Am. Midl. Nat. 155:188–196

Delineating the Range of a Disjunct Population of Southern Flying Squirrels (Glaucomys volans) AMANDA J. LAVERS, STEPHEN D. PETERSEN, DONALD T. STEWART1 AND TOM B. HERMAN Department of Biology, Acadia University, Wolfville, Nova Scotia B4P 2R6 Canada ABSTRACT.—The Southern flying squirrel (Glaucomys volans) is a species designated at risk in Canada where its range is restricted to parts of Ontario, Quebec and Nova Scotia. Before this study, its distribution in Nova Scotia was poorly documented, with only seven site records. Based on live-trapping and intact and partial specimens provided by the public, we present data for 28 additional locations; these combined with historic records delineate a disjunct range that is more extensive than previously believed, but limited to southwest Nova Scotia. To identify specimens that were not fully intact, simple morphological and molecular techniques were employed. The latter, which consisted of PCR amplification and then restriction enzyme digestion of the cytochrome-b gene, allowed reliable species identification of tree squirrels from Nova Scotia by use of partial specimens.

INTRODUCTION The range of the southern flying squirrel [Glaucomys volans (Linnaeus)] extends over eastern North America from Florida to southern Ontario and Quebec (Dolan and Carter, 1977) with disjunct populations in Nova Scotia and Central America (Wood and Tessier, 1974; Diersing, 1980). Glaucomys volans was first recorded in Nova Scotia in 1971 from near Grafton lakes in Kejimkujik National Park (Wood and Tessier, 1974). Since that initial report approximately 20 additional records from seven sites in Nova Scotia (Fig. 1) have been documented (Elderkin, 1987; Davis, 1998; Hope, 2000). Glaucomys volans is designated a ‘‘species at risk’’ in Canada (COSEWIC, 2004). Southern and northern flying squirrels (Glaucomys sabrinus), both of which are present in Nova Scotia, are difficult to distinguish. For example, a specimen collected at Pebbleoggitch Lake, Kejimkujik National Park in 1962, and reported by Wood and Tessier (1974) as G. volans, is actually a small G. sabrinus (Scott and Hebda, 2004). The diagnostic test to distinguish Glaucomys species requires close examination of ventral hairs (Dolan and Carter, 1977). Most mammal inventories use trapping techniques inadequate for sampling Glaucomys species. The public rarely reports flying squirrel sightings and never differentiates the two species. These difficulties, combined with the nocturnal habits of flying squirrels, have resulted in a poor understanding of the basic distribution of G. volans in Nova Scotia. Advances in DNA extraction techniques and increased access to PCR technology allow use of molecular methods to quickly and inexpensively confirm species identification from trace evidence. Various molecular methods have been used to identify species, races and sexes (for review see Symondson, 2002). These methodologies range from amplification of a specific distinguishing marker (Hoelzel, 2001; Jarman et al., 2002; Palomares, 2002) to amplification of a general marker followed by restriction enzyme digestion (Paxinos et al., 1997) or sequencing (Farrell et al., 2000). 1

e-mail: [email protected]

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FIG. 1.—Seven sites of Glaucomys volans historic records in Nova Scotia

STUDY AREA Glaucomys volans in Nova Scotia is disjunct from the nearest extant population in Maine by about 400 km across the Bay of Fundy. Nova Scotia supports diverse mixed wood forests comprising varying amounts of red spruce, Picea rubens; white spruce, P. glauca; black spruce, P. mariana; white pine, Pinus strobus; red pine, P. resinosa; eastern hemlock, Tsuga canadensis; white birch, Betula papyrifera; yellow birch, Betula alleghaniensis; red maple, Acer rubrum; sugar maple, A. saccharum; American beech, Fagus grandifolia; white ash, Fraxinus americana; and red oak, Quercus rubra (Davis and Browne, 1996). Oak-hickory or pine-oak forests typically associated with G. volans in the United States (Weigl, 1969; Sonenshine and Levy, 1981; Fridell and Litvaitis, 1991; Taulman and Seaman, 2000) are uncommon in eastern Canada. In southwestern Nova Scotia maple-oak-birch associations are typical of hardwood and mixedwood forests (Davis and Browne, 1996). Full habitat details and site descriptions are provided in Lavers (2004). METHODS Sample collection.—To collect flying squirrel specimens, we ran a province-wide public reporting network targeting domestic cat owners, fur trappers and amateur naturalists from March 2001 to March 2003. We distinguished many Glaucomys specimens by examining hairs on the venter; animals (including juveniles) with pure white ventral hair were identified as G. volans (Dolan and Carter, 1977). We measured mass and hindfoot length to corroborate identifications; animals with a mass greater than 80 g or a hindfoot longer than 35 mm were identified as G. sabrinus (Dolan and Carter, 1977). We collected partial specimens (mostly tails) and tested techniques to identify them to species. At locations where we collected photographs of apparent Glaucomys volans we subsequently trapped with Model 201 Tomahawk (Tomahawk, Wisconsin) and/or folding Sherman (HB Sherman Traps Inc., Florida) live-traps. We modified Sherman traps by

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removing one side panel and fitting the trap into a small wooden chamber. We covered all traps with waxed cardboard, provided them with 100% cotton batting and baited them with peanut butter and apple. We wired traps to or wedged them between trees 1–2 m above the ground. For all live captures, we marked individuals with a Number 1 Monel ear tag (National Band and Tag Co., Kentucky) or with a unique mark of picric acid. We collected blood samples by puncturing the caudal vein on the ventral side of the tail and absorbing the blood onto FTAÒ Cards (Canadian Life Technologies, Ontario). We dried and stored blood samples at room temperature until we extracted DNA. We obtained geographic coordinates at all Glaucomys volans capture locations with a handheld Garmin GPS 12 unit (Garmin International Inc., Kansas) using map datum WGS84. We recorded coordinates when the unit reported error less than 10 m. We mapped geographic locations for all verifiable Glaucomys reports using ArcMap 8.2 (Environmental Systems Research Institute, California). We digitized capture locations of all G. sabrinus specimens, including points from historical records and from Nova Scotia Department of Natural Resource’s voluntary trapper return program, using ArcMap and topographic survey map sheets. Morphological discrimination.—We measured tail length, tail width, and individual tail hair length in 37 whole adult specimens (14 Glaucomys volans and 23 G. sabrinus). With data from whole adult specimens, we compared parameters for each species by use of Student’s t-test. Molecular discrimination.—To minimize development time and cost, we explored the use of a general marker. The cytochrome-b gene has been well characterized in mammals and sequence data for most common species are available from GenBank (http:// www.ncbi.nlm.nih.gov). To develop a restriction digest assay, we obtained cytochromeb sequences for all five squirrel species found in Nova Scotia: Glaucomys volans (GenBank accession number AJ39531.1), G. sabrinus (GenBank accession number AF359221.1), Marmota monax (GenBank accession number AF100719.1), Tamias striatus (GenBank accession number AF147673.1) and Tamiasciurus hudsonicus (GenBank accession number AB030029.1). We aligned sequences using the online program Clustal W (Thompson et al., 1994), imported them into GeneRunner (v. 3.05, Hastings Software, Inc.) and scanned them for restriction sites. We investigated eight common and inexpensive restriction enzymes (Alu I, Eco RI, Hinf I, Rsa I, Msp I, Sau I, Taq I and Sau 3AI ¼ Mbo I). We extracted DNA using a NaCl salt extraction protocol (Miller et al., 1988), Chelex 100 resin (BioRad) protocol (Walsh et al., 1991) or from FTAÒ Cards (Whatman); approximately 25 ng of DNA was used per reaction. We amplified the entire cytochrome-b gene using primers L14724 and H15915 (Irwin et al., 1991). We carried out PCR reactions in 20 ll volumes with the following concentration of ingredients: 10 mM Tris-HCl (pH 9), 50 mM KCl, 0.1% TritonÒX-100, 2.5 mM MgCl2, 2.5 mM of each dNTP, 10 mM of each primer and 1.5 units of Taq DNA polymerase (Promega, Madison, WI). Approximately 25 ng of DNA was used per reaction. We conducted amplifications in a PTC-100 thermocycler (MJ Research, Inc., MA) under the following conditions: 3 min at 94 C, followed by 45 cycles of 30 s at 94 C, 1 min at 50 C, and 2 min at 72 C. We added an extension time of 4 min at 72 C to the end of the run. We performed restriction digests using 5 ll of PCR product, 3.75 ll ddH2O, 1.0 ll reaction buffer, and 2.5 units of restriction enzyme. We incubated reactions at 37 C for 2 h and visualized them on 2% agarose gels. RESULTS Collecting samples.—Between March 2001 and March 2003, 393 reports of flying squirrels were received from the public. Only 135 reports (34%) could be substantiated with a

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FIG. 2.—Tail length of Glaucomys volans (n ¼ 14) and G. sabrinus (n ¼ 23) in Nova Scotia

photograph or specimen. Of these, 68 were whole specimens, 25 were partial specimens and 42 were photographs. From the whole specimens, Glaucomys volans were identified at 10 new locations. Live trapping resulted in the capture of G. volans at 11 additional new locations. One hundred and four intact Glaucomys specimens were submitted to the Nova Scotia Department of Natural Resource’s voluntary trapper return program between 1997 and 2002. Among trapper returns, specimens from three new locations were identified as G. volans. Morphological discrimination.—Glaucomys volans tails were significantly shorter (mean ¼ 81.1 6 5.5 mm, n ¼ 14) than G. sabrinus tails (mean ¼ 105.8 6 9.6mm, n ¼ 23) in Nova Scotia (t ¼
9.98, P ¼ 0.000, d.f. ¼ 35; Fig. 2). There was little overlap in tail length; no G. volans tails exceeded 90 mm and no intact adult G. sabrinus tail was less than 90 mm. Molecular discrimination.—Three of the eight restriction enzymes investigated using published sequences produced unique banding patterns for each tree squirrel: EcoR1, Msp1 and Rsa1. Sixty-five squirrel samples, including 44 samples from known specimens used as controls and to assess possible variation, were tested using the three aforementioned restriction enzymes. The most informative restriction enzyme, RsaI, produced unique banding patterns for each species of squirrel (Fig. 3). Two bands were produced for Glaucomys volans, G. sabrinus and Tamias striatus; band lengths for each of these species were unique (Fig. 3). For

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FIG. 3.—Agarose gel visualizing the restriction digest of cytochrome-b gene with RsaI. Lanes 1 (far left) and lane 8 are ladders with each band representing 100 bp difference in product size. Sample codes are as follows: Glvo ¼ Glaucomys volans, Glsa ¼ G. sabrinus, Tast ¼ Tamias striatus and Tahu ¼ Tamiasciurus hudsonicus

Tamiasciurus hudsonicus, the full cytochrome-b fragment was cut approximately in half resulting in only one apparent band. MspI and EcoRI distinguished G. volans (one cut site) from other tree squirrels (absence of cut site), but could not distinguish G. sabrinus from other tree squirrels. Of 21 unidentified samples, 11 samples from four additional new locations were identified as G. volans by use of EcoR1, Msp1 and Rsa1. Delineating the range.—Glaucomys volans was live-trapped and specimens were collected from 28 new locations (by definition, locations are separated by .2 km). In conjunction with the seven previously documented locations, these new sites delineate a disjunct geographic range that is limited to southwestern Nova Scotia (Fig. 4). The most northern location, Falmouth, Hants County, lies at 458029270N, 648119;050W and the most southern location, Ponhook Lake, Queens County, at 448179390N, 648519060W.

DISCUSSION Distribution.—Our results indicate that Glaucomys volans in Nova Scotia is not limited to Kejimkujik National Park and Gaspereau, Kings County, as previously reported (Stabb, 1987), but occurs within a 6500 km2 area of southwestern Nova Scotia. This range is largely coincident with the regional distribution of abundant masting red oak, Quercus rubra, (Lavers, 2004) used by G. volans for storable food (Giacalone-Madden, 1976; Harlow and Doyle, 1990; Thomas and Weigl, 1998; Ivan and Swihart, 2000). In southwestern Nova Scotia, where current forestry management is for even-aged coniferous forests, red oak and shade-tolerant mixedwood forests are being replaced at a lower rate than they are disappearing from the landscape (Colville and Rozalska, 2000). This trend is also apparent in other parts of eastern Canada where forestry management is intense (Betts et al., 2003). The conversion of mature mixedwood forests with masting trees such as red oak and American beech to less diverse, even-aged stands of shade-intolerant trees is likely to have important consequences for the conservation of flying squirrels and other wildlife. The southwestern distribution exhibited by Glaucomys volans is typical of more than 60 species of southern-affiliated flora and fauna that have disjunct populations in southwestern Nova Scotia (review in Davis and Browne, 1996). These populations likely colonized Nova Scotia during a hypsithermal period after the last ice age (Wisconsin) approximately 8000 y

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FIG. 4.—Total known sites of Glaucomys volans in Nova Scotia (includes 28 new and 7 historic records) and area sampled for flying squirrels

ago (Pielou, 1991). Since that time, Nova Scotia’s climate has cooled and the ranges of these southern affiliates have likely contracted to the warmest portion of peninsular Nova Scotia: the interior southwest (Pielou, 1991; Davis and Browne, 1996). These species, including G. volans, have been effectively isolated from the main part of their ranges and have likely adapted to live at the limit of their physiological tolerance. A detailed understanding of the ranges of rare species, including peripheral populations, is critical for long-term conservation. Glaucomys volans in Nova Scotia is isolated from continental populations but may, as a peripheral population, protect genetic diversity relevant to coping with rapid environmental change (Lomolino and Channell, 1995; Mockford et al., 1999) and may host future speciation events (Lesica and Allendorf, 1995). To fully characterize genetic variability, phylogeographic patterns and gene flow across the range of this disjunct population, a detailed knowledge of its distribution is essential. The cost of large-scale surveys for rare species is prohibitive (Odom et al., 2001). The public, however, can contribute to the research on rare species more quickly, cost-effectively and over a larger scale than researchers employing conventional survey methods (Scott and Herman, 1995). Employing the public for sampling effort may, however, bias results because sampling is disproportionately higher in areas of human activity. Absence data for research aided by the public must be considered in light of this bias. In our study, the public indiscriminately collected both species of flying squirrels from many parts of Nova Scotia (Lavers, 2004), but wilderness areas and the interior of the province where few people live require more sampling. Species identification.—Venter hair color, total mass, and hindfoot length are widely used as diagnostic tools to identify Glaucomys species (Dolan and Carter, 1977). These features are available when animals are live-trapped and when dead animals are intact. After consumption by wild or domestic predators, however, there may be little left of a flying

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squirrel except the unpalatable tail. It is possible to discriminate Glaucomys species from partial specimens using both morphological measurements (in the case of tails) and molecular techniques. When complete tails are available, our analyses suggest that tails shorter than 90 mm are from either G. volans or juvenile G. sabrinus. Quick morphological measurements can help prioritize specimens but verification with molecular techniques is necessary to ascertain the species identity of tail fragments or tails of adult G. volans from juvenile G. sabrinus. Although the development of the molecular technique described here was driven by a need to identity flying squirrels from tails discarded by domestic cats, it could be applied to other investigations. For example, owl pellets and feces from other predators could be collected and examined for flying squirrel DNA. Identifying prey species in predator diets by use of PCR has been successful for terrestrial (Farrell et al., 2000) and marine vertebrate predators (Jarman et al., 2002) and also may be of interest in the southern Appalachian Mountains where Glaucomys sabrinus is a species at risk (Payne et al., 1989; Loeb et al., 2000; Odom et al., 2001). The molecular assay used here is simple, relatively inexpensive, and can be readily expanded to other rodent species. Acknowledgments.—We would like to thank all the people who contributed information about flying squirrels and acknowledge the voluntary participation of fur trappers. Thanks to Kasia Rozalska and the Centre of Geographic Sciences for providing GIS services and maps, Torre Eaton for help in the field and with the public reporting network and Fred Scott and two reviewers for comments on an earlier version of the manuscript. The Nova Scotia Department of Natural Resources generously shared its flying squirrel database. Financial support was provided by the Government of Canada’s Habitat Stewardship Program for Species at Risk – Atlantic Region, the Nova Scotia Department of Natural Resources Wildlife Division, the World Wildlife Fund’s Macnaughton Scholarship, the Endangered Species Recovery Fund, the Hants West Wildlife Association and NSERC (operating grant 217175 to DTS).

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ACCEPTED 22 APRIL 2005