly to microsatellites, and precision and accuracy of the estimates of ID increased as more loci were considered. Although dependent on the demographic history of the population considered, these results illustrate that genome-wide heterozygosity, and therefore HFCs, are best measured by a large number of markers, a feat now more realistically accomplished with SNPs than microsatellites.

Effective population size, genetic variation, and their relevance for conservation: the bighorn sheep in Tiburon Island and comparisons with managed artiodactyls.

The amount of genetic diversity in a finite biological population mostly depends on the interactions among evolutionary forces and the effective population size (N(e)) as well as the time since population establishment. Because the N(e) estimation helps to explore population demographic history, and allows one to predict the behavior of genetic diversity through time, N(e) is a key parameter for the genetic management of small and isolated populations. Here, we explored an N(e)-based approach using a bighorn sheep population on Tiburon Island, Mexico (TI) as a model. We estimated the current (N(crnt)) and ancestral stable (N(stbl)) inbreeding effective population sizes as well as summary statistics to assess genetic diversity and the demographic scenarios that could explain such diversity. Then, we evaluated the feasibility of using TI as a source population for reintroduction programs. We also included data from other bighorn sheep and artiodactyl populations in the analysis to compare their inbreeding effective size estimates. The TI population showed high levels of genetic diversity with respect to other managed populations. However, our analysis suggested that TI has been under a genetic bottleneck, indicating that using individuals from this population as the only source for reintroduction could lead to a severe genetic diversity reduction. Analyses of the published data did not show a strict correlation between H(E) and N(crnt) estimates. Moreover, we detected that ancient anthropogenic and climatic pressures affected all studied populations. We conclude that the estimation of N(crnt) and N(stbl) are informative genetic diversity estimators and should be used in addition to summary statistics for conservation and population management planning.