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Comparative Study
, 10 (8), e0136329

Mitochondrial Analysis of the Most Basal Canid Reveals Deep Divergence Between Eastern and Western North American Gray Foxes (Urocyon Spp.) and Ancient Roots in Pleistocene California

Comparative Study

Mitochondrial Analysis of the Most Basal Canid Reveals Deep Divergence Between Eastern and Western North American Gray Foxes (Urocyon Spp.) and Ancient Roots in Pleistocene California

Natalie S Goddard et al. PLoS One.


Pleistocene aridification in central North America caused many temperate forest-associated vertebrates to split into eastern and western lineages. Such divisions can be cryptic when Holocene expansions have closed the gaps between once-disjunct ranges or when local morphological variation obscures deeper regional divergences. We investigated such cryptic divergence in the gray fox (Urocyon cinereoargenteus), the most basal extant canid in the world. We also investigated the phylogeography of this species and its diminutive relative, the island fox (U. littoralis), in California. The California Floristic Province was a significant source of Pleistocene diversification for a wide range of taxa and, we hypothesized, for the gray fox as well. Alternatively, gray foxes in California potentially reflected a recent Holocene expansion from further south. We sequenced mitochondrial DNA from 169 gray foxes from the southeastern and southwestern United States and 11 island foxes from three of the Channel Islands. We estimated a 1.3% sequence divergence in the cytochrome b gene between eastern and western foxes and used coalescent simulations to date the divergence to approximately 500,000 years before present (YBP), which is comparable to that between recognized sister species within the Canidae. Gray fox samples collected from throughout California exhibited high haplotype diversity, phylogeographic structure, and genetic signatures of a late-Holocene population decline. Bayesian skyline analysis also indicated an earlier population increase dating to the early Wisconsin glaciation (~70,000 YBP) and a root height extending back to the previous interglacial (~100,000 YBP). Together these findings support California's role as a long-term Pleistocene refugium for western Urocyon. Lastly, based both on our results and re-interpretation of those of another study, we conclude that island foxes of the Channel Islands trace their origins to at least 3 distinct female founders from the mainland rather than to a single matriline, as previously suggested.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Fig 1
Fig 1. Approximate range of the gray fox (Urocyon cinereoargenteus).
Dashed line indicates hypothetical northern boundary at the last glacial maximum and arrows denote hypothetical post-Pleistocene expansion [11]. Filled circles represent approximate locations of gray and island (U. littoralis) foxes used in the present study from the southwestern (n = 131 gray fox; 11 island fox) and southeastern (n = 38) portions of the range. Southeastern samples were from a 300 km2 region, represented by a single large filled circle.
Fig 2
Fig 2. Maximum likelihood tree of concatenated 785 bp cytochrome b and D loop sequences.
Tree was constructed using a HKY+ Γ model of DNA substitution. Bootstrap values >75 are shown at nodes, although values on inner nodes are shown only on the Urocyon clade expansion. Asterisks denote haplotypes of island foxes.
Fig 3
Fig 3. Bayesian phylogeny of concatenated 785 bp cytochrome b and D loop haplotypes.
Tree was constructed using HKY (cytochrome b) and HKY+Γ (D loop) models of DNA substitution. Bayesian posterior probability values >0.75 are shown at nodes. Asterisks denote haplotypes of island foxes.
Fig 4
Fig 4. Coalescent analysis of mitochondrial cytochrome b sequences of 180 Urocyon in IMa2, providing (A) an estimate of splitting time separating eastern and western Urocyon and (B) estimates of effective population sizes (N e) of western, eastern, and ancestral Urocyon spp.
Fig 5
Fig 5. Bayesian skyline plot illustrating Pleistocene increase and Holocene decline in population size of western Urocyon based on 785 bp of concatenated cytochrome b and D loop sequence data.
The black line represents the median population size, while the lines above and below represent the 95% highest posterior density (HPD). Dashed lines indicate glacial-interglacial boundaries and the blue shaded rectangle indicates the last glacial maximum (LGM) for reference to the population sizes. The estimated root height (coalescence time) was 96,860 years (95% HPD: 55,318–144,274 years) or 48,380 generations (assuming a 2-year generation time).
Fig 6
Fig 6. Median-joining network of 785-bp composite cytochrome b and D loop haplotypes.
Haplogroups are color-coded for reference to map. Numbered samples correspond to those listed in Table 1. Haplotypes in green correspond to island foxes from Santa Cruz Island (A-4), San Nicholas Island (A-7), and San Clemente Island (A-23). All others represent gray foxes from the mainland. Dashed ellipses on network enclose nested tip clades and dotted arcs denote nesting clades according to the Bayesian tree (Fig 3), with Bayesian Posterior Support indicated with numerals in italics. Positioning of the root is in reference to the eastern haplotypes as indicated on the Bayesian tree (Fig 3). Nodes are approximately proportional to samples size for n < 10 (i.e., circle size was the same for n = 10–30). Inferred, unsampled haplotypes are marked with black and red circles.

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The authors received no specific funding for this work.