Abstract

We examined intraspecific relationships in the eastern fox squirrel (Sciurus niger) and the eastern gray squirrel (S. carolinensis) using sequence variation in a portion of the mitochondrial DNA cytochrome-b gene and part of the D-loop in the control region. These closely related species are codistributed temperate forest obligates that have similar generation time and population ecologies. For both species, we documented high haplotype diversity, low nucleotide variation, and several groups of divergent haplotypes. However, there is a general lack of spatial structure in maternal lineages within each species. For S. carolinensis, we observed a pattern of population genetic structure that suggests the presence of at least 2 distinct refugiai populations that evolved in isolation during the Pleistocene (approximately 98.3–266.3 thousand years ago [kya]) and subsequently expanded to the species' current range following the last glacial maximum. For S. niger, the genetic structure was much less pronounced, with fewer strongly diverged haplotypes. This finding suggests that eastern fox squirrels persisted in either a single population in a glacial refugium or as several refugiai populations that maintained gene flow throughout the Pleistocene. For both species, there is evidence that scattered populations were present in multiple, small refugia close to the Laurentide Ice Sheet, allowing rapid range expansion following glacial recession. Taken together, our results indicate that S. niger and S. carolinensis underwent multiple episodes of genetic divergence during isolation in glacial refugia, followed by range expansion and contact that resulted in admixture of divergent maternal lineages within each species during interglacials. Examination of our data further indicates that the most recent range expansion in both species occurred within the past 12–20 kya.

Climate change, and the associated changes in size and position of ice sheets, dramatically shifted the distributions of temperate plants and animals in eastern North America during the Pleistocene (Pielou 1991). Evidence from fossil pollen and plant macrofossils indicates that many temperate tree species underwent episodes of being pushed into southern refugia during glacial advances, followed by northward expansion after the glaciers receded (Davis 1981; Delcourt and Delcourt 1981; Soltis et al. 2006). Phylogeographic data often are used to examine current patterns of genetic connectivity and differentiation to gain insight into historical factors, such as climate change, that have altered the distributions of plants and animals. Analyses of annual plants and forest trees indicate that Pleistocene glacial refugia of forest trees existed in central Texas, southern Florida, and the southern Appalachians (Jaramillo-Correa et al. 2009). Alternative scenarios for postglacial spread of these forests suggest that temperate species were present (in “cryptic” refugia) in low densities across much of the continent, even during the most severe glacial periods (Bennett 1985; McLachlan et al. 2005).

Many tree species have low chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) mutation rates, thereby providing limited polymorphism for phylogeographic analyses (Hu et al. 2009). Hu et al. (2009) recently observed that the short generation times and rapidly evolving mtDNA of animals closely associated with forests can act as a “magnifying glass” to help illuminate the population history of trees, which evolve at a relatively slow rate. Hu et al. (2009) also noted that the increased level of mtDNA polymorphisms in many animals can be analyzed with sophisticated statistical techniques, which can be used to infer paleopopulation information such as the timing of divergence and estimates of refugiai population sizes for tree species. Hu et al. (2009) further noted that the most reliable “magnifying glasses” of tree population histories are phylogeographic surveys of specialist species.

In the temperate forests of eastern North America, 2 of the most notable tree specialists are the eastern gray squirrel (Sciurus carolinensis) and the eastern fox squirrel (S. niger). Both of these species are obligates of mature forests that depend on mature trees for food, nests, and cover from predators (Koprowski 2005). Both species coevolved with seed, fruit, and nut trees in the temperate forests of eastern North America in their dual role as seed predators and seed dispersers (Steele 2008).

Sciurus carolinensis is one of the most familiar wild mammals in eastern North America, occurring naturally over most of eastern North America in temperate forests east of about 100°W and south of about 47°N (Hall 1981; Koprowski 1994b). The eastern gray squirrel also is a conspicuous (diurnal) and common resident of urban parks and suburban neighborhoods. Although S. carolinensis has been the subject of numerous studies of ecology and behavior (Steele and Koprowski 2001), few studies have examined geographic variation and population genetics in this species (Moncrief 1993, 1998).

The closely related eastern fox squirrel (S. niger) also occurs naturally in temperate forests over most of eastern North America south of about 49°N and east of about 105°W (Hall 1981; Koprowski 1994a). S. carolinensis and S. niger are sympatric, if not syntopic, over broad portions of their range (Edwards et al. 2003). A recent range-wide phylogeographic study of S. niger using 402 base pairs (bp) of the mtDNA cytochrome-b (Cytb) gene found evidence for a recent and rapid range expansion in this species following the last glacial maximum (LGM) of the Pleistocene and failed to detect regional differentiation (Moncrief et al. 2010). This is in sharp contrast to the well-documented regional differences the eastern fox squirrel exhibits in morphology (Moncrief 1993; Turner and Laerm 1993; Weigl et al. 1998) and ecology (Ditgen et al. 2007; Edwards et al. 1998; Jodice and Humphrey 1992; Kantola and Humphrey 1990; Perkins and Conner 2004; United States Fish and Wildlife Service 1993; Weigl et al. 1989).

Eastern fox squirrels and eastern gray squirrels have similar life histories (Wood et al. 2007). Females of both species typically do not reproduce until >1.25 years of age, and reproductive longevity in females may exceed 12 years (Koprowski 1994a, 1994b). Adult females (>1.0 year old) may produce 2 litters in the same year (early spring and late summer), although reproduction in both species is highly dependent on food availability (Edwards et al. 2003). Average litter size ranges from 1.7 to 3.0 in eastern fox squirrels and 2.3 to 2.9 in eastern gray squirrels (Koprowski 1994a, 1994b). Sex ratios in litters of both species approximate 1:1 (Edwards et al. 2003).

These species also have similar ecological requirements. Both species feed heavily on the acorns, nuts, flowers, and buds of oaks (Quercus), hickories and pecans (Carya), walnuts (Juglans), and beech (FagusEdwards et al. 2003; Koprowski 1994a, 1994b). Other foods for both species include fruits, seeds, buds, and flowers of a variety of other trees including maples (Acer) and Alleghany chinkapin (Castanea pumilaEdwards et al. 2003). Both species construct leaf nests and use tree cavities to escape from predators, for protection from inclement weather, and for rearing young (Edwards et al. 2003; Koprowski 1994a, 1994b).

Studies of codistributed, closely related species with similar generation time and population ecologies can provide insight into how ecological, geological, and historical processes have shaped regional communities (Arbogast and Kenagy 2001; Austin and Zamudio 2008). In this study we use variation in mtDNA sequences to investigate intraspecific relationships in S. niger and S. carolinensis to increase our understanding of the evolutionary histories of these 2 ecologically similar, closely related sciurids. We augment existing Cytb data for S. niger (Moncrief et al. 2010) with sequence data from the D-loop in the control region, which is the most rapidly evolving portion of the mitochondrial genome (Moritz et al. 1987). We also report and analyze comparable Cytb and D-loop data for S. carolinensis to address the following questions for each species: Does phylogeographic structure exist? What historical evolutionary processes (e.g., isolation in glacial refugia and range expansion) have led to the contemporary distribution of genetic diversity?

Materials and Methods

Sample collection.—We obtained tissue samples from 81 individuals of S. niger collected at 16 localities in Arkansas, Georgia, Indiana, Kansas, Louisiana, Maryland, Mississippi, South Dakota, Texas, and Virginia from 1984 through 1998 (Fig. 1). We also obtained tissue samples from 69 individuals of S. carolinensis collected at 14 localities in Alabama, Georgia, Indiana, Louisiana, Maryland, Mississippi, Tennessee, and Virginia from 1983 through 1993 (Fig. 1). Nine localities (in Georgia, Indiana, Louisiana, Maryland, Mississippi, and Virginia) yielded samples of both species (Fig. 1). All samples were collected following guidelines approved by the American Society of Mammalogists (Sikes et al. 2011). All samples (0.5 g of liver) were obtained from the frozen tissue collections of the Virginia Museum of Natural History (VMNH) and Louisiana State University Museum of Natural Sciences (LSUMZ); voucher specimens are deposited in their respective mammal collections. Voucher specimens and tissues of S. n. cinereus were transported to and are housed at VMNH under Regional Blanket Permit 697823, issued to NDM.

Fig. 1

Sampling localities for the eastern fox squirrel (Sciurus niger) and the eastern gray squirrel (S. carolinensis). Codes for sampling localities (3 letters) correspond to those defined in Tables 1–3 and Appendices I and II. Samples of both species were obtained at localities shown as open circles; samples of only S. niger were obtained at localities shown as closed triangles; and samples of only S. carolinensis were obtained at localities shown as closed squares.

Fig. 1

Sampling localities for the eastern fox squirrel (Sciurus niger) and the eastern gray squirrel (S. carolinensis). Codes for sampling localities (3 letters) correspond to those defined in Tables 1–3 and Appendices I and II. Samples of both species were obtained at localities shown as open circles; samples of only S. niger were obtained at localities shown as closed triangles; and samples of only S. carolinensis were obtained at localities shown as closed squares.

DNA extraction, amplification, and sequencing.—Total genomic DNA was isolated using standard phenol–chloroform protocols (Longmire et al. 1997). A 486-bp fragment of the D-loop was amplified using polymerase chain reaction with primers DFSloopF (5′CGCAATACTCGACCAATCC-3′) and DFSloopR 5′TGATGATTTCACGGAGGTAGG-3′—Lance et al. 2003). Also, for S. carolinensis, a fragment consisting of the first 402 bp of Cytb was amplified with primers MVZ05 and MVZ04 (Smith and Patton 1991). Double-stranded products were purified using the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, Wisconsin), and both strands of the purified polymerase chain reaction products were sequenced using Big Dye chain terminators and a 3130 Genetic Analyzer (Applied Biosystems, Inc., Foster City, California). AssemblyLIGN 1.0.9 (1998—Oxford Molecular Group, Oxford, United Kingdom) was used to assemble contigs for each individual, and CLUSTAL X (Thompson et al. 1997) was used to generate multiple sequence alignments. MacClade 4.0 (Maddison and Maddison 2000) was used for visual inspection of the multiple sequence alignment and to determine unique haplotypes using the REDUNDANT TAXA option.

Data analysis.—We used Arlequin version 3.5.1.21 (Excoffier and Lischer 2010) to estimate measures of intraspecific genetic variation (haplotype diversity and nucleotide diversity) in D-loop sequence data for both S. niger and S. carolinensis and in Cytb sequence data for S. carolinensis. For the remainder of the analyses, we combined both fragments for S. carolinensis into a segment that totaled 888 bp. For S. niger we combined a 402-bp fragment of Cytb sequence published previously (18 haplotypes—Moncrief et al. 2010) with D-loop sequences for each individual, also yielding a segment that totaled 888 bp.

For each species, we used BEAST version 1.6.1 (Drummond and Rambaut 2007) to estimate divergence times and to generate Bayesian skyline plots (Drummond et al. 2005). The Bayesian skyline plot estimates effective sample size (Drummond et al. 2002) through time, providing a graphical representation of past population dynamics while simultaneously sampling phylogenies and node ages (Drummond et al. 2002). A likelihood ratio test was unable to reject the molecular clock hypothesis for concatenated Cytb and D-loop for both species (S. niger,L = 4.0284, P = 0.6728; S. carolinensis,L = 82.4083, P = 0.0972); therefore, to produce time estimates in years we used a strict molecular clock. For Cytb, we utilized a pairwise divergence of 7.5–12% per million years, as had been estimated previously for rodents (Arbogast et al. 2001). Past estimates of D-loop substitution rates have been extremely variable (Parsons et al. 1997), making it essentially impossible to identify in the literature a consistent substitution rate for the rodent D-loop. To overcome this, the substitution rate for the D-loop was left with an uninformative prior in all analyses and estimated relative to the Cytb rate. All BEAST analyses utilized the HKY + 1 + Γ model of nucleotide substitution, as was indicated to be most appropriate by MrModeltest version 2.3 (Nylander 2004). Cytb and D-loop sequences were concatenated for all BEAST analyses, but partitioned to allow a distinct substitution model for the entire D-loop, and for each codon position within the Cytb alignment (in addition to the substitution rate partitioning described above). Tree topologies were linked across the Cytb and D-loop loci, due to the permanent link among mtDNA loci, and utilized the Bayesian skyline plot tree prior (Drummond et al. 2005). For each species, BEAST analyses consisted of an initial run of 5 × 107 generations, following which operator values were adjusted to optimize search settings. Two final runs of 5 × 107 generations were run with optimized search settings, and the resulting log and tree files were combined to produce final estimates of demographic parameters, to generate Bayesian skyline plots, and to obtain time-calibrated phylogenies (following removal of 10% of samples as burn-in). All runs were checked for sufficient mixing, stable convergence on a unimodal posterior, and effective sample size >200 for all parameters using TRACER version 1.4 (Drummond and Rambaut 2003). One assumption of the Bayesian skyline plot analysis is the absence of strong population structure. Although this is a reasonable assumption for S. niger based on previous work (Moncrief et al. 2010) and analyses herein, network and phylogenetic analyses of S. carolinensis in this study recovered 2 highly divergent clades, with some structure within these clades. To minimize the structure in the Bayesian skyline plot analysis of S. carolinensis, we analyzed the 2 divergent clades separately with the same search parameters as described above.

We also constructed a minimum spanning network at a 95% confidence level using TCS version 2.1 (Clement et al. 2000). Finally, we used DNAsp version 4.20.2 to generate mismatch distributions, which plot the distribution of pairwise genetic differences between pairs of individuals (Rogers and Harpending 1992). The plots generated from the observed data set were compared to expected distributions under models of sudden expansion (Rogers and Harpending 1992). For each data set, we used Arlequin version 3.5.1.21 to calculate sum of squared deviation and Harpending's raggedness index (Harpending 1994) to assess the fit of the observed data to a model of sudden expansion.

Results

Sciurus niger.—Among the 81 individuals of S. niger examined, we detected 55 unique D-loop haplotypes. Representative sequences of these 55 haplotypes have been deposited in GenBank (accession numbers JX104344JX104398; Appendix I). Five haplotypes (f, i, k, n, and o) were shared between 2 or more localities; the remaining 50 haplotypes were private, occurring at only a single locality (Table 1; Appendix I). Seven haplotypes (when reduced by approximately 200 bp) were identical to haplotypes reported by the only other study of S. niger that used D-loop sequences (Lance et al. 2003) as follows: haplotype f is the same as GenBank accession number AF533258, ll is AF533254, mm is AF533256, nn is AF533255, pp is AF533260, ooo is AF533268, and rr is AF533272. Five of these (f, ll, mm, nn, and pp) are restricted in both studies to samples from Maryland. Haplotype ooo in our study was from South Dakota; AF533268 in Lance et al. (2003) was from South Carolina. Haplotype rr in our study was from Georgia; AF 533272 in Lance et al. (2003) was from South Carolina.

Table 1

—Geographic information and distribution of D-loop haplotypes among 16 sampling localities of the eastern fox squirrel (Sciurus niger). Locality codes (code) and haplotype designations correspond to those shown in Fig. 1 and Appendix I. For each locality, we provide the number of individuals sequenced (n), number of individuals with a haplotype shared by 2 or more localities (F, I, K, N, and O), private haplotypes (with number of individuals in parentheses), haplotype diversity (h, with standard deviation in parentheses), and nucleotide diversity (π, with standard deviation in parentheses).

   Haplotypes   
 County(ies) or parish(es) and state  Shared    
Code n Private h π 
ARG Greene, Arkansas      u(l) 0.000 0.000 
GAJ Jasper, Georgia 10      e (3), g (1), p (2), rr (1), ss (1), tt (1), uu (1) 0.911 (0.077) 0.019 (0.011) 
IND Dubois, Indiana      0.000 0.000 
KSR Rooks and Ellis, Kansas    b (4), ggg (1), hhh (1) 0.786 (0.151) 0.011 (0.007) 
LAA Acadia, Louisiana      bb (1), d (3), h (2) 0.733 (0.155) 0.015 (0.010) 
LAB Bossier, Louisiana      w (1), x (1), y (1) 1.000 (0.272) 0.009 (0.008) 
LAI East Baton Rouge and Iberville, Louisiana      gg (1), q (2), r (1), s (1), v (1), z (1) 0.952 (0.096) 0.014 (0.008) 
LAM Madison, Louisiana      hh (1), j (2), 1 (2) 0.800 (0.164) 0.013 (0.009) 
LAS St. Tammany, Louisiana      ii (1), jj (1), kk (1) 1.000 (0.272) 0.029 (0.022) 
MDA Allegany, Maryland  c (2), ccc (1), zz (1) 0.964 (0.077) 0.017 (0.010) 
MDD Dorchester, Maryland    11 (1), mm (1), nn (1), pp (1) 0.893 (0.111) 0.015 (0.009) 
MSH Holmes, Mississippi     cc (1), ee (1), ff (1) 1.000 (0.177) 0.024 (0.017) 
SDC Clay, South Dakota      nnn (1), ooo (1) 1.000 (0.500) 0.021 (0.022) 
TXT Tom Green, Texas 10      a (7), kkk (1), 111 (1), mmm (1) 0.533 (0.180) 0.006 (0.005) 
VAA Alleghany, Virginia      ww (1), xx (1), yy (1) 1.000 (0.272) 0.018 (0.014) 
VAS Sussex, Virginia      ppp (i), qqq (i) 1.000 (0.500) 0.008 (0.009) 
   Haplotypes   
 County(ies) or parish(es) and state  Shared    
Code n Private h π 
ARG Greene, Arkansas      u(l) 0.000 0.000 
GAJ Jasper, Georgia 10      e (3), g (1), p (2), rr (1), ss (1), tt (1), uu (1) 0.911 (0.077) 0.019 (0.011) 
IND Dubois, Indiana      0.000 0.000 
KSR Rooks and Ellis, Kansas    b (4), ggg (1), hhh (1) 0.786 (0.151) 0.011 (0.007) 
LAA Acadia, Louisiana      bb (1), d (3), h (2) 0.733 (0.155) 0.015 (0.010) 
LAB Bossier, Louisiana      w (1), x (1), y (1) 1.000 (0.272) 0.009 (0.008) 
LAI East Baton Rouge and Iberville, Louisiana      gg (1), q (2), r (1), s (1), v (1), z (1) 0.952 (0.096) 0.014 (0.008) 
LAM Madison, Louisiana      hh (1), j (2), 1 (2) 0.800 (0.164) 0.013 (0.009) 
LAS St. Tammany, Louisiana      ii (1), jj (1), kk (1) 1.000 (0.272) 0.029 (0.022) 
MDA Allegany, Maryland  c (2), ccc (1), zz (1) 0.964 (0.077) 0.017 (0.010) 
MDD Dorchester, Maryland    11 (1), mm (1), nn (1), pp (1) 0.893 (0.111) 0.015 (0.009) 
MSH Holmes, Mississippi     cc (1), ee (1), ff (1) 1.000 (0.177) 0.024 (0.017) 
SDC Clay, South Dakota      nnn (1), ooo (1) 1.000 (0.500) 0.021 (0.022) 
TXT Tom Green, Texas 10      a (7), kkk (1), 111 (1), mmm (1) 0.533 (0.180) 0.006 (0.005) 
VAA Alleghany, Virginia      ww (1), xx (1), yy (1) 1.000 (0.272) 0.018 (0.014) 
VAS Sussex, Virginia      ppp (i), qqq (i) 1.000 (0.500) 0.008 (0.009) 

Haplotype diversity for the 16 sampling localities of S. niger in our study ranged from 0 to 1.00, and nucleotide diversity ranged from 0 to 0.029 (Table 1). Overall haplotype diversity was high (0.985 ± 0.006), and overall nucleotide diversity was low (0.023 ± 0.012). This latter estimate was similar to values (0.019−0.031) reported by Lance et al. (2003).

Combining Cytb and D-loop sequences for the 81 individuals of S. niger from 16 localities yielded 55 unique combined haplotypes (Appendix I). The BEAST chronogram indicated all sampled haplotypes of S. niger coalesce on a common ancestor approximately 33.3–91 thousand years ago (kya; Fig. 2). Three major groups of haplotypes that occurred at 2 or more localities (consisting of 16, 6, and 9 haplotypes, respectively) were supported by posterior probability values ≥ 0.95. Haplotypes in group 1 were widely distributed across the species’ range and occurred in 10 of 16 localities (Figs. 1 and 2). Haplotypes in group 2 were absent from the southwest and lower Mississippi River valley, whereas those in group 3 were restricted to the lower Mississippi River valley (Figs. 1 and 2). The remaining 24 haplotypes occurred throughout the species’ range (Figs. 1 and 2). Each of these major groups of S. niger coalesce on a common ancestor approximately 9.9–48 kya (Fig. 2). The upper end of the estimated range of dates at which each of these major groups of S. niger coalesces on a common ancestor (48 kya) overlaps the lower end of the range of dates for the root of the chronogram (33.3 kya; Fig. 2).

Fig. 2

BEAST chronogram for the 55 combined cytochrome-b and D-loop mitochondrial DNA haplotypes of the eastern fox squirrel (Sciurus niger). Gray bars indicate 95% highest posterior density estimates for major nodes. Numbers designate nodes supported by posterior probabilities of greater than or equal to 95% for groups of haplotypes that occur at 2 or more localities. Heavy vertical lines to the right of the tree indicate 3 groups of haplotypes. Haplotype labels and codes for sampling localities correspond to those shown in Appendix I and Fig. 1.

Fig. 2

BEAST chronogram for the 55 combined cytochrome-b and D-loop mitochondrial DNA haplotypes of the eastern fox squirrel (Sciurus niger). Gray bars indicate 95% highest posterior density estimates for major nodes. Numbers designate nodes supported by posterior probabilities of greater than or equal to 95% for groups of haplotypes that occur at 2 or more localities. Heavy vertical lines to the right of the tree indicate 3 groups of haplotypes. Haplotype labels and codes for sampling localities correspond to those shown in Appendix I and Fig. 1.

The minimum spanning network (Fig. 3) revealed several divergent clusters of haplotypes, but these clusters did not correspond to any geographic grouping of localities. Clusters of 16, 6, and 9 haplotypes correspond to groups 1, 2, and 3, respectively, of the phylogenetic tree generated using BEAST (Fig. 2). Five haplotypes were shared between localities: N52 (KSR and MSH), N32 (IND and MDA), N17 (KSR and MDA), N31 (MDA and MDD), and N13 (MDA and MDD).

Fig. 3

Parsimony network showing phylogenetic relationships among 55 combined cytochrome-b and D-loop mitochondrial DNA haplotypes of the eastern fox squirrel (Sciurus niger). Haplotype labels and codes for sampling localities correspond to those shown in Appendix I and Fig. 1. The small, filled circles represent unsampled or extinct haplotypes. Each line between circles, regardless of length, represents a single mutational change. Size of the labeled circles is proportionate to the number of individuals possessing that haplotype. Groups of haplotypes labeled as 1,2, and 3 correspond to those shown in Fig. 2.

Fig. 3

Parsimony network showing phylogenetic relationships among 55 combined cytochrome-b and D-loop mitochondrial DNA haplotypes of the eastern fox squirrel (Sciurus niger). Haplotype labels and codes for sampling localities correspond to those shown in Appendix I and Fig. 1. The small, filled circles represent unsampled or extinct haplotypes. Each line between circles, regardless of length, represents a single mutational change. Size of the labeled circles is proportionate to the number of individuals possessing that haplotype. Groups of haplotypes labeled as 1,2, and 3 correspond to those shown in Fig. 2.

Mismatch analysis exhibited a unimodal distribution that did not differ significantly from the distribution expected under population expansion (Fig. 4). The Bayesian skyline plot analysis also indicated a significant population expansion, which began approximately 15 kya (Fig. 5a). This is in agreement with the expansion date previously reported by Moncrief et al. (2010).

Fig. 4

Mismatch distribution for 55 combined cytochrome-b and D-loop mitochondrial DNA haplotypes of the eastern fox squirrel (Sciurus niger). The observed distribution is represented by a dotted line, and the expected distribution based on a model of exponential population growth is represented by a solid line. The sum of squared deviation (SSD) and Harpending's raggedness index (HRI), with the respective P-values, are provided.

Fig. 4

Mismatch distribution for 55 combined cytochrome-b and D-loop mitochondrial DNA haplotypes of the eastern fox squirrel (Sciurus niger). The observed distribution is represented by a dotted line, and the expected distribution based on a model of exponential population growth is represented by a solid line. The sum of squared deviation (SSD) and Harpending's raggedness index (HRI), with the respective P-values, are provided.

Fig. 5

Bayesian skyline plots of effective female population size × generation time, Nefτ (logarithmic scale), based on combined cytochrome-b and D-loop mitochondrial DNA haplotypes. a) Fifty-five haplotypes of the eastern fox squirrel (Sciurus niger); b) 23 haplotypes of the eastern gray squirrel (Sciurus carolinensis) identified as subset 1 in Fig. 7; and c) 28 haplotypes of S. carolinensis identified as subset 2 in Fig. 7. In each plot, the thick black line is the median estimate, and the thin gray lines correspond to the 95% highest posterior density estimate. Note that the scales on the y-axes are different.

Fig. 5

Bayesian skyline plots of effective female population size × generation time, Nefτ (logarithmic scale), based on combined cytochrome-b and D-loop mitochondrial DNA haplotypes. a) Fifty-five haplotypes of the eastern fox squirrel (Sciurus niger); b) 23 haplotypes of the eastern gray squirrel (Sciurus carolinensis) identified as subset 1 in Fig. 7; and c) 28 haplotypes of S. carolinensis identified as subset 2 in Fig. 7. In each plot, the thick black line is the median estimate, and the thin gray lines correspond to the 95% highest posterior density estimate. Note that the scales on the y-axes are different.

Fig. 7

BEAST chronogram for the 51 combined cytochrome-b and D-loop mitochondrial DNA haplotypes of the eastern gray squirrel (Sciurus carolinensis). Gray bars indicate 95% highest posterior density estimates for major nodes. Numbers designate nodes supported by posterior probabilities of greater than or equal to 95% for groups of haplotypes that occur at 2 or more localities. Heavy vertical lines to the right of the tree indicate 2 major subsets of haplotypes and 3 groups of haplotypes. Group 1 includes the 15 haplotypes labeled as network 1 in Fig. 6 plus 2 additional haplotypes (C42 and C18). Groups 2 and 3 correspond to networks 2 and 3, respectively, in Fig. 6. Haplotype labels and codes for sampling localities correspond to those shown in Appendix II and Fig. 1.

Fig. 7

BEAST chronogram for the 51 combined cytochrome-b and D-loop mitochondrial DNA haplotypes of the eastern gray squirrel (Sciurus carolinensis). Gray bars indicate 95% highest posterior density estimates for major nodes. Numbers designate nodes supported by posterior probabilities of greater than or equal to 95% for groups of haplotypes that occur at 2 or more localities. Heavy vertical lines to the right of the tree indicate 2 major subsets of haplotypes and 3 groups of haplotypes. Group 1 includes the 15 haplotypes labeled as network 1 in Fig. 6 plus 2 additional haplotypes (C42 and C18). Groups 2 and 3 correspond to networks 2 and 3, respectively, in Fig. 6. Haplotype labels and codes for sampling localities correspond to those shown in Appendix II and Fig. 1.

Fig. 6

Parsimony networks showing phylogenetic relationships among 51 combined cytochrome-b and D-loop mitochondrial DNA haplotypes of the eastern gray squirrel (Sciurus carolinensis). Haplotype labels and codes for sampling localities correspond to those shown in Appendix II and Fig. 1. The small, filled circles represent unsampled or extinct haplotypes. Each line between circles, regardless of length, represents a single mutational change. Size of the labeled circles is proportionate to the number of individuals possessing that haplotype. Networks of more than 3 haplotypes are labeled 1, 2, and 3.

Fig. 6

Parsimony networks showing phylogenetic relationships among 51 combined cytochrome-b and D-loop mitochondrial DNA haplotypes of the eastern gray squirrel (Sciurus carolinensis). Haplotype labels and codes for sampling localities correspond to those shown in Appendix II and Fig. 1. The small, filled circles represent unsampled or extinct haplotypes. Each line between circles, regardless of length, represents a single mutational change. Size of the labeled circles is proportionate to the number of individuals possessing that haplotype. Networks of more than 3 haplotypes are labeled 1, 2, and 3.

Sciurus carolinensis.—We detected 16 Cytb haplotypes (Table 2) among the 69 individuals of S. carolinensis examined. Representative sequences have been deposited in GenBank (accession numbers JX104399JX104414; Appendix II). Haplotype diversity for 14 sampling localities of S. carolinensis ranged from 0.00 to 0.810, and nucleotide diversity ranged from 0.00 to 0.020 (Table 2). Overall haplotype diversity was high (0.853 ± 0.025), and nucleotide diversity was low (0.017 ± 0.009). Two Cytb haplotypes (A and B) were widely distributed; each occurred at 7 of 14 localities, although they occurred together at only 4 of 14 localities (Table 2). Those 2 haplotypes also predominated in the entire data set, occurring in 18 and 17 animals, respectively (Table 2). Eleven Cytb haplotypes (in a total of 19 individuals) were restricted to a single locality (Table 2).

Table 2

—Geographic information and distribution of cytochrome-b haplotypes among 14 sampling localities of the eastern gray squirrel (Sciurus carolinensis). Locality codes (code) and haplotype designations correspond to those shown in Fig. 1 and Appendix II. For each locality, we provide the number of individuals sequenced (n), number of individuals with a haplotype shared by 2 or more localites (A, B, C, J, and M), private haplotypes (with number of individuals in parentheses), haplotype diversity (h, with standard deviation in parentheses), and nucleotide diversity (π, with standard deviation in parentheses).

   Haplotypes   
 County(ies) or parish(es) and state  Shared    
Code n Private h π 
ALC Covington, Alabama    E (4) 0.600 (0.215) 0.011 (0.007) 
GAJ Jasper, Georgia    K (3), L(1) 0.810 (0.130) 0.011 (0.007) 
IND Dubois, Indiana     0.389 (0.164) 0.012 (0.007) 
LAA Acadia, Louisiana      I (1) 0.000 (0.000) 0.000 (0.000) 
LAF West Feliciana, Louisiana      0.000 (0.000) 0.000 (0.000) 
LAI East Baton Rouge, Louisiana     0.333 (0.215) 0.001 (0.001) 
LAS St. Tammany, Louisiana     O (1) 0.667 (0.314) 0.003 (0.003) 
LAV Vernon, Louisiana      F (2) 0.000 (0.000) 0.000 (0.000) 
MDA Allegany, Maryland    P (1) 0.679 (0.122) 0.017 (0.010) 
MDD Dorchester, Maryland    0.667 (0.160) 0.011 (0.007) 
MSH Holmes, Mississippi     D (3), H (1), N (1) 0.800 (0.170) 0.005 (0.004) 
TNS Shelby, Tennessee      G(l) 0.000 (0.000) 0.000 (0.000) 
VAA Alleghany and Augusta, Virginia     0.500 (0.265) 0.015 (0.011) 
VAH Henry, Virginia    0.733 (0.155) 0.020 (0.012) 
   Haplotypes   
 County(ies) or parish(es) and state  Shared    
Code n Private h π 
ALC Covington, Alabama    E (4) 0.600 (0.215) 0.011 (0.007) 
GAJ Jasper, Georgia    K (3), L(1) 0.810 (0.130) 0.011 (0.007) 
IND Dubois, Indiana     0.389 (0.164) 0.012 (0.007) 
LAA Acadia, Louisiana      I (1) 0.000 (0.000) 0.000 (0.000) 
LAF West Feliciana, Louisiana      0.000 (0.000) 0.000 (0.000) 
LAI East Baton Rouge, Louisiana     0.333 (0.215) 0.001 (0.001) 
LAS St. Tammany, Louisiana     O (1) 0.667 (0.314) 0.003 (0.003) 
LAV Vernon, Louisiana      F (2) 0.000 (0.000) 0.000 (0.000) 
MDA Allegany, Maryland    P (1) 0.679 (0.122) 0.017 (0.010) 
MDD Dorchester, Maryland    0.667 (0.160) 0.011 (0.007) 
MSH Holmes, Mississippi     D (3), H (1), N (1) 0.800 (0.170) 0.005 (0.004) 
TNS Shelby, Tennessee      G(l) 0.000 (0.000) 0.000 (0.000) 
VAA Alleghany and Augusta, Virginia     0.500 (0.265) 0.015 (0.011) 
VAH Henry, Virginia    0.733 (0.155) 0.020 (0.012) 

Among the 69 individuals of S. carolinensis examined, we detected 51 D-loop haplotypes (Table 3). Representative sequences have been deposited in GenBank (accession numbers JX104415JX104465; Appendix II). Haplotype diversity for the 14 sampling localities of S. carolinensis ranged from 0.00 to 1.0, and nucleotide diversity ranged from 0.00 to 0.039 (Table 3). Overall haplotype diversity was high (0.984 ± 0.007), and nucleotide diversity was low (0.032 ± 0.016). Only 1 of 51 D-loop haplotypes (f) occurred at more than 1 locality (Table 3).

Table 3

—Geographic information and distribution of D-loop haplotypes among 14 sampling localities of the eastern gray squirrel (Sciurus carolinensis). Locality codes (code) and haplotype designations correspond to those shown in Fig. 1 and Appendix II. For each locality, we provide the number of individuals sequenced (n), number of individuals with a haplotype shared by 2 or more localites (F), private haplotypes (with number of individuals in parentheses), haplotype diversity (h, with standard deviation in parentheses), and nucleotide diversity (π, with standard deviation in parentheses).

   Haplotypes   
Code County(ies) or parish(es) and state n Shared Private h π 
ALC Covington, Alabama  eee (1), ff (1), 111 (1), nnn (1), ss (1), yy (1) 1.000 (0.096) 0.032 (0.019) 
GAJ Jasper, Georgia  aa (1), bb (1), dd (1), ddd (1), ee (2), iii (1) 0.952 (0.096) 0.023 (0.014) 
IND Dubois, Indiana  a (7), ggg (1), t (1) 0.417 (0.191) 0.023 (0.013) 
LAA Acadia, Louisiana  pp (1) 0.000 (0.000) 0.000 (0.000) 
LAF West Feliciana, Louisiana  c (2), 1 (1) 0.667 (0.314) 0.001 (0.002) 
LAI East Baton Rouge, Louisiana  b(4), hh(l), i(l) 0.600 (0.215) 0.023 (0.014) 
LAS St. Tammany, Louisiana  kkk(2), m(l) 0.667 (0.314) 0.032 (0.025) 
LAV Vernon, Louisiana  ii (1), mmm (1) 1.000 (0.500) 0.039 (0.040) 
MDA Allegany, Maryland  e(2), fff(1), u(1), v(2), w(1), y(1) 0.929 (0.084) 0.031 (0.018) 
MDD Dorchester, Maryland F (2) gg (1), n (2), o (1), vv (1) 0.905 (0.103) 0.007 (0.004) 
MSH Holmes, Mississippi  jj (1), 11 (1), mm (1), nn (1), oo (1), rr (1) 1.000 (0.096) 0.026 (0.016) 
TNS Shelby, Tennessee  j (1) 0.000 (0.000) 0.000 (0.000) 
VAA Alleghany and Augusta, Virginia  hhh(1), p(1), q(1), zz(1) 1.000 (0.177) 0.039 (0.026) 
VAH Henry, Virginia F(l) bbb(l), d(2), r(l), ww (1) 0.933 (0.122) 0.025 (0.015) 
   Haplotypes   
Code County(ies) or parish(es) and state n Shared Private h π 
ALC Covington, Alabama  eee (1), ff (1), 111 (1), nnn (1), ss (1), yy (1) 1.000 (0.096) 0.032 (0.019) 
GAJ Jasper, Georgia  aa (1), bb (1), dd (1), ddd (1), ee (2), iii (1) 0.952 (0.096) 0.023 (0.014) 
IND Dubois, Indiana  a (7), ggg (1), t (1) 0.417 (0.191) 0.023 (0.013) 
LAA Acadia, Louisiana  pp (1) 0.000 (0.000) 0.000 (0.000) 
LAF West Feliciana, Louisiana  c (2), 1 (1) 0.667 (0.314) 0.001 (0.002) 
LAI East Baton Rouge, Louisiana  b(4), hh(l), i(l) 0.600 (0.215) 0.023 (0.014) 
LAS St. Tammany, Louisiana  kkk(2), m(l) 0.667 (0.314) 0.032 (0.025) 
LAV Vernon, Louisiana  ii (1), mmm (1) 1.000 (0.500) 0.039 (0.040) 
MDA Allegany, Maryland  e(2), fff(1), u(1), v(2), w(1), y(1) 0.929 (0.084) 0.031 (0.018) 
MDD Dorchester, Maryland F (2) gg (1), n (2), o (1), vv (1) 0.905 (0.103) 0.007 (0.004) 
MSH Holmes, Mississippi  jj (1), 11 (1), mm (1), nn (1), oo (1), rr (1) 1.000 (0.096) 0.026 (0.016) 
TNS Shelby, Tennessee  j (1) 0.000 (0.000) 0.000 (0.000) 
VAA Alleghany and Augusta, Virginia  hhh(1), p(1), q(1), zz(1) 1.000 (0.177) 0.039 (0.026) 
VAH Henry, Virginia F(l) bbb(l), d(2), r(l), ww (1) 0.933 (0.122) 0.025 (0.015) 

Combining Cytb and D-loop sequences for the 69 individuals of S. carolinensis from 14 localities yielded 51 unique combined haplotypes (Appendix II). The minimum spanning network (Fig. 6) revealed 3 unlinked networks, where the 95% confidence interval for parsimony analysis allowed up to 9 mutational steps to link haplotypes, but there is an overall lack of spatial structure in these data. Network 1 consisted of 15 haplotypes, from all localities except those in Louisiana, Mississippi, and Tennessee. Networks 2 and 3 consisted of 11 and 10 haplotypes, respectively. The remaining 15 haplotypes were either singletons or linked together in pairs or triplets. Only 1 haplotype (C42) was shared by 2 localities (MDD and VAH); it was not part of a network.

The BEAST chronogram indicated that all sampled haplotypes of S. carolinensis coalesce on a common ancestor approximately 98.3–266.3 kya (Fig. 7). Two major subsets of haplotypes (consisting of 23 and 28 haplotypes, respectively) were supported by posterior probability values ≥0.99. Haplotypes in subset 1 were not present at any locality in Louisiana, Mississippi, or Tennessee; subset 2 included haplotypes from all 14 localities (Figs. 1 and 7). Group 1 includes 17 haplotypes, which are the 15 haplotypes in network 1 in Fig. 6, plus C42 and C18. Groups 2 and 3 correspond exactly to networks 2 and 3, respectively, in Fig. 6. Each of these major groups of S. carolinensis coalesce on a common ancestor approximately 26.1–79.7 kya (Fig. 7). The upper end of the estimated range of dates at which each of these major groups of S. carolinensis coalesces on a common ancestor (79.7 kya) does not overlap the lower end of the range of dates for the root of the chronogram (98.3 kya; Fig. 7).

Mismatch analyses of the entire data set (Fig. 8a) and subsets 1 and 2 shown in Fig. 7 (Figs. 8b and 8c) exhibited distributions that did not differ significantly from the distribution expected under population expansion. Mismatch analysis of each of the 3 networks in Fig. 6 (Figs. 8d–f) exhibited a unimodal distribution that did not differ significantly from the distribution expected under population expansion. All 6 plots show peaks at 7 pairwise differences, suggesting that these data sets share the same expansion event. The Bayesian skyline plot analysis for each of the 2 subsets in Fig. 7 indicated that a significant population expansion occurred in S. carolinensis between 12 and 25 kya (Figs. 5b and 5c).

Fig. 8

Mismatch distribution for combined cytochrome-b and D-loop haplotypes of the eastern gray squirrel (Sciurus carolinensis): a) 51 haplotypes; b) 23 haplotypes identified as subset 1 in Fig. 7; c) 28 haplotypes identified as subset 2 in Fig. 7; d) 15 haplotypes clustered in network 1 in Fig. 6; e) 11 haplotypes clustered in network 2 in Fig. 6; and f) 10 haplotypes clustered in network 3 in Fig. 6. In each plot, the observed distribution is represented by a dotted line, and the expected distribution based on a model of exponential population growth is represented by a solid line. The sum of squared deviation (SSD) and Harpending's raggedness index (HRI), with the respective P-values, are provided for each plot.

Fig. 8

Mismatch distribution for combined cytochrome-b and D-loop haplotypes of the eastern gray squirrel (Sciurus carolinensis): a) 51 haplotypes; b) 23 haplotypes identified as subset 1 in Fig. 7; c) 28 haplotypes identified as subset 2 in Fig. 7; d) 15 haplotypes clustered in network 1 in Fig. 6; e) 11 haplotypes clustered in network 2 in Fig. 6; and f) 10 haplotypes clustered in network 3 in Fig. 6. In each plot, the observed distribution is represented by a dotted line, and the expected distribution based on a model of exponential population growth is represented by a solid line. The sum of squared deviation (SSD) and Harpending's raggedness index (HRI), with the respective P-values, are provided for each plot.

Discussion

There is a general lack of spatial structure in the maternal lineages of both S. niger and S. carolinensis, even though we observed several clusters of divergent haplotypes in each species (Figs. 3 and 6). Examination of our data revealed high haplotype diversity and low nucleotide diversity in the D-loop and Cytb sequences of both S. niger and S. carolinensis (Tables 1–3; Moncrief et al. 2010). We also observed in S. carolinensis 2 Cytb haplotypes that are present at high frequency in the overall data set and are shared by populations separated by large geographic distances (Table 2). These 2 haplotypes were internal nodes for 2 separate parsimony networks (results not shown), suggesting that they represent divergent ancestral maternal lineages resulting from distinct glacial refugia during the Pleistocene.

We also documented evidence for rapid, recent population expansion in each species. Bayesian skyline plot estimates indicate expansion in both species within the past 12–20 kya (Fig. 5). Mismatch distribution of pairwise differences in the combined Cytb and D-loop sequences of S. niger (Fig. 4) and S. carolinensis (Figs. 8d–f) were unimodal. All of these results are in agreement with the findings of a previous study of Cytb sequences of S. niger (Moncrief et al. 2010) and all are indicative of a rapid population expansion (Avise 2000; Rogers and Harpending 1992). Similarly, Moncrief (1993, 1998) reported relatively low levels of allozymic variation in both species, and Lance et al. (2003) reported low levels of microsatellite polymorphism in S. niger, providing evidence from unlinked nuclear markers that these species have undergone recent population bottlenecks or range expansion, or both. The high haplotype diversity observed here and the low nuclear diversity presented in previous studies suggest that S. niger and S. carolinensis underwent a recent population expansion from glacial refugia, and mtDNA diversity has recovered whereas nuclear diversity has not.

This rapid expansion in populations of eastern fox squirrels and eastern gray squirrels is consistent with a scenario of rapid postglacial range expansion of deciduous tree species following the LGM in eastern North America (Williams 2003). These results are not surprising: both S. niger and S. carolinensis are temperate forest obligates that coevolved with seed, fruit, and nut trees (Koprowski 2005; Steele 2008). Because eastern fox squirrels and eastern gray squirrels do not hibernate, the presence and persistence of populations of these animals are especially dependent on the presence of mature trees that produce winter-storable foods (acorns and nuts), including Quercus and Juglans (Edwards et al. 2003; Koprowski 1994a, 1994b). The American chestnut (Castanea dentata) also was an important cacheable food source until the early 1900s, when it was almost eliminated by a fungus (Dane 2009). Other important foods for S. niger and S. carolinensis, include the seeds and nuts of Fagus, Acer, and C. pumila (Edwards et al. 2003; Koprowski 1994a, 1994b).

Molecular analyses of many animals and plants have supported hypotheses that southern glacial refugia existed along the Gulf and Atlantic coasts in eastern North America during the Pleistocene (Soltis et al. 2006). Our data provide tentative evidence for a refugium on the Gulf Coast for S. niger: all haplotypes of the eastern fox squirrel in group 3 are restricted to the lower Mississippi River valley (localities LAI, LAM, LAS, and MSH; Figs. 1–3), which corresponds to glacial refugiai area “G” in Swenson and Howard (2005). Additionally, haplotype N52 of S. niger, which is shared by localities MSH and KSR (Figs. 1–3), provides some evidence of postglacial expansion from a refugium in the Gulf Coast state of Mississippi (MSH) to areas north and west, including Kansas (KSR). Recent molecular support for a Pleistocene refugium for trees in the lower Mississippi River valley includes studies of J.nigra (Victory et al. 2006), Acer (Saeki et al. 2011), F. grandifolia (Morris et al. 2010), and Q. rubra (Magni et al. 2005).

Several recent studies of cpDNA variation in trees also have provided evidence supporting the existence of interior glacial refugia in the southern Appalachian Mountains and on the interior plateaus to the west of those mountains. These include analyses of Q. rubra (Birchenko et al. 2009; Magni et al. 2005), Acer (McLachlan et al. 2005; Saeki et al. 2011), F. grandifolia (McLachlan et al. 2005), and C. dentata and C. pumila (Dane 2009). In one of the 1st studies to use cpDNA for phylogeographic analyses of trees, McLachlan et al. (2005) concluded that F. grandifolia and A. rubrum persisted during the LGM as low-density populations in the Appalachians and on interior plateaus, much farther north (and much closer to modern range limits) than previously hypothesized. Similarly, Magni et al. (2005) suggested that oak stands grew close to the Laurentide Ice Sheet (which extended south to about 39°N— Soltis et al. 2006) shortly after the LGM and that northward recolonization was limited to a few hundred kilometers. Magni et al. (2005) concluded that their results are consistent with palynological evidence that Quercus was abundant during the LGM in the lower Mississippi River valley and Florida, but that scattered populations also were present farther north, between these 2 regions and the ice sheet (Jackson et al. 2000; Williams 2003).

For both species of Sciurus in our study, we found tentative evidence for 1 or several interior glacial refugia. In eastern fox squirrels, group 2 consists of haplotypes that are present only in localities KSR, SDC, GAJ, and MDD (Figs. 1–3). Also, haplotype N32 is shared by localities in Indiana (IND) and Maryland (MDA; Figs. 1–3), and haplotype N17 is an internal node in group 1 that is shared by localities in Kansas (KSR) and Maryland (MDA; Figs. 1 and 3). In each case, we suggest that these haplotypes of S. niger originated in 1 or more interior refugia and that the current distribution of haplotypes is the result of postglacial range expansion. Similarly, haplotypes that comprise subset 1 in eastern gray squirrels are restricted to localities IND, ALC, GAJ, VAA, VAH, MDA, and MDD (Figs. 1 and 7), providing evidence for an interior refugium west of the southern Appalachian Mountains.

Scenarios for Sciurus that include expansion from scattered populations in interior refugia presume survival of populations of squirrels in numerous small, low-density fragments of thermophilous forests. The ecology of modern populations of eastern fox squirrels and eastern gray squirrels suggests that ancestral populations of these animals could have persisted in relatively small forest fragments (<40 ha—Koprowski 2005) during the LGM. Koprowski (2005) reported that, in both S. niger and S. carolinensis, density is negatively related to fragment size, and the size of home ranges is positively related to forest fragment size. Therefore, compaction of home ranges can provide a mechanism by which population densities may increase (or hold steady) in small forest fragments.

Wood et al. (2007) conducted population viability analyses for S. niger and S. carolinensis and found that populations of both species could be successfully established by as few as 35 individuals. Wood et al. (2007) concluded that the high biotic potential of tree squirrels and lack of density-dependent reproduction at low population densities allows even a small population to increase during a year of good or modest food availability. Wood et al. (2007) also noted that tree squirrels such as S. niger and S. carolinensis possess good dispersal capability, can colonize and use novel habitats, and can make their own nests. Furthermore, these animals eat many different foods, which permits persistence in a variety of forest types and allows them to survive during years of low seed production.

Our study revealed similar timing of recent population expansion in these 2 species (Fig. 5), but we documented more genetic structure in maternal lineages of eastern gray squirrels than in eastern fox squirrels (Figs. 2, 3, 6, and 7). This may reflect differences in the ecology of these animals. Female eastern gray squirrels are more philopatric than female eastern fox squirrels (Koprowski 2005). Female eastern gray squirrels tend to remain in their natal areas to form overlapping generations of kin (Koprowski 2005). Additionally, dispersal distances in the eastern gray squirrel tend to be shorter than in the eastern fox squirrel; dispersal distances in eastern gray squirrels rarely exceed about 3.5 km, whereas eastern fox squirrels have dispersed more than 60 km (Edwards et al. 2003).

Both S. niger and S. carolinensis exhibited genetic structure that predates the LGM (Figs. 2 and 7). The timing of divergence of major subsets of eastern gray squirrels (approximately 57.5–170.2 kya; Fig. 7) includes the Sanga-monian interglacial (approximately 130–100 kya—Gibbard and Van Kolfschoten 2004). The timing of divergence within subsets of S. carolinensis (26.1–79.7 kya; Fig. 7) is roughly coincident with timing of coalescence of S. niger (33.3-91 kya; Fig. 2). This may reflect fragmentation of populations during glacial advances in the Wisconsinan (Gradstein 2004), which would cause habitat fragmentation into refugia. Expansion events for both species (15–20 kya; Fig. 5) are coincident with the recession of glaciers that followed the LGM.

Clearly, cycles of Pleistocene glaciation were major determinants of historical range contractions and expansions, which shaped current patterns of genetic diversity in eastern fox squirrels and eastern gray squirrels. These cycles also undoubtedly affected the demographic histories of other organisms in eastern North America. Indeed, phylogeographic analyses of several other codistributed, nonvolant mammals, including the common gray fox (Urocyon einereoargenteusBozarth et al. 2011), the red fox (Vulpes vulpesAubry et al. 2009), the northern raccoon (Procyon lotorCullingham et al. 2008), the American black bear (Ursus americanusWooding and Ward 1997), the northern short-tailed shrew (Blarina brevicaudaBrant and Orti 2003), the northern flying squirrel (Glaucomys sabrinusArbogast 1999), the white-footed mouse (Peromyscus leucopusRowe et al. 2006), the eastern chipmunk (Tamias striatusRowe et al. 2006), and the eastern woodrat (Neotoma floridanaHayes and Harrison 1992), all reported weak genetic structure in these species in eastern North America. Moreover, studies of the southern flying squirrel (G. volans), another nonhibernating sciurid that is considered to be a temperate forest obligate, reported that this species displays very little genetic variation, despite broad geographic sampling over much of eastern North America (Arbogast 1999; Arbogast et al. 2005; Petersen and Stewart 2006).

Taken together, examination of our data indicates that S. niger and S. carolinensis underwent multiple episodes of genetic divergence during isolation in glacial refugia, followed by range expansion and contact that resulted in admixture of divergent maternal lineages within each species during interglacials. The location of refugia for both species of these squirrels probably shifted geographically through time, and animals that comprised populations in successive iterations of refugia were descendants of different combinations of source populations. Examination of our data further indicates that the most recent range expansion in both species occurred within the past 12V–20 kya. This estimate is consistent with analyses of historical tree-cover density. Williams (2003) found that density of Quercus increased in southeastern North America between 21 and 16 kya and further increased after 14 kya, followed by a northward range expansion from 13 to 9 kya.

For both species, examination of our data revealed several clusters of divergent haplotypes, but a general lack of spatial structure (Figs. 3 and 6). For 5. carolinensis, we observed a pattern that suggests the presence of at least 2 distinct refugiai populations that evolved in isolation during the Pleistocene (approximately 98.3–266.3 kya; Figs. 6 and 7) and expanded to the species’ current range following the LGM. For S. niger, structure was much less pronounced, with fewer strongly diverged groups of haplotypes (Fig. 3). Additionally, there was overlap between the estimated dates of coalescence for major groups and the root of the chronogram in S. niger, but not S. carolinensis (Figs. 2 and 7). These findings all suggest that eastern fox squirrels persisted in either a single population in a glacial refugium or as several refugiai populations that maintained gene flow throughout the Pleistocene. The highly dispersed haplotypes of S. niger relative to the stronger geographic structure of haplotypes of S. carolinensis suggests that dispersal and gene flow following glacial recession and range expansion has occurred to a greater extent in eastern fox squirrels than in eastern gray squirrels.

The relatively frequent climatic oscillations throughout the Pleistocene likely resulted in numerous cycles of range expansions and contractions, overlaying multiple evolutionary signals on phylogeographic structure in these squirrels and temperate tree species (Morris et al. 2008). We suggest a scenario by which temperate forests (and the eastern fox squirrels and eastern gray squirrels that used them) were concentrated at various times in coastal refugia (including the lower Mississippi River valley) and interior refugia (including areas west of the southern Appalachian Mountains), but that scattered populations of trees and squirrels also were present in multiple, small refugia between these regions and the Laurentide Ice Sheet. Because populations of trees and squirrels persisted so close to the ice sheet, the most recent recolonization (following the LGM) occurred rapidly.

Our data support evidence presented by McLachlan et al. (2005) and Morris et al. (2010) that thermophilous trees persisted close to the ice sheets during the LGM. This evidence, accumulated from molecular and fossil data for trees, and now augmented by data from this phylogeographic study of 2 closely related sympatric species that are nonhibernating temperate forest obligates, points to potential losses of dominant tree species over much or all of their ranges (as a result of rapid climate change that has been predicted due to 21st century warming [McLachlan et al. 2005]). Together with recent findings from studies of other nonvolant mammals and temperate tree species in eastern North America (Aubry et al. 2009; Birchenko et al. 2009; Bozarth et al. 2011; Cullingham et al. 2008; Dane 2009; Morris et al. 2008, 2010; Rowe et al. 2006; Saeki et al. 2011; Soltis et al. 2006; Victory et al. 2006), our study indicates that demographic histories of species in glaciated landscapes are often more complex and variable than previously suggested. Additional studies of temperate forest obligates and analyses that include finer-scale genetic data are necessary to more confidently infer the locations of glacial refugia for the trees that compose the temperate forests of eastern North America and the animals that inhabit those forests.

Acknowledgments

We thank M. Hafner for allowing access to tissue samples from Arkansas, Louisiana, Mississippi, and Tennessee that are housed at LSUMZ. S. Donnelson, T. Henry, D. Onorato, and J. Streicher provided assistance in the laboratory. We thank J. Davenport and M. Cartwright for valuable advice and assistance in producing the figures. We thank P. Weigl and A. Dooley, Jr., for helpful discussions about the ecology of tree squirrels and Pleistocene glaciations.

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Appendix I

Specimens examined.—Locality code, state, county or parish, haplotype designations, and voucher number for the 81 eastern fox squirrels (Sciurus niger) included in this study. Samples without vouchers are marked with an asterisk (*). GenBank accession numbers are provided for each of the 55 D-loop haplotypes; Moncrief et al. (2010) reported GenBank accession numbers for the cytochrome-b haplotypes. Vouchers are housed in the Virginia Museum of Natural History (VMNH) and Louisiana State University Museum of Natural Sciences (LSUMZ).

Locality code State County or parish Combined haplotype Cytochrome-b haplotype D-loop haplotype GenBank accession no. for D-loop haplotype Voucher no. 
ARG Arkansas Greene N51 JX104388 JAH 0467* 
GAJ Georgia Jasper N19 JX104378 VMNH 1261 
GAJ Georgia Jasper N19 JX104378 VMNH 1263 
GAJ Georgia Jasper N28 JX104355 VMNH 1253 
GAJ Georgia Jasper N37 JX104351 VMNH 1254 
GAJ Georgia Jasper N37 JX104351 VMNH 1260 
GAJ Georgia Jasper N37 JX104351 VMNH 1264 
GAJ Georgia Jasper N40 ss JX104386 VMNH 1258 
GAJ Georgia Jasper N41 uu JX104389 VMNH 1255 
GAJ Georgia Jasper N46 rr JX104384 VMNH 1257 
GAJ Georgia Jasper N54 tt JX104387 VMNH 1262 
IND Indiana Dubois N32 JX104376 VMNH 0326 
KSR Kansas Rooks N17 JX104373 VMNH 0296 
KSR Kansas Ellis N24 JX104345 VMNH 0289 
KSR Kansas Rooks N24 JX104345 VMNH 0300 
KSR Kansas Rooks N24 JX104345 VMNH 0301 
KSR Kansas Rooks N24 JX104345 VMNH 0302 
KSR Kansas Rooks N29 ggg JX104357 VMNH 0297 
KSR Kansas Rooks N44 hhh JX104360 VMNH 0298 
KSR Kansas Ellis N52 JX104361 VMNH 0290 
LAA Louisiana Acadia N27 JX104350 LSU M-7487 
LAA Louisiana Acadia N27 JX104350 LSU M-7488 
LAA Louisiana Acadia N27 JX104350 LSU M-7491 
LAA Louisiana Acadia N30 JX104358 LSU M-5914 
LAA Louisiana Acadia N30 JX104358 LSU M-7492 
LAA Louisiana Acadia N47 bb JX104346 LSU M-7489 
LAB Louisiana Bossier N33 JX104391 LSU M-2093 
LAB Louisiana Bossier N34 X JX104393 LSU M-7494 
LAB Louisiana Bossier N36 JX104395 LSU M-2313 
LAI Louisiana Iberville N3 gg JX104356 LSU M-7498 
LAI Louisiana Iberville N9 JX104381 LSU M-3454 
LAI Louisiana Iberville N9 JX104381 LSU M-3462 
LAI Louisiana Iberville N10 JX104383 LSU M-3455 
LAI Louisiana East Baton Rouge N11 V JX104390 LSU M-7495 
LAI Louisiana Iberville N12 JX104397 LSU M-2316 
LAI Louisiana Iberville N22 JX104385 LSU M-3461 
LAM Louisiana Madison N4 hh JX104359 LSU M-2363 
LAM Louisiana Madison N5 JX104363 LSU M-2362 
LAM Louisiana Madison N5 JX104363 LSU M-2364 
LAM Louisiana Madison N42 JX104368 LSU M-2366 
LAM Louisiana Madison N42 JX104368 LSU M-7499 
LAS Louisiana St. Tammany N6 jj JX104364 LSU M-2430 
LAS Louisiana St. Tammany N15 ii JX104362 LSU M-2429 
LAS Louisiana St. Tammany N43 kk JX104366 LSU M-2431 
MDA Maryland Allegany N13 JX104353 VMNH 0253 
MDA Maryland Allegany N17 JX104373 VMNH 0247 
MDA Maryland Allegany N25 JX104347 VMNH 0249 
MDA Maryland Allegany N25 JX104347 VMNH 0255 
MDA Maryland Allegany N26 ccc JX104349 VMNH 0252 
MDA Maryland Allegany N31 JX104365 VMNH 0254 
MDA Maryland Allegany N32 JX104376 VMNH 0256 
MDA Maryland Allegany N49 zz JX104398 VMNH 0248 
MDD Maryland Dorchester N13 JX104353 VMNH 1116 
MDD Maryland Dorchester N13 JX104353 VMNH 1121 
MDD Maryland Dorchester N13 JX104353 VMNH 1124 
MDD Maryland Dorchester N16 ll JX104369 VMNH 1115 
MDD Maryland Dorchester N18 nn JX104374 VMNH 1119 
MDD Maryland Dorchester N20 pp JX104379 VMNH 1122 
MDD Maryland Dorchester N31 JX104365 VMNH 1118 
MDD Maryland Dorchester N38 mm JX104371 VMNH 1117 
MSH Mississippi Holmes N2 ee JX104352 LSU M-2327 
MSH Mississippi Holmes N14 ff JX104354 LSU M-2330 
MSH Mississippi Holmes N52 JX104361 LSU M-2322 
MSH Mississippi Holmes N55 ce JX104348 LSU M-2325 
SDC South Dakota Clay N39 ooo JX104377 VMNH 2385 
SDC South Dakota Clay N53 nnn JX104375 VMNH 2384 
TXT Texas Tom Green N1 JX104344 VMNH 0266 
TXT Texas Tom Green N1 JX104344 VMNH 0276 
TXT Texas Tom Green N1 JX104344 VMNH 0277 
TXT Texas Tom Green N1 JX104344 VMNH 0279 
TXT Texas Tom Green N1 JX104344 VMNH 0281 
TXT Texas Tom Green N1 JX104344 VMNH 0282 
TXT Texas Tom Green N1 JX104344 VMNH 0284 
TXT Texas Tom Green N7 kkk JX104367 VMNH 0278 
TXT Texas Tom Green N8 111 JX104370 VMNH 0280 
TXT Texas Tom Green N50 mmm JX104372 VMNH 0283 
VAA Virginia Alleghany N23 yy JX104396 VMNH 0450 
VAA Virginia Alleghany N35 XX JX104394 VMNH 0454 
VAA Virginia Alleghany N48 ww JX104392 VMNH 0449 
VAS Virginia Sussex N21 ppp JX104380 VMNH 2275 
VAS Virginia Sussex N45 qqq JX104382 VMNH 2276 
Locality code State County or parish Combined haplotype Cytochrome-b haplotype D-loop haplotype GenBank accession no. for D-loop haplotype Voucher no. 
ARG Arkansas Greene N51 JX104388 JAH 0467* 
GAJ Georgia Jasper N19 JX104378 VMNH 1261 
GAJ Georgia Jasper N19 JX104378 VMNH 1263 
GAJ Georgia Jasper N28 JX104355 VMNH 1253 
GAJ Georgia Jasper N37 JX104351 VMNH 1254 
GAJ Georgia Jasper N37 JX104351 VMNH 1260 
GAJ Georgia Jasper N37 JX104351 VMNH 1264 
GAJ Georgia Jasper N40 ss JX104386 VMNH 1258 
GAJ Georgia Jasper N41 uu JX104389 VMNH 1255 
GAJ Georgia Jasper N46 rr JX104384 VMNH 1257 
GAJ Georgia Jasper N54 tt JX104387 VMNH 1262 
IND Indiana Dubois N32 JX104376 VMNH 0326 
KSR Kansas Rooks N17 JX104373 VMNH 0296 
KSR Kansas Ellis N24 JX104345 VMNH 0289 
KSR Kansas Rooks N24 JX104345 VMNH 0300 
KSR Kansas Rooks N24 JX104345 VMNH 0301 
KSR Kansas Rooks N24 JX104345 VMNH 0302 
KSR Kansas Rooks N29 ggg JX104357 VMNH 0297 
KSR Kansas Rooks N44 hhh JX104360 VMNH 0298 
KSR Kansas Ellis N52 JX104361 VMNH 0290 
LAA Louisiana Acadia N27 JX104350 LSU M-7487 
LAA Louisiana Acadia N27 JX104350 LSU M-7488 
LAA Louisiana Acadia N27 JX104350 LSU M-7491 
LAA Louisiana Acadia N30 JX104358 LSU M-5914 
LAA Louisiana Acadia N30 JX104358 LSU M-7492 
LAA Louisiana Acadia N47 bb JX104346 LSU M-7489 
LAB Louisiana Bossier N33 JX104391 LSU M-2093 
LAB Louisiana Bossier N34 X JX104393 LSU M-7494 
LAB Louisiana Bossier N36 JX104395 LSU M-2313 
LAI Louisiana Iberville N3 gg JX104356 LSU M-7498 
LAI Louisiana Iberville N9 JX104381 LSU M-3454 
LAI Louisiana Iberville N9 JX104381 LSU M-3462 
LAI Louisiana Iberville N10 JX104383 LSU M-3455 
LAI Louisiana East Baton Rouge N11 V JX104390 LSU M-7495 
LAI Louisiana Iberville N12 JX104397 LSU M-2316 
LAI Louisiana Iberville N22 JX104385 LSU M-3461 
LAM Louisiana Madison N4 hh JX104359 LSU M-2363 
LAM Louisiana Madison N5 JX104363 LSU M-2362 
LAM Louisiana Madison N5 JX104363 LSU M-2364 
LAM Louisiana Madison N42 JX104368 LSU M-2366 
LAM Louisiana Madison N42 JX104368 LSU M-7499 
LAS Louisiana St. Tammany N6 jj JX104364 LSU M-2430 
LAS Louisiana St. Tammany N15 ii JX104362 LSU M-2429 
LAS Louisiana St. Tammany N43 kk JX104366 LSU M-2431 
MDA Maryland Allegany N13 JX104353 VMNH 0253 
MDA Maryland Allegany N17 JX104373 VMNH 0247 
MDA Maryland Allegany N25 JX104347 VMNH 0249 
MDA Maryland Allegany N25 JX104347 VMNH 0255 
MDA Maryland Allegany N26 ccc JX104349 VMNH 0252 
MDA Maryland Allegany N31 JX104365 VMNH 0254 
MDA Maryland Allegany N32 JX104376 VMNH 0256 
MDA Maryland Allegany N49 zz JX104398 VMNH 0248 
MDD Maryland Dorchester N13 JX104353 VMNH 1116 
MDD Maryland Dorchester N13 JX104353 VMNH 1121 
MDD Maryland Dorchester N13 JX104353 VMNH 1124 
MDD Maryland Dorchester N16 ll JX104369 VMNH 1115 
MDD Maryland Dorchester N18 nn JX104374 VMNH 1119 
MDD Maryland Dorchester N20 pp JX104379 VMNH 1122 
MDD Maryland Dorchester N31 JX104365 VMNH 1118 
MDD Maryland Dorchester N38 mm JX104371 VMNH 1117 
MSH Mississippi Holmes N2 ee JX104352 LSU M-2327 
MSH Mississippi Holmes N14 ff JX104354 LSU M-2330 
MSH Mississippi Holmes N52 JX104361 LSU M-2322 
MSH Mississippi Holmes N55 ce JX104348 LSU M-2325 
SDC South Dakota Clay N39 ooo JX104377 VMNH 2385 
SDC South Dakota Clay N53 nnn JX104375 VMNH 2384 
TXT Texas Tom Green N1 JX104344 VMNH 0266 
TXT Texas Tom Green N1 JX104344 VMNH 0276 
TXT Texas Tom Green N1 JX104344 VMNH 0277 
TXT Texas Tom Green N1 JX104344 VMNH 0279 
TXT Texas Tom Green N1 JX104344 VMNH 0281 
TXT Texas Tom Green N1 JX104344 VMNH 0282 
TXT Texas Tom Green N1 JX104344 VMNH 0284 
TXT Texas Tom Green N7 kkk JX104367 VMNH 0278 
TXT Texas Tom Green N8 111 JX104370 VMNH 0280 
TXT Texas Tom Green N50 mmm JX104372 VMNH 0283 
VAA Virginia Alleghany N23 yy JX104396 VMNH 0450 
VAA Virginia Alleghany N35 XX JX104394 VMNH 0454 
VAA Virginia Alleghany N48 ww JX104392 VMNH 0449 
VAS Virginia Sussex N21 ppp JX104380 VMNH 2275 
VAS Virginia Sussex N45 qqq JX104382 VMNH 2276 

Appendix II

Specimens examined.—Locality code, state, county or parish, haplotype designations, and voucher number for the 69 eastern gray squirrels (Sciurus carolinensis) included in this study. Samples without vouchers are marked with an asterisk (*). GenBank accession numbers are provided for each of the 16 cytochrome-b haplotypes and each of the 51 D-loop haplotypes. Vouchers are housed in the Virginia Museum of Natural History (VMNH) and Louisiana State University Museum of Natural Sciences (LSUMZ).

Locality code State County or parish Combined haplotype Cytochrome-b haplotype GenBank accession no. for cytochrome-b haplotype D-loop haplotype GenBank accession no. for D-loop haplotype Voucher no. 
ALC Alabama Covington C12 JX104399 yy JX104464 VMNH 2251 
ALC Alabama Covington C20 JX104400 lll JX104442 VMNH 2249 
ALC Alabama Covington C33 JX104403 eee JX104426 VMNH 2248 
ALC Alabama Covington C34 JX104403 ff JX104428 VMNH 2246 
ALC Alabama Covington C35 JX104403 nnn JX104448 VMNH 2250 
ALC Alabama Covington C36 JX104403 ss JX104456 VMNH 2247 
GAJ Georgia Jasper C13 JX104400 bb JX104418 VMNH 1190 
GAJ Georgia Jasper C15 JX104400 ddd JX104423 VMNH 1184 
GAJ Georgia Jasper C43 JX104409 ee JX104425 VMNH 1180 
GAJ Georgia Jasper C43 JX104409 ee JX104425 VMNH 1181 
GAJ Georgia Jasper C44 JX104409 iii JX104436 VMNH 1183 
GAJ Georgia Jasper C45 JX104410 dd JX104422 VMNH 1189 
GAJ Georgia Jasper C47 JX104411 aa JX104416 VMNH 1187 
IND Indiana Dubois C18 JX104400 ggg JX104431 VMNH 0315 
IND Indiana Dubois C25 JX104400 JX104457 VMNH 0314 
IND Indiana Dubois C46 JX104411 JX104415 VMNH 0307 
IND Indiana Dubois C46 JX104411 JX104415 VMNH 0308 
IND Indiana Dubois C46 JX104411 JX104415 VMNH 0309 
IND Indiana Dubois C46 JX104411 JX104415 VMNH 0310 
IND Indiana Dubois C46 JX104411 JX104415 VMNH 0311 
IND Indiana Dubois C46 JX104411 JX104415 VMNH 0312 
IND Indiana Dubois C46 JX104411 JX104415 VMNH 0313 
LAA Louisiana Acadia C41 JX104407 pp JX104452 NDM 1057* 
LAF Louisiana West Feliciana C2 JX104399 JX104420 LSU M-1968 
LAF Louisiana West Feliciana C2 JX104399 JX104420 LSU M-2079 
LAF Louisiana West Feliciana C6 JX104399 JX104440 LSU M-1967 
LAI Louisiana East Baton Rouge C1 JX104399 JX104417 LSU M-2412 
LAI Louisiana East Baton Rouge C1 JX104399 JX104417 LSU M-2413 
LAI Louisiana East Baton Rouge C1 JX104399 JX104417 LSU M-2414 
LAI Louisiana East Baton Rouge C1 JX104399 JX104417 LSU M-2416 
LAI Louisiana East Baton Rouge C5 JX104399 hh JX104432 LSU M-2415 
LAI Louisiana East Baton Rouge C28 JX104401 JX104434 LSU M-1960 
LAS Louisiana St. Tammany C29 JX104401 kkk JX104439 LSU M-1964 
LAS Louisiana St. Tammany C29 JX104401 kkk JX104439 LSU M-1966 
LAS Louisiana St. Tammany C50 JX104413 JX104443 LSUM-1135 
LAV Louisiana Vernon C37 JX104404 ii JX104435 LSU M-2395 
LAV Louisiana Vernon C38 JX104404 mmm JX104445 LSU M-2393 
MDA Maryland Allegany C4 JX104399 fff JX104429 VMNH 0243 
MDA Maryland Allegany C8 JX104399 JX104458 VMNH 0244 
MDA Maryland Allegany C9 JX104399 V JX104459 VMNH 0241 
MDA Maryland Allegany C9 JX104399 V JX104459 VMNH 0242 
MDA Maryland Allegany C16 JX104400 JX104424 VMNH 0236 
MDA Maryland Allegany C16 JX104400 JX104424 VMNH 0238 
MDA Maryland Allegany C26 JX104400 JX104463 VMNH 0237 
MDA Maryland Allegany C51 JX104414 JX104461 VMNH 0240 
MDD Maryland Dorchester C10 JX104399 vv JX104460 VMNH 0411 
MDD Maryland Dorchester C17 JX104400 gg JX104430 VMNH 1114 
MDD Maryland Dorchester C21 JX104400 JX104446 VMNH 0365 
MDD Maryland Dorchester C21 JX104400 JX104446 VMNH 0410 
MDD Maryland Dorchester C22 JX104400 JX104449 VMNH 0408 
MDD Maryland Dorchester C42 JX104408 JX104427 VMNH 0412 
MDD Maryland Dorchester C42 JX104408 JX104427 VMNH 0413 
MSH Mississippi Holmes C7 JX104399 ll JX104441 LSU M-2343 
MSH Mississippi Holmes C30 JX104402 mm JX104444 LSU M-2336 
MSH Mississippi Holmes C31 JX104402 nn JX104447 LSU M-2333 
MSH Mississippi Holmes C32 JX104402 oo JX104450 LSU M-2331 
MSH Mississippi Holmes C40 JX104406 jj JX104438 LSU M-2348 
MSH Mississippi Holmes C49 JX104412 rr JX104455 LSU M-2341 
TNS Tennessee Shelby C39 JX104405 JX104437 LSU M-2091 
VAA Virginia Augusta C19 JX104400 hhh JX104433 VMNH 0528 
VAA Virginia Alleghany C23 JX104400 JX104453 VMNH 0526 
VAA Virginia Alleghany C27 JX104400 zz JX104465 VMNH 0490 
VAA Virginia Augusta C48 JX104411 JX104451 VMNH 0529 
VAH Virginia Henry C3 JX104399 JX104421 VMNH 1557 
VAH Virginia Henry C3 JX104399 JX104421 VMNH 1559 
VAH Virginia Henry C11 JX104399 ww JX104462 VMNH 1551 
VAH Virginia Henry C14 JX104400 bbb JX104419 VMNH 1554 
VAH Virginia Henry C24 JX104400 JX104454 VMNH 1558 
VAH Virginia Henry C42 JX104408 JX104427 VMNH 2243 
Locality code State County or parish Combined haplotype Cytochrome-b haplotype GenBank accession no. for cytochrome-b haplotype D-loop haplotype GenBank accession no. for D-loop haplotype Voucher no. 
ALC Alabama Covington C12 JX104399 yy JX104464 VMNH 2251 
ALC Alabama Covington C20 JX104400 lll JX104442 VMNH 2249 
ALC Alabama Covington C33 JX104403 eee JX104426 VMNH 2248 
ALC Alabama Covington C34 JX104403 ff JX104428 VMNH 2246 
ALC Alabama Covington C35 JX104403 nnn JX104448 VMNH 2250 
ALC Alabama Covington C36 JX104403 ss JX104456 VMNH 2247 
GAJ Georgia Jasper C13 JX104400 bb JX104418 VMNH 1190 
GAJ Georgia Jasper C15 JX104400 ddd JX104423 VMNH 1184 
GAJ Georgia Jasper C43 JX104409 ee JX104425 VMNH 1180 
GAJ Georgia Jasper C43 JX104409 ee JX104425 VMNH 1181 
GAJ Georgia Jasper C44 JX104409 iii JX104436 VMNH 1183 
GAJ Georgia Jasper C45 JX104410 dd JX104422 VMNH 1189 
GAJ Georgia Jasper C47 JX104411 aa JX104416 VMNH 1187 
IND Indiana Dubois C18 JX104400 ggg JX104431 VMNH 0315 
IND Indiana Dubois C25 JX104400 JX104457 VMNH 0314 
IND Indiana Dubois C46 JX104411 JX104415 VMNH 0307 
IND Indiana Dubois C46 JX104411 JX104415 VMNH 0308 
IND Indiana Dubois C46 JX104411 JX104415 VMNH 0309 
IND Indiana Dubois C46 JX104411 JX104415 VMNH 0310 
IND Indiana Dubois C46 JX104411 JX104415 VMNH 0311 
IND Indiana Dubois C46 JX104411 JX104415 VMNH 0312 
IND Indiana Dubois C46 JX104411 JX104415 VMNH 0313 
LAA Louisiana Acadia C41 JX104407 pp JX104452 NDM 1057* 
LAF Louisiana West Feliciana C2 JX104399 JX104420 LSU M-1968 
LAF Louisiana West Feliciana C2 JX104399 JX104420 LSU M-2079 
LAF Louisiana West Feliciana C6 JX104399 JX104440 LSU M-1967 
LAI Louisiana East Baton Rouge C1 JX104399 JX104417 LSU M-2412 
LAI Louisiana East Baton Rouge C1 JX104399 JX104417 LSU M-2413 
LAI Louisiana East Baton Rouge C1 JX104399 JX104417 LSU M-2414 
LAI Louisiana East Baton Rouge C1 JX104399 JX104417 LSU M-2416 
LAI Louisiana East Baton Rouge C5 JX104399 hh JX104432 LSU M-2415 
LAI Louisiana East Baton Rouge C28 JX104401 JX104434 LSU M-1960 
LAS Louisiana St. Tammany C29 JX104401 kkk JX104439 LSU M-1964 
LAS Louisiana St. Tammany C29 JX104401 kkk JX104439 LSU M-1966 
LAS Louisiana St. Tammany C50 JX104413 JX104443 LSUM-1135 
LAV Louisiana Vernon C37 JX104404 ii JX104435 LSU M-2395 
LAV Louisiana Vernon C38 JX104404 mmm JX104445 LSU M-2393 
MDA Maryland Allegany C4 JX104399 fff JX104429 VMNH 0243 
MDA Maryland Allegany C8 JX104399 JX104458 VMNH 0244 
MDA Maryland Allegany C9 JX104399 V JX104459 VMNH 0241 
MDA Maryland Allegany C9 JX104399 V JX104459 VMNH 0242 
MDA Maryland Allegany C16 JX104400 JX104424 VMNH 0236 
MDA Maryland Allegany C16 JX104400 JX104424 VMNH 0238 
MDA Maryland Allegany C26 JX104400 JX104463 VMNH 0237 
MDA Maryland Allegany C51 JX104414 JX104461 VMNH 0240 
MDD Maryland Dorchester C10 JX104399 vv JX104460 VMNH 0411 
MDD Maryland Dorchester C17 JX104400 gg JX104430 VMNH 1114 
MDD Maryland Dorchester C21 JX104400 JX104446 VMNH 0365 
MDD Maryland Dorchester C21 JX104400 JX104446 VMNH 0410 
MDD Maryland Dorchester C22 JX104400 JX104449 VMNH 0408 
MDD Maryland Dorchester C42 JX104408 JX104427 VMNH 0412 
MDD Maryland Dorchester C42 JX104408 JX104427 VMNH 0413 
MSH Mississippi Holmes C7 JX104399 ll JX104441 LSU M-2343 
MSH Mississippi Holmes C30 JX104402 mm JX104444 LSU M-2336 
MSH Mississippi Holmes C31 JX104402 nn JX104447 LSU M-2333 
MSH Mississippi Holmes C32 JX104402 oo JX104450 LSU M-2331 
MSH Mississippi Holmes C40 JX104406 jj JX104438 LSU M-2348 
MSH Mississippi Holmes C49 JX104412 rr JX104455 LSU M-2341 
TNS Tennessee Shelby C39 JX104405 JX104437 LSU M-2091 
VAA Virginia Augusta C19 JX104400 hhh JX104433 VMNH 0528 
VAA Virginia Alleghany C23 JX104400 JX104453 VMNH 0526 
VAA Virginia Alleghany C27 JX104400 zz JX104465 VMNH 0490 
VAA Virginia Augusta C48 JX104411 JX104451 VMNH 0529 
VAH Virginia Henry C3 JX104399 JX104421 VMNH 1557 
VAH Virginia Henry C3 JX104399 JX104421 VMNH 1559 
VAH Virginia Henry C11 JX104399 ww JX104462 VMNH 1551 
VAH Virginia Henry C14 JX104400 bbb JX104419 VMNH 1554 
VAH Virginia Henry C24 JX104400 JX104454 VMNH 1558 
VAH Virginia Henry C42 JX104408 JX104427 VMNH 2243 

Author notes

Associate Editor was Ryan W. Norris.