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.