2011 NORTHEASTERN NATURALIST 18(3):253–264 Using Stable Carbon Isotopes to Distinguish Wild from Captive Wolves Roland Kays1,* and Robert S. Feranec1 Abstract - Morphological and genetic techniques for distinguishing captive vs. wild stock are often insufficient. We found differences in carbon isotope values from Canis latrans (Coyote) and Canis lupus (Wolf) eating wild vs. domestic diets. Wild canids in the Northeast have lower δ13C values because they eat prey that mainly feed on C3 plants. However, canids eating typical domestic diets have more positive δ13C values (≈+6‰) because of the Zea mays (Corn; a C4 plant) fed to domestic stock and used in dog foods. We applied this technique to hair and bone samples from eight Wolves in the northeastern USA, where no natural Wolf populations are known. Three Wolves had strongly negative δ13C values, typical of a wild-food diet, while the other five Wolves had more positive values typical of captive animals. As expected, we found no significant difference in δ15N isotope values between captive and wild animals. This new evidence suggests that, while some Wolves are escaping from captivity, at least three animals have apparently dispersed into the area. This finding adds new urgency to the preparation of conservation plans for the potential natural recovery of this endangered species in the region. Introduction Evaluating the status of rare species in the wild is complicated when they, or a closely related species, are abundant in captivity. This confusion is caused after captive individuals escape or are released into the wild and then mistaken for a local population. For example, the occasional records of Puma concolor L. (Cougar) in the eastern USA, outside of their established range, has sparked controversy between those believing they represent a relict native population and those suggesting they are merely a handful of escaped pets (Cardoza and Langlois 2002, Kurta et al. 2007, Swanson and Rusz 2006). This type of problem is often more than an isolated escaped animal, for example, when hunting organizations captive-rear and release thousands of game birds and thereby complicate the conservation of native wild stock (Brennan 1999). In the northeast United States, Canis lupus L. (Wolf) is widely considered extirpated, and is listed as an endangered species by the Federal and many state governments (Coleman 2004). However, in the last 25 years, hunters targeting C. latrans Say (Coyote) have occasionally killed Wolves in the region (USFWS 1993, 1997, 2002, 2004b). Determining the provenance of these Wolves is important for understanding their ecological significance, as well as in conservation and management policy. If established in the wild, these individuals signal that Wolves are capable of naturally recolonizing the northeastern USA from surviving populations such as those present in eastern Canada. This result would urge additional study to determine if the Northeast already supports resident, cryptic, Wolf 1New York State Museum, Albany, NY 12230. *Corresponding author - rkays@mail. nysed.gov. 254 Northeastern Naturalist Vol. 18, No. 3 populations. Confirmation of natural Wolf populations would further encourage additional measures to protect this endangered species and plan for their recovery. On the other hand, if these animals are all escaped pets, the natural recovery of Wolves to the region would not appear as imminent, and planning for their recovery would then be a lower priority than for other endangered species. Traditionally, classifying Wolves as pets relies on physical signs of an animal being penned up. These signs include overgrown claws, being overweight, and/or having tarter buildup on the teeth. However, these characters are not perfect since some captive situations and diets may not affect claws or cause tarter build up, while some wild animals may naturally show different levels of these conditions (USFWS 2004a). Isotopic analysis offers an additional tool for determining the origin of a specimen because it gives an indication of animal diet. Herbivores acquire an isotopic value from the plants they eat, and this value is reflected up the food chain as predators consume herbivores (DeNiro and Epstein 1978a, Koch 1998, Lee-Thorp et al. 2000, Tieszen et al. 1979, Vogel 1978). This technique has been used to document the evolution of domestication by comparing isotopic values of archeological specimens (Barton et al. 2009, Hu et al. 2009, Minagawa et al. 2005, Noe-Nygaard et al. 2005). We suggest that, by establishing isotopic differences between domestic and wild foods from samples of known origin, this tool could also be applied to modern conservation questions. In particular, we suggest that the isotopic values of hair and bone samples could help determine if an animal had spent substantial time in captivity. Isotopic values in mammalian tissues are a reflection of an individual's diet averaged over the metabolically active time for a specific tissue (Ambrose 1991; DeNiro and Epstein 1978b, 1981; Sealy et al. 1987, 1995). Accordingly, the isotopes in hair represent the average diet of an individual since its last molt, while bone collagen typically represents a longer-term average, possibly over 20 years (Stenhouse and Baxter 1979, Wild et al. 2000), which would typically be the full life of a Wolf (Mech 1974). In this study, we focused on addressing whether the analysis of stable carbon and nitrogen isotope ratios can be used as an additional test to determine whether Wolves from northeastern USA were captive (e.g., pets) or likely from a wild stock. Our goal was to identify discrete differences in diet between captive and wild canids, not necessarily the more detailed composition of the specific items in their diet. We focused on carbon and nitrogen stable isotopes because we suspected that they should be different between typical wild animal and captive animal diets. Carbon, in particular, should show dramatic differences because native foods in the northeastern USA are primarily C3 plants (with more negative C-isotope values), while typical captive diets are based on C4 agricultural foods, primarily Zea mays L. (Corn), and generally have more positive C-isotope values (Cormie and Schwartz 1994; Keelan and Keelan 2009; Sage et al. 1999a, b; Wolfe 2008). Nitrogen stable isotopes are influenced by trophic position and conditions of the local habitat (e.g., climate and vegetation type), as well as whether commercial fertilizer has been used to grow the food (Ambrose 1991, DeNiro and Epstein 1981, Fox-Dobbs et al. 2007, Heaton 1986, Minagawa and Wada 1984, Roth and Hobson 2000, Sealy et al. 1987). In addition to testing this 2011 R. Kays and R.S. Feranec 255 method with samples of known origin, we also tested a series of potentially recolonizing Wolves from the northeast USA, and herein discuss the implications of our results for the conservation of Wolves in the region. Methods To test the accuracy of using stable carbon and nitrogen isotope values to distinguish captive from wild animals, we analyzed the isotopic value of bone collagen of 24 wild canids from natural areas in New York (14 Coyotes), New Hampshire (5 Coyotes), and Ontario (5 Wolves), as well as 6 captive canids from New Hampshire (Silver and Silver 1969) whose remains are now archived at the Museum of Comparative Zoology. The captive animals were fed a scrap beef and kibbled dog food diet typical for captive Coyotes and Wolves. Because urban canids often eat different diets (Fedriani et al. 2001), we also sampled 8 wild canids from urban and suburban Massachusetts. To quantify the isotopic values of foods for northeastern canids, we analyzed three species of wild fruit (many canids eat fruit in the fall) as well as the bone collagen isotope values of 5 species (30 total samples) of medium and large mammals that typically dominate their wild diet (Hamilton 1974, Potvin et al. 1988, Sears et al. 2003). As representatives of domestic foods, we analyzed a variety of store-bought meat and commercial dog foods typical of captive canid diet (Silver and Silver 1969). The species and locations for all samples are detailed in Supplemental Appendix 1 (available online at https://www.eaglehill.us/NENAonline/ suppl-files/n18-3-974-Kays-s1, and, for BioOne subscribers, at http:// dx.doi.org/10.1656/N974.s1). We sampled the bone collagen and/or hair samples of 8 Wolves shot in the northeastern USA between 1984 and 2008 (Table 1). Each of these animals was substantially larger than eastern Coyotes (average = 12–18 kg [Kays and Wilson 2009], max = 23 kg [R. Kays, unpubl. data]) and classified as a Wolf (C. lupus) or possible Wolf hybrid by state and/or federal wildlife officials. In two cases (NY84, NY05; Table 1), physical conditions suggested the animal had been in captivity, but no evidence of a captive past was noted for the others (USFWS 1993, 1997, 2002, 2004b). In one case (ME93), the hunters who shot the Wolf were prosecuted for taking an endangered species (USFWS 1993). To obtain bone collagen stable isotope values, we followed the general methodology of Sealy et al. (1987). Briefly, pieces of bone were placed into 0.5 N hydrochloric acid at room temperature to remove the mineral portion of the bone, generally 24 to 48 hours. Samples were rinsed with distilled water, then decanted once the mineral portion of the bone was fully dissolved. To remove any lipids, these samples were placed in a mixture of chloroform, methanol, and water (ratio of 2:1:0.8 by volume) following Bligh and Dyer (1959). Once the lipids were removed, the samples were washed and freeze-dried. Hair samples were first mechanically cleaned and washed with distilled water. To remove lipids, the chloroform, methanol, water mixture was used as done for the collagen samples. Hair samples were then air dried. Both collagen and hair samples were then loaded into tin cups and analyzed using a Carlo Erba elemental analyzer attached to a Micromass Europa Mass Spectrometer at the Center for Stable Isotope Biogeochemistry at the University of California, Berkeley, CA. Samples were corrected to NIST 1577b (bovine liver). 256 Northeastern Naturalist Vol. 18, No. 3 The results of the isotopic analyses are expressed in standard δ-notation: X = [(Rsample/Rstandard) - 1] × 1000, where X is the δ13C or δ15N value, and R = 13C/12C or 15N/14N, respectively. In this study, δ13C values are reported relative to the V-PDB standard, while δ15N values are reported relative to atmospheric N2. The average difference for replicate analyses was 0.2‰ for carbon, and 0.4‰ for nitrogen. Because there is a discrimination of isotope values as food is consumed, we adjusted the δ13C and δ15N values to more directly compare the results of the Wolves in our study to their food sources. To make the correction, we converted isotope values using isotopic discriminations for both collagen and hair. Collagen has been shown to be +5.0‰ enriched in δ13C from food, while the δ15N value was +3.0‰ enriched (Minegawa and Wada 1984, Schoeninger and Deniro 1984). For hair samples, we followed Roth and Hobson (2000), who showed an enrichment in canid δ13C values of +2.6‰ from consumed food, while δ15N was +3.2‰. We compared the isotope values for groups of specimens using Tukey’s HSD test. Results We observed statistically significant differences between the δ13C values of captive and wild canids (P < 0.0001) and between domestic and wild foods (P less than 0.0001). No statistical differences were observed between the δ13C values of captive canids and domestic foods (P = 0.094), or between wild canids and wild foods (P = 0.115). More important than statistical differences, however, is the lack of overlap in the δ13C values between known wild and known captive canids (Fig. 1A). As expected, wild canids and wild food had more negative carbon isotope values, reflecting the incorporation of the native C3 plant signal. In comparison, the captive canids and domestic foods had more positive carbon isotope values (Table 1), reflecting the inclusion of C4 plants (primarily Corn) in the commonly available prepared food (e.g., pet foods) or in the feed for domestic livestock. A sample of suburban and coastal canids from MA (Table 1) showed δ13C values that were statistically similar to the captive canids (P = 0.327) and statistically different from the wild canid group (P < 0.0001). When comparing nitrogen isotope values, each group was statistically similar to each other, except for wild foods, which were statistically different from all Table 1. Stable carbon and nitrogen isotope values for captive and wild canids and their food. Isotope values reported in permil (‰). Samples Mean δ13C δ13C range Mean δ15N δ15N range Category analyzed (n) (SD) (min, max) (SD) (min, max) Food Domestic 14 -18.2 (3.4) -25.6, -10.7 5.1 (1.2) 2.5, 7.2 Wild 42 -27.8 (1.8) -33.3, -24.0 1.0 (1.7) -3.5, 4.3 Canids Captive 6 -20.6 (1.1) -21.6, -18.9 6.5 (1.1) 5.4, 8.5 Urban 8 -22.5 (1.5) -24.9, -20.9 6.1 (1.2) 4.5, 7.4 Wild 24 -26.7 (1.1) -30.2, -24.6 4.7 (1.3) 0.9, 7.0 2011 R. Kays and R.S. Feranec 257 other groups. Domestic foods and wild foods were statistically different (P less than 0.0001), while there was no difference between captive canids and wild canids (P < 0.0534). The captive canids and domestic foods displayed δ15N values more positive than wild canids and wild foods (Table 1, Fig. 2B). However, there is overlap among samples in δ15N values from all analyzed groups. To isotopically identify Wolves with unknown provenance, we compared carbon isotope values obtained from their hair or bone collagen to the known groups (i.e., captive or wild). We defined the two groups as any values within Figure 1. (A) Stable carbon isotope values for canids of known provenance and their food. (B) Stable carbon isotope values for canids of unknown provenance. 258 Northeastern Naturalist Vol. 18, No. 3 two standard deviations of the mean, and values greater than two standard deviations outside both groups were identified as ambiguous. Among the Wolves of unknown provenance, five (ME 93, ME 96, NY 08, NY 05, NY 84) had more positive δ13C values, implying a history of eating domestic foods (Fig. 1B). Therefore, we categorize these as having been held in captivity for a substantial portion of their life. Interestingly, while the bone collagen value (-18.4‰) from Wolf ME 96 shows that the diet for most of its life was likely developed in captivity, the hair sample had a much more negative δ13C value (-24.8‰), suggesting it had a diet of wild food at the time of its last molt. Three other Wolves (NY 01, Figure 2. (A) Stable nitrogen isotope values for canids of known provenance and their food. (B) Stable nitrogen isotope values for canids of unknown provenance. 2011 R. Kays and R.S. Feranec 259 VT 98, VT 06) had δ13C values of bone collagen indicative of a wild diet (i.e., predominantly C3), and are classified as being wild canids. Two of the three wolves (NY 01, VT 98) that we classified as being wild canids had hair δ13C values that fell between the groupings, and these individuals would be classified as ambiguous if we were to use hair alone. However, for NY 01, the average hair δ13C value was -24.5‰, which falls into the wild canid category. For VT 98, while the hair value is considered ambiguous, two collagen samples clearly fall within the wild canid category. For this canid, we suggest that the more positive δ13C values in the hair sample may result from having a slightly more mixed history of eating domestic and wild foods since its last molt. Because there was a lack of significant differences observed among canid groups in δ15N values (Fig 2A), it was not possible to distinguish which Wolves of unknown origin were captive or wild based on the observed nitrogen isotope values (Fig 2B). Discussion There are limited options for determining if animals have been held in captivity. Physical signs of captivity that may result from animals walking on concrete, or from limited exercise or food choices (Duman 2001), may not be evident depending on the exact conditions of its captivity, and may also appear in wild populations (USFWS 2004a). By examining hair (i.e., a record of recent dietary history) and bone collagen (i.e., a record of diet over many years), we found that the analysis of carbon stable isotopes can be used as an additional tool to evaluate the history of an animal. We show that this method can help distinguish Wolves of unknown provenance as being either captive or of wild origin. In our sample of eight unknowns, isotope data imply that five individuals were primarily raised in captivity, and three others were of wild origin. Interestingly, our data suggests that one of the Wolves was raised in captivity (ME 96) but later survived on wild foods at least during its last molt. This finding matches observations of large Wolf footprints in the area approximately one year before the animal was shot (W. Jakubas, Maine Department of Inland Fisheries and Wildlife, Bangor, ME, pers. comm.). Thus, our results support the idea of using stable isotope analysis, particularly of carbon isotopes, as an additional tool to distinguish wild animals from those with a captive history. Limitations of the method It is important to understand the reasons carbon isotopes are different in our samples of wild and captive canids in order to recognize the situations in which this test will work, and those in which it will not. In this study, we are able to distinguish wild from domestic Wolves because Corn is the only substantial food source of C4 plant material in this region (Keelan and Keelan 2009; Sage et al. 1999a, b). Where native C4 plants are more abundant, such as more arid biomes, this technique may not be as discriminating. Further, in some urban and suburban areas, even where C3 plants are naturally dominant, wild animals may incorporate substantial amounts of typically domestic food in their diet, and thus could have a more positive carbon isotope value (Fedriani et al. 2001). For example, our sample of coastal and suburban canids from MA displayed higher carbon isotope 260 Northeastern Naturalist Vol. 18, No. 3 values more typical of captive animals. This result does not affect the test of potential immigrant Wolves since they were not living in urban areas, but does point out when this type of analysis can be problematic. Although it is possible that native wild herbivores consuming Corn in the region could acquire more positive carbon isotope values, all available data suggest this is not happening at levels significant enough to affect our test. None of the Odocoileus virginianus Zimmermann (White-tailed Deer) we tested show any isotopic signature of eating substantial amounts of Corn. Although it is apparent that deer eat Corn, these isotopic data, as well as data from other more comprehensive studies (Cormie and Schwartz 1994, Maynard et al. 1935), have shown that Corn is, at most, a seasonal resource for deer in the northeast USA. In the most wide-ranging study of deer isotope values across North America, Cormie and Schwartz (1994) showed that deer typically eat a diet of native plants and very rarely have isotopic values typical of having consumed agricultural Corn. Finally, even if one or two odd Corn-fed deer were occasionally consumed by a wild Wolf in the northeast, its average isotope value would not change substantially since Wolf packs typically kill and eat ca. 100 deer each year, not to mention other prey species (Fuller 1986, Kolenosky 1972). Wild Wolves and Coyotes do occasionally hunt livestock, which could also increase their carbon isotope values if livestock was a substantial portion of their diet. However, such a diet is not typical for canids, and even in areas with few native prey and abundant livestock, most (85%) Wolf diet consists of wild animals (Chavez and Gese 2005). Furthermore, individual predators that target domestic animals are typically killed by ranchers before they can consume enough livestock to alter their carbon isotope values (Mech 1998, Musiani et al. 2003). Alternatively, Wolves kept in captivity could have more negative δ13C values if they were fed wild foods all their lives. We did not test this possibility, but note that it would be quite a challenge to provide the 635–1011 kg of wild meat needed annually by a single Wolf (Glowacinski and Profus 1997). It would also be possible to obtain more negative carbon isotope values from certain dog foods, since, from our data, a very limited number of dog foods do have depleted δ13C values. However, we feel that this scenario is unlikely due to the amount of food needed by a Wolf; the less expensive dry dog foods, which generally have corn as a major ingredient, would be more typical for a captive Wolf. Thus, our results show that the analysis of carbon isotopes from mammal tissues can be used to distinguish typical captive diets from typical wild diets in C3 ecosystems. This technique is not foolproof; it could be confounded by wild animals specializing on corn-fed prey, or captive animals fed only wild prey. However, these are exceptional situations that our data show are rare. This technique could be useful for clarifying other muddled conservation situations, such as the status of Cougars in the northeastern USA (Cardoza and Langlois 2002, Swanson and Rusz 2006) and the effect of captive-reared game birds on native populations (Brennan 1999). Implications for regional Wolf conservation Wolves were extirpated from the northeastern USA by the late 1800s (Coleman 2004), but survive to the north in Ontario and Québec, and have recently 2011 R. Kays and R.S. Feranec 261 been expanding to the west in the Great Lakes (Cook et al. 1999, Potovin et al. 2005). Our results suggest that at least three wild Wolves were living in Vermont and New York in the last 10 years. Given these relatively few Wolf records scattered over many years and states, we do not think these represent an established breeding population, but are probably natural migrants. Another five recent eastern Wolves had isotope values typical of animals held in captivity, showing that not all Wolf records represent natural migrants. There is substantial suitable Wolf habitat in northern New York and New England that could support a viable population of Wolves (Harrison and Chapin 1998, Mladenoff and Sickley 1998). The distance between surviving Wolf populations in Canada and this suitable habitat in northeastern states is relatively small when compared with potential Wolf dispersal distances (70–230 km; Harrison and Chapin 1998). Our data suggest that three animals may have successfully dispersed into the area. The recent recovery of Wolves throughout much of the Great Lakes region (USFWS 2007), and increased protection of Wolves in Ontario (Patterson and Murray 2008), make it likely that even more Wolves will migrate into the northeastern USA in the near future. It is important to note that there is essentially no conservation plan for northeastern Wolves, and thus no guidelines for promoting their recovery. The most relevant plan is over 18 years old and actually focuses on Great Lakes Wolves (Eastern Timber Wolf Recovery Team 1992). Our data suggest that Wolves have been naturally dispersing into the northeastern USA. If these were to become established, this new top predator would probably reduce Coyote populations in the region, and change the behavior and densities of prey (Berger et al. 2001, Ripple and Beschta 2004). Based on this information, we suggest that state and federal wildlife managers should recognize that Wolves are likely dispersing into the area, and that there is a need for a revised conservation plan. Conclusions Carbon isotope values showed statistically significant differences among captive and wild canids and their food in northeastern USA. Importantly, the δ13C values between known captive canids and known wild canids did not overlap, showing that this technique can help distinguish whether individual Wolves occurring in northeastern USA were wild immigrants or escaped pets. Analysis of nitrogen isotope values did not aid in distinguishing between captive and wild canids. Of the eight Wolf specimens that had unknown provenance, five individuals displayed more positive δ13C values typical of having spent much of their lives in captivity while three others showed more negative δ13C values typical of a wild diet. These data suggest that at least three Wolves naturally dispersed into the northeastern USA. This recent Wolf dispersal, combined with earlier work showing that suitable Wolf habitat exists, suggest a pressing need for the development of Wolf conservation plans for the region. Acknowledgments Thanks to all the state wildlife officials who helped us obtain and sample specimens including Will Staats, Kim Royar, Mike Amaral, Gordon Batcheller, and Walter Jakubas. 262 Northeastern Naturalist Vol. 18, No. 3 Thanks to the trappers who provided us specimens from New York, and the Museum staff and volunteers who prepared them: Joe Bopp, Daniel Bogan, Abigail Curtis, and Paul Gallery. 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