Mark J. Statham

Phylogeography of the North American red fox: vicariance in Pleistocene forest refugia

Fossil, archaeological, and morphometric data suggest that indigenous red foxes in North America were derived from vicariance in two disjunct refugia during the last glaciation: one in Beringia and one in the contiguous USA. To test this hypothesis, we conducted a phylogeographical analysis of the North American red fox within its presettlement range. We sequenced portions of the mitochondrial cytochrome b (354 bp) gene and D‐loop (342 bp) from 220 historical red fox specimens. Phylogenetic analysis of the cytochrome b gene produced two clades that diverged c. 400 000 years before present (bp): a Holarctic and a Nearctic clade. D‐loop analyses of the Nearctic clade indicated three distinct subclades (≥ 99% Bayesian posterior probability); two that were more recently derived (rho estimate c. 20 000 bp) and were restricted to the southwestern mountains and the eastern portion of North America, and one that was older (rho estimate c. 45 000 bp) and more widespread in North America. Populations that migrated north from the southern refugium following deglaciation were derived from the colonization of North America during or prior to the Illinoian glaciation (300 000–130 000 bp), whereas populations that migrated south from the northern refugium represent a more recent colonization event during the Wisconsin glaciation (100 000–10 000 bp). Our findings indicate that Nearctic clade red foxes are phylogenetically distinct from their Holarctic counterparts, and reflect long‐term isolation in two disjunct forest refugia during the Pleistocene. The montane lineage, which includes endangered populations, may be ecologically and evolutionarily distinct.

A restricted hybrid zone between native and introduced red fox (Vulpes vulpes) populations suggests reproductive barriers and competitive exclusion.

Introduced species can threaten native taxa in multiple ways, including competition and hybridization, which can reduce fitness, alter ecological niches or swamp native genomes. Encroachment and hybridization by introduced species also provide opportunities to study the dynamics of invasiveness and hybridization during early stages following contact. We used 33 microsatellites, 51 single nucleotide polymorphisms and a mtDNA marker to characterize the extent and spatial pattern of encroachment and hybridization between a native, endemic subspecies of red fox (Vulpes vulpes patwin) and an introduced red fox population composed of highly admixed, phylogenetically divergent stock, resulting from a century of domestication. Both nuclear and mtDNA markers indicated that hybridization was primarily restricted to a narrow zone where the two populations came into contact. Although a few introgressed genotypes were detected in the interior of the native range, we found no immigrant foxes or F1 or F2 hybrids there, suggesting native foxes excluded introduced individuals. We speculate that the observed interbreeding at the periphery was facilitated by low densities. In total, 98% of mtDNA haplotypes in the native range were native and 96% of the nuclear ancestry was estimated to be native. Although the introduced range had expanded fivefold over the past four decades, native and non‐native haplotypes from museum samples collected in and near the native range three decades earlier showed a similar geographic distribution as today, suggesting that the native range and hybrid zone were relatively stable. We hypothesize that the monogamous mating system of red foxes and other wild canids may enhance their resistance to hybridization because of greater fitness consequences associated with mate discrimination.

North American montane red foxes: expansion, fragmentation, and the origin of the Sacramento Valley red fox

Most native red foxes (Vulpes vulpes) in the western contiguous United States appear to be climatically restricted to colder regions in the major mountain ranges and, in some areas, have suffered precipitous declines in abundance that may be linked to warming trends. However, another population of unknown origin has occurred in arid habitats in the Sacramento Valley of California well outside this narrow bioclimatic niche since at least 1880. If native, this population would be ecologically distinct among indigenous North American red foxes. We used mitochondrial and microsatellite markers from historical and modern samples (modes: 1910–1930 and 2000–2008, respectively) obtained throughout the western United States to determine the origins of the Sacramento Valley red fox, and assess the historical and modern connectivity and genetic effective population sizes of Sacramento Valley and montane red foxes. We found clear and consistent evidence supporting the indigenous origin of the Sacramento Valley population, including the phylogenetic positioning of the dominant, endemic mtDNA clade and microsatellite clustering of the Sacramento Valley population with the nearest montane population. Based on both mitochondrial and microsatellite AMOVAs, connectivity among Western populations of red foxes declined substantially between historical and modern time periods. Estimates based on temporal losses in gene diversity for both marker types suggest that both the Sierra Nevada (including the Southern Cascades population) and the Sacramento Valley populations have small genetic effective population sizes. Significant heterozygote excesses also indicate the occurrence of recent bottlenecks in these populations. Both substitutions distinguishing the 2 endemic Sacramento Valley haplotypes from the dominant montane haplotype were in the coding region and nonsynonymous, consistent with adaptive differences. These findings along with previously reported body size distinctions between Sacramento Valley and montane red foxes argue for distinct subspecific status for the Sacramento Valley red fox, for which we propose the designation V. v. patwin n. subsp. The small genetic effective population size estimates for the Sierra Nevada red fox and Sacramento Valley red fox are cause for concern, as is the possibility of genetic introgression into the latter population from an adjacent, recently established nonnative population.

The origin of recently established red fox populations in the United States: translocations or natural range expansions?

Abstract Red foxes (Vulpes vulpes) are native to boreal and western montane portions of North America but their origins are unknown in many lowland areas of the United States. Red foxes were historically absent from much of the East Coast at the time of European settlement and did not become common until the mid-1800s. Some early naturalists described an apparent southward expansion of native foxes that coincided with anthropogenic habitat changes in the region. Alternatively, red foxes introduced from Europe during Colonial times may have become established in the east and subsequently expanded their range westward. The red fox also was absent historically from most lowland areas of the western United States. Extant populations of red foxes in those areas are considered to have arisen from intentional introductions from the east (and by extension are putatively European), escapes or releases from fur farms, or range expansions by native populations. To test these hypotheses we compared mitochondrial DNA sequences (cytochrome b and D-loop) from 110 individuals from 6 recently established populations to 327 native (primarily historical) individuals from Eurasia, Alaska, Canada, the northeastern United States, and montane areas in the western contiguous United States, and to 38 individuals from fur farms. We found no Eurasian haplotypes in North America, but found native haplotypes in recently established populations in the southeastern United States and in parts of the western United States. Red foxes from the southeastern United States were closely related to native populations in eastern Canada and the northeastern United States, suggesting that they originated from natural range expansions, not from translocation of European lineages, as was widely believed prior to this study. Similarly, recently established populations in the Great Basin and in western Oregon originated primarily from native populations in western montane regions, but also contained a few nonnative North American haplotypes. In contrast, populations in western Washington and southern California contained nonnative, highly admixed stock that clearly resulted from intracontinental translocations. Several common haplotypes in these populations originated in regions where fur-farm stocks originated. Although European red foxes translocated to the eastern United States during Colonial times may have contributed genetically to extant populations in that region, our findings suggest that most of the matrilineal ancestry of eastern red foxes originated in North America.

Efficient species identification of pine marten (Martes martes) and red fox (Vulpes vulpes) scats using a 5′ nuclease real-time PCR assay

Monitoring wildlife species by DNA identification of samples collected non-invasively is an important tool in conservation management. DNA identification of species from faecal (scat) samples is problematic due to the small quantities and poor quality of the DNA isolated from such samples. This study demonstrates the use of real-time PCR technology in the identification of red fox (Vulpes vulpes) and pine marten (Martes martes). It is shown that real-time PCR can be used to identify fox and pine marten by either melting curve analysis (Tm determination) with SYBR Green 1 detection or by the use of species specific fluorogenic probes. The technique is shown to work efficiently with scat DNA.

Range-wide multilocus phylogeography of the red fox reveals ancient continental divergence, minimal genomic exchange and distinct demographic histories

Widely distributed taxa provide an opportunity to compare biogeographic responses to climatic fluctuations on multiple continents and to investigate speciation. We conducted the most geographically and genomically comprehensive study to date of the red fox (Vulpes vulpes), the worlds most widely distributed wild terrestrial carnivore. Analyses of 697 bp of mitochondrial sequence in ~1000 individuals suggested an ancient Middle Eastern origin for all extant red foxes and a 400 kya (SD = 139 kya) origin of the primary North American (Nearctic) clade. Demographic analyses indicated a major expansion in Eurasia during the last glaciation (~50 kya), coinciding with a previously described secondary transfer of a single matriline (Holarctic) to North America. In contrast, North American matrilines (including the transferred portion of Holarctic clade) exhibited no signatures of expansion until the end of the Pleistocene (~12 kya). Analyses of 11 autosomal loci from a subset of foxes supported the colonization time frame suggested by mtDNA (and the fossil record) but, in contrast, reflected no detectable secondary transfer, resulting in the most fundamental genomic division of red foxes at the Bering Strait. Endemic continental Y‐chromosome clades further supported this pattern. Thus, intercontinental genomic exchange was overall very limited, consistent with long‐term reproductive isolation since the initial colonization of North America. Based on continental divergence times in other carnivoran species pairs, our findings support a model of peripatric speciation and are consistent with the previous classification of the North American red fox as a distinct species, V. fulva.

Temporal genetic variation of the red fox, Vulpes vulpes, across western Europe and the British Isles

Quaternary climatic fluctuations have had profound effects on the phylogeographic structure of many species. Classically, species were thought to have become isolated in peninsular refugia, but there is limited evidence that large, non-polar species survived outside traditional refugial areas. We examined the phylogeographic structure of the red fox (Vulpes vulpes), a species that shows high ecological adaptability in the western Palaearctic region. We compared mitochondrial DNA sequences (cytochrome b and control region) from 399 modern and 31 ancient individuals from across Europe. Our objective was to test whether red foxes colonised the British Isles from mainland Europe in the late Pleistocene, or whether there is evidence that they persisted in the region through the Last Glacial Maximum. We found red foxes to show a high degree of phylogeographic structuring across Europe and, consistent with palaeontological and ancient DNA evidence, confirmed via phylogenetic indicators that red foxes were persistent in areas outside peninsular refugia during the last ice age. Bayesian analyses and tests of neutrality indicated population expansion. We conclude that there is evidence that red foxes from the British Isles derived from central European populations that became isolated after the closure of the landbridge with Europe.

Pleistocene Refugia and Holocene Expansion of a Grassland-Dependent Species, the Black-Footed Ferret (Mustela nigripes)

Abstract Climate change during the late Quaternary has been implicated as the cause of both massive range shifts and extinction events. We combined molecular marker data and previously published fossil data to reconstruct the late Quaternary history of a grassland-dependent species, the black-footed ferret (Mustela nigripes), and to determine whether populations from Pleistocene refugia in the Columbia Basin, eastern Beringia, and Great Plains persisted into the Holocene and Recent eras. Using DNA extracted from 97 museum specimens of extirpated populations, we amplified 309 bp of the mtDNA control region, and 8 microsatellite markers from the nuclear genome. Overall haplotype diversity from 309 base pairs (bp) of mitochondrial DNA (mtDNA) control region was low (5 haplotypes, nucleotide diversity = 0.001 ± 0.001 SD) and was contained within a single phylogenetic clade. The star phylogeny and unimodal mismatch distribution indicated that a rapid range expansion from a single Pleistocene refugium occurred. Microsatellite data corroborated this genetic pattern: populations from the mixed grasslands of the Great Plains had significantly higher expected heterozygosity and allelic richness than populations to the west (HE = 0.66 versus 0.41, AR = 4.3 versus 2.7, respectively), and 𝜰, a measure of relative population size, was substantially greater in the east than west (2.4 versus 0.7). We infer from these data that black-footed ferrets rapidly colonized western ecoregions in a stepwise fashion from the Great Plains to the intermountain regions of the Rocky Mountains and the Colorado Plateau after the last ice age. It appears that glacial retreat and global warming caused both range expansion and localized extinction in this North American mustelid species.

Discovery of a Remnant Population of Sierra Nevada Red Fox (Vulpes vulpes necator)

Abstract The Sierra Nevada red fox (Vulpes vulpes necator) occurred historically throughout the high elevations of Californias Sierra Nevada and southern Cascade Mountains. Before this study, the only known remaining population in California consisted of ≤ 20 individuals restricted to the Lassen Peak region in the southern Cascades. In August 2010, we photographed a red fox in the Sonora Pass area of the Sierra Nevada, > 100 km from the Lassen Peak region. To determine if multiple individuals were present and were indigenous, we set up additional camera stations, collected genetic samples (saliva, scat, hair, and a carcass), and conducted a comparative genetic analysis between these individuals and historical and modern reference samples. Photo-detections identified at least three individuals based on pelage characteristics. Genetic analyses identified two females and one male, whose microsatellite profiles suggested they were closely related. A genetic assignment analysis indicated that all three individuals clustered most closely (> 95%) with historical samples from the Sierra Nevada, and were distinct from those in the Lassen Peak region. Additionally, mtDNA and microsatellite alleles unique to each population confirmed that the Sonora Pass individuals represent a second remnant California population of Sierra Nevada red fox. Reduced genetic diversity relative to historical levels in both remnant populations was consistent with small populations. Follow-up surveys are needed to determine the abundance and distributional extent of the Sonora Pass population, combined with research on both populations to assess demographic trajectories, determine threats, and to inform conservation efforts.

Fate of the other redcoat: remnants of colonial British foxes in the Eastern United States

Red foxes were absent or rare in the southeastern United States until the late 1800s. Their origins potentially include natural population increase/expansion, translocations from Europe, and, eventually, 20th century fur farming. Previous studies have found no European haplotypes in North America, but few samples were sourced from the Atlantic coastal plain, closer to the source of putative introductions. Through analysis of mitochondrial DNA in 584 red foxes from this region, we identified indigenous haplotypes in ≥ 35% of foxes, 1 of 2 European haplotypes in 17% of foxes and fur farm haplotypes in ≥ 13% of foxes; another 35% of foxes had haplotypes potentially indigenous or native. In contrast, only 3 of 135 (2%) male foxes carried a single European Y chromosome haplotype. Most European and fur farm haplotypes were found near the densely human-populated coastal plain and Hudson River lowlands; most red foxes of the Appalachians and Piedmont had native eastern haplotypes. Our findings suggest that the more remote, upland populations primarily reflect indigenous red fox matrilines, whereas urban-associated populations in and around the mid-Atlantic coastal plain and Hudson lowlands reflect an admixture of native and nonnative maternal sources. Autosomal markers are needed to further elucidate the extent of European and fur farm introgression in the Appalachians and further west.