2013 NORTHEASTERN NATURALIST 20(3):383–396 Genetic Confirmation of Cougars (Puma concolor) in Eastern Canada Le Duing Lang1, Nathalie Tessier1, Marc Gauthier2, Renee Wissink3, Hélène Jolicoeur4, and François-Joseph Lapointe1,* Abstract - This paper presents the results of a long-term study to detect the presence of Puma concolor (Cougar) in eastern Canada. We installed 38 scratching posts to attract wild Cougars and collect hair samples in several national and provincial parks in Québec, New Brunswick, and Nova Scotia. A set of semi-nested primers was used to discriminate Cougar samples from other mammalian species based on variation in the16S rRNA gene of the mitochondrial DNA. Our analyses performed on 476 hair samples revealed 19 positive identifications of Cougars in Québec and New Brunswick. Sequencing further showed that some specimens were from South America, whereas others had a North American origin. We discuss the implications of these results for the conservation of Cougars in eastern Canada. Introduction The last Puma concolor couguar Kerr (Eastern Cougar), which once flourished in North America (Hall 1981), was reportedly killed near the Maine/ Québec border in 1938 (Wright 1961). Yet, in the last three decades, sightings, tracks, and mortalities of wild Cougars have increased steadily (van Dyke and Brocke 1987). From 1970 to 1993, 180 and 315 reports have been filed in New Brunswick and Nova Scotia, respectively (Stocek 1995). In Québec, 1061 sightings were reported from 1955 to 2005 (Jolicoeur et al. 2006, Tardif 1997), whereas 497 pieces of evidence were collated in Ontario from 1991 to 2010 (Rosatte 2011). Since 1983, more than 2000 observations have been gathered in the eastern US (Bolgiano et al. 2000, Cardoza and Langlois 2002, Lutz and Lutz 1996). Confirmed occurrences include tracks discovered in Maine, Virginia, West Virginia, Ontario, and New Brunswick (Bolgiano et al. 2000, Cumberland and Dempsey 1994, Rosatte 2011), DNA-based identification of scat or hair samples from New Brunswick, Ontario, Michigan, and Louisiana (Bertrand et al. 2006, Harris 2007, Leberg et al. 2004, Rosatte 2011, Swanson and Rusz 2006), and specimens killed in West Virginia and Illinois (Bolgiano et al. 2000, Heist et al. 2001). In Québec, three Cougar mortalities have also been documented: a male Cougar was shot in May 1992, a second Cougar was hit by a truck in April 1996 and subsequently mounted by a taxidermist, and a third Cougar was hit by a car in September 2002. 1Département de Sciences Biologiques, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, QC, H3C 3J7, Canada. 2Genivar, Inc., 171 Rue Léger, Sherbrooke, QC, J1L 1M2, Canada. 3Fundy National Park, PO Box 1001, Alma, NB, E4H 1B4, Canada. 4Direction du Développement de la Faune, Ministère des Ressources naturelles et de la Faune, 930 Chemin Sainte-Foy, 3e étage, Québec, QC, G1S 2L4, Canada. *Corresponding author - francois-joseph.lapointe@umontreal.ca. L.D. Lang, N. Tessier, M. Gauthier, R. Wissink, H. Jolicoeur, and F-J. Lapointe 2013 Northeastern Naturalist Vol. 20, No. 3 384 Controversies surrounding the Cougar sighting reports in eastern North America have been conflated by a number of conflicting issues related to the identification, taxonomy, and conservation status of Cougars. Several methods have been developed to detect the presence of Cougars in the wild, including the analysis of tracks (Beier and Cunningham 1996, Lewinson et al. 2001, Smallwood and Fitzhugh 1993), the chromatography of bile acid from scats (Fernandez et al. 1997), the macroscopic and microscopic analysis of hair (Harrison 2002), and the use of DNA-based identification (Mills et al. 2000). Based on these techniques, some studies authenticated the species’ presence in Michigan, Ontario, and New Brunswick (Bertrand et al. 2006, Bolgiano et al. 2000, Cumberland and Dempsey 1994, Swanson and Rusz 2006, Rosatte 2011, Wright 1953), some could not confirm it (Belant et al. 2006, Brown 2005, Downing 1984, Gerson 1988), while others suggested that it was presumably extinct (Culver et al. 2000, Parker 1998, Scott 1998, Young and Goldman 1946). Taxonomic debate has further complicated interpretations of the status of the Eastern Cougar. Young and Goldman (1946) described 15 subspecies of Cougar in North and South America, respectively, with two subspecies in eastern North America: P. c. couguar (Eastern Cougar) and P. c. coryi (Florida Panther). Molecular methods have informed this taxonomy more recently. Numerous microsatellite loci have been designed and employed to assess the genetic structure of Cougar populations (Culver et al. 2001, Holbrook et al. 2012, Kurushima et al. 2006, Rodzen et al. 2007), and mitochondrial DNA (mtDNA) has been used to determine the genomic ancestry and clarify the taxonomy of the Cougar. Namely, the analysis of three mitochondrial genes revealed that it is impossible to distinguish the Eastern Cougar from other North American Cougars (Culver et al. 2000). In other words, all North American Cougars are the same subspecies, Puma concolor couguar. Here, we follow this recommendation and consider North American Cougars as a single subspecies (see also Wilson and Reeder 2005), which can be differentiated from Central and South American subspecies on the basis of mtDNA variation. The molecular classification, however, is not agreed upon by all biologists and has not been adopted by management agencies in the US and Canada. The taxonomy of Puma concolor has important implications for the conservation and management of Cougars in North America. Since 1978, the US Fish and Wildlife Service (USFWS), which maintains the classification of Young and Goldman (1946), had listed the Eastern Cougar as endangered, although it was not actively protected (Cardoza and Langlois 2002). After a five-year review, the subspecies P. c. couguar (Eastern Cougar, following Young and Goldman 1946) was, however, delisted in the US, based upon extinction (McCollough 2011). In Canada, the Committee on the Status of Endangered Wildlife (COSEWIC) initially listed P. c. couguar as endangered (Van Zyll de Jong and van Ingen 1978). In 1998, COSEWIC reassigned the Eastern Cougar to the data deficient or indeterminate category (Scott 1998). Different provinces and states have granted conservation status to the Cougar, but Québec is the only jurisdiction in eastern 385 L.D. Lang, N. Tessier, M. Gauthier, R. Wissink, H. Jolicoeur, and F-J. Lapointe 2013 Northeastern Naturalist Vol. 20, No. 3 North America that still considers the Eastern Cougar present on its territory (Jolicoeur et al. 2006). In this paper, we present a broad-scale application of molecular identification techniques to detect the presence of Cougars in eastern Canada. Scent-baited scratching posts were installed to collect hair samples in the southern part of Québec and two localities in New Brunswick and Nova Scotia. Species-specific mtDNA primers were designed to quickly screen a large number of samples and discriminate Cougars from other mammalian species. Each positive sample was further sequenced using one of three mtDNA regions to determine its corresponding geographical ancestry (Central and South America or North America). Based on our results, we discuss the conservation status and provide recommendations on the management of Cougars in eastern Canada. Methods Sample collection Since 2001, a large number of scratching posts (n = 38) have been installed at various sites in the provinces of Québec, New Brunswick, and Nova Scotia. The selected sites were located in national parks (Cape Breton Highlands, Forillon, Frontenac, Fundy, Mauricie), provincial parks (Gaspésie, Mont-Tremblant), and some other localities (Estrie, Montérégie) where Cougar sightings have been reported (Fig. 1). In order to maximize pheromone dispersion, local topography and predominant wind direction were considered, and the posts were placed in remote forested areas to avoid human disturbance. Forest composition varied among areas and was mainly composed of Acer saccharum Marsh. (Sugar Maple), Carya cordiformis (Wang.) K. Koch (Bitternut Hickory), Tilia americana L. (American Basswood), Betula alleghaniensis Britt. (Yellow Birch), Betula espapyrifera Marsh. (Paper Birch), Abies balsamea (L.) P. Mill. (Balsam Fir), or Picea rubens Sarg. (Red Spruce). Each scratching post consisted of a 2-m PVC pipe covered with a plastic boot mat and wrapped in barbed wire to increase the probability of sampling hairs. Several field teams working in the different areas visited the posts once a month to apply a scent lure based on Cougar urine and to collect hair samples. The samples were stored at room temperature in paper envelopes or plastic bags and sent to the lab for genetic identification. From 2001 to 2012, a total of 476 hair samples were collected from the scratching posts. Three known Cougar samples from Québec were also obtained and analyzed to confirm their identification and assess the geographical origin of the specimens: (1) a muscle sample from a specimen that was shot in 1992, (2) a skin sample from a stuffed Cougar that was hit by a truck in 1996, and (3) hair and dried blood samples collected from the bumper of the car that hit a Cougar in 2002. Molecular identification of field samples Hair samples collected from the same post and that had similar macroscopic characteristics were pooled to increase the quantity of extracted DNA (Alpers et L.D. Lang, N. Tessier, M. Gauthier, R. Wissink, H. Jolicoeur, and F-J. Lapointe 2013 Northeastern Naturalist Vol. 20, No. 3 386 al. 2003, Frantz et al. 2004, Goossens et al. 1998, Roon et al. 2003). These hairs were cut into smaller pieces (0.5–1 cm) and soaked in 150 μL of a lysis buffer with 40 mM Tris-HCl pH 9.0, 50 mM KCl, 0.5% Tween 20, and proteinase K (0.1 mg/mL concentration) at 37 °C overnight followed by 15 min at 95 °C (Olsen et al. 1996). The DNA extract was then centrifuged for 12 min at 14,000 rpm. The supernatant was transferred to a new tube, centrifuged at 14,000 rpm for another 12 min, and subsequently used for DNA amplification. Due to its low divergence rate, the 16S rRNA region of mtDNA was selected to design species-specific primers for Cougar identification (Lopez et al. 1997). Sequences from 11 species from five mammalian families (one Bovidae, two Cervidae, two Ursidae, one Canidae, and five Felidae) were retrieved from GenBank and aligned using ClustalX (Thompson et al. 1997) to identify divergent sites. Figure 1. Sampling sites and positive identifications of Cougars in eastern Canada. Black circles represent the scratching posts installed at different localities, and grey circles represent scratching posts with positively identified Cougar samples. The letters refer to the labels in Table 2. QC = Québec, NB = New Brunswick, NS = Nova Scotia. 387 L.D. Lang, N. Tessier, M. Gauthier, R. Wissink, H. Jolicoeur, and F-J. Lapointe 2013 Northeastern Naturalist Vol. 20, No. 3 The sizes of the targeted regions were minimized to ensure efficiency and repeatability (Frantz et al. 2004, Roon et al. 2003). A first primer set (16S-Universal-F: GAG GG(C/T) TT(A/T) ACT GTC TCT TAC and 16S-Universal-R: GGG TAA CTT GTT CCG TTG ATC) was designed to amplify a fragment of about 300 bp in all mammals, with slight differences across species. These primers were thus used as a positive control indicating PCR success. A species-specific primer (16S-Cougar-F: AGA GAC CCA TTA ATT TC) was also designed to amplify an additional internal 138-bp fragment only for the Cougar, when used in combination with the previous reverse primer (16S-Universal-R). Consequently, the analysis of a Cougar sample should produce two bands in a multiplex PCR reaction using our semi-nested sets of primers. Other mammals should only exhibit the universal fragment of 300 bp. Multiplex PCR was carried out in 50-μL volumes containing 10 μL of DNA extract, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.1% Triton X-100, 2.5 mM of MgCl2, 0.3 μM of each of the three primers (16S-Universal-F, 16S-Cougar- F, and 16S-Universal-R), 250 μM dNTP, and 1U of Ampli Taq Gold DNA polymerase (Roche Applied Science, Indianapolis, IN). Amplifications were performed in a GeneAmp® PCR System 9700 thermal cycler (Applied Biosystem, Foster City, CA) with an initial incubation at 94 °C for 2 min, followed by a PCR profile of 40 cycles of 94 °C for 60 sec, 53 °C for 90 sec, and 72 °C for 90 sec, with a final extension step at 72 °C for 10 min. PCR products were run in a 2% agarose gel using Vistra Green Nucleic Acid Stain (Amersham Biosciences, Buckinghamshire, UK) to visualize the DNA fragments. Extractions and amplifications were conducted in separate areas of the lab (Taberlet et al. 1999), and negative controls (blank PCR) were always used to avoid contamination (Kwok and Higuchi 1989). The performance of the universal and species-specific primers was assessed using 57 hair samples of known identity representing 22 different species of mammals, including four Cougar samples collected from zoos or museums (Table 1). As expected, all samples exhibited the universal fragment, but the species-specific primers accurately amplified the second fragment in known Cougar samples. More importantly, this fragment was only observed in Cougars. Geographical ancestry Each sample positively identified as a Cougar with the species-specific primers was further analyzed to determine the geographical origin of the corresponding specimen, based on a subset of the haplotypes identified in Culver et al. (2000). To do so, three genes were successively employed until we were able to obtain a long enough sequence for assigning the corresponding mtDNA region to a unique haplotype. We used the 16S rRNA, NADH-dehydrogenase-5 (ND5) and ATPase-8 (ATP8) primers defined in Johnson et al. (1998) to amplify a 376-bp, a 315-bp, and a 162-bp region, respectively. These fragments were then sequenced in both directions using Big Dye® Terminator v1.1 (Applied Biosystems) on an ABI 3100 automated sequencer (Applied Biosystems). The sequences were visualized with 4Peaks (version 1.6), and identification was L.D. Lang, N. Tessier, M. Gauthier, R. Wissink, H. Jolicoeur, and F-J. Lapointe 2013 Northeastern Naturalist Vol. 20, No. 3 388 further confirmed with respect to the 14 haplotypes in Culver et al. (2000). We were only interested in assessing the geographical ancestry of Cougars to discriminate South and Central American Cougars (haplotypes A–L in Culver et al. 2000) from North American Cougars (haplotypes M–N in Culver et al. 2000). This determination was made by identifying single mutations in the mtDNA Table 1. Species used to assess the performance of the universal primers and to validate the species-specific primer designed for the Cougar. MRNF = Ministère des Ressources Naturelles et de la Faune du Québec Family/species Common name n Sample Source Hominidae Homo sapiens L. Human 4 Hair Lab members Cervidae Odocoileus virginianus Zimmermann White-tailed Deer 4 Hair MRNF, QC Muridae Ondatra zibethicus (L.) Muskrat 1 Hair MRNF, QC Peromyscus leucopus (Rafinesque) White-footed Mouse 4 Muscle MRNF, QC Peromyscus maniculatus (Wagner) Deer Mouse 4 Muscle MRNF, QC Mustelidae Gulo gulo (L.) Wolverine 1 Muscle U. of Alberta, AB Lutra canadensis (Schreber) River Otter 1 Hair MRNF, QC Martes americana (Turton) Marten 3 Hair MRNF, QC Mustela erminea (L.) Shorttail Weasel 1 Hair MRNF, QC Mustela frenata Lichtenstein Longtail Weasel 1 Hair MRNF, QC Mustela vison (Schreber) Mink 1 Hair MRNF, QC 2 Muscle U. of Alberta, AB Procyonidae Procyon lotor (L.) Raccoon 1 Hair MRNF, QC 1 Muscle U. of Alberta, AB Ursidae Ursus americanus (Pallas) Black Bear 2 Hair MRNF, QC 2 Muscle U. of Alberta, AB Canidae Canis lupus familiaris L. Domestic Dog 2 Hair Lab members Canis latrans Say Coyote 2 Hair MRNF, QC Canis lupus L. Gray Wolf 2 Muscle U. of Alberta, AB Alopex lagopus (L.) Arctic Fox 1 Hair MRNF, QC Vulpes velox (Say) Swift Fox 2 Muscle U. of Alberta, AB Felidae Felis catus L. Domestic Cat 4 Hair Lab members Lynx canadensis Kerr Canada Lynx 1 Hair Granby Zoo, QC 1 Hair MRNF, QC 2 Muscle U. of Alberta, AB Lynx rufus (Schreber) Bobcat 1 Hair Granby Zoo, QC 1 Hair MRNF, QC 1 Muscle MRNF, QC Puma concolor (L.) Cougar 1 Hair Granby Zoo, QC 1 Hair Bioparc, QC 2 Muscle U. of Alberta, AB 389 L.D. Lang, N. Tessier, M. Gauthier, R. Wissink, H. Jolicoeur, and F-J. Lapointe 2013 Northeastern Naturalist Vol. 20, No. 3 genes at positions 3094 (16S), 8681 (ND5), or 12,751 (ATP8). We relied on two haplotypes to discriminate specimens from Central and South America (haplotype I) from specimens originating from North America (haplotype II). Results The 476 hair samples collected from scratching posts, as well as the three Cougar mortality samples were analyzed. Nineteen field samples were positively identified as Cougars, and the identity of the three known Cougar mortality samples was also confirmed. Of the remaining samples, 291 were identified as non-Cougars and 166 (35% of the total) did not successfully amplify because of low DNA quantity and quality. Some non-Cougar samples (n = 95) were further selected at random and sequenced to determine their taxonomic identity using a BLAST search in GenBank. These analyses indicated that Ursus americanus (American Black Bear, n = 38), Odocoileus virginianus (White-tailed Deer, n = 15), Canis lupus (Gray Wolf, n = 13), Canis latrans (Coyote, n = 12), Alces alces L. (Moose, n = 4), and Lynx canadensis (Canada Lynx, n = 2) among others, were also attracted by the scratching posts. The geographic origin of the field samples identified as Cougars was determined on the basis of the corresponding haplotypes (Table 2). Out of these 19 positive field samples, six were assigned to haplotype I (South and Central America) and ten to haplotype II (North America) using one of three mtDNA regions (16S, ND5, ATP8); the remaining three samples were not sequenced due to lack of material. Some of the samples collected at nearby localities at different times may have originated from the same individual (Fig. 1). Namely, four samples (b, c, e, g) with haplotype II were collected in Forillon National Park (QC) over five years (2004–2009). Likewise, the two specimens (n, o) collected in Frontenac National Park in 2009 and 2011 share the same haplotype II. On the other hand, two samples (p, q) collected in Fundy National Park (NB) from nearby scratching posts in 2003 were assigned to different haplotypes, thus certainly representing distinct specimens. Three other Cougar samples (k, l, m) with different haplotypes (I and II) were collected in the Montérégie (QC) from 2009 to 2011, likely from two different individuals. Sequence analysis was also performed in order to determine the origin of the three known Cougar mortality samples. Haplotype data indicate that the specimen killed in 1992 (t) is from South America, whereas the sample hit by a car in 1996 (u) is from North America. The sequence of the third Cougar killed in 2002 (v) was not conclusive and could not confirm its origin. Discussion The use of DNA in wildlife conservation and management has gained popularity in recent years (Gupta 2012, Linacre and Tobe 2011, Ogden 2010 ), not only to detect elusive animals (Verma et al. 2003), but also to track down poachers (Tobe et al. 2011). A wide range of molecular techniques are routinely applied L.D. Lang, N. Tessier, M. Gauthier, R. Wissink, H. Jolicoeur, and F-J. Lapointe 2013 Northeastern Naturalist Vol. 20, No. 3 390 Table 2. Details of the 22 samples identified as Cougars (Puma concolor) in eastern Canada. Haplotype I = South and Central America, haplotype II = North America, n/a: not sequenced. 16S = 16S rRNA region, ND5 = NADH-dehydrogenase-5 gene, ATP8 = ATPase-8 gene. QC = Québec, NB = New Brunswick. The last three specimens (t, u, and v) are mortalities. ***Sequ ence < 200 bp (not allowed in Genbank). Label Province Locality Date Sample type Haplotype Genbank# Region a QC Gaspésie, Montagne Blanche 10 Sept 2001 Hair sample II DQ493937 16S b QC Gaspésie, Forillon National Park 25 Aug 2004 Hair sample II EF028637 16S c QC Gaspésie, Forillon National Park 26 Nov 2004 Hair sample II KC407690 16S d QC Gaspésie, Forillon National Park 3 Jul 2007 Hair sample n/a e QC Gaspésie, Forillon National Park 9 Sept 2007 Hair sample II EU655706 16S f QC Gaspésie, Forillon National Park 23 May 2009 Hair sample n/a g QC Gaspésie, Forillon National Park 28 Aug 2009 Hair sample II KC407696 ND5 h QC Gaspésie, ZEC des Anses 12 Jun 2006 Hair sample I *** ATP8 i QC Estrie, Ruiter Valley 30 Jan 2002 Hair sample n/a j QC Estrie, Ruiter Valley 6 Mar 2002 Hair sample I EF028636 16S k QC Montérégie, Massif des monts Sutton 26 May 2009 Hair sample II KC407692 ND5 l QC Montérégie, Massif des monts Sutton 18 Jul 2010 Hair sample I KC407697 ND5 m QC Montérégie, Massif des monts Sutton 2 May 2011 Hair sample I KC407691 16S n QC Estrie, Frontenac National Park 17 Jun 2009 Hair sample II KC407695 ND5 o QC Estrie, Frontenac National Park 20 Aug 2010 Hair sample II KC407693 ND5 p NB Alma, Fundy National Park 22 July 2003 Hair sample I DQ493939 16S q NB Alma, Fundy National Park 22 Oct 2003 Hair sample II DQ493940 16S r QC Lac-Saint-Jean, ZEC Martin Valin 31 Oct 2002 Hair sample I DQ493938 16S s QC Bas-St-Laurent, ZEC Casault 25 Oct 2007 Hair sample II KC407694 ND5 t QC Abitibi, Saint-Lambert 27 May 1992 Muscle I DQ493935 16S u QC Estrie, East-Hereford Apr 1996 Tanned hair II DQ493936 16S v QC Québec, Réserve des Laurentides 15 Sept 2002 Hair and blood n/a 391 L.D. Lang, N. Tessier, M. Gauthier, R. Wissink, H. Jolicoeur, and F-J. Lapointe 2013 Northeastern Naturalist Vol. 20, No. 3 to identify specimens from scats or hair samples (Foran et al. 1997, Haag et al. 2009, Onorato et al. 2006, Sawaya et al. 2011, Swanson and Rusz 2006). In spite of evidence provided by molecular data, the debate is still raging about the presence of the Cougar in eastern Canada (see Lemelin 2009). On one side are those who criticize it, reject it, or ignore it altogether (Carney 2006, Kurta et al. 2007, Larivière 2012). On the other side stand those who endorse DNA evidence and accept the verdict of molecular identification techniques. Indeed, several independent studies (e.g., Bertrand et al. 2006, Harris 2007, Jung and Merchant 2005, Leberg et al. 2004, Swanson and Rusz 2006) have relied on genetics to confirm the presence of Cougars in different states (Michigan, Louisiana), provinces (Ontario, New Brunswick), and territories (Yukon). In this study, we designed a set of semi-nested primers to discriminate Cougars from other mammal species. With 19 positive samples collected on a broad-scale over an 11-year period, our results add to the evidence that Cougars are present in eastern Canada. According to Culver et al. (2000), it is impossible to distinguish the Eastern Cougar from the other North American subspecies on the basis of mtDNA variation. In this study, our approach enabled us to distinguish two haplotypes of Cougars— consistent with a North American or South American origin. Eleven samples identified in the present study shared the North American haplotype. These Cougars could have been migrants from the western or southern parts of the continent, namely South Dakota (Nero and Wrigley 1977) or Florida (Maehr et al. 2002, 2003). Our results also showed that seven Cougar samples shared the South American haplotype. This finding suggests that these were either exotic individuals that were released or escaped from captivity (see also Bertrand et al. 2006), or had a mixed ancestry. Yet, if the North American subspecies actually occurs in eastern Canada, there may be a possibility for outbreeding with exotic subspecies, as suggested by Scott (1998), and this poses an additional challenge for conservation. In light of the different hypotheses presented in McCollough (2011), it cannot be concluded whether our positive identifications are (1) remnant individuals of a persisting population of the Eastern Cougar, (2) escapees from captivity, or (3) dispersers from western or southern populations. Additional analyses should thus be completed using different markers (i.e., microsatellites) to determine the dispersal patterns (Ernest et al. 2000, McRae et al. 2005) and the genetic structure of Cougar populations (Anderson et al. 2004, Castilho et al. 2012, Ernest et al. 2003, Holbrook et al. 2012). Future work should also include molecular sexing of Cougar samples, as this might allow identification of a haplotype associated with dispersing males. Indeed, if the haplotype II individuals are predominantly dispersing males, whereas the majority of the females are of haplotype I, then outbreeding will be an even greater conservation challenge. Further work should also use improved DNA-extraction methods to increase PCR success of degraded hair samples collected in the field (e.g., Bekaert et al. 2012), as we found that molecular identification was not always successful with our method because many of the collected samples were mainly shed hairs, exempt of follicles (see Gagneux et al. 1997, Goossens et al. 1998, Higuchi et al. 1988) . L.D. Lang, N. Tessier, M. Gauthier, R. Wissink, H. Jolicoeur, and F-J. Lapointe 2013 Northeastern Naturalist Vol. 20, No. 3 392 Conservation biology requires not only scientific knowledge, it also relies on social, technical, economical, legal, and political considerations (Clark et al. 1996). The conservation and management of Cougars in eastern North America is a perfect example of such intricate problems. It not only suffers from the lack of information on its presence in the wild (Bolgiano 1995), but also from inappropriate assessment of biopolitical issues (Cardoza and Langlois 2002). Both the USFWS (McCollough 2011) and Canadian Wildlife Service (Scott 1998) have concluded there is no evidence that population(s) of the Eastern Cougar have persisted continuously in northern New England/eastern Canada—or anywhere else in the historic range of the Eastern Cougar. Nevertheless, Cougars are recolonizing the Midwest (LaRue et al. 2012) and the species is indisputably present in eastern Canada. Upon confirmation of its presence, education and conservation programs must be implemented, and recovery plans should be established. Different states and provinces already have management plans, but Québec still lacks one. Given that it is possible that a viable population of Cougars exists in eastern Canada (Brocke and Van Dyke 1985), we believe that COSEWIC should revisit the status of the species, currently listed in the data deficient category. All thirteen species of wild felids in the New World are currently listed as near threatened, threatened, endangered, or vulnerable (IUCN 2011, Janczewski et al. 1995). Although public attitude can be unstable towards conservation of large carnivores (Mech 1996), protection of the Cougar is warranted in eastern Canada for maintaining biodiversity as well as for the ecological role of the species. Acknowledgments We thank Clément Lanthier from the Calgary Zoo as well as Claude Daigle and Pierre Canac-Marquis from the Ministère des Ressources naturelles et de la Faune (MRNF) for providing hair samples of different mammal species. The Société Zoologique de Granby, Ruiter Valley Land Trust, Fédération des Gestionnaires de Zecs de la Gaspésie et du Bas-St-Laurent, FAPAQ, SEPAQ, Corridor Appalachien, Société de Conservation de la Rivière au Saumon, Parcs Canada (Québec), Parcs Canada (Maritimes), Fondation du Parc du Mont-Tremblant, Warden Service of Fundy, and Cape Breton Highlands National Parks were instrumental in collecting hair samples from the scratching posts in the field. Thanks to Dr. Cyrille Barrette, from Université Laval, who provided a sample from the Cougar hit in Abitibi in 1992 and Jean Tanguay, from the MRNF, who recovered the bumper of the car that killed the Cougar in Réserve des Laurentides in 2002. 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