Introduction
Isolation and subsequent local adaptation of populations are considered
common processes that lead to speciation. Changes in habitat, barriers
to dispersal, or stochastic demographic events can cause population
isolation and diversification (Slatkin 1987; Steinberg et al.2000). In this context, environmental variation plays an important role
in facilitating or hindering connectivity, and thus in promoting the
persistence of populations, their vicariance, or even extinction (Waitset al. 2016). On one hand, increased structural and functional
connectivity facilitates the persistence of small populations that are
highly susceptible to demographic stochasticity, genetic drift and
density-dependent effects (Hanski 1998; Lopes & de Freitas 2012;
Wittmann et al. 2018). On the other hand, high levels of
connectivity result in more genetically homogenous populations, with
less propensity for local adaptation (Kawecki & Ebert 2004). With loss
of connectivity between populations, allele frequencies tend to diverge
due to genetic drift, ultimately leading to neutral genetic
differentiation (Rundell & Price 2009). Additionally, spatial variation
in local selection pressures within a species’ range, particularly when
there is habitat fragmentation, can lead to changes in allele
frequencies and fixation of new adaptive mutations, resulting in the
emergence of adaptive differences (Orsini et al. 2013). This is
especially important for small and isolated populations that are
restricted to increasingly unfavorable habitat, for which studies have
shown that local adaptation tends to occur more rapidly (Wood et
al. 2016; Hoffmann et al. 2017). Recently diverged populations
represent a great opportunity to study the process of genetic
differentiation (Fišer et al. 2018; Fletcher et al. 2019;
Marques et al. 2019). The comparison of neutral and adaptive
variation should provide evidence for distinguishing which processes are
contributing most to differentiation, and what can be done to circumvent
or sustain that diversification, depending on specific conservation
goals (Orsini et al. 2013).
Northern and southern Idaho ground squirrels (Urocitellusbrunneus and U. endemicus , respectively) are a recently diverged
pair of sister species, which currently have an allopatric distribution
(Figure 1) (Yensen 1991). Northern Idaho ground squirrels (hereafter
NIDGS) and southern Idaho ground squirrels (hereafter SIDGS) were
formerly considered two subspecies of Spermophilus brunneus and
have been distinguished as separate species on the basis of ecological
niche modelling, morphology and genetics (Gill & Yensen 1992; Yensen &
Sherman 1997; Helgen et al. 2009; Hoisington-Lopez et al.2012; USFWS 2015). Both species are rare, endemic to Idaho, and are of
high conservation concern (IUCN 2000, 2018). Ecologically, both species
are semi-colonial and patchily distributed, representing classic
examples of metapopulation structure whereby dispersal among populations
is uncommon and tends to occur in a ‘stepping stone’ manner (Yensen
1991; Yensen & Sherman 1997; USFWS 2003). NIDGS live in open meadows,
grassy scabs and small rocky outcroppings at an elevation of 1100 to
2300 m within coniferous forests of central Idaho (Burak 2011; Goldberg,
Conway, Mack, et al. 2020), and they persist within only a small
fraction of their former range likely due to habitat loss and reduced
population connectivity, mostly as a result of forest encroachment
(Sherman & Runge 2002; Suronen & Newingham 2013; Yensen & Dyni 2020;
Helmstetter et al. 2021). SIDGS live in sagebrush steppe and
rolling hill slopes at an elevation of 630 to 1400 m in southwestern
Idaho, and are currently threatened by urban and agricultural
development, as well as the spreading of invasive annual plants (USFWS
2000; Lohr et al. 2013). Morphological differences between the
two species include coat color, which tends to mimic differences in soil
color between the species’ geographic ranges (Yensen 1991), pelage
(longer in SIDGS), and baculum characteristics (longer with more spines
in SIDGS) (Yensen 1991). Previous genetic work on NIDGS and SIDGS
estimated that the divergence between NIDGS and SIDGS occurred about
32.5 (18.3-63.5) thousand years ago, during the Quaternary climate
cycles, and found no subsequent gene flow between the two species
(Hoisington-Lopez et al. 2012). Vicariant events of this
magnitude have been found to be sufficient for distinct evolutionary
lineages to become different species, a pattern frequently found in
several North American small mammals (Hope et al. 2014, 2016,
2020).
Local adaptation is likely to be an important factor for
ground-dwelling, hibernating, small mammals like NIDGS and SIDGS with
limited dispersal abilities. Both ground squirrel species hibernate but
the timing of hibernation differs, likely due to differences in
elevation and climate (Yensen 1991; Goldberg & Conway 2021). This
difference in emergence timing between NIDGS and SIDGS could have a
genomic basis, or may simply result from a plastic response (Yensen
1991; Hut & Beersma 2011; Santos et al. 2017). Typically,
adaptations are associated with the habitat variables that affect
fitness the most, which in the case of the Idaho ground squirrels
(hereafter IDGS) are likely variables associated with energy
consumption, timing of food availability, soil temperature, forage
quality, and risk of predation, which may vary between the active season
and hibernation (Goldberg, Conway, Mack, et al. 2020; Goldberg,
Conway, Tank, et al. 2020). Variation in ground squirrel
hibernation emergence timing has been associated with food availability
and snowpack and thus, site productivity appears to dictate differences
within and possibly between species differentiation (French 1982;
Goldberg & Conway 2021). These differences may be determinant for
species divergence, but may also lead to intraspecific local adaptation
if environmental differences are found across the species range (Kawecki
& Ebert 2004; Savolainen et al. 2013). Previous intraspecific
genetic studies on Idaho ground squirrels have found that genetic
differentiation was low to moderate among NIDGS populations, with one
disjunct population (Round Valley) being completely isolated (Yensen &
Sherman 1997; Garner et al. 2005; Hoisington 2007;
Hoisington-Lopez et al. 2012). In SIDGS, the Weiser River was
documented as a barrier to dispersal between populations, but
connectivity among populations on either side of the river was
relatively high (Garner et al. 2005; Hoisington 2007).
Additionally, there are reports of human-mediated translocations between
localities east of the Weiser River, particularly from southern
localities close to Van Deussen to the vicinity of the Weiser River
population (Yensen et al. 2010; Yensen & Tarifa 2012). However,
translocation success in SIDGS has been very limited, especially into
areas without established populations, for which the majority of the
translocated individuals did not survive the first winter (Busscher
2009; Yensen et al. 2010). Given the currently observed low
levels of gene flow among some populations within both species,
population persistence might be highly dependent on locally adapted
genotypes for many isolated populations (USFWS 2000; Rundell & Price
2009). Thus, to better understand the probability of population
persistence under habitat and climate change, it is essential to
determine the role of neutral processes in maintaining population
connectivity and overall genetic diversity, and the role of adaptive
processes in improving population resilience through local adaptation
(Macdonald et al. 2018).
In this study, we aimed to develop and use genomic tools to provide
novel information on neutral and adaptive genetic diversity and
differentiation within and among NIDGS and SIDGS to address the
following five questions: 1) how do genetic patterns of adaptive and
neutral variation compare between and within species? 2) are there any
populations that have elevated levels of adaptive differentiation? 3)
what landscape variables are associated with loci under selection? 4)
can we identify specific genes under selection and what are their
putative functions? and 5) can we identify conservation units based on
genomic data to inform management? To address these questions, we tested
the following hypotheses: (a) geographic distance will be the main
driver of differentiation for neutral loci in NIDGS and SIDGS, possibly
exacerbated by geographic barriers to gene flow; (b) populations within
each species will exhibit signatures of adaptation to local
environmental conditions ; (c) local adaptation between populations
within species will be highest in NIDGS because this species occupies a
more topographically diverse area (Yensen & Sherman 1997); and (d)
adaptive variation will be associated with timing of hibernation,
production and storage of fat, and increased metabolism at higher
elevation for NIDGS (Faherty et al. 2018; Garcia-Elfring et
al. 2019). Finally, we combine all of this information to identify
Evolutionarily Significant Units (ESUs), Management Units (MUs) and
Adaptive Units (AUs) that could warrant heightened protection due to
genetic isolation or the emergence of local adaptation.