1 | Introduction
Threatened and endangered species often exist in small isolated populations that theory predicts should suffer elevated risks of extinction due to impacts on their genetic makeup (Frankham et al., 2017; van Oosterhout, 2020). Populations with these characteristics experience increased genetic drift and inbreeding, which lead to a loss of genetic variation, reducing the efficacy of natural selection resulting in increased frequency and expression of deleterious recessive mutations termed genetic load (Charlesworth, 2009; Keller & Waller, 2002). However, the precise genetic endpoint of this process in terms of observed levels of genetic load in natural populations depends on complex interactions between the levels of inbreeding and effectiveness of purifying selection that are present (Grossen, Guillaume, Keller, & Croll, 2020; Hedrick & Garcia-Dorado, 2016).
Specifically, inbreeding results in the expression of recessive deleterious mutations creating the potential for selection to reduce the frequency of these mutations depending on the degree of dominance and the magnitude of the deleterious effects (Glémin, 2003). This process, termed genetic purging is most effective at high levels of inbreeding and so bottlenecks tend to purge highly deleterious, recessive mutations unless population sizes are extremely small (Garcia-Dorado, 2012; Kirkpatrick & Jarne, 2000). However, reduced population size also increases genetic drift and reduces the efficacy of selection which allows deleterious mutations to drift to higher frequency (Renaut & Rieseberg, 2015; Robinson et al., 2016). Thus, the type and level of genetic load present in a given population reflects a balance of the relative magnitudes of these distinct processes (inbreeding versus reduced selection due to drift) with opposite effects on mutation frequencies. As a result, assessing levels of inbreeding, effective population size, and levels of genetic load are an important goal of empirical studies that seek to assess the genetic risks impacting long term viability of threatened species (Mathur & DeWoody, 2021) and for assessing the appropriateness of specific management activities such as genetic rescue for mitigating these risks (Kyriazis, Wayne, & Lohmueller, 2020; but see Ralls, Sunnucks, Lacy, & Frankham, 2020).
In addition, van der Valk, de Manuel, Marques-Bonet, and Guschanski (2019) have recently emphasized the previously underappreciated role of historical demography on the observed level of genetic load of deleterious mutations in contemporary populations. They show that estimates of genetic load vary widely across mammal species with small effective sizes and are only weakly correlated with genome-wide levels of inbreeding. They argue that this is because long term reductions in effective size can result in sustained purging of deleterious mutations leading to reduced genetic load in present-day populations. Such a pattern has been observed in populations of endangered species which have experienced long-term bottlenecks (Benazzo et al., 2017) suggesting the importance of incorporating historical patterns of population size in explanations for genetic load.
Whole genome resequencing offers new data and methods of analysis to evaluate inbreeding and genetic load in endangered species (Brüniche-Olsen, Kellner, Anderson, & DeWoody, 2018). For assessing inbreeding, individual genome sequences provide information on runs of homozygosity (ROHs) which are genomic regions that contains significantly less nucleotide variation than expected based on the genome–wide average for an individual (Ceballos, Joshi, Clark, Ramsay, & Wilson, 2018). These ROH regions arise in a genome due to the transmission of genome segments that are identical by descent (IBD) from parents to offspring (Ceballos et al., 2018). ROH segments can be used to quantify inbreeding reflected by genomic autozygosity because ROH distributions of regions that are IBD often match pedigree histories (Kardos, Qvarnström, & Ellegren, 2017). Distributions of different size ROHs also provide insights into recent demography because under random mating the length of ROH regions is expected to decrease with increasing number of generations to the most recent common ancestor due to recombination and de novo mutations (Bosse et al., 2012). Therefore, the number and length of ROH reflect the timing and intensity of a population bottleneck, with longer ROH originating from recent inbreeding, and shorter ROH from ancestral bottlenecks (Ceballos et al., 2018).
For assessing genetic load, new analytical methods (Adzhubei et al., 2010; Choi, Sims, Murphy, Miller, & Chan, 2012) now permit strong inference of the mutational effects of substitutions in coding sequences assumed to be under strong purifying selection. As such data from these approaches can be used to estimate levels of accumulation of putatively deleterious mutations at the population or species level in non-model species and explore the process that drive variation in genetic load in natural populations (Allendorf, 2017; Kardos, Taylor, Ellegren, Luikart, & Allendorf, 2016).
The Eastern Massasauga (Sistrurus catenatus ) is a small rattlesnake found in eastern North America. Population declines throughout its range due to habitat fragmentation and destruction have led to the listing of this species as threatened under the United States Endangered Species Act (U.S. Fish and Wildlife Service, 2016) and as a Species at Risk in Canada (Government of Canada, 2009). This species exhibits little phylogeographic structure across its range (Sovic, Fries, & Gibbs, 2016), and high levels of population genetic structure (Chiucchi & Gibbs, 2010; Sovic, Fries, Martin, & Gibbs, 2019). Thus, the relevant management units within this species are individual populations.
The negative genetic impacts drift on population viability are potentially a significant conservation issue for S. catenatus.Sovic et al. (2019) have recently shown that the contemporary effective values of most populations of S. catenatus are < 50, suggesting that genetic drift and the negative genetic impacts of inbreeding could be significant yet evidence in support of this possibility is unclear. Specifically, heterozygosity-fitness correlations based on neutral genetic markers and body condition show few positive relationships consistent with the negative effects of drift and inbreeding but the power of these tests may be limited because of body condition is an indirect measure of individual fitness (Gibbs & Chiucchi, 2012; Sovic et al., 2019). Resolving how genetic factors impact the future viability of S. catenatus at the species and individual population level is an important goal of management plans at state and federal levels (Szymanski et al., 2016). Genome-level quantification of levels of inbreeding and genetic load similar to those conducted in other threatened species in similar conservation situations would address this need.
To this end, we use data from resequenced genomes from 90 individualS. catenatus to conducted a detailed analyses of inbreeding, population demography and levels of deleterious genetic load at the species and population level. To provide necessary context for our results, we also generated similar data for 10 individuals from a single population of a closely-related yet non-threatened species, the Western Massasauga rattlesnake (S. tergeminus ). In contrast, to S. catenatus , S. tergeminus is relatively common through much of its range and genetic studies show no evidence for high levels of population genetic structure supporting the idea that it represents an outbred species that has experienced far fewer anthropogenic impacts on its genetic makeup (Bylsma, 2020; McCluskey & Bender, 2015; Ryberg, Harvey, Blick, Hibbitts, & Voelker, 2015).
We used these data to address the following questions: What are the genome-wide levels of inbreeding and is inbreeding higher S. catenatus as compared to S. tergeminus ? Based demographic analyses that use ROH size and abundance distributions, what are trends in population size over recent timescales and how do these aid in interpreting observed levels of inbreeding and levels of genetic load in each species? What are levels of different types of deleterious mutations in both species and among S. catenatus populations and what evolutionary processes underlie differences in load between species and S. catenatus populations? Our results support an emerging idea that the historical demography of a threatened species has a significant impact on the type of genetic load present which impacts conservation actions such as genetic rescue and also identify populations of S. catenatus whose viability may be at risk due to genetic factors.