Northeastern Naturalist 202 B.E.Sturgis, A.A. Aispuro, and K. Vulinec 22001199 NORTHEASTERN NATURALIST 2V6(o1l). :2260,2 N–2o1. 31 Variation in Deuterium Levels of Non-migratory Eptesicus fuscus (Big Brown Bat) along the Delmarva Peninsula Brittany Elizabeth Sturgis1,2, Armando Alberto Aispuro1,3, and Kevina Vulinec1,* Abstract - Stable-isotope analysis can address fundamental questions about the ecology and life history of mobile organisms. The hydrogen isotope, deuterium (δ2H), follows distinct and predictable patterns along environmental gradients, enabling migratory-origin assignments in vagile animals such as bats. However, it is unclear to what degree deuterium levels vary within non-migratory bat populations in fixed areas. To understand this, we compared deuterium signatures among adult female bats in maternity colonies of non-migratory Mid-Atlantic Eptesicus fuscus (Big Brown Bat). We sampled from 5 different locations along the Delmarva Peninsula. We also compared differences in signatures between males and females within colonies. Despite relative proximity to each other, deuterium signatures of females among colony locations differed significantly. Deuterium signatures did not differ among males and females or between 2010 and 2011. We suggest that variation in foraging and roosting behavior, as well as changes in water sources along the peninsula influence deuterium levels on a small geographic scale. These and other factors should be considered when interpreting sampled deuterium levels. Introduction Bats play an integral role in the functioning of ecosystems in the northeastern US, but face several threats including white-nose syndrome transmission and windturbine collisions (Frick et al. 2010, Kunz et al. 2007). Because of these threats, it is imperative to better understand bat-movement patterns (Sullivan et al. 2012) and roosting ecology. Mark–recapture, radio-tracking, and indirect methods used to study night-flying animals have primarily yielded an understanding of bat behavior at local scales (Cryan et al. 2004, Limpert et al. 2007). Stable-isotope analysis can be used to supplement these conventional techniques and provide a more precise picture of regional bat ecology (Britzke et al. 2012, Cryan et al. 2012, Hobson and Wassenaar 1997, Rubenstein et al. 2002). Deuterium (δ2H), an isotope of hydrogen, is a particularly convenient tool for assessing movement patterns. Rainwater concentrations (or signature) of δ2H vary predictably across North America; rainwater in the Southeast is depleted of δ2H relative to rainwater in the Northwest (reviewed in Hobson 1999). This pattern is related to the clinal variation in growing-season precipitation across the landscape 1Department of Agriculture and Natural Resources, Delaware State University, Dover, DE 19901-2277. 2Current address - Delaware Department of Natural Resources and Environmental Control, 100 West Water Street, Suite 6B, Dover, DE 19904. 3Konrad Lorenz Institute of Ethology, University of Veterinary Medicine Vienna, Savoyenstraße 1a, A-1160 Vienna, Austria. Corresponding author - kvulinec@desu.edu. Manuscript Editor: Adrienne Kovach Northeastern Naturalist Vol. 26, No. 1 B.E.Sturgis, A.A. Aispuro, and K. Vulinec 2019 203 and the resulting patterns of hydrogen isotopes within the vegetation. These isotopes are in turn detectable in animals at higher trophic levels (Cormie et al. 1994) and can be used to infer relative geographic positions of sampled-tissue origins. This technique has been widely and successfully used to elucidate breeding and natal origins of migratory bats and birds sampled away from those sites (e.g., Cryan et al 2004, Fraser et al. 2012, Hobson 2005, Popa-Lisseanu et al. 2012). However, it remains unclear how much variation in δ2H isotopic signatures should be expected within highly vagile but non-migratory animals. A potential confounding factor in understanding life histories may exist if δ2H variation in migratory bats is different from that of non-migratory bats. Cryan et al. (2012) found that urban bats from proximate roosting sites in Colorado had high within-species δ2H variation, though mean values for 2 maternal nesting colonies were not significantly different. Voigt et al. (2013) also found a high degree of within-species δ2H variation among bats in tropical Costa Rica, though in addition they found significant differences in mean δ2H values between maternal nesting colonies. These results came from a large city and tropical forest, respectively, and they may not be representative of a semi-rural region in the Mid- Atlantic states. Furthermore, it is unknown if the sources of this variation (such as sex, age, year, or season) are similar between geographically distant populations. Therefore, we investigated factors that may contribute to variation in δ2H concentrations of non-migratory bats within the Delmarva Peninsula. Understanding variation among colonies and among years may allow for better identification of roosting sites or reveal the inability to determine sites if the variation among sites or years is too large. We analyzed the δ2H signatures of bat fur in Eptesicus fuscus (Palisot de Beauvois) (Big Brown Bat), a non-migratory bat in Delaware, Maryland, and Pennsylvania. In the US, Big Brown Bats do not travel far to their winter roosts (usually not more than 80 km) and the species is considered non-migratory (Neubaum et al. 2006). These bats go through an annual molt at their roosting site in mid- to late summer (Fraser et al. 2013), when new fur is grown and local δ2H signatures are incorporated (Britzke et al. 2009; Cryan et al. 2004, 2014). We compared the δ2H signature of Big Brown Bat fur (1) between males and females, (2) among females from 5 maternity colonies, and (3) between 2010 and 2011. Following Cryan et al. (2012), we expected to find differences between males and females, but we did not expect substantial variation among the maternity colonies or between years because of the non-migratory behavior of Big Brown Bats. The δ2H signatures for individuals and maternity colonies may help determine patterns in local variation in δ2H of a non-migratory but highly vagile bat species. Understanding the extent of local δ2H variation in non-migratory bats on the Delmarva Peninsula may improve the efficacy of using δ2H as an intrinsic marker, especially in the context of migratory-origin assignments (Langin et al. 2007, Voigt et al. 2013). Northeastern Naturalist 204 B.E.Sturgis, A.A. Aispuro, and K. Vulinec 2019 Vol. 26, No. 1 Methods Study sites The Delmarva Peninsula is characterized as a low-lying, semi-rural landscape in a matrix of abundant agricultural land with natural parks and few large population centers. Study sites (Fig. 1, Appendix A) consisted of known bat maternity colonies (K. Vulinec and B.E. Sturgis, pers. observ.; H. Niederriter, Delaware Department of Natural Resources and Environmental Control [DNREC], Dover, DE, pers. comm.). Field methods We conducted our fieldwork during the 2010 and 2011 maternity season (April– June) by netting bats from maternity colonies throughout Delaware and 1 location in Pennsylvania (Fig. 1). We captured bats with mist nets or harp traps positioned at colony egresses (Kunz and Fenton 2003). We erected nets or traps ~15 min prior to dusk and left them operational for ~3 h, checking nets every 15 min. We followed the protocol for capturing and handling live bats as established by the American Society of Mammalogists (Sikes and Gannon 2011). The Delaware State University Institutional Animal Care and Use Committee (IACUC) approved these methods in 2010. We wore latex gloves during bat handling to reduce possible white-nose syndrome contamination, according to the National White-Nose Syndrome Decontamination Protocol (USFWS 2010). Once bats were removed from the net, we placed individuals in separate, numbered, brown paper bags. We recorded species, weight, age, sex, wing health, and Figure 1. Big Brown Bat sampling locations in Delaware and Pennsylvania in 2010 and 2011. Appendix A identifies the labels and the corresponding location coordinate s. Northeastern Naturalist Vol. 26, No. 1 B.E.Sturgis, A.A. Aispuro, and K. Vulinec 2019 205 trapping location. We considered individuals that were born the year of sampling to be juveniles, as determined by examining the metacarpal-phalangeal joint. Juvenile joints are less calcified and more cartilaginous when compared to adults (Brunet-Rossinni and Wilkinson 2009). We then cut a fur sample as close to the skin as possible from the dorsal region between the scapulae, placed it in a labeled plastic 1.5-ml microcentrifuge tube, and stored tubes in a dry location for further laboratory processing.To avoid confusing molting periods and new fur growth on juveniles, we excluded juveniles and all captures after the first week of July from the analysis (Cryan et al 2012). Stable-isotope analysis Fur samples were analyzed at the Colorado Plateau Stable Isotope Laboratory (CPSIL) at Northern Arizona University with an isotope mass-spectrometer using standardized methods. All stable hydrogen-isotope (δ2Hfur) values from fur were reported in parts per thousand (‰) relative to the internationally accepted Vienna Standard Mean Ocean Water (VSMOW). Six in-house standards were used as part of the quality-assurance protocol at the Colorado Plateau Stable Isotope Laboratory, including Rangifer tarandus (L.) (Caribou) hoof, Tragelaphus strepsiceros (Pallas) (Kudu) horn, Alces alces (L.) (Moose) hair, Cervus canadensis (Erxleben) (Elk) hair, Urus arctos (L.) (Grizzly Bear) hair, and Meleagris gallopavo L. (Wild Turkey) feathers. Our bat-fur samples were analyzed alongside these standards, and their δ2Hfur signatures were standardized to VSMOW using comparative equilibration techniques (Wassenar and Hobson 2003). The CPSIL lab uses keratin standards throughout their sample runs as a measure of within-run precision. Samples that had very little material were analyzed alongside a series of standards at similarly low weights to determine if the chromatography was accurate (Britzke et al. 2009, 2012; Cryan et al 2004; Fraser et al. 2012; Popa-Lisseanu et al. 2012). Discrimination factors account for the observed difference between source and tissue isotopic signatures (Del Rio et al. 2009). Discrimination factors have been used to correct isotope data in order to make it comparable across groups. We analyzed raw data because we do not have an experimental basis for discrimination factors and because these values for Big Brown Bat on the Delmarva Peninsula are not known (Cryan et al. 2012). This approach likely did not influence our results because we sampled and analyzed the same tissue type from the same species. Therefore, comparisons within this defined group are not influenced by correction. Statistical analyses We performed statistical analyses in version 25 of IBM SPSS Statistics software (SPSS Inc. 2017). We fit a general linear model (GLM; UNIANOVA) to compare adult female’s δ2Hfur signatures among locations and between years. Following Cryan et al. (2012), we excluded from the location analysis juveniles, males, and bats not caught at the maternity colonies’ egress in order to determine a more accurate picture of the δ2Hfur levels of non-migratory Big Brown Bat maternity colonies. We excluded outliers of less than 20‰ as samples with inadequate material. Data on other bats and juvenile Big Brown Bats are available in Sturgis (2013). Northeastern Naturalist 206 B.E.Sturgis, A.A. Aispuro, and K. Vulinec 2019 Vol. 26, No. 1 δ 2H values did not deviate from normal (Kolmogorov–Smirnov: P = 0.200) and the variances were homogeneous (Levene’s test for mean and median = 0.750); therefore, we used a generalized linear model (GLM) with a normal distribution to examine differences of adult female δ2Hfur values among locations and years, with location and year as fixed effects. The interaction of location*year was not significant and so we removed this interaction from the model. We used Type III sum of squares and pairwise comparisons of the estimated marginal means of locations through least significant differences (LSD) for multiple comparisons. We assessed effect sizes using the proportion of variation explained for a certain effect ([effect SS] / [effect SS + error SS]). This statistic is analogous to R2 in multiple regression and is used to examine effect size when there is more than 1 independent variable. A partial Eta squared value >0.2 is considered a large effect (Fritz et al. 2012). We employed a GLM to examine the difference between δ2Hfur values of males and females captured from the same maternity colonies. Results We collected and analyzed 55 fur samples in 2010 and 54 fur samples in 2011 from females, for a total of 109 samples for both years (Appendix B). We also collected and analyzed the fur from 16 males during 2010. No samples from males were collected during 2011. A table of all sampled individuals is provided in Supplemental File 1 (available online at http://www.eaglehill.us/NENAonline/ suppl-files/n26-1-N1644-Vulinec-s1, and, for BioOne subscribers, at https:// dx.doi.org/10.1656/N1664.s1). The mean δ2Hfur for adult females caught at maternity colonies (-44.76‰, SD = 9.67, n = 109) did not differ significantly from the mean for adult males at the same maternity colonies (-42.82‰, SD = 6.20, n = 16) (Sex: F = 0.607, df = 1, P = 0.437). δ 2Hfur values of Big Brown Bat adult females differed significantly among maternity- colony sites located throughout the study area (F = 7.286, df = 4, P < 0.001; partial Eta squared = 0.221; Table 1, Fig. 2). Pairwise comparisons of the estimated marginal means by LSD procedure (Table 2) revealed no differences among Dover and Georgetown, Killens Pond and White Clay Creek, and White Clay Creek and Bellevue (Fig. 2). The greatest differences were between and White Clay Creek and Georgetown (P < 0.001) and Bellevue and Georgetown (P < 0.001); the latter 2 sites were the farthest apart (Fig. 1). Table 1. General linear model tests of between-subjects effects with the dependent variable, deuterium. Test of effect size (partial Eta squared) is included. Partial Source Sum of squares df Mean square F P value Eta squared Location 1788.035 4 447.009 7.286 less than 0.001 0.221 Year 0.750 1 0.750 0.012 0.912 0.000 Error 6319.317 103 61.353 Total 228524.620 109 Northeastern Naturalist Vol. 26, No. 1 B.E.Sturgis, A.A. Aispuro, and K. Vulinec 2019 207 δ2Hfur values did not change significantly between both study seasons for Big Brown Bat (F = 0.012, df = 1, P = 0.912). Mean δ2Hfur in 2010 was -49.02‰ (SD = 9.82; n = 55) and in 2011, mean δ2Hfur was -40.43‰ (SD = 7.40; n = 54). Discussion We compared the effect of sex, population location, and year on the δ2H signature of Big Brown Bat fur on the Delmarva Peninsula. We collected fur in the same general geographic region for all individuals and established a baseline for expected variation of a non-migratory bat in the region. The variation in δ2H of a population has been described by Cryan et al. (2012) for the same species in a western urban center. Here we report similarly high levels of variation in a much different habitat type. Big Brown Bats are non-migratory, opportunistic and generalist feeders, and rarely move farther than 80 km between their summer and winter roosting sites (Mills et al. 1975, Sullivan et al. 2006). The generalist feeding style of Big Brown Bats probably explains some of the variation in δ2H signatures of fur observed in this study, as was also suggested by Cryan et al. (2012) in a Colorado urban center. The δ2Hfur signatures for Big Brown Bats were within the range associated with precipitation signatures in Delaware and the surrounding areas (Meehan et al. 2004), supporting the proposed relationship between the δ2H signatures of fur in an Figure 2. δ2Hfur values of female Big Brown Bats in 5 locations sampled in 2010 and 2011. Boxes enclose the 25th to 75th quartiles. Central line indicates median and whiskers encompass the range of the data. The circle represents an outlier (extreme values that fall more than 1.5 interquartile ranges beyond the 25th and 75th percentile). Boxes sharing a letter are not significantly different. Sample sizes for each location are below the location n ame. Northeastern Naturalist 208 B.E.Sturgis, A.A. Aispuro, and K. Vulinec 2019 Vol. 26, No. 1 insectivorous bat and local water sources (Popa-Lisseanu et al. 2012). Therefore, while foraging behavior may cause high variation in δ2Hfur signatures for this species, these values appear to be primarily influenced by local wa ter sources. Big Brown Bat δ2Hfur values were very different from those reported by Cryan et al. (2012), perhaps partially related to differences in geographic variation of δ2H. Adult males captured in mid-Atlantic maternity colonies did not differ significantly from females, contrary to findings of Cryan et al. (2012) of sex-related δ2Hfur patterns in a western urban center that is likely more chemically complex than our semi-rural mid-Atlantic sites. Our results showing no difference between males and females may be due to small sample size of males or potentially because males and females have similar foraging behaviors on the Delmarva Peninsula. We found differences in the δ2Hfur signatures among maternity colonies within the Delmarva Peninsula. This finding is in contrast to prior work showing that, while there may be large differences in the δ2H levels among individual bats within a relatively small geographical area (9.25 km maximum distance), there was no significant difference in the mean level between 2 Colorado maternity colonies (Cryan et al. 2012). Different results came from a study in Costa Rica by Voigt et al. (2013), who found a large variation (20‰) in the δ2H makeup of sedentary neotropical bats from 2 colonies 4 km apart. Our study sites were up to 100 km apart (Fig. 1), therefore greater differences might be expected among our colonies Table 2. Pairwise comparisons of female Big Brown Bat mean δ2Hfur values among 5 locations sampled in Delaware and surrounding areas in 2010 and 2011, based on estimated marginal means. An asterisk (*) indicates that the difference in mean δ2Hfur values is significant at the 0.05 alpha level.. 95% CI = 95 % confidence interval for differrence. Mean 95% CI difference Std Lower Upper Location I Location J (I - J) error P-value Bound Bound Dover, DE Georgetown DE -7.951 4.070 0.053 -16.024 0.121 Killens Pond 7.190* 3.085 0.022 1.072 13.308 Bellevue 13.178* 2.656 0.001 7.911 18.445 White Clay Creek Preserve 9.301* 3.159 0.004 3.036 15.567 Georgetown DE Dover, DE 7.951 4.070 0.053 -0.121 16.024 Killens Pond 15.141* 5.091 0.004 5.045 25.238 Bellevue 21.129* 4.831 0.001 11.548 30.711 White Clay Creek Preserve 17.253* 5.136 0.001 7.066 27.440 Killens Pond Dover, DE -7.190* 3.085 0.022 -13.308 -1.072 Georgetown DE -15.141* 5.091 0.004 -25.238 -5.045 Bellevue 5.988* 2.863 0.039 0.311 11.665 White Clay Creek Preserve 2.111 3.270 0.520 -4.373 8.596 Bellevue Dover, DE -13.178* 2.656 0.001 -18.445 -7.911 Georgetown DE -21.129* 4.831 0.001 -30.711 -11.548 Killens Pond -5.988* 2.863 0.039 -11.665 -0.311 White Clay Creek Preserve -3.877 2.943 0.191 -9.712 1.959 White Clay Creek Dover, DE -9.301* 3.159 0.004 -15.567 -3.036 Preserve Georgetown DE -17.253* 5.136 0.001 -27.440 -7.066 Killens Pond -2.111 3.270 0.520 -8.596 4.373 Bellevue 3.877 2.943 0.191 -1.959 9.712 Northeastern Naturalist Vol. 26, No. 1 B.E.Sturgis, A.A. Aispuro, and K. Vulinec 2019 209 than those in the study of Voigt et al. (2013); yet we found similar differentiation of δ2Hfur values among colonies to those in Costa Rica (largest δ2Hfur differences approximately 20‰; Appendix B). The δ2Hfur variance may be attributed to 2 different factors: the local water sources may have different δ2Hprecipitation levels because of the surrounding landscape or maternity colonies may have different diets and food preferences (Agosta 2002). The Piedmont and Coastal Plain regions make up the 2 physiographic regions found in Delaware and the surrounding areas. The Piedmont is found in the hilly northernmost part of the state where the parent material is made up of metamorphic and igneous rocks. Conversely, the Coastal Plain physiographic region covers most of the state and is flatter and underlain with Appalachian sediments that are relatively young (Polsky et al. 2000). The difference in the surrounding geology may change the groundwater signatures (Bowen et al. 2005), and that may alter the stable δ2H signatures of bat fur within the locales. Furthermore, water composition changes because of runoff and leaching, which may cause differences among localities (Good et al. 2015). Our study revealed δ2Hfur signatures among Big Brown Bat that showed considerable variation within and among maternity colonies. Two of the locations with similar δ2Hfur profiles were on the Piedmont portion of the peninsula (Bellevue and White Clay Creek Preserve). The other 3 locations were on the Coastal Plain (structure in Dover, Killens Pond, and a residence in Georgetown). Only 2 of these Coastal Plain locations showed slightly similar δ2Hfur levels—Dover and Georgetown (P = 0.053). Both of these sites are heavily urbanized, while Killens Pond is a wooded park set within a matrix of agricultural land. We suggest that there is a difference of the δ2Hfur mean among locations because of differences in local water composition (Kendall and Coplan 2001). In addition, the opportunistic feeding ecology and generalist diet of this species probably contributed to relatively wide variation in δ2Hfur values. As mentioned above, influence δ2Hfur levels may be influenced by local diet, but we did not analyze stable isotopes in food sources to relate it to δ2Hfur levels. The difference in δ2Hfur from insect diet and surface water would be important to examine in future studies in this area (V oigt et al. 2013). While variation can be extensive, bats of the northeastern US are assumed to molt once a year during mid- to late summer (Fraser et al. 2015). For this reason, we excluded any bats caught after 3 July. We did not find differences in δ2Hfur between 2010 and 2011 in Big Brown Bat females from maternity colonies. We collected fur samples from 1 May through 3 July in 2010 and 11 April through 23 June in 2011. We hypothesized that the mean δ2Hfur level might vary throughout the year as the bats (1) migrated from their winter roosting sites or other locations or (2) molted during the summer. The δ2Hfur level did not change between years, suggesting that our methods avoided confounding molting with fur from the previous year. Furthermore, as there was no interaction between locations and years, our results indicate that individual female bats may return to different maternity colonies in the summer, as is suggested in Vonhof et al. (2008). Big Brown Bats appear to molt and grow new fur while in the Mid-Atlantic region. The process to grow new fur is not well understood (Fraser et al. 2015), and the timing of fur replacement may differ among individuals as well as species. Northeastern Naturalist 210 B.E.Sturgis, A.A. Aispuro, and K. Vulinec 2019 Vol. 26, No. 1 While collecting fur samples, we noticed an overall “fuzzy” appearance of bats throughout August. The fur also pulled out more readily than earlier in the season; it was particularly easy to remove fur follicles in August compared to the April– July period. In conclusion, our study showed that (1) Big Brown Bats differ in δ2Hfur signatures depending on where they roost, and (2) there is high regional δ2Hfur variation within a small rural peninsular region of the northeastern US. We suggest that diet, sex, age, water sourcing, roosting ecology, molting patterns, and thermoregulation strategies may contribute to variation in δ2Hfur signatures of bats even at a small local scale (Cryan et al. 2012, Weller 2009). Microsatellite and mitochondrial DNA analyses of colonies showed complex genetic structure, suggesting that females may not be as philopatric as previously thought (Vonhof et al. 2008). Accordingly, intercolony migration may account for the wide variation in δ2H. These and other pertinent ecological factors should be considered when using δ2H values to inform bat ecology, especially in the context of migratory and colonizing patterns. Further sampling of these 5 colonies, local water sources, and local insects may parse out specific factors that influence bat δ 2H values. Acknowledgments This project was funded by Delaware State University and a USDA Evans–Allen grant to K. Vulinec (PROJ NO: DELX0029-10-03). We thank Virginia Balke, Richard Barczewski, Christopher (Kitt) Heckscher, Lori Brown, and Kimmi Swift for providing input on this project and the manuscript. We also thank Megan Wallrichs, Kesha Braunskill, Ileana Mayes, and Dr. Virginia Balke’s crew at Delaware Technical Community College. We thank Holly Niederriter, Erin Adams, and volunteers with the Bat Program of the Delaware Department of Natural Resources and Environmental Control. We thank the biologists from Bombay Hook National Wildlife Refuge and the managers within the Delaware State Parks (Killens Pond, White Clay Creek State Park, Bellevue State Park), and homeowners. We extend special thanks to Matt Sturgis and Dave Mellow for continual help and support. Literature Cited Agosta, S.J. 2002. Habitat use, diet, and roost selection by the Big Brown Bat (Eptesicus fuscus) in North America: A case for conserving an abundant species. Mammal Review 32:179–198. Bowen, G.J., L.L. Wassenaar, and K.A. Hobson. 2005. Global application of stable hydrogen and oxygen isotopes to wildlife forensics. Oecologia 143:33 7–348. Britzke, E.R., S.C. Loeb, K.A. Hobson, C.S. Romanek, and M.J. Vonhof. 2009. Using hydrogen isotopes to assign origins of bats in the Eastern United States. Journal of Mammalogy 90:743–751. Britzke, E.R., S.C. Loeb, C.S. Romanek, K.A. Hobson, and M.J. Vonhof. 2012. Variation in catchment areas of Indiana Bat (Myotis sodalis) hibernacula inferred from stable hydrogen (δ2H) isotope analysis. Canadian Journal of Zoology 90:1243–1250. Brunet-Rossinni, A.K., and G.S. Wilkinson. 2009. Methods for age estimation and the study of senescence in bats. Pp. 315–325, In T.H. Kunz and S. Parsons (Eds.). Ecological and Behavioral methods for the Study of Bats. Johns Hopkins University Press, Baltimore, MD. 556 pp. Northeastern Naturalist Vol. 26, No. 1 B.E.Sturgis, A.A. Aispuro, and K. Vulinec 2019 211 Cormie, A.B., H.P. Schwarcz, and J. Gray. 1994. Determination of the hydrogen isotopic composition of bone collagen and correction for hydrogen exchange. Geochimica et Cosmochimica Acta 58:365–375. Cryan, P.M., M.A. Bogan, R.O. Rye, G.P. Landis, and C.L. Kester. 2004. Stable hydrogen isotope analysis of bat hair as evidence for seasonal molt and long-distance migration. Journal of Mammalogy 85:995–1001. Cryan, P.M., C.A. Stricker, and M.B. Wunder. 2012. Evidence of cryptic individual specialization in an opportunistic insectivorous bat. Journal of Mammalogy 93:381–389. Cryan, P.M., C.A. Stricker, and M.B. Wunder. 2014. Continental-scale, seasonal movements of a heterothermic migratory tree bat. Ecological Applications 24:602–616. Del Rio, C.M., P. Sabat, R. Anderson-Sprecher, and S.P. Gonzalez. 2009. Dietary and isotopic specialization: The isotopic niche of three Cinclodes ovenbirds. Oecologia 161:149–159. Fraser, E.E., L.P. McGuire, J.L. Eger, F.J. Longstaffe, and M.B. Fenton. 2012. Evidence of latitudinal migration in Tri-colored Bats, Perimyotis subflavus. PLoS ONE 7:e31419. Fraser, E.E., F.J. Longstaffe, and M.B. Fenton. 2013. Moulting matters: The importance of understanding moulting cycles in bats when using fur for endogenous marker analysis. Canadian Journal of Zoology 91:533–544. Fraser, E.E., J.F. Miller, F.J. Longstaffe, and M.B. Fenton. 2015. Systematic variation in the stable hydrogen isotope (δ2H) composition of fur from summer populations of two species of temperate insectivorous bats. Mammalian Biology - Zeitschrift für Säugetierkunde 80:278–284. Frick, W.F., J.F. Pollock, A.C. Hicks, K.E. Langwig, D.S. Reynolds, G.G. Turner, C.M. Butchkoski, and T.H. Kunz. 2010. An emerging disease causes regional population collapse of a common North American bat species. Science 329:679–82. Fritz, C.O., P.E. Morris, and J.J. Richler. 2012. Effect-size estimates: Current use, calculations, and interpretation. Journal of Experimental Psychology: General 141:2–18. DOI:10.1037/a0024338. Good, S.P., D. Noone, and G. Bowen. 2015. Hydrologic connectivity constrains partitioning of global terrestrial water fluxes. Science 349:175–177. Hobson, K.A. 1999. Tracing origins and migration of wildlife using stable isotopes: A review. Oecologia 120:314–326. Hobson, K.A. 2005. Stable isotopes and the determination of avian migratory connectivity and seasonal interactions. The Auk 122:1037–1048. Hobson, K.A., and L.I. Wassenaar. 1997. Linking breeding and wintering grounds of neotropical migrant songbirds using stable hydrogen isotopic analysis of feathers. Oecologia 109:142–148. Kendall, C., and T.B. Coplen. 2001. Distribution of oxygen-18 and deuterium in river waters across the United States. Hydrological Processes 15:1363–1393. Kunz, T.H., and M.B. Fenton. 2003. Bat Ecology. University of Chicago Press, Chicago, Illinois. 798 pp. Kunz, T.H., E.B. Arnett, B.M. Cooper, W.P. Erickson, R.P. Larkin, T. Mabee, M.L. Morrison, M.D. Strickland, and J.M. Szewczak. 2007. Assessing impacts of wind-energy development on nocturnally active birds and bats: A guidance document. Journal of Wildlife Management 71:2449–2486. Langin, K.M, M.W. Reudink, P.P. Marra, E.R. Norris, T.K. Kyser, and L.M. Ratcliffe. 2007. Hydrogen isotopic variation in migratory bird tissues of known origin: Implications for geographic assignment. Oecologia 152:449–457. Northeastern Naturalist 212 B.E.Sturgis, A.A. Aispuro, and K. Vulinec 2019 Vol. 26, No. 1 Limpert, D.L., D.L. Birch, M.S. Scott, M. Andre, and E. Gillam. 2007. Tree selection and landscape analysis of Eastern Red Bat day roosts. The Journal of Wildlife Management 71:478–86. Meehan, T.D., J.T. Giermakowski, and P.M. Cryan. 2004. GIS-based model of stable hydrogen-isotope rations in North American growing-season precipitation for use in animal-movement studies. Isotopes in Environmental and Health Studies 40:291–300. Mills, R.S., G.W. Barret, and M.P. Farrell. 1975. Population dynamics of the Big Brown Bat (Eptesicus fuscus) is southwestern Ohio. Journal of Mammalogy 56:591–604. Neubaum, D.J., T.J. O’Shea, and K.R. Wilson. 2006. Autumn migration and selection of rock crevices as hibernacula by Big Brown Bats in Colorado. Journal of Mammalogy 87:470–479. Polsky, C., J. Allard, N. Currit, R. Crane, and B. Yarnal. 2000. The Mid-Atlantic region and its climate: Past, present, and future. Climate Research 14:161–173. Popa-Lisseanu A.G., K. Sörgel, A. Luckner, L.I. Wassenaar, C. Ibáñez, and S. Kramer- Schadt. 2012. A triple-isotope approach to predict the breeding origins of European bats. PLoS ONE 7:e30388. Rubenstein, D.R., C.P. Chamberlain, R.T. Holmes, M.P. Ayres, J.R. Waldbauer, G.R. Graces, and N.C. Tuross. 2002. Linking breeding and wintering ranges of a migratory songbird using stable isotopes. Science 295:1062–1065. Sikes, R., and W.L. Gannon. 2011. Guidelines of the American Society of Mammalogists for the use of wild mammals in research. Journal of Mammology 92:235–253. Sturgis, B.E. 2013. Stable-isotope analysis of mid-Atlantic bats during spring and summer months. M.Sc. Thesis. Delaware State University, Dover, DE. Sullivan, J.C., K.J. Buscetta, R.H. Michener, J.O. Whitaker Jr, J.R. Finnerty, and T.H. Kunz. 2006. Models developed from δ13C and δ15N of skin tissue indicate non-specific habitat use by the Big Brown Bat (Eptesicus fuscus). Ecoscience 13:11–22. Sullivan, A.R., J.K. Bump, L.A. Kruger, and R.O. Peterson. 2012. Bat-cave catchment areas: Using stable isotopes to determine the probable origins of hibernating bats. Ecological Applications 22:1428–1434. US Fish and Wildlife Service (USFWS). 2010. Decontamination protocol for bat field studies. July 2010. Available online at https://www.fws.gov/Midwest/endangered/mammals/ BatDisinfectionProtocol.html. Accessed 17 February 2010. Voigt, C., K. Schneeberger, and A. Luckner. 2013. Ecological and dietary correlates of stable hydrogen-isotope ratios in fur and body water of syntopic tropical bats. Ecology 94:346–355. Vonhof, M.J., C. Strobeck, and M.B. Fenton. 2008. Genetic variation and population structure in Big Brown Bats (Eptesicus fuscus): Is female dispersal important? Journal of Mammalogy 89:1411–1419. Wassenar, L.I., and K.A. Hobson. 2003. Comparative equilibration and online technique for determination of non-exchangeable hydrogen of keratins for use in animal-migration studies. Isotopes in Environmental and Health Studies 39:211–217. Weller, T.J., P.M Cryan, and T.J. O’Shea. 2009. Broadening the focus of bat conservation and research in the USA for the 21st century. Endangered Species Research 8:129–145. Northeastern Naturalist Vol. 26, No. 1 B.E.Sturgis, A.A. Aispuro, and K. Vulinec 2019 213 Appendix A. Locations and latitude and longitude of bat-trapping sites on Delmarva. Location Latitude and Longitude Structure, Bellevue, DE 39°46'42.0024''N, 75°30'20.7612''W Industrial structure, Dover, DE 39°9'42.822''N, 75°32'2.7528''W Killens Pond State Park, Felton, DE 38°59'17.2104''N, 75°32'41.8128''W House, Georgetown, DE 38°41'26.5704''N, 75°23'1.4244''W White Clay Creek Preserve, London Britain, PA 39°44'16.7568''N, 75°46'0.9696''W Appendix B. The year, location, mean, standard deviation, and sample size of stable hydrogen-isotope signature (δ2H) of Big Brown Bat female individuals (n = 109) sampled and analyzed in 2010 and 2011 at various trapping locations in Delaware and Pennsylvania. Year Location Mean Standard deviation n 2010 Dover, DE -40.75 9.19 13 Killens Pond -48.23 7.86 12 Bellevue -54.41 7.27 19 White Clay Creek Preserve -50.34 10.44 11 Total -49.02 9.82 55 2011 Dover, DE -40.85 7.14 49 Georgetown DE -32.83 6.40 4 Bellevue -50.20 - 1 Total -40.43 7.40 54 Total Dover, DE -40.83 7.53 62 Georgetown DE -32.83 6.40 4 Killens Pond -48.23 7.86 12 Bellevue -54.20 7.14 20 White Clay Creek Preserve -50.34 10.44 11 Total -44.76 9.68 109