Contrasting genetic trajectories of endangered and expanding red fox populations in the western U.S

Abstract

As anthropogenic disturbances continue to drive habitat loss and range contractions, the maintenance of evolutionary processes will increasingly require targeting measures to the population level, even for common and widespread species. Doing so requires detailed knowledge of population genetic structure, both to identify populations of conservation need and value, as well as to evaluate suitability of potential donor populations. We conducted a range-wide analysis of the genetic structure of red foxes in the contiguous western U.S., including a federally endangered distinct population segment of the Sierra Nevada subspecies, with the objectives of contextualizing field observations of relative scarcity in the Pacific mountains and increasing abundance in the cold desert basins of the Intermountain West. Using 31 autosomal microsatellites, along with mitochondrial and Y-chromosome markers, we found that populations of the Pacific mountains were isolated from one another and genetically depauperate (e.g., estimated Ne range = 3–9). In contrast, red foxes in the Intermountain regions showed relatively high connectivity and genetic diversity. Although most Intermountain red foxes carried indigenous western matrilines (78%) and patrilines (85%), the presence of nonindigenous haplotypes at lower elevations indicated admixture with fur-farm foxes and possibly expanding midcontinent populations as well. Our findings suggest that some Pacific mountain populations could likely benefit from increased connectivity (i.e., genetic rescue) but that nonnative admixture makes expanding populations in the Intermountain basins a non-ideal source. However, our results also suggest contact between Pacific mountain and Intermountain basin populations is likely to increase regardless, warranting consideration of risks and benefits of proactive measures to mitigate against unwanted effects of Intermountain gene flow.

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ARTICLE OPEN
Contrasting genetic trajectories of endangered and expanding
red fox populations in the western U.S
Cate B. Quinn
1
✉, Sophie Preckler-Quisquater
1
, Jocelyn R. Akins
2
, Patrick R. Cross
3
, Preston B. Alden
1
, Stevi L. Vanderzwan
1
,
John A. Stephenson
4
, Pete J. Figura
5
, Gregory A. Green
6
, Tim L. Hiller
7
and Benjamin N. Sacks
1,8
© The Author(s) 2022
As anthropogenic disturbances continue to drive habitat loss and range contractions, the maintenance of evolutionary processes
will increasingly require targeting measures to the population level, even for common and widespread species. Doing so requires
detailed knowledge of population genetic structure, both to identify populations of conservation need and value, as well as to
evaluate suitability of potential donor populations. We conducted a range-wide analysis of the genetic structure of red foxes in the
contiguous western U.S., including a federally endangered distinct population segment of the Sierra Nevada subspecies, with the
objectives of contextualizing field observations of relative scarcity in the Pacific mountains and increasing abundance in the cold
desert basins of the Intermountain West. Using 31 autosomal microsatellites, along with mitochondrial and Y-chromosome markers,
we found that populations of the Pacific mountains were isolated from one another and genetically depauperate (e.g., estimated
Ne range =3–9). In contrast, red foxes in the Intermountain regions showed relatively high connectivity and genetic diversity.
Although most Intermountain red foxes carried indigenous western matrilines (78%) and patrilines (85%), the presence of
nonindigenous haplotypes at lower elevations indicated admixture with fur-farm foxes and possibly expanding midcontinent
populations as well. Our findings suggest that some Pacific mountain populations could likely benefit from increased connectivity
(i.e., genetic rescue) but that nonnative admixture makes expanding populations in the Intermountain basins a non-ideal source.
However, our results also suggest contact between Pacific mountain and Intermountain basin populations is likely to increase
regardless, warranting consideration of risks and benefits of proactive measures to mitigate against unwanted effects of
Intermountain gene flow.
Heredity; https://doi.org/10.1038/s41437-022-00522-4
INTRODUCTION
Although conservation has historically targeted the species level,
modern awareness that evolution is continuous highlights that
populations can also be evolutionarily distinct, sometimes posses-
sing local adaptations or transitioning toward speciation (Moritz
2002; Roux et al. 2016). In certain cases, long-isolated populations
that evolved in distinct habitats arguably warrant greater
conservation attention due to their evolutionary distinctiveness
than larger or more connected populations, which may harbor
greater redundancy (Lesica and Allendorf 1995; Hampe and Petit
2005). In addition to their potentially greater evolutionary value,
historically small and isolated populations also tend to have lower
genetic diversity (Frankham 1996) and thus face an elevated risk of
extirpation due to demographic and genetic stochasticity (Frank-
ham 2015). Anthropogenic stressors, such as development and
exploitation, further exacerbate extinction risk because rapid and
recent declines may disproportionately increase inbreeding
depression (Kyriazis et al. 2021; van der Valk et al. 2021).
As human activities continue to contribute to habitat loss and
range contractions, maintaining evolutionary processes will
increasingly require targeting interventions to the population
level, even for common and widespread species. Deliberate
translocations of small numbers of individuals from one population
to another has been advocated as a conservation tool to rescue
genetically depauperate populations from the most severe fitness
effects of genetic erosion (Frankham 2015; Whiteley et al. 2015;
Ralls et al. 2018). However, doing so without sufficient under-
standing of evolutionary relationships or demographic history
could potentially thwart evolutionary trajectories, risk outbreeding
depression, or, depending on the genetic load of donor popula-
tions, exacerbate inbreeding depression (Edmands 2007; Bell et al.
2019; Wilder et al. 2020; Kyriazis et al. 2021). Targeting conservation
measures to specific populations therefore requires detailed
knowledge of population structure, both to identify populations
of conservation need and value as well as to assess the suitability
of potential donor populations (e.g., Frankham et al. 2011).
Received: 2 February 2021 Revised: 24 February 2022 Accepted: 25 February 2022
1
Mammalian Ecology and Conservation Unit, Veterinary Genetics Laboratory, University of California, Davis, 1 Shields Avenue, Davis, CA 95616, USA.
2
Cascades Carnivore Project,
309 Oak Street, Suite 201, Hood River, OR 97301, USA.
3
Yellowstone Ecological Research Center, 4135 Valley Commons Drive Suite D, Bozeman, MT 59718, USA.
4
Grand Teton
National Park, PO Drawer 170, Moose, WY 83012, USA.
5
California Department of Fish and Wildlife, 601 Locust Street, Redding, CA 96001, USA.
6
Huxley College of the
Environment, Western Washington University, Bellingham, WA 98225, USA.
7
Wildlife Ecology Institute, P.O. Box 4725, Helena, MT 59604, USA.
8
Department of Population Health
and Reproduction, School of Veterinary Medicine, University of California, Davis, 1 Shields Avenue, Davis, CA 95616, USA. Associate editor Paul Sunnucks.
✉email: cbquinn@ucdavis.edu
www.nature.com/hdy
1234567890();,:

As a species, red foxes (Vulpes vulpes) have the widest
distribution of any non-domestic terrestrial carnivore, ranging
naturally over three continents and introduced to Australia
(Larivière and Pasitschniak-Arts 1996). The IUCN considers the
species conservation status of “least concern”(Hoffmann and
Sillero-Zubiri 2021), yet their global distribution belies variation in
morphology, ecology, and demography at local levels (e.g.,
Gortázar et al. 2000; Szuma 2008; Devenish-Nelson et al. 2013).
The population structure of red foxes is further complicated by a
20th century history of translocating red foxes (Saunders et al.
1995; Long 2003), often for fur-farming (Petersen 1914; Ashbrook
1928). Without means to discern ancestry, it can be difficult to
distinguish the demographic trends of indigenous, locally adapted
populations from recently translocated, genetically divergent
populations (Champagnon et al. 2012). Genetic analyses that
simultaneously characterize ancestry and connectivity are thus
vital in determining conservation needs of specific red fox
populations, as well as informing decisions on how to manage
connectivity.
In the western contiguous United States (hereafter, western U.
S.), naturalists and trappers from the early 20th century reported
that red foxes were largely restricted to the upper montane and
subalpine life zones of the major western mountain ranges
(Seton 1929;Bailey1936a,1936b;Grinnelletal.1937; Dalquest
1948; Hall and Kelson 1959). Although recognized as four distinct
subspecies, indigenous western red foxes form a single lineage
that is >20,000 years diverged from red foxes in the remainder of
the continent (see Supplementary Text SI for details on
taxonomy). Available information on phenotype suggests that
montane members of the lineage are also smaller, have
proportionally larger foot surfaces, and breed considerably later
in the year, all of which have been suggested to represent local
adaptations to the subalpine environment (Grinnell et al. 1937;
Roest 1977; Fuhrmann 1998; Cross et al. 2018;SCAT,2022).
Following European colonization of North America, native
western red foxes declined in portions of their range presumably
due to a combination of unregulated harvest and poisoning
associated with predator control programs (Perrine et al. 2010;
Sacks et al. 2010). Despite decades of regulation or protection,
native western red foxes in some areas have yet to recover the
full extent of their historical range, particularly in the Pacific
mountains of the Cascades and Sierra Nevada where detections
remain relatively rare (Sierra Nevada Red Fox Conservation
Advisory Team [SCAT] 2022; Washington Department of Fish
and Wildlife 2015).
Low abundance, limited distributions, and increased research
and outreach have led to conservation attention of multiple high-
elevation populations in the Pacific mountains, including listing at
both state and federal levels (see Supplementary Text for details
on state listing status). Most recently, the U.S. Fish and Wildlife
Service designated the population of the Sierra Nevada subspecies
(V. v. necator) residing in the Sierra Nevada as a federally
endangered distinct population segment (DPS; U.S. Fish and
Wildlife Service 2021). The U.S. Fish and Wildlife Service has also
recognized the Southern Cascade DPS of the Sierra Nevada
subspecies, composed of populations in the vicinity of Lassen
Peak of northern California and the Oregon Cascades, but
determined the DPS not warranted for listing, largely due to an
absence of data in the Oregon Cascades at that time (U.S. Fish and
Wildlife Service 2015). In all Pacific mountain regions, conservation
efforts have generally been hindered by uncertainty about basic
population status and limiting factors (SCAT, 2022). Previous
genetic assessments have considered Pacific mountain popula-
tions individually (e.g., Akins et al. 2018, Quinn et al. 2019), but
only limited attempts have been made to conduct genetic
analyses at a broad scale (e.g., Aubry et al. 2009) that could both
highlight important commonalities as well as facilitate conserva-
tion prioritization.
Concurrent with apparent declines in the Pacific mountains, red
foxes reportedly began increasing in abundance in the Inter-
mountain West during 1960–2000s, including expansion into
habitat types and elevation bands not historically attributed to the
native montane subspecies. Such increases have been observed in
the Snake River Plain of Idaho, low elevations of the Basin and
Range ecoregions of Nevada, Utah and southeastern Oregon, and
the Columbia River Plateau of Washington and northern Oregon
(Fichter and Williams 1967; Mace 1970; Verts and Carraway 1998;
Hoffmann et al. 1969; Kamler and Ballard 2003; Green et al. 2017).
Red foxes in these cold desert basins occupy a range of vegetative
zones, including river plains, shrub-steppe, arid grasslands, and
agricultural fields. Both the departure from historical montane
habitat and apparent rapid increase have led some to posit that
red foxes in the Intermountain basins are not members of native
western montane subspecies, but rather descendants of non-
native red foxes that escaped or were released from fur farms (e.g.,
Fichter and Williams 1967; Mace 1970), similar to feral populations
documented in western Washington and California (Aubry 1984;
Lewis et al. 1999). Alternatively, these lower elevation red foxes
may have also originated from a continental-scale expansion from
eastern or northwestern North America (hereafter referred to as
midcontinent) (e.g., Hoffmann et al. 1969; Kamler and Ballard
2002; Cross et al. 2018).
The degree to which red foxes in the cold desert basins of the
Intermountain West derive from downslope expansions of the
native montane subspecies, descendants of fur-farm foxes, or
post-glacial expansions from the midcontinent carry different
implications for their management. Fur-farm stock originated
primarily from wild-caught red foxes of eastern Canada and Alaska
(Laut 1921), similar to the expected composition of midcontinent
red foxes (Aubry et al. 2009; Black et al. 2018). In both cases, red
fox lineages from northwestern and eastern North America
diverged before the Last Glacial Maximum and occupy different
long-term niches (Aubry et al. 2009; Statham et al. 2014). In
addition, nonindigenous fur-farm stock underwent generations of
selective breeding in captivity (Laut 1921; Lord et al. 2020),
potentially resulting in traits that are maladaptive in wild
environments (Rhymer and Simberloff 1996; Laikre et al. 2010).
Previous genetic investigations have shown largely native western
mitochondrial haplotypes in the expansion zone (e.g., Statham
et al. 2012; Green et al. 2017), but low sampling resolution and the
absence of nuclear data has limited their conclusions.
Here, we conduct the most comprehensive range-wide
analysis of the contemporary genetic structure of red foxes in
the western U.S to date, with the intent of contextualizing field
observations of relative scarcity in the Pacific mountains and
increasing abundance in the arid basins to their east. To
accomplish this, we filled critical sampling gaps in the high-
elevation Oregon Cascades and low-elevation interior western
U.S., where few nuclear data have been available until now. Our
questions were threefold. First, do red foxes show genetic
signatures of isolated, remnant populations throughout the
Pacific mountains, or is it a phenomenon limited to the federally
protected Sierra Nevada DPS? We were specifically interested in
the southern Cascades of Oregon and California, where the
conservation status has been poorly defined due to a lack of
data. Second, to what extent can the apparent increases in red
fox abundance and distribution in the cold desert basins be
explained by nonindigenous red foxes and, if applicable, do
nonindigenous foxes originate from anthropogenic transloca-
tions of the past or natural expansion from the midcontinent?
Finally, we sought to characterize any contact between Pacific
and Intermountain populations, as novel gene flow can rapidly
alter the trajectories of small, isolated populations (Hedrick et al.
2014), the consequences of which are particularly complex when
populations differ in their evolutionary histories. Results lay the
foundation for conservation of the native western red fox
C.B. Quinn et al.
2
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lineage generally, and future recovery planning for the
endangered Sierra Nevada red fox DPS specifically.
METHODS
Study area and samples
We analyzed DNA from 730 individual red foxes collected throughout the
western U.S. between 1986 and 2018 (Table S1; Fig. 1). For convenience,
we refer to two broad regions of the study area: (1) the “Far West,”which
includes the high-elevation Pacific ranges (Cascades, Sierra Nevada) and
the low-elevation valley and coastal areas to their west, and (2) the
“Intermountain West,”which includes the high-elevation Rocky Mountain
and Great Basin ranges and the surrounding lower elevation, cold desert
basins (i.e., the Columbia Plateau, the Snake River Plain, and the lower
elevations of the Great Basin). Generally, the high-elevation mountain
ranges encompass the historical distribution of indigenous western red
foxes (Hall and Kelson 1959), whereas lower elevations correspond to
either populations known to originate from fur farms (California; Sacks
et al. 2016) or those of poorly characterized ancestry (cold desert basins;
Fichter and Williams 1967; Verts and Carraway 1998; Kamler and Ballard
2003). The Sacramento Valley subspecies (V. v. patwin) is an exception to
this elevational characterization, in that it is sister to the high-elevation
Sierra Nevada subspecies but occupied the grassland-dominated Sacra-
mento Valley prior to European colonization (Sacks et al. 2010; Volkmann
et al. 2015).
Most of the 730 DNA samples were used in previous studies but typed at
only a subset of the genetic markers used here (e.g., mitochondrial only;
see Table S1 for details); 251 samples were newly collected. The majority
(62%) of samples were tissue or blood collected opportunistically from
mortalities (e.g., foxes killed via vehicle strikes), pelts with permission of
trappers, or live captures for other studies whose trapping and handling
methods followed American Society of Mammalogists animal care
guidelines (Sikes et al. 2011) and were approved by the University of
California, Davis Animal Care and Use Committee (IACUC No. 17860). From
more remote montane regions, we additionally incorporated noninvasively
collected hair and fecal samples (e.g., Hiller et al. 2015; Akins et al. 2018).
Tissue and hair were stored in desiccant or frozen at −20 °C and fecal
samples were stored in >95% ethanol. We extracted DNA from feces using
QIAamp Stool Kit (Qiagen Inc., Valencia CA), and from hair, urine, and tissue
using DNeasy Blood and Tissue Kits (Qiagen Inc.) using previously
described protocols (e.g., Quinn et al. 2019).
Autosomal markers
We used autosomal allele frequencies to characterize genetic structure,
quantify diversity, and infer demographic trends. We attempted PCR
amplification of 31 microsatellite loci originally ascertained in domestic
dogs but with primers revised based on red fox sequences where
appropriate (Moore et al. 2010). We multiplexed loci using primers and PCR
conditions previously described (e.g., Quinn et al. 2019). Amplification of
each locus was attempted 1–2× for tissue and blood samples and 2–5× for
hair and fecal samples, based on previously calculated allelic dropout rates
of <1% for tissue- and blood-extracted DNA (Sacks et al. 2010) and <5% for
fecal extracted DNA (Sacks et al. 2011; Quinn et al. 2019). For fecal and hair
samples, we assigned sample genotypes to distinct individuals using
previously described matching criteria (Quinn et al. 2019) and used only
one genotype/individual sampled in final analyses. We excluded any final
genotypes composed of <29 loci to minimize effects of missing data.
Based on this dataset, we calculated per-locus heterozygosity, observed
number of alleles, and degree of deviance from Hardy–Weinberg
equilibrium (F
IS
) and its statistical significance using packages hierfstat
(Goudet 2005) and pegas (Paradis 2010) in R v.4.0.2 (R Development Core).
Population structure. We used three individual-based clustering methods
to explore population structure: nonspatial Bayesian clustering in Structure
Fig. 1 Distribution of DNA samples from red foxes (Vulpes vulpes;n=730) in the western contiguous U.S. Circles indicate the location of
collection, with dark fill specifying those known from previous studies to be introduced via fur farms (Sacks et al. 2011,2016). Colored
polygons depict coarse historical ranges of native western subspecies according to Hall and Kelson (1959) and modified by the genetic
findings of Sacks et al. (2010). Finer-scaled historical habitat associations are approximated by gray shading, which depicts a merged version of
Kuchler’s(1964) vegetation categories, “conifer forest”and “alpine meadows or barren”. The Far West, referred to throughout the main text,
includes red foxes in the Pacific mountains (Cascades, Sierra Nevada) and westward, whereas the Intermountain West includes red foxes in the
Rocky Mountains and surrounding cold desert basins (Great Basin, Columbia Plateau, Snake River Plain).
C.B. Quinn et al.
3
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v. 2.3 (Pritchard et al. 2000; Falush et al. 2003), spatial Bayesian clustering in
Tess v. 2.3 (Chen et al. 2007; Durand et al. 2009), and multivariate
discriminant analysis of principal components (DAPC; Jombart et al. 2010).
We compared results across methods, as each one has its own strengths:
Structure serves as a standard, having the most widespread use; Tess
accounts explicitly for spatial autocorrelation among samples and there-
fore should outperform other methods in the presence of isolation-by-
distance (Chen et al. 2007); finally, DAPC is a multivariate method that
assumes no particular underlying population processes such as
Hardy–Weinberg equilibrium or linkage equilibrium (Jombart et al. 2010).
In Structure, we used an admixture model with correlated allele
frequencies and no prior information to run 20 simulations with the
number of genetic clusters (K) ranging from 1 to 20 for 200,000 Markov
chain Monte Carlo (MCMC) repetitions, discarding the first 100,000 as burn-
in. After removing 1% of the runs with the lowest values, we calculated the
mean log posterior probability of the data in Structure Harvester (Earl and
vonHoldt 2012) and identified a range of reasonable Kvalues based on
where likelihoods approached a plateau. In Tess, we first ran ten models
with no admixture for K=2–20 to obtain an upper bound on the number
of clusters in the data. We then plotted the deviance information criterion
(DIC) for each Kvalue, and ran admixture models for Kvalues between two
and the number of clusters for which the DIC reached a plateau in the non-
admixture models. We ran 30 admixture models using the conditional
autoregressive (CAR) model and used DIC to select plausible K values for
comparison with other methods. For all runs we used Euclidian geographic
distances for weighting, 100,000 iterations, and a 25,000 iteration burn-in
period. For DAPC assignments, we used the K-means clustering algorithm
and Bayesian Information Criteria in R package adegenet (Jombart 2008)to
determine the number of clusters that most efficiently summarized the
data. The DAPC results did not meaningfully differ when the number of
principal components retained was decided using the α-score method
(n=14) or explanation of 80% cumulative variance (n=75); we therefore
present results based on retention of 75 principal components and all
discriminant functions.
We tested for concordance among the three clustering algorithms and
used a total evidence approach to characterize population structure (e.g.,
Ball et al. 2010). We chose not to average models across multiple runs (e.g.,
Jakobsson and Rosenberg 2007), as model averaging can falsely suggest
admixture or ambiguous assignment when none exists. Instead, we
visualized individual runs with the highest support (based on DIC and
posterior probability) for a range of K values that seemed to describe the
majority of the structure in the data (Pritchard et al. 2003). We then
evaluated genetic groupings based on three criteria: stability of member-
ship across Kvalues (hierarchal stability), stability of membership across
methods (model stability), and the average magnitude of qvalues (i.e.,
membership coefficients, admixture proportions, etc., indicative of cluster
distinctiveness). We interpreted genetic clusters composed predominantly
of high qvalues (e.g., >0.9) that were robust to the choice of algorithm and
Kas “discrete clusters”. In contrast, clusters displaying a broad distribution
of qvalues (e.g., 0.25–0.75) that varied by model and choice of Ksuggested
continuous genetic structure not easily delineated with discrete spatial
boundaries (e.g., clines, admixture zones).
When isolation-by-distance contributes to population structure, the
magnitude of isolation increases with distance, which can overwhelm and
obscure discrete subdivisions operating locally. Due to our interest in such
subdivisions, particularly the relationships between red foxes in the upper
elevation zones and the more recently colonized Intermountain basins, we
therefore performed additional clustering analyses at a finer spatial scale in
Oregon and southeastern Washington. Within a few hundred kilometers,
this region contained samples from the historical ranges of two native
subspecies (Sierra Nevada red fox in the Cascades and Rocky Mountain red
fox in the Blue Mountain and Wallowa ranges) as well as intermediary
basins where red foxes colonized more recently (Fig. 1; Bailey 1936b; Verts
and Carraway 1998). The expected variation in population histories
combined with high sampling resolution suggested this region offered
the greatest power to elucidate the role that montane populations played
in founding the basin populations, as well as detect and assess
directionality of any subsequent gene flow between low and high
elevations.
Genetic dissimilarity. To evaluate relative degrees of genetic differentia-
tion among clusters, we used DAPC to visualize the relationship of clusters
in ordinal space. Because in this case our objective was to evaluate the
genetic dissimilarity of clusters rather than designate them de novo (Miller
et al. 2020), we defined the genetic groups using Tess assignments.
Otherwise we conducted DAPC using the same number of PCs and
discriminant functions as in the de novo cluster analysis. Next, using the
same clustering results from Tess to define genetic groups (i.e., based on a
maximum qvalue > 0.5), we calculated pairwise F
ST
according to Weir and
Cockerham (1984) in the R package hierfstat (Goudet 2005). We
bootstrapped F
ST
values across loci for 5000 iterations to estimate 95%
confidence intervals.
To visualize spatial variation in rates of gene flow, we used the estimated
effective migration surface algorithm (EEMS; Petkova et al. 2016). The EEMS
approach highlights geographic regions where genetic dissimilarity decays
faster or slower than expected under a strict model of isolation-by-
distance. Briefly, a dense grid is overlain to assign individuals to demes,
and effective migration rates are estimated among demes and adjusted so
that the observed genetic dissimilarities reflect fitted values under a
stepping-stone model. To ensure results were independent of grid size, we
combined analyses using 500, 1000, and 1500 demes. In all cases we ran
three independent MCMC chains of 1,000,000 iterations following a burn-
in of 100,000 iterations, sampling every 5000 iterations. Algorithm
parameters were optimized to produce the recommended 20–30%
acceptance rates and we inspected log posterior plots to verify
convergence. We visualized results using the R package reemsplots2
(Petkova 2020).
Geographic patterns of genetic diversity and effective population size. For
all populations, we calculated observed (H
O
) and expected (H
E
) hetero-
zygosities and rarefied allelic richness (AR) in the R package hierfstat
(Goudet 2005). We estimated genetic effective population sizes (N
e
) using
a bias-corrected version of the linkage disequilibrium method (Waples
2006; Waples and Do 2008) in the software program NeEstimator v2 (Do
et al. 2014). We estimated N
e
under the assumption of random mating
because although red foxes have strong pair bonds, they exhibit polygyny
in some circumstances (Zabel and Taggart 1989). We excluded alleles with
frequencies <0.05 to balance tradeoffs of precision and bias (Waples and
Do 2010) and used jackknife-based confidence intervals. Finally, while the
linkage disequilibrium method has high precision when N
e
is small (Waples
and Do 2010), sources of linkage disequilibrium other than drift, such as
gene flow and overlapping generations, can downwardly bias estimates
(Waples and England 2011; Waples et al. 2014). We therefore used
subsampling to explore the sensitivity of N
e
estimates under different
sampling schemes. We used a custom R script to randomly sample without
replacement 10, 20, or 30 individuals from multiple geographic groups and
then iterated estimation in NeEstimator 1000 times for each subsampling
scheme and geographic group.
We delineated how individuals were aggregated for computing the
above summary statistics in two ways, depending on the results of
clustering analyses. For genetic clusters that were categorized as “discrete”
(see “Population structure”), we drew minimum convex polygons around
their members and incorporated all individuals within, including those that
were assigned to different clusters and were presumed to be immigrants.
For genetic clusters deemed to have more continuous or complex
structure, rather than partitioning data with arbitrary geographic
boundaries, we estimated summary statistics using Wright’s(1946)
concept of a genetic neighborhood, which describes the local area in
which most matings occur. We implemented this approach using the R
package sGD (Shirk and Cushman 2011), which groups individuals into
overlapping neighborhoods based on a defined radius and centered on
each individual. We considered only neighborhoods with at least ten
individuals and we used a radius of 85 km, based on a previous study that
showed ~90% of red foxes in North Dakota disperse less than this distance
(Allen and Sargeant 1993). We also tested radii of 75 and 100 km and
observed no qualitative difference in results.
To visualize spatial patterns of diversity, we interpolated a continuous
spatially explicit surface for each diversity and N
e
metric using inverse
distance weighting in the R package gstat (Pebesma 2004). All R scripts
used to modify the sGD function and visualize genetic diversity and
effective population size estimates as an interpolated surface are available
at https://github.com/cbquinn/westernRFstructure.
Uniparentally inherited markers
We primarily used uniparentally inherited markers on the mitochondrial
genome and Y chromosome to investigate the contribution of non-
indigenous lineages to the genetic composition of red foxes in the western
U.S. The absence of recombination and their structuring at phylogeo-
graphic timescales (e.g., thousands to hundreds of thousands of years)
allowed us to discriminate matrilines and patrilines indigenous to the
C.B. Quinn et al.
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western U.S. from those that originated in other parts of the continent (i.e.,
eastern and northwestern lineages; Aubry et al. 2009). Secondarily, we
used uniparentally inherited markers to test the geographic patterns of
diversity and structure inferred using autosomal markers. With one-quarter
of the effective population sizes of autosomal markers, Y chromosomes
and mitochondrial DNA can be sensitive indicators of loss of diversity
through drift or founder effect (Moritz 1994).
Mitochondria. For each individual, we amplified a 354 bp segment of
the cytochrome b gene (primers RF14724 and RF15149; Perrine et al.
2007) and a 343 bp segment of the D-loop (primers VVDL1 and VVDL6;
Aubry et al. 2009). We concatenated the two fragments as “C-D,”where C
indicates the name of the cytochrome bhaplotype and D indicates the
numeric name of the D-loop haplotype, according to the convention of
previous studies (e.g., Sacks et al. 2010). Because of widespread use of
these markers in previous studies (e.g., Aubry et al. 2009; Sacks et al.
2010;Mersonetal.2017), we could reasonably impute one of the two
fragments in cases where only a single combination had been previously
documented (noted in Table S1). For A-19, a widespread haplotype
ancestral to all other haplotypes in the Mountain subclade (e.g., Sacks
et al. 2010), we additionally amplified a 200 bp fragment of cytochrome b
(primers VVmc-780F and VVmc-980R; Volkmann et al. 2015) containing a
single SNP that resolves A-19 into two subhaplotypes (A-19a, A-19b). For
all mitochondrial fragments, we used previously published PCR condi-
tions and reagent mixtures (Perrine et al. 2007;Aubryetal.2009;Quinn
et al. 2019).
Multiple previous studies have described mitochondrial variation in red
foxes in wild North American populations and globally distributed fur
farms (e.g., Aubry et al. 2009; Sacks et al. 2010; Statham et al.
2011,2012,2014; Kasprowicz et al. 2016; Lounsberry et al. 2017; Merson
et al. 2017; Black et al. 2018; Cross et al. 2018). Using all known published
homologous mtDNA haplotypes sampled in wild populations in North
America or fur farms, we recreated a median joining network in PopART
(Leigh and Bryant 2015) and identified haplotypes as belonging to one of
four previously described matrilineal groups: the Holarctic clade corre-
sponding to Alaska and northwestern Canada, the Eastern subclade
corresponding to eastern North America, the Mountain subclade
corresponding to the western contiguous U.S., and the Widespread
subclade that is ancestral to both the Mountain and Eastern subclades (i.e.,
member haplotypes occur in either eastern North America or the western
U.S. but not in both; Aubry et al. 2009; Sacks et al. 2010). We conservatively
assumed all haplotypes belonging to the Mountain and Widespread
subclades were indigenous western matrilines, with the exceptions of two
haplotypes (K-36 and O-24) previously documented in fur farms
(Lounsberry et al. 2017; Black et al. 2018). Historically, the O-24 haplotype
was exclusive to the Washington Cascades, so we considered it native in
Washington and fur farm-derived elsewhere. For estimates of matrilineal
diversity, we calculated gene diversity (the equivalent of expected
heterozygosity for diploid data) according to Nei (1987). For continuous
populations, we modified the sGD function to calculate gene diversity
using haplotype frequency data (i.e., according to Nei 1987) with the same
neighborhood approach as described for autosomal diversity.
Y chromosome. A Y-chromosome phylogeographic network for North
American red foxes has yet to be resolved. We therefore utilized faster-
mutating Y-microsatellites to assess diversity in the patriline and slower-
mutating Y-SNPs to evaluate lineage introgression. First, we determined a
Y-chromosome microsatellite haplotype consisting of 14 linked loci
(Statham et al. 2014; Rando et al. 2017). We allowed for missing data at
≤3 loci and used linkage to impute them based on an exclusive 100%
match of the 11–14 alleles present with another complete haplotype, for a
total of 282 Y-microsatellite haplotypes in the western U.S. and 359
reference haplotypes sampled from non-western North American popula-
tions. The PCR protocols were identical to those for autosomal
microsatellites and are described in full by Quinn et al. (2019). We then
used Y-microsatellite haplotypes to estimate gene diversity for the patriline
in the study area using the same combination of discrete and
neighborhood approaches as for mitochondrial sequences.
Because high rates of homoplasy in Y-microsatellites can obscure a
deeper phylogenetic signal (Wei et al. 2013), we used slower mutating
Y-SNPs to determine the origin of the patriline. We assayed all Y-SNPs with
the Sequenom MassARRAY iPLEX platform (Agena Biosciences, Inc., San
Diego, California, USA) using cycling conditions described by Sacks et al.
(2021). The sequences and concentrations for PCR and extension primers
are presented in Table S3 and the flanking sequences used in primer
design in Table S4. To assess the degree to which Y-SNPs could
discriminate western from other North American lineages (i.e., eastern,
northwestern) and identify the western allele, we extended our Y analysis
to include 361 reference samples collected from the eastern U.S. (n=198),
eastern Canada (n=44), Alaska and northern Canada (n=17), and five
globally distributed fur farms (n=102; Table S2). For each distinct
Y-microsatellite haplotype (including reference samples), we selected 1–2
representative tissue samples to SNP-type (n=128) and imputed SNP
alleles for all other samples bearing an identical Y-microsatellite haplotype.
We screened 21 candidate SNPs, four of which were previously shown to
segregate within North America (Sacks et al. 2021); however, only one SNP
was polymorphic in our dataset outside of Alaska (SNP-39) and was
therefore the only one retained for final analysis. Upon identifying the
putative native western allele for this Y-SNP, we then used a Fisher Exact
test to compare the proportions of patrilines and matrilines in the
Intermountain West that were western.
RESULTS
We obtained autosomal genotypes for 642 distinct individuals
across nine western states (Table S1). All but three of the 31 loci
significantly deviated from Hardy–Weinberg equilibrium as
expected due to structure (Table S5). We also obtained 673
concatenated mitochondrial sequences that corresponded to 25
distinct haplotypes, two of which had novel D-loop sequences
(A-278, sampled 5× in Utah, Genbank accession number
OM810161; A-280, sampled 1× in Idaho, Genbank accession
number OM810162) (Table S1). For the Y chromosome, we
successfully typed 11–14 Y-microsatellites for 282 males and
imputed Y-SNPs for 281 males in the western U.S., which
corresponded to 32 distinct Y-haplotypes (Table S1).
Autosomal population structure
All three clustering algorithms (Structure, Tess, DAPC) indicated
broadly similar solutions, with 8–10 clusters encompassing the
majority of structuring in genotype frequencies (Fig. S1). The
qvalues assigned to individuals with respect to six of the clusters
were stable across runs of K=8, 9, or 10 clusters and, regardless of
algorithm, were consistently >0.9 (Figs. S2–S6). The remaining
ancestry was apportioned less consistently across runs to the
other 2–4 clusters, with qvalues frequently falling between 0.25
and 0.75. To maximize resolution and because higher levels of K
were consistently nested within lower levels of K, we based
subsequent analyses on K=10 (Fig. 2).
The six highly stable clusters with high qvalues corresponded to
geographically discrete populations in the Far West: the Cascade
subspecies in the Washington Cascades, each of the three
populations of the Sierra Nevada subspecies (Oregon Cascades,
Lassen, Sierra Nevada), the Sacramento Valley subspecies in
California, and the known nonnative population of central and
coastal California (Fig. 2). The four more variable clusters
corresponded to red foxes primarily from the Intermountain West,
with two exceptions: (1) several individuals from the Sierra Nevada
DPS of Sierra Nevada red fox, and (2) all individuals from the Mount
Hood (northernmost) region of the Oregon Cascades, both of
which had qvalues >0.1 associated with the four Intermountain
clusters. The former finding was expected based on previous
studies that documented immigration from the Great Basin (Quinn
et al. 2019). Otherwise, individuals in the Intermountain West were
composed of mixed ancestry for which contribution varied
continuously across geography: one cluster extended from eastern
Oregon into western Idaho, another cluster primarily in the Greater
Yellowstone area of Wyoming and Montana, a third cluster was
most represented in Colorado and northeastern Utah, and a fourth
cluster concentrated near the Ruby Mountains of northeastern
Nevada (Fig. 2). In their totality, these patterns suggested discrete
biological populations in the Far West and, by comparison,
relatively continuous genetic structure across the
Intermountain West.
C.B. Quinn et al.
5
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Genetic dissimilarity
For clusters defined using Tess at K=10, DAPC indicated higher
levels of genetic dissimilarity for the six discrete clusters in the Far
West relative to those in the Intermountain West (Fig. 3A, B). The
first linear discriminant function separated the two low-elevation
California populations (nonnative California, native Sacramento
Valley) from all others (Fig. 3A), whereas the second through
fourth discriminant functions isolated the four montane popula-
tions that reside in the Pacific mountains (Fig. 3B). Similarly,
estimates of FST showed Pacific mountain clusters to be the most
differentiated in all pairwise comparisons, especially in the
Cascades (WAC =0.12–0.25; ORC =0.14–0.31; LAS =0.16–0.31;
SN =0.10–0.24; Fig. 3C, Table S6). In contrast, clusters of the
Intermountain West were completely overlapping in the first four
linear functions of the DAPC and pairwise FST values among them
were consistently low (0.04–0.10; Fig. 3A–C).
Results from EEMS corroborated the stronger degree of spatial
structuring in the Far West, with substantially lower effective
migration rates and particularly strong bands of resistance
surrounding the four high-elevation populations in the Pacific
mountains (Fig. 4). In the Intermountain West, the EEMS analysis
indicated no significant resistance to gene flow between the
higher elevation native historical range and more recent lower
elevation expansion zones in the Intermountain West, suggesting
genetic continuity.
Geographic patterns of genetic diversity
Genetic diversity followed a similar overarching spatial pattern
across marker types, in which the lowest values occurred in the
Cascade Range of the Pacific mountains (Figs. 5, S7). Mitochon-
drial diversity showed the sharpest spatial contrasts between low-
and high-diversity populations, as expected given female
philopatry of red foxes (Gosselink et al. 2010) and the smaller
effective population size of mitochondrial relative to autosomal
loci (Fig. 5A). In particular, the Lassen Cascade and Oregon
Cascade populations were nearly fixed for a single mitochondrial
haplotype. The Y-chromosome haplotypes showed a similar
pattern to mitochondrial haplotypes, although with the scale
shifted higher, presumably due to a faster mutation rate of
Y-microsatellites (Wei et al. 2013); In addition, the Washington
Cascade population was the most depauperate in Y-chromosome
haplotype diversity (Fig. 5B). Expected heterozygosity for auto-
somal microsatellites, an approximation of the genome average,
similarly showed the populations in the Cascades to be the most
Fig. 2 Population genetic structure of red foxes (Vulpes vulpes;n=642) across the western contiguous U.S. based on 31 autosomal
microsatellites, estimated by the spatially-explicit Bayesian clustering algorithm Tess at K=10 genetic clusters. Admixture proportions
for each individual are shown as bar plots (above) and spatially explicit pie charts (below). We categorized six clusters as discrete in the Far
West and four clusters as continuous in the Intermountain West. Cluster abbreviations are CANN =California nonnative, GYE =Greater
Yellowstone, LAS =Lassen Cascades, ORC =Oregon Cascades, ORE =eastern Oregon, NV =Nevada, UT =Utah, SN =Sierra Nevada, SV =
Sacramento Valley, WAC =Washington Cascades.
C.B. Quinn et al.
6
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genetically impoverished: Lassen with the lowest values (H
E
=
0.51 ± 0.03 SE), followed by the Oregon Cascades (H
E
=0.55 ±
0.03 SE), and the Washington Cascades (H
E
=0.58 ± 0.03 SE;
Fig. 5C, Table S7). For comparison, autosomal heterozygosities in
the California lowlands and the Intermountain West ranged
between 0.63 and 0.73.
Commensurate with genetic diversity, estimates of N
e
were
strikingly low in the Pacific mountains with point estimates of <10
for all four populations (Fig. 5D, Table S7). In contrast, estimates of
N
e
in the Intermountain West were higher and spatially
heterogenous (x=65, range =3–352). Subsampling analyses
indicated estimates within the Pacific mountains to be consis-
tently small across subsampling trials, whereas estimates within
the Intermountain West were highly sensitive to sampling scheme
(Fig. S8). This suggests the heterogeneity of N
e
estimates within
the Intermountain West reflects a combination of true spatial
variance and biases induced by gene flow and sample size
(Waples and England 2011; Neel et al. 2013).
Lineage introgression
Across the study area, most individuals possessed native
mitochondrial matrilines belonging to either the Mountain or
Widespread subclades (Fig. 6A, Table S8). Non-western matri-
lines were especially rare (3%) in Pacific mountain populations,
the only exceptions being five individuals with a Holarctic
matriline (G-38) in the northern Oregon Cascades that did not
assign with more southern Cascade red foxes based on
microsatellites, and two presumed fur-farm matrilines (G-38
and O-24) in the Sierra Nevada. Most samples in the
Intermountain West also possessed native western matrilines,
but nonnative haplotypes were comparatively more frequent
(23%) there than in the Pacificmountains.
The Y-chromosome SNP-39 was nearly fixed (99%) for the T
allele in eastern and fur-farm reference samples and of high
frequency (76%) in the northern Alaska and northern Canada
populations (Fig. 6B, Table S9). The nonnative population in
California, known to be sourced from the eastern and north-
western lineages (Sacks et al. 2016), was also nearly fixed (99%) for
the T allele. In contrast, most (72%) samples from the western U.S.,
excluding the nonnative California population, carried the A allele,
suggesting SNP-39 acts as a reasonable discriminator of western
ancestry. Under this assumption, the spatial patterning and
frequency of introgressed patrilines were similar to those of the
mitochondrial haplotypes (Fig. 6, Table S9), with the notable
exceptions of the Washington Cascades and Sacramento Valley,
where most males carried the “eastern-like”T allele despite the
nearly exclusive occurrence of western matrilines in both regions.
In the Intermountain West, non-western matrilines and patrilines
occurred in the same localities and there was no significant
difference in their frequencies (mtDNA =23%, Y =18%, p=0.31).
Importantly, only eight males in the western U.S. (outside the
range of nonnative red foxes in California) carried both a non-
western matriline and patriline (Table S1), supporting genetic
admixture of indigenous and nonindigenous lineages in localities
where non-western markers were clustered as opposed to pure
nonindigenous red foxes.
Fine-scaled structure in Oregon
Although we observed high connectivity across the broad
Intermountain region, we sought to assess the possibility that
Fig. 4 Effective migration surface is based on 637 samples (i.e.,
excluding five samples from Colorado) typed at 31 autosomal
microsatellites. Effective migration surface for red foxes (Vulpes
vulpes) in the western contiguous U.S. estimated using EEMS. Cool
colors indicate areas with higher migration rates than expected
under isolation-by-distance, warm colors indicate lower migration
rates than expected, and white areas represent expectations under
isolation-by-distance.
Fig. 3 Genetic differentiation of red fox genetic clusters in the Western U.S. DAPC results according to (A) the first two linear discriminants
(LD) and (B) the third and fourth LDs; (C) a matrix of pairwise F
ST
, with blues and reds indicating lower and higher F
ST
values, respectively. In
both analyses, abbreviations and colors correspond to cluster membership that was assigned using Tess admixture proportions in Fig. 2.
C.B. Quinn et al.
7
Heredity

the historical portion of the Rocky Mountain red fox range in
Oregon retained its historical integrity, despite contributing gene
flow to the lower-elevation expansion zone. In support of this
scenario, analysis of fine-scaled autosomal variation suggested a
distinction in the genetic composition of the lower and upper
elevation populations that was not evident in the broader analysis.
In contrast to the semi-continental-scale analysis that split Oregon
into two genetically differentiated populations (southern Cascades
versus all other locations), the fine-scale analysis of Oregon
indicated greatest support for three genetic clusters (Fig. S9),
corresponding to three geographic groups: (1) a cluster strongly
associated with and restricted to the southern Cascades
population, as in the broader analysis, (2) a cluster with high
assignment values centered in the Wallowa and Blue Mountain
ranges that correspond to the historical Rocky Mountain
subspecific range (Bailey 1936b), and (3) a cluster restricted to
lower elevations and the Mount Hood region (Fig. 7A). The low-
elevation individuals additionally had a portion of ancestry
attributed to the cluster corresponding with the historical Rocky
Mountain red fox native range, whereas those in the native range
generally did not have qvalues corresponding to the low-
elevation cluster, suggesting the connectivity was unidirectional
from high to low elevation. The enhanced resolution of this
regional Oregon analysis also helped to clarify the relationship of
Fig. 5 Spatiallly interpolated metrics of genetic diversity of red foxes (Vulpes vulpes) in the western contiguous U.S. A mitochondrial
(mtDNA) gene diversity based on cytochrome b(including VVMC amplicon) and D-loop haplotypes (n=626); (B) Y-chromosome gene
diversity based on microsatellite haplotypes (n=282); (C) expected heterozygosity (H
E
) for 31 autosomal microsatellites (n=642); (D) genetic
effective population sizes (N
e
) estimated using the bias-corrected linkage disequilibrium estimator with the same autosomal microsatellites.
Diversity metrics were calculated for populations categorized as discrete according to spatial delineation of genetic clusters (dashed lines) and
for all other populations using an overlapping neighborhood approach. White circles indicate neighborhoods with <10 samples (<5 for
Y-chromosome diversity), for which estimations were not attempted.
C.B. Quinn et al.
8
Heredity

the Mount Hood individuals with Intermountain ancestry. These
individuals clustered with those in the lower elevation portion of
Oregon rather than those in the historical native range of the
Rocky Mountains, suggesting that the connectivity is a recent
consequence of expansion across the Intermountain West rather
than a reflection of ancient connectivity between native Rocky
Mountain and Sierra Nevada red foxes.
Analysis of mitochondrial DNA also supported unidirectional
gene flow from the historical range of the Rocky Mountain
subspecies to the low-elevation basins. The two eastern Oregon
clusters shared a native montane haplotype, but nearly all non-
western haplotypes were relegated to individuals belonging to
the low-elevation cluster (Fig. 7B). The Y chromosome similarly
indicated gene flow between the historical and expansion zones
in the form of shared Y-microsatellite haplotypes, but suggested
differing compositions of patrilines (Fig. 7C). Red foxes in the
native Rocky Mountain range contained three western patrilines
not found at lower elevations, whereas the only patriline unique to
the low-elevation expansion zone was indicated by its Y-SNP to be
nonnative in origin.
Uniparentally inherited markers also supported autosomal
analyses that showed red foxes in the Cascades as highly distinct
from those found in the rest of the state. Most individuals in the
core range shared matrilines and patrilines found only in the
Cascades (Fig. 7B, C). As with autosomal DNA, the exception was
near Mount Hood, where individuals shared matrilines and
patrilines with red foxes at lower elevations in the expansion
zone, consistent with recent gene flow.
DISCUSSION
The overarching impetus for our study was to inform the
conservation of imperiled or potentially imperiled montane red
fox populations of the Pacific mountains, which involved direct
Fig. 6 Lineage introgression of red foxes (Vulpes vulpes) in the western contiguous U.S. A mitochondrial matrilines (n=673), with colors
indicating phylogeographic clade and dots indicating matrilines sampled from fur farms. Inset shows median joining network used to
determine phylogeographic clades. Matrilines belonging to the Mountain and Widespread subclades are assumed indigenous unless also
sampled in fur farms; (B) Y-chromosome patrilines (n=281), as informed by a single ancestry-informative SNP. Inset shows the frequency of
the western-like allele in reference samples (n=361) from globally distributed fur farms and wild North American populations.
Fig. 7 Genetic structuring of red foxes (Vulpes vulpes) in Oregon and southern Washington. Genetic structure according to (A) clustering of
120 autosomal microsatellite genotypes at K=3 according to the spatially-explicit Bayesian clustering algorithm Tess; (B) 110 mitochondrial
haplotypes with matrilineal clade indicated, and (C) 46 Y-microsatellite haplotypes. Shared Y-microsatellite haplotypes are connected by lines
of the same color. Mitochondrial and Y-microsatellite haplotypes that are not native to the western U.S. are indicated with black dots.
C.B. Quinn et al.
9
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questions about those populations as well as about their
relationship to and characteristics of adjacent populations in the
Intermountain West. Broadly, the Pacific mountains contained
isolated populations that were genetically distinct and depaupe-
rate of diversity, whereas red foxes in the Intermountain West
showed low differentiation among regions and high genetic
diversity, suggestive of higher gene flow, larger effective
population sizes, or both. We found the transition from strong
genetic structure in the Pacific mountains to the more weakly
structured Intermountain West to be abrupt, with only low levels
of genetic exchange between the two regional populations
despite increasing proximity of expanding red fox populations in
the low-elevation basins.
In contrast to the genetic discontinuities observed between
Pacific and Intermountain regional populations of other montane
species, which typically reflect isolation in different glacial refugia
(Barrowclough et al. 2004; Manthey et al. 2012; Hope et al. 2016;
Arbogast et al. 2017), the divisions observed in the present study
were comparatively shallow and recent (e.g., <20,000 years; Aubry
et al. 2009). Thus, our data suggest a contemporary genetic
structure of western red foxes caused in large part by distinct
anthropogenic factors. Native Pacific mountain populations likely
became isolated as part of a general population decline
precipitated by overharvest in the 19th and early 20th centuries
(Perrine et al. 2010), whereas native montane populations in the
Intermountain region likely colonized the cold desert basins and
received admixture from translocated nonnative foxes during the
mid-to late 1900s. Below, we discuss our findings and interpreta-
tions in the context of each of these regions separately.
Genetic bottlenecks in the Pacific mountains
Our first objective was to identify whether red foxes in the
southern Cascades (i.e., Oregon Cascade and Lassen populations)
had low genetic diversity and effective population sizes, similarly
to those previously documented in the Sierra Nevada and the
northern Cascades of Washington (Akins et al. 2018; Quinn et al.
2019). All populations in the Pacific mountains, including Lassen
and Oregon, had strikingly small genetic effective population sizes
(N
e
< 10). Microsatellite heterozygosities were also much lower
(H
E
=0.51–0.58) in the Pacific mountains than other western
regions. The exception was the Sierra Nevada DPS (H
E
=0.66), for
which heterozygosity was only recently elevated by admixture
with low-elevation foxes from the Great Basin (Quinn et al. 2019).
Prior to immigration of foxes from the Intermountain region
beginning in 2013, its heterozygosity estimated using the same
marker set was the lowest of all western populations (H
E
=0.43,
Quinn et al. 2019). As a benchmark for historical diversities,
heterozygosities estimated from museum samples collected
during 1850–1950 (based on a subset of 12 microsatellites) were
0.70 for the southern Cascades and 0.65 for the Sierra Nevada
(Sacks et al. 2010). Together, the small effective population size
and reduced heterozygosities imply a shared history of recent and
dramatic bottlenecks, consistent with the hypothesis that
unregulated harvest and poisoning associated with predator
eradication activities during the 19th and 20th centuries were
major contributors to population declines of montane red foxes
(Perrine et al. 2010).
Evidence for severe genetic bottlenecks in all Pacific mountain
populations has particular relevance for conservation of the Sierra
Nevada subspecies, for which federal protection is currently
restricted to the Sierra Nevada DPS (U.S. Fish and Wildlife Service
2021). At the time of initial review, no nuclear data were available
from the Oregon Cascades and listing of the Southern Cascade
DPS was declined based solely on a distribution of sighting
records spanning the length of the Oregon Cascades (U.S. Fish and
Wildlife Service 2015). Our findings raise a question as to what
degree low genetic effective population sizes also reflect current
abundance (i.e., census population size). At least two scenarios are
possible with respect to contemporary populations in the Pacific
mountains: (1) they are small and possibly decreasing in
abundance, or (2) they are larger than suggested by their genetic
effective population sizes and possibly increasing in abundance. In
California, camera-based and noninvasive genetic scat surveys
clearly indicate that Lassen and Sierra Nevada populations are
composed of few individuals, relegated to a fraction of their
historical range (SCAT, 2022). In Washington and Oregon,
contemporary census population sizes are less certain, both in
terms of abundance and trajectory (U.S. Fish and Wildlife Service
2015; Washington Department of Fish and Wildlife 2015). In the
absence of abundance data, the low genetic effective population
sizes and diversities observed in this study signal that the
conservation status may not be all that more robust for the
Cascade subspecies in Washington or the Southern Cascade DPS
of the Sierra Nevada subspecies than it is for the endangered
Sierra Nevada DPS.
In the meantime, our findings raise the possibility that
regardless of poorly understood contemporary stressors (Perrine
et al. 2010; SCAT, 2022), the genetic legacy of past declines may
itself be contributing to a sluggish recovery in some or all Pacific
mountain populations. Harvest is not considered to be a primary
stressor because no harvest occurs in California and harvest is
regulated in Oregon (SCAT, 2022). Furthermore, inbreeding
depression has previously been demonstrated in the Sierra
Nevada DPS (Quinn et al. 2019), raising the possibility that other
Pacific populations with only slightly higher N
e
may also
experience reduced fitness, particularly relative to more outbred
neighbors. Understanding the risks of inbreeding depression in
other Pacific populations should be a research priority. Future
study could involve pairing more precise estimates of inbreeding
(e.g., using runs of homozygosity; Kardos et al. 2016) with variation
in fitness-related traits among western populations
However, even without additional study, the current data are
sufficient to implicate small population effects as a credible and
immediate threat to the persistence of the Lassen population
(e.g., following guidelines in Frankham et al. 2017). The genetic
data definitively show that the Lassen and Oregon Cascade
populations are not connected by long-distance dispersal, as
suggested in previous assessments (U.S. Fish and Wildlife
Service 2015), supporting near-total isolation of the Lassen
population from other Pacific mountain populations. Combined
with already diminished genetic diversity, as well as observa-
tions of interbreeding among first-order relatives and few
documented individuals (SCAT, 2022), a strong case can be
made for the likely benefits of genetic rescue to the Lassen
population. In terms of safeguarding the “3Rs”(resiliency,
representation, redundancy; Shaffer and Stein 2000), a founda-
tional principle of the U.S. Endangered Species Act, extirpation
of the Lassen population would compromise the redundancy
and representation of the Sierra Nevada subspecies and Pacific
mountain populations as a whole.
Origin of red foxes in intermountain basins
Our second objective was to determine whether the recent
appearance of red foxes in the cold desert basins of the
Intermountain West could be explained by colonization of non-
western lineages, as might be expected from habitat associations
that differ from both historical and Pacific-mountain counterparts.
We found that while inter-lineage admixture was present and at
higher rates than in the Pacific mountains, the genomic
composition of red foxes in the desert basins was predominantly
native in origin. Frequencies of native western matrilines and
patrilines were both high and roughly equivalent throughout low-
elevation basins. In addition, we observed only weak nuclear
differentiation between foxes in the historical range of the Rocky
Mountain subspecies and those in expansion zones. Neither
Bayesian clustering nor EEMS analyses differentiated these zones
C.B. Quinn et al.
10
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when the entire dataset was analyzed, implying either a high level
of contemporary connectivity or a common founding source.
These findings are consistent with conclusions of previous studies
(e.g., Statham et al. 2012; Volkmann et al. 2015), but are based on a
more comprehensive geographic and genomic dataset. Such
downslope expansion of populations from the Rocky Mountains
may have been facilitated by agricultural (e.g., irrigation) and other
habitat conversion practices affecting prey abundance at lower
elevations of the Intermountain region (Fichter and Williams 1967;
Green et al. 2017). In addition, or alternatively, admixture itself (i.e.,
the exchange of particular genes) could have contributed to
subsequent expansions of Rocky Mountain red foxes throughout
lower elevation habitats.
Regardless of the driver, downslope expansion of populations
likely increased proximity and opportunity for contact between
native and nonnative red foxes in the Intermountain basins.
Fine-scaled genetic analyses suggested low-level, but pervasive
inter-lineage admixture in the expansion zone. When we
repeated Bayesian clustering analyses at a local scale within
Oregon (the region where higher sampling density allowed for
more direct comparison of low- and high-elevation regions)
structuring between historical and expansion zones was
evidentinbothmitochondrialand nuclear genomes. These
findings suggest the possibility that admixture was largely
restricted to lower elevations, leaving genomes from higher
elevation, montane regions in relatively “pure”native form. A
negative correlation between elevation and admixture has
been documented at a local scale in Colorado (Merson et al.
2017), but has not yet been tested throughout the Intermoun-
tain West. Increased sampling of more high-elevation montane
regions in the Rocky Mountains and higher-powered genomic
datasets (e.g., reduced representation-sequencing) could help
elucidate more precisely how landscape features correspond to
nonnative admixture.
The compositions of nonnative matrilines and patrilines were
insufficient on their own to discriminate between fur-farm and
wild midcontinent populations as the principal source of
admixture. Both fur-farm stock and wild populations nearest the
Rocky Mountains (e.g., Great Plains, Canadian taiga) are them-
selves expected to be composed of an admixture of northern and
eastern lineages (Aubry et al. 2009; Statham et al. 2012; Black et al.
2018). Thus, while we sampled some matrilines common in fur
farms (e.g., F-9 in the vicinity of Salt Lake City, UT; Fig. 6A), others
(e.g., G-38; Table S8) were sampled in both translocated and wild
midcontinent populations and were thus ambiguous in origin
(Black et al. 2018). Tentatively, the spatial distribution of nonnative
haplotypes supported fur-farm admixture over natural expansions.
Nonnative haplotypes seemed more clustered than clinal, which is
more congruent with localized pockets of feral fur-farm foxes near
developed areas, as observed in lowland regions of California
(Sacks et al. 2016) and Colorado (Merson et al. 2017), than an
advancing directional wave (Kamler and Ballard 2002). The two
hypotheses are not mutually exclusive; both natural and anthro-
pogenic hybridization could be contributing to higher rates of
inter-lineage admixture in the cold desert basins relative to the
historical range.
Conservation implications
Red foxes of the Intermountain West seem to be thriving and
therefore are not of direct conservation concern. However, the
potential effects of expanding low-elevation populations on
Pacific mountain populations, both positive and negative, bear
consideration. On one hand, low-to-moderate gene flow might be
expected to alleviate inbreeding depression and boost short-term
fitness levels (Frankham 2015;Whiteleyetal.2015), as observed
in the first few generations following gene flow from the Great
Basin into the Sierra Nevada population (Quinn et al. 2019).
Conversely, the extreme disparity in population sizes makes the
smaller Pacific mountain populations susceptible to swamping
and disruption of local adaptations (Rhymer and Simberloff 1996;
Harris et al. 2019; though see Fitzpatrick et al. 2020). A critical
knowledge gap is how introgression from non-western lineages
has altered the phenotype of admixed red foxes in the low-
elevation Intermountain basins. Studies in the Greater Yellow-
stone Ecosystem found red foxes there to be considerably larger
in body size than their Pacific mountain counterparts, suggesting
a departure from the historical morphotype of montane red foxes
(Roest 1977; Fuhrmann 1998; SCAT, 2022). Inter-lineage admix-
ture could have introduced variation in other traits relevant to
fitness in high-elevation environments as well (e.g., breeding
phenology; Cross et al. 2018).
Given admixture in the Intermountain basins and its unknown
effects on phenotype, we recommend that connectivity not be
actively facilitated between Pacific mountain and low-elevation
Intermountain regions. However, the expanding trajectory of red
foxes in the Intermountain West suggests that contact with small
populations in the Pacific mountains may be inevitable. As of yet,
most Pacific mountain populations have retained their genetic
distinctiveness despite the increasing proximity of low-elevation
populations to their east. The isolation is especially notable in the
southern portion of the Cascade Mountains in Oregon, where the
native population showed little introgression from adjacent
admixed basin red foxes well-within dispersal distance. In contrast,
the federally endangered Sierra Nevada DPS showed more
extensive gene flow with low-elevation Intermountain red foxes.
Continued monitoring of contact zones will be necessary to
determine whether differentiation is actively maintained by
biological processes (e.g., Sacks et al. 2011) or represents a
snapshot in time during the process of genetic homogenization.
More broadly, our findings highlight new complexities that
can affect decisions about managing the genetic health and
composition of endangered populations. A central paradigm in
conservation genetics relates to balancing the risks of inbreed-
ing and outbreeding depression in planned genetic rescue (Love
Stowell et al. 2017;Rallsetal.2018). Conversations about how to
select donor individuals that will minimize outbreeding depres-
sion (e.g., swamping of local adaptations, increasing genetic
load) while maximizing heterozygosity typically presuppose that
managers have complete control of whether to conduct
translocations, which populations to use as sources, and the
rate at which to introduce individuals (e.g., Frankham et al.
2011,2017; Kyriazis et al. 2021; Ralls et al. 2020). For managing
potentially inbred Pacific mountain populations of red foxes, we
face a more dynamic situation whereby an adjacent, increasing
population currently or may soon contribute gene flow,
regardless of its genetic suitability. The decision to intervene
and actively translocate individuals from donor populations into
Pacific mountain populations must weigh the usual risks,
benefits,andcosts,butalsodosoexplicitlyinthecontextof
unmanaged gene flow from admixed Intermountain red foxes.
Decision analyses will need to take into consideration rates of
gene flow relative to effective population sizes, the importance
of adaptive differences between Pacific mountain and low-
elevation populations, and the capacity for selection to counter
infusion of maladaptive alleles or allele combinations. Such
assessments will ultimately inform the genetic management of
Pacific mountain populations, requiring innovative modifications
to a traditional conservation dilemma.
DATA AVAILABILITY
We have deposited the primary data underlying these analyses as follows: Autosomal
microsatellite genotypes, Y-microsatellite genotypes, Y-SNP genotypes: Dryad
(https://doi.org/10.25338/B87P8K). Novel mitochondrial sequences: GenBank
(OM810161–OM810162). Locations, Y-SNP identifiers, Y-microsatellite genotype
identifiers, mitochondrial haplotype identifiers for samples in study area and
reference samples: Supplementary Material Tables S1, S2.
C.B. Quinn et al.
11
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ACKNOWLEDGEMENTS
We thank two anonymous reviewers and the handling editor for helpful comments
on an earlier draft of this paper. Many collaborators contributed samples: Oregon
Department of Fish and Wildlife (J. Bowles, J. Vaughn, L. Erickson); California
Department of Fish and Wildlife (B. Hatfield, C. Stermer); Nevada Department of Fish
and Wildlife (P. Jackson, R. Woolstenhulme); Idaho Department of Fish and Game
C.B. Quinn et al.
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(D. Kemner); the Deschutes, Willamette, and Mt. Hood national forests (J. Doerr,
A. Dyck, C. Ferland, R. Seitz, L. Turner); the USDA Pacific Northwest Research Station
(K. Moriarty); USDA Wildlife Services (J. Bennett, M. Bodenchuk, M. Collinge,
M. Jensen, E. McDonald, T. Pitlick, and J. Wiscomb); Crater Lake, Mount Rainier,
Great Basin, Lassen, Yosemite, and Grand Teton national parks (S. Mohren,
B. Hamilton, M. Magnuson, S. Stock); Oregon State University-Cascades
(D. Gumtow-Farrior, K. Gumtow-Farrior); Cascadia Wild; High Desert Museum; Central
Sierra Environmental Resource Center; R. Beach; and R. Stroeberl. Funding for this
research was provided by many sources, primarily the US Fish and Wildlife Service
(Agreement No. F18AC00276), Oregon Department of Fish and Wildlife (Agreement
379-15) through a Pittman Robertson grant, USDA Forest Service (Agreement No. 15-
CR- 11060120-029), Nevada Department of Wildlife through the state’s $3 Predator
Fee Program and Wildlife Heritage Trust Account Grant (No. 15-17), a Western
National Parks Association Scientific Research Grant, and Great Basin Heritage Area
Partnership. Additional funding was provided by multiple agreements with the US
Fish and Wildlife Service, USDA Forest Service, National Park Service, Cascade
Carnivore Project, Cascadia Wild, and the Mammalian Ecology and Conservation Unit
of the Veterinary Genetics Laboratory in the UC Davis School of Veterinary Medicine.
AUTHOR CONTRIBUTIONS
CBQ and BNS conceived the study. CBQ, SPQ, PA, and SLV conducted the research.
CBQ, SPQ, JRA, PRC, PBA, JAS, PJF, GAG, TLH, and BNS contributed samples and
information about them. CBQ wrote the initial paper with BNS and all authors
assisted in critique and revising the paper.
COMPETING INTERESTS
The authors declare no competing interests.
ADDITIONAL INFORMATION
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41437-022-00522-4.
Correspondence and requests for materials should be addressed to Cate B. Quinn.
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