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Miller, Joshua M.; Coltman, David W. 2014-02-26 Heterozygosity fitness correlations (HFCs) have often been used to detect inbreeding depression, under the assumption that genome-wide heterozygosity is a good proxy for inbreeding. However, meta-analyses of the association between fitness measures and individual heterozygosity have shown that often either no correlation is observed, or the effect sizes are small. One of the reasons for this may be the absence of variance in inbreeding, a requisite for generating general-effect HFCs. Recent work has highlighted identity disequilibrium (ID) as a measure that may capture variance in the level of inbreeding within a population, however, no thorough assessment of ID in natural populations has been conducted. In this meta-analysis we assess the magnitude of ID (as measured by the g2 statistic) from 50 previously published HFC studies and its relationship to the observed effect sizes of those studies. We then assess how much power the studies had to detect general-effect HFCs, and the number of markers that would have been needed to generate an expected high correlation (r2 = 0.9) between observed heterozygosity and inbreeding. Across the majority of studies g2 values were not significantly different than zero. Despite this, we found that the magnitude of g2 was associated with the average effect sizes observed in a population, even when point estimates were non-significant. These low values of g2 translated into low expected correlations between heterozygosity and inbreeding, and suggest that many more markers than typically used are needed to robustly detect HFCs.
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Malenfant, René M.; Coltman, David W.; Davis, Corey S. 2014-09-04 Single-nucleotide polymorphisms (SNPs) offer numerous advantages over anonymous markers such as microsatellites, including improved estimation of population parameters, finer-scale resolution of population structure and more precise genomic dissection of quantitative traits. However, many SNPs are needed to equal the resolution of a single microsatellite, and reliable large-scale genotyping of SNPs remains a challenge in nonmodel species. Here, we document the creation of a 9K Illumina Infinium BeadChip for polar bears (Ursus maritimus), which will be used to investigate: (i) the fine-scale population structure among Canadian polar bears and (ii) the genomic architecture of phenotypic traits in the Western Hudson Bay subpopulation. To this end, we used restriction-site associated DNA (RAD) sequencing from 38 bears across their circumpolar range, as well as blood/fat transcriptome sequencing of 10 individuals from Western Hudson Bay. Six-thousand RAD SNPs and 3000 transcriptomic SNPs were selected for the chip, based primarily on genomic spacing and gene function respectively. Of the 9000 SNPs ordered from Illumina, 8042 were successfully printed, and – after genotyping 1450 polar bears – 5441 of these SNPs were found to be well clustered and polymorphic. Using this array, we show rapid linkage disequilibrium decay among polar bears, we demonstrate that in a subsample of 78 individuals, our SNPs detect known genetic structure more clearly than 24 microsatellites genotyped for the same individuals and that these results are not driven by the SNP ascertainment scheme. Here, we present one of the first large-scale genotyping resources designed for a threatened species.
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Malenfant, René M.; Coltman, David L.; Richardson, Evan S.; Lunn, Nicholas J.; Davis, Corey S.; Coltman, David W.; Lunn, Nick J. 2013-11-12 Polar bears (Ursus maritimus) in the Western Hudson Bay subpopulation have been declining in size and body condition for decades, as climate change causes earlier sea ice breakup, reduced hunting time on the ice, and an increasingly long fasting season. As Western Hudson Bay females have decreased in size, rates of litter production and average litter size have also decreased, while cub mortality and average time to independence have increased. Although these changes have potential evolutionary consequences, little is yet known about the adaptive genetic variation in body size or fat accumulation that would have to underlie any such change. In this study, we used high-throughput Illumina sequencing to develop SNPs from pooled blood and fat transcriptomes, using samples from five adult female polar bears and five (unrelated) dependent cubs. In total, we generated 371,258 transcripts of which 36,755 were deemed to be “full length” (i.e., covered more than 90% of their best BLAST hit), and we identified 63,020 SNPs. Since this study was conducted, we have used a subset of these SNPs to develop an Illumina BeadArray for quantitative genetics research in Western Hudson Bay.
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Martin, Alexandre M.; Festa-Bianchet, Marco; Coltman, David W.; Pelletier, Fanie 2016-06-02 Sexual selection has a critical role in evolution, and it is fundamental to identify what ecological factors drive its variation. Disentangling the ecological correlates of sexual selection over the long term, however, is challenging and has rarely been done in nature. We sought to assess how demographic changes influenced the intensity, direction and form of sexual selection and whether selective pressures varied with age. We tested whether breeder sex ratio, number of competitors and age structure influenced selection differentials on horn length of wild bighorn rams (Ovis canadensis) of different age classes on Ram Mountain, Alberta. We used 21 years of data including a detailed pedigree, demographic parameters and repeated morphological measurements. Sexual selection on horn length of males of all ages was directional and positive. Selection intensity increased with the number of competitors, reflecting male–male encounter rate during the rut, but was independent of breeder sex ratio or age structure. This result can also be linked to changes in population size because the number of competitors was highly correlated to total number of sheep. This demographic effect likely arises from age-dependent mating tactics. Males aged 2–4 years are weakly competitive and experienced stronger sexual selection as they accounted for a greater proportion of all males. Selection experienced by mature males appeared independent of demography. Our study provides a rare description of the demographic determinants of sexual selection in nature.
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Samarasekera, G. D. N. Gayathri; Bartell, Nicholas V.; Lindgren, B. Staffan; Cooke, Janice E. K.; Davis, Corey S.; James, Patrick M. A.; Coltman, David W.; Mock, Karen E.; Murray, Brent W. 2011-10-17 Environmental change has a wide range of ecological consequences, including species extinction and range expansion. Many studies have shown that insect species respond rapidly to climatic change. A mountain pine beetle epidemic of record size in North America has led to unprecedented mortality of lodgepole pine, and a significant range expansion to the northeast of its historic range. Our goal was to determine the spatial genetic variation found among outbreak population from which genetic structure, and dispersal patterns may be inferred. Beetles from 49 sampling locations throughout the outbreak area in western Canada were analysed at 13 microsatellite loci. We found significant north-south population structure as evidenced by: (i) Bayesian-based analyses, (ii) north-south genetic relationships and diversity gradients; and (iii) a lack of isolation-by-distance in the northernmost cluster. The north-south structure is proposed to have arisen from the processes of postglacial colonization as well as recent climate-driven changes in population dynamics. Our data support the hypothesis of multiple sources of origin for the outbreak and point to the need for population specific information to improve our understanding and management of outbreaks. The recent range expansion across the Rocky Mountains into the jack/lodgepole hybrid and pure jack pine zones of northern Alberta is consistent with a northern British Columbia origin. We detected no loss of genetic variability in these populations, indicating that the evolutionary potential of mountain pine beetle to adapt has not been reduced by founder events. This study illustrates a rapid range-wide response to the removal of climatic constraints, and the potential for range expansion of a regional population.
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Cullingham, Catherine I.; James, Patrick M. A.; Cooke, Janice E. K.; Coltman, David W. 2012-04-10 Understanding the physical and genetic structure of hybrid zones can illuminate factors affecting their formation and stability. In north central Alberta, lodgepole pine (P. contorta Dougl. ex Loud. var. latifolia) and jack pine (P. banksiana Lamb) form a complex and poorly defined hybrid zone. Better knowledge of this zone is relevant, given the recent host expansion of mountain pine beetle into jack pine. We characterized the zone by genotyping 1998 lodgepole, jack pine and hybrids from British Columbia, Alberta, Saskatchewan, Ontario and Minnesota at 11 microsatellites. Using Bayesian algorithms, we calculated genetic ancestry and used this to model the relationship between species occurrence and environment. In addition, we analyzed the ancestry of hybrids to calculate the genetic contribution of lodgepole and jack pine. Finally we measured the amount of gene flow between the pure species. We found the distribution of the pine classes is explained by environmental variables, and these distributions differ from classic distribution maps. Hybrid ancestry was biased towards lodgepole pine; however gene flow between the two species was equal. The results of this study suggest that the hybrid zone is complex and influenced by environmental constraints. As a result of this analysis, range limits should be redefined. https://creativecommons.org/publicdomain/zero/1.0/
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Roe, Amanda D.; Rice, Adrianne V.; Coltman, David W.; Cooke, Janice E. K.; Sperling, Felix A. H. 2010-11-08 Multipartite symbioses are complex symbiotic relationships involving multiple interacting partners. These types of partnerships provide excellent opportunities in which to apply a comparative approach to identify common historical patterns of population differentiation and species-specific life history traits. Using three symbiotic blue stain fungal species (Ophiostomatacea) associated with outbreaking populations of the mountain pine beetle (Dendroctonus ponderosae Hopkins) in western Canada, we applied phylogenetic, population genetic, and demographic approaches to clarify phylogeographic patterns among the three fungal species. Broadly, the three species showed significant population differentiation, forming northern and southern populations, despite dramatic differences in haplotype diversity. Finer scale structuring and population demographic patterns were less consistent, showing some interspecific incongruence. By contrasting these species simultaneously, we were able to identify differences in recombination rate and ecological traits that can explain the observed patterns of incongruence among the fungal species. By applying a comparative approach to partners of a multipartite symbiosis we were able to distinguish congruent population structuring and species-specific differences that help us to understand the complexity and evolution of this symbiotic system.
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Martin, Alexandre M.; Festa-Bianchet, Marco; Coltman, David W.; Pelletier, Fanie 2014-11-25 The reliability and consistency of the many measures proposed to quantify sexual selection have been questioned for decades. Realized selection on quantitative characters measured by the selection differential i was approximated by metrics based on variance in breeding success, using either the opportunity for sexual selection Is or indices of inequality. There is no consensus about which metric best approximates realized selection on sexual characters. Recently, the opportunity for selection on character mean OSM was proposed to quantify the maximum potential selection on characters. Using 21 years of data on bighorn sheep (Ovis canadensis), we investigated the correlations between seven indices of inequality, Is, OSM and i on horn length of males. Bighorn sheep are ideal for this comparison because they are highly polygynous, sexually dimorphic, ram horn length is under strong sexual selection, and we have detailed knowledge of individual breeding success. Different metrics provided conflicting information, potentially leading to spurious conclusions about selection patterns. Iδ, an index of breeding inequality, and to a lesser extent Is, showed the highest correlation with i on horn length, suggesting that these indices document breeding inequality in a selection context. OSM on horn length was strongly correlated with i, Is, and indices of inequality. By integrating information on both realized sexual selection and breeding inequality, OSM appeared to be the best proxy of sexual selection and may be best suited to explore its ecological bases.
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Poirier, Marc-Antoine; Coltman, David W.; Pelletier, Fanie; Jorgenson, Jon; Festa-Bianchet, Marco 2018-08-31 Isolation of small populations is expected to reduce fitness through inbreeding and loss of genetic variation, impeding population growth and compromising population persistence. Species with long generation time are the least likely to be rescued by evolution alone. Management interventions that maintain or restore genetic variation to assure population viability are consequently of significant importance. We investigated, over 27 years, the genetic and demographic consequences of a demographic bottleneck followed by artificial supplementation in an isolated population of bighorn sheep (Ovis canadensis). Based on a long-term pedigree and individual monitoring, we documented the genetic decline, restoration and rescue of the population. Microsatellite analyses revealed that the demographic bottleneck reduced expected heterozygosity and allelic diversity by 6.2 and 11.3%, respectively, over two generations. Following supplementation, first-generation admixed lambs were 6.4% heavier at weaning and had 28.3% higher survival to 1 year compared to lambs of endemic ancestry. Expected heterozygosity and allelic diversity increased by 4.6 and 14.3% after two generations through new alleles contributed by translocated individuals. We found no evidence for outbreeding depression and did not see immediate evidence of swamping of local genes. Rapid intervention following the demographic bottleneck allowed the genetic restoration and rescue of this bighorn sheep population, likely preventing further losses at both the genetic and demographic levels. Our results provide further empirical evidence that translocation can be used to reduce inbreeding depression in nature and has the potential to mitigate the effect of human-driven environmental changes on wild population.
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Miller, Joshua M.; Hogg, John T.; Coltman, David W. 2013-07-19 In this work we present the development of a large set of single nucleotide polymorphisms (SNPs) discovered in two populations of bighorn sheep (Ovis canadensis). To do so we used restriction-site associated DNA (RAD) sequencing of four individuals from each population. Through alignment of reads to the domestic sheep (Ovis aries) genome we discovered >83,000 SNPs, of which >38,000 are suitable for assays such as an Illumina SNP chip. These loci will allow for fine-mapping of loci associated with horn size, and examination of the consequences of the genetic rescue including mapping genes underling differences in life-history characteristics.
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Thompson, Megan J.; Dobson, F. Stephen; Coltman, David W.; Murie, Jan O.; Raveh, Shirley; Lane, Jeffrey E. 2023-10-23 <p>Desynchrony of phenological responses to climate change is a major concern in 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 (<em>Urocitellus</em> <em>columbianus</em>). 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. Older males also tended to emerge earlier and secure territories, and so it is unclear whether selection acts indirectly through age. Together, our results provide evidence that the sexes should remain synchronised, 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.</p>
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Fletcher, Quinn E.; Speakman, John R.; Boutin, Stan; Lane, Jeffrey E.; McAdam, Andrew G.; Gorrell, Jamieson C.; Coltman, David W.; Humphries, Murray M. 2015-06-13 1. Energy expenditure is a trait of central importance in ecological and evolutionary theory. We examined the correlates of, the strength of selection on, and the heritability of, daily energy expenditure (DEE; kJ/day) during lactation in free-ranging North American red squirrels (Tamiasciurus hudsonicus). 2. Over seven years, lactating squirrels with greater DEE had higher annual reproductive success (ARS; standardized selection gradient: β’ = 0.47; top 12% of published estimates). Surprisingly, positive fecundity selection on lactation DEE for increased ARS did not result because lactation DEE was correlated with typical measures of reproductive performance and/or investment. 3. We found no evidence of costs of elevated lactation DEE acting through female survival, subsequent year lactation DEE, or subsequent year reproduction. 4. Lactation DEE was not significantly repeatable and heritability was not significantly different from zero. 5. Elevated lactation DEE enhances ARS through a link between DEE and an unidentified measure of maternal or environmental quality, but there is limited evolutionary potential for lactation DEE to respond to our documented selection.
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Douhard, Mathieu; Festa-Bianchet, Marco; Coltman, Dave W.; Pelletier, Fanie; Coltman, David W. 2016-01-13 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.
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Douhard, Mathieu; Pigeon, Gabriel; Festa-Bianchet, Marco; Coltmann, Dave W.; Guillemette, Simon; Pelletier, Fanie; Coltman, David W. 2016-11-30 The development of male secondary sexual characters such as antlers or horns has substantial biological and socio-economic importance because in many species these traits affect male fitness positively through sexual selection and negatively through trophy hunting. Both environmental conditions and selective hunting can affect horn growth but their relative importance remains unexplored. We first examined how a large-scale climate index, the Pacific Decadal Oscillation (PDO), local weather and population density influenced both absolute and relative annual horn growth from birth to 3 years of male bighorn sheep (Ovis canadensis) over 42 years. We then examined the relative influence of environmental conditions and evolution mainly driven by trophy hunting on male horn length at 3 years of age. Horn growth was positively influenced by low population density and warm spring temperature, suggesting that ongoing climate change should lead to larger horns. Seasonal values of PDO were highly correlated. Horn growth increased with PDO in spring or summer at low density, but was weak at high density regardless of PDO. The interaction between population density and PDO in spring or summer accounted for a similar proportion of the observed annual variation in horn growth (32% or 37%) as did the additive effects of spring temperature and density (34%). When environmental conditions deteriorated, males allocated relatively more resources to summer mass gain than to horn growth, suggesting a conservative strategy favoring maintenance of condition over allocation to secondary sexual characters. Population density explained 27% of the variation in horn length, while evolutionary effects explained 9% of the variance. Thus, our study underlines the importance of both evolution and phenotypic plasticity on the development of a secondary sexual trait.
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Miller, Joshua M.; Malenfant, René M.; David, Patrice; Davis, Corey S.; Poissant, Jocelyn; Hogg, John T.; Festa-Bianchet, Marco; Coltman, David W. 2013-09-17 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.
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Miller, Joshua M.; Poissant, Jocelyn; Hogg, John T.; Coltman, David W. 2011-12-01 Genetic rescue is a management intervention whereby a small population is supplemented with individuals from other populations in an attempt to reverse the effects of inbreeding and increased genetic load. One such rescue was recently documented in the population of bighorn sheep (Ovis canadensis) within the National Bison Range wildlife refuge (Montana, USA). Here we examine the locus-specific effects of rescue in this population using a newly developed genome-wide set of 195 microsatellite loci and first-generation linkage map. We found that the rate of introgression varied among loci, and that 111 loci, 57% of those examined, deviated from patterns of neutral inheritance. The most common deviation was an excess of homozygous genotypes relative to neutral expectations, indicative of directional selection. As in previous study of this rescue, individuals with more introduced alleles had higher lifetime reproductive success and longevity. In addition, we found 30 loci, spread throughout the genome, which seem to have individual effects on these life history traits. Though the potential for outbreeding depression is a major concern when translocating individuals between populations, we found no evidence of such effects in this population.
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Shafer, Aaron B. A.; Northrup, Joseph M.; White, Kevin S.; Boyce, Mark S.; Côté, Steeve D.; Coltman, David W. 2012-06-14 Landscape heterogeneity plays an integral role in shaping ecological and evolutionary processes. Despite links between the two disciplines, ecologists and population geneticists have taken different approaches to evaluating habitat selection, animal movement, and gene flow across the landscape. Ecologists commonly use statistical models such as resource selection functions (RSFs) to identify habitat features disproportionately selected by animals, while population genetic approaches model genetic differentiation according to the distribution of habitat variables. We combined ecological and genetic approaches by using RSFs and step-selection functions (SSFs) to predict genetic relatedness across a heterogeneous landscape. We constructed sex and season-specific resistance surfaces based on RSFs and SSFs estimated using data from 102 GPS radiocollared mountain goats (Oreamnos americanus) in southeast Alaska. Based on mountain goat ecology, we hypothesized that summer and male surfaces would be the best predictors of relatedness. All individuals were genotyped at 22 microsatellite loci, which we used to estimate genetic relatedness. Summer resistance surfaces derived from RSFs were the best predictors of genetic relatedness, and winter models the poorest. Male and female specific surfaces were similar, except for winter where male habitat selection better predicted genetic relatedness. The null models of isolation-by-distance and barrier only outperformed the winter models. This study merges high-resolution individual locations through GPS telemetry and genetic data, that can be used to validate and parameterize landscape genetics models, and further elucidates the relationship between landscape heterogeneity and genetic differentiation.
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Malenfant, René M.; Davis, Corey S.; Richardson, Evan S.; Lunn, Nicholas J.; Coltman, David W. 2022-04-26 Among polar bears (Ursus maritimus), fitness is dependent on body size through males' abilities to win mates, females' abilities to provide for their young, and all bears' abilities to survive increasingly longer fasting periods caused by climate change. In the Western Hudson Bay subpopulation (near Churchill, Manitoba, Canada), polar bears have declined in body size and condition, but nothing is known about the genetic underpinnings of body size variation, which may be subject to natural selection. Here, we combine a 4449-individual pedigree and an array of 5433 single-nucleotide polymorphisms (SNPs) to provide the first quantitative genetics study of polar bears. We used animal models to estimate heritability (h2) among polar bears handled between 1966 and 2011, obtaining h2 estimates of 0.34-0.48 for strictly skeletal traits and 0.18 for axillary girth (which is also dependent on fatness). We genotyped 859 individuals with the SNP array to test for marker-trait association and combined p-values over genetic pathways using gene-set analysis. Variation in all traits appeared to be polygenic, but we detected one region of moderately large effect size in body length near a putative noncoding RNA in an unannotated region of the genome. Gene-set analysis suggested that variation in body length was associated with genes in the regulatory cascade of cyclin expression, which has previously been associated with body size in mice. A greater understanding of the genetic architecture of body size variation will be valuable in understanding the potential for adaptation in polar bear populations challenged by climate change.
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Martin, Alexandre M.; Festa-Bianchet, Marco; Coltman, David W.; Pelletier, Fanie 2014-02-17 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.
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Miller, Joshua M.; Poissant, Jocelyn; Malenfant, René M.; Hogg, John T.; Coltman, David W. 2016-07-14 Linkage disequilibrium (LD) is the nonrandom association of alleles at two markers. Patterns of LD have biological implications as well as practical ones when designing association studies or conservation programs aimed at identifying the genetic basis of fitness differences within and among populations. However, the temporal dynamics of LD in wild populations has received little empirical attention. In this study, we examined the overall extent of LD, the effect of sample size on the accuracy and precision of LD estimates, and the temporal dynamics of LD in two populations of bighorn sheep (Ovis canadensis) with different demographic histories. Using over 200 microsatellite loci, we assessed two metrics of multi-allelic LD, D′, and χ′2. We found that both populations exhibited high levels of LD, although the extent was much shorter in a native population than one that was founded via translocation, experienced a prolonged bottleneck post founding, followed by recent admixture. In addition, we observed significant variation in LD in relation to the sample size used, with small sample sizes leading to depressed estimates of the extent of LD but inflated estimates of background levels of LD. In contrast, there was not much variation in LD among yearly cross-sections within either population once sample size was accounted for. Lack of pronounced interannual variability suggests that researchers may not have to worry about interannual variation when estimating LD in a population and can instead focus on obtaining the largest sample size possible.

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