Cougars in Guadalupe Mountains National Park, Texas: estimates of occurrence and distribution using analysis of DNA.
As indicated by Krausman and Bowyer (2003), translocations are more likely to succeed when disturbance by humans is minimized; Guadalupe Mountains National Park is a site where such disturbance likely will be less severe. Another potential factor that can impact reintroduction is predation, which as noted by Berger (1990) can threaten small populations. The cougar (Puma concolor) is a natural predator of bighorn sheep and is a potential threat to small, reintroduced populations of bighorn sheep (e.g., Leopold and Krausman, 1983; Rominger and Weisenberger, 2000; Ernest et al., 2002). In many instances, extreme measures have been used to minimize the impact of such predation (United States Fish and Wildlife Service, 2000, 2003; New Mexico Department of Game and Fish, in litt.).
Status of populations of cougars within Guadalupe Mountains National Park and the surrounding area has been studied twice in the past 20 years. T. E. smith et al. (in litt.) indicated a high density of cougars as a result of hunting pressure and reported 30-50% mortality of adults and 58-83% mortality of subadults when they leave the park, which might lead to a large number of transients. Harveson et al. (1999) used multiple-evidence surveys to study trends in populations within the park and reported a decrease in evidence of cougars during 1987-1991 and an increase in 1992-1996. Both studies suggested negative impacts of predator control on populations outside the park. At the time data were collected and analyzed in our study, predator control outside the park included systematic removal of cougars entering the sierra Diablo Management Area, Texas, ca. 50 km south of the park, random killing by local ranchers in Texas and New Mexico, and killing by Wildlife Services in Texas and New Mexico.
In preparation for reintroduction of bighorn sheep into Guadalupe Mountains National Park, a study of cougars was conducted during an 8-year period (1997-2004). Our primary objectives were to determine number and distribution of cougars in areas where bighorn sheep were to be reintroduced, to compare patterns of genetic variation in the population within the park to populations outside the park, and to use noninvasive genetic techniques as methods to attain these objectives.
MATERIALS AND METHODS--Bordering New Mexico and 177 km E El Paso is Guadalupe Mountains National Park, Texas (encompasses 350 [km.sup.2]). The Guadalupe Mountains are the southern extreme of the Rocky Mountains. Fecal samples of cougars (n = 98) were collected during 1997-2004 along transects on 74 km of trails, encompassing 230 [km.sup.2] within the park (Table 1).
Park personnel collected and preserved samples during 1997-2004 (Table 1) and prior to involvement of the authors. Methods were described in Harveson et al. (1999). Based on data from radiotelemetry and other information on movements (T. E. smith et al., in litt.), park personnel established transects along canyons, ridges, and trails throughout six watersheds within the park. individuals used a compass and map to navigate transects each spring (April-May) and autumn (October-November). Two to five observers, trained in identification of evidence of cougars, surveyed 8-15 km/day along transects and recorded location (1-km increments) of scats (fecal samples) suspected to be from a cougar. scats were segmented, contained hair, and were >29 mm in diameter. Park personnel placed individual samples into sealed plastic bags stored at -20[degrees]C, where they remained until 2005. We transferred samples to a -80[degrees]C freezer at Texas A&M University until extractions of DNA were performed.
We used QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) to extract DNA from 98 fecal samples. initially, we removed samples of fecal material from external portions of each sample and 200 mg of this material was used to isolate DNA by following the protocol outlined in the QiAamp DNA stool Mini Kit. Processing was performed in groups of 12-14 samples. DNA was extracted twice for those samples that either did not provide positive results for DNA or showed only small amounts of polymerase-chain-reaction (PCR) product after initial amplification of a fragment of mitochondrial DNA (mtDNA). All extractions included negative-extraction controls containing all reagents but no fecal material. such controls minimized risk of contamination.
DNA from fecal samples was identified to species by sequencing a ca. 200 base-pair fragment of the mitochondrial-control region. All sets of PCR reactions included a positive control (DNA isolated from tissue) for verification that failure to amplify was not the result of the set of reactions performed. All sets of PCR reactions also included a negative control (without extracted fecal DNA) to confirm that amplification of samples was not an artifact of contamination of reagents. sequencing primers (PDL-1 and PDL-6/PDL-6del) were the same as reported by Uphyrkina et al. (2001). PCR reactions were performed in 20-[micro]l volumes containing: 10 [micor]M of each primer, 10X Hotmaster Taq buffer with 25 mM [Mg.sup.2+], 10mM of each dNTP, 10 mg/[micro]l BSA, 0.5 U/[micro]l HotMaster Taq DNA polymerase (5 Prime, inc., Gaithersburg, Maryland), dd[H.sub.2]O, and template DNA. We performed PCR at 94[degrees]C for 1 min, 48 cycles of 94[degrees]C for 15 s, 52[degrees]C for 30 s, 72[degrees]C for 1 min, and a final step at 72[degrees]C for 2 min. Excess primers were removed with ExosAP-iT (UsB Corporation, Cleveland, Ohio). PCR fragments were sequenced on an automated sequencer with the ABi Big Dye termination technique (Applied Biosystems, Foster City, California). Genotyping was performed only on samples of DNA identified as cougars.
Minimum number of microsatellite loci required for identification of individual cougars was determined using allele-frequency data from a reference group consisting of 68 cougars (samples of tissues or blood) collected across western Texas and outside the park. This reference group represented five localities (Fig. 1), containing 7, 12, 28, 14, and 7 individuals, respectively. Genotypes for these individuals were determined initially for 18 microsatellite loci by J. E. Janecka (in litt.). All loci were derived from the domestic cat Felis catus (Menotti-Raymond and O'Brien, 1995; Menotti-Raymond et al., 1999). Data from the present study and that of J. E. Janecka (in litt.) were analyzed under the same conditions. To minimize errors associated with scoring alleles, we simultaneously regenotyped individuals from the reference populations along with new samples of DNA obtained from scats.
We used the program API-CALC 1.0 (Ayres and overall, 2004) to compute probabilities of identity (Waits et al., 2001) for various combinations of the 18 loci in an effort to identify a panel with high levels of heterozygosity and low probabilities of identity. We identified six loci (FCA23, FCA26, FCA35, FCA43, FCA82, and FCA96) that were used to establish multilocus genotypes of samples. These six loci had low expected probabilities of identity (2.0 X [10.sup.-5], range 0.106-0.385 X [10.sup.-5]) and high expected heterozygosity (0.672, range 0.523-0.785; Table 2); thus, providing sufficient power for discriminating among individuals (Waits et al., 2001). Therefore, we interpreted samples identical at all six loci as being the same individual.
[FIGURE 1 OMITTED]
PCR amplification was performed separately for each of the six loci in a 20-[micro]l reaction containing 10 mM for each forward and reverse primer, 10X Hotmaster Taq buffer with 25 mM [Mg.sup.2+], 10 mM of each dinucleotide triphosphates (dNTP), 10 mg/[micro]l BSA, 5 U/[micro]l HotMaster Taq DNA polymerase, dd[H.sub.2]O, and 0.3 [micro]l of template DNA/reaction. PCR reactions included an initial step of 94[degrees]C for 1 min, 48 cycles of 94[degrees]C for 15 s, 54[degrees]C for 30 s, 72[degrees]C for 45 s, and a final step at 72[degrees]C for 2 min. As described, we included a positive and a negative control in all sets of PCR reactions. Amplified fragments were prepared for genotyping by first diluting the sample 1:20 followed by mixing 1 [micro]l of diluted DNA product with a 10-[micro]l solution of 9.7-[micro]l formamide and 0.3 [micro]l size-standard (GeneScan 400HD ROX; Applied Biosystems, Foster City, California). Genotyping was performed using an ABi 3,100 automated sequencer (Applied Biosystems, Foster City, California).
We treated a total of 15 samples, 10 that failed to amplify for one or more microsatellite loci and five that tested positive for mtDNA but failed to amplify for all microsatellite loci (totaling 45 reactions), with an experimental DNA repair kit (PreCR-A Repair Mix, New England BioLabs, Ipswich, Massachusetts). The Repair Mix is a cocktail of enzymes that targets damaged template DNA and repairs various damages of DNA including some that block PCR. We placed 5 [micro]l of template DNA in 50 [micro]l reaction containing 38 [micro]l of dd[H.sub.2]O, 5 [micro]l of 10X ThermoPol RX Buffer, 0.5 [micro]l of each 10 mM DNTP, 0.5 [micro]l 100X NAD+, and 1[micro]l PreCR Repair Mix-A, and allowed the mixture to stand at room temperature for 15 min (T. Evans, in litt.). We used 5 [micro]l of the repaired mixture of DNA as a template for regenotyping, following the PCR protocol.
We used multilocus microsatellite genotypes to identify individuals. We accepted an individual as homozygous at a locus after repeating the genotyping 3-5 times in independent reactions, and we scored individuals as heterozygous only after each allele was observed at least twice in independent reactions. Using Pedant (version1.0; Johnson and Haydon, 2007), we estimated rates of dropout and false alleles per genotype for each of the loci by running 100,000 simulations on two duplicate genotypes per locus per sample. in addition, we followed McKelvey and Schwartz (2004) for evaluating genotypic error from noninvasive sampling by estimating minimum number of differences for all pairs of genotypes. According to Mowat and Paetkau (2002), this method helps evaluate whether samples were scored erroneously as a result of a high percentage of genotypes differing from each other by a low number of loci. in addition, we compared allelic variation in our samples to that seen in the five reference populations in an effort to assess whether samples of scats had significant levels of allelic dropout.
We used the program GenAlEx version 5.1.1 (Peakall and Smouse, 2001) to calculate genetic variability at the six loci and we tested for deviation from Hardy-Weinberg equilibrium using a Chi-square test with pooling (Hartl and Clark, 1989) and the Markov Chain method (Guo and Thompson, 1992). We calculated average number of alleles, mean number of effective alleles per locus, expected heterozygosity, and observed heterozygosity for each locus, and these estimates were compared to other populations in Texas. We obtained values of genotypic and genic differentiation between our population and reference populations by estimating [F.sub.ST] with GENEPOP 3.4 (Raymond and Rousset, 1995), and we used the program GeneClass2 (Piry et al., 2004) to assign individuals to either the park or one of the reference populations and to estimate the probability of assignments of individuals. Our criterion for assignment of individuals was based on the Bayesian algorithm by Rannala and Mountain (1997) as implemented in GeneClass2, and exclusion probabilities (99.9% confidence level) were computed using 10,000 simulations of the Monte Carlo resampling algorithm described by Paetkau et al. (2004).
We used a 30-m-resolution, digital-elevation model from the National Elevation Dataset ArcGIS 9.0 software (Environmental Systems Research Institute, Inc., Redlands, California) to plot the distribution of confirmed samples. We correlated locations of cougars and habitat of bighorn sheep (Gilad, 2006) to evaluate possible overlap in habitat for these species.
RESULTS--DNA was isolated from 98 fecal samples and 54 samples (55%) provided mtDNA-amplification products. The 54 fragments of mtDNA were sequenced and 44 (44 of 54; 80%) samples tested positive for mtDNA from cougars. Only one haplotype of mtDNA was in the population and this haplotype was identical to the haplotype in the 68 individuals in the reference populations (J. E. Janecka, in litt.) and identical to the common haplotype identified for North American cougars (Culver et al., 2000).
The 44 samples that tested positive for mtDNA of cougars were amplified across all six microsatellite loci. Of these 44, only 32 successfully amplified for nuclear DNA across 5-6 loci. Based on these results, 31 unique genotypes, one of which occurred in two samples, were observed. The two samples that were identical across all loci were assumed to be from the same cougar.
Initially, only 27 individuals of the 44 samples (61.4%) could be genotyped for all loci. In some instances, samples failed to amplify for several loci. Subsequent to treatment of these failed PCR reactions with PreCR-A, five additional samples were successfully amplified across 5-6 loci; thus, increasing the sample to 32.
Rate of allelic dropout averaged 0.089 (range 0-0.180) and average rate of false alleles was 0.034 (range 0-0.031; Table 2). Pairwise estimates of genotypes observed indicated differences at an average of 4.9 loci and reference populations had an average difference at 4.36 loci/genotype. All six loci were polymorphic with a fixation index or [F.sub.ST] (differentiation between populations based on genetic distance) close to zero. Average number of alleles was 6.5 (range 5.0-9.0, SE 5 0.62) and mean number of effective alleles per locus was 3.29 (range 2.09-4.65, SE = 0.39). The expected and observed mean estimates of heterozygosity were 0.67 and 0.68, respectively (Table 2). Individual comparisons revealed two loci (FCA26, FCA96) out of Hardy-Weinberg Equilibrium, whereas among all loci the population was in equilibrium (P = 5 0.343).
Overall, the genotyping provided little evidence ofallelic dropout and error in genotyping. Genotypes identified within the population were not skewed toward differences at a single locus (McKelvey and Schwartz, 2004) and differed at an average of 4.9 loci, similar to reference populations in western Texas. Estimates of the probability of identity for the population also were similar to reference populations in western Texas.
Although samples were small, estimates of [F.sub.ST] for the population in the park relative to the five reference populations averaged 0.130 (ranged from 0.103 relative to population 1 and 0.168 for population 5; Fig. 1), suggesting structure in the population with some restrictions of gene flow between the population in the park and the other populations in Texas. Assignment tests were used to place individuals in either the park or one of the five reference populations (Fig. 1). All 31 individuals (100%, [alpha] = 0.001) were assigned to the park, indicating genetic divergence of the population within the park from other areas of Texas.
The PreCR-A Repair Mix increased successful amplification and genotyping by 19.5% and proved to be an effective method where noninvasive sampling of DNA is derived from fecal material. Therefore, this method provided a marked improvement for noninvasive genetic studies that rely on fecal samples, which can yield DNA of low quantity that is more prone to error as a result of allelic dropout (Hedmark and Ellegren, 2005).
DISCUSSION--Guadalupe Mountains National Park contains ca. 80-98 [km.sup.2] of suitable habitat for bighorn sheep (Gilad, 2006). Based on locations where fecal samples of cougars were collected and a comparison to suitable habitat of bighorn sheep within the park (Gilad, 2006), a potential for overlap in habitat exists between cougars and bighorn sheep. Adult cougars are selective predators (Emmons, 1987; Novaro et al., 2000), but they also are highly skilled opportunistic predators and are capable of preying on bighorn sheep (e.g., Cunningham et al., 1999; Ernest et al., 2002). An analysis of content of scats determined that 82% of diet consisted of mule deer (Odocoileus hemionus), with North American porcupines (Erethizon dorsatum), lagomorphs (Sylvilagus and Lepus), elk (Cervus elaphus), rodents, and ca. 8% domestic livestock (T. E. Smith et al., in litt.). The extent to which cougars would prey on bighorn sheep in the event of a reintroduction is unknown.
Although suitable habitat for bighorn sheep exists at Guadalupe Mountains National Park (Gilad, 2006), any artificial reestablishment of the population probably will involve a small initial population, and as the population of adult females decreases, risk of extinction increases, particularly if predation by cougars is considered (Ernest et al., 2002). Therefore, any management plan for the re-introduction of bighorn sheep must consider the number of cougars inhabiting the area.
Although patterns of dispersal on a larger scale can be complicated, cougars are not restricted to the park (T. E. Smith et al., in litt.). Based on genotypic data, [greater than or equal to]31 cougars have visited the park over an 8-year period (1997-2004). During a 3-year period (2002-2004), [greater than or equal to]26 different cougars were using the study area (ca. 230 [km.sup.2]) and corresponds to an average of 8.67 cougars/year or 3.77 cougars/100 [km.sup.2]. In 2002, [greater than or equal to]15 adults were using the park, corresponding to 6.5 adults/100 [km.sup.2] . This estimated density is comparatively high. T. E. Smith et al. (in litt.) calculated 2.3 cougars/100 [km.sup.2] in the park, Guzman (1998) estimated 0.26-0.59 cougars/ 100 [km.sup.2] in Big Bend State Park, Texas, and K. A. Logan and L. L. Sweanor (in litt.) estimated 0.94-2.01 cougars/100 [km.sup.2] in the San Andres Mountains, New Mexico.
For the six loci we examined, the population had an average heterozygosity of 0.68 and an average of 3.29 alleles/locus (Table 2). The overall pattern of genetic variation in the population is similar to values obtained in other genetic studies of cougars. For example, Walker et al. (2000), using seven polymorphic microsatellite loci, reported a heterozygosity of 0.67 and an average of 4.0 alleles/locus for a population in western Texas. Populations of cougars in Wyoming Basin (Anderson et al., 2004), California (Ernest et al., 2003), and several southwestern states (McRae et al., 2005) also had similar levels of heterozygosity and alleles per locus as those reported herein.
Although the cougar is a large mammal with an average distance of dispersal of 102-116 km for males and 13.1-34.6 km for nonphilopatric females (Sweanor et al., 2000; K A. Logan and L. L. Sweanor, in litt.), patterns of genetic variation in different regions of western North America provide evidence of genetic subdivision. For example, Walker et al. (2000) compared a representative population of cougars from western and southern Texas and detected high genetic subdivision ([F.sub.ST] = 0.11). Although Anderson et al. (2004) reported little evidence of structure in Wyoming Basin, McRae et al. (2005) observed a north to south subdivision (average [F.sub.ST] = 0.15) between northern Arizona and Utah, and northern and southern New Mexico. Similarly, Ernest et al. (2003) observed an average [F.sub.ST] of 0.12 between regions of California. Based on assignment tests and estimates of [F.sub.ST], gene flow between our population and other populations in Texas seems to be restricted, with cougars in the park having an average [F.sub.ST] of 0.13 in comparison to five populations from western Texas (J. E. Janecka, in litt.).
Noninvasive genetic sampling has proven an effective means of providing a reasonable estimate of relative abundance of populations of evasive species (e.g., Ernest et al., 2000; Apps et al., 2004; Proctor et al., 2006). With necessary precautions (Waits et al., 2001), these techniques provide an additional management tool for studies like ours that focus on an evaluation of abundance of predators that might interfere with reintroduction of a potential prey. The protocol described herein provides a set of mitochondrial and nuclear markers that allow for detection of individual cougars, and based on comparisons to other studies, these markers provide an estimate of abundance that is important to consider prior to reintroduction of bighorn sheep.
There may be various reasons for the uniqueness of cougars in Guadalupe Mountains National Park. As indicated by McRae et al. (2005), connectivity of the landscape influences patterns of genetic subdivision in cougars. This may partially explain the divergence between cougars in the park and those in other regions in western Texas. In addition, unregulated hunting and predator-control activities in New Mexico and the proximity of the park to the Texas-New Mexico border may explain alleles not detected in other populations in Texas, but that are present in our population; it is possible that these unique alleles are a result of cougars dispersing from New Mexico. T. E. Smith et al. (in litt.) determined that 66% of cougars with established home ranges that included parts of Guadalupe Mountains National Park also moved into New Mexico (16% established a home range extending south of the park and 16% never left the park during their study). As Guadalupe Mountains National Park represents the southern part of the Guadalupe Mountains that extends into New Mexico, it is possible that many cougars within the park are migrants from New Mexico. Further research is needed to test this hypothesis; this should involve both genetic comparisons and radiotelemetry.
Results of this study support observations of T. E. Smith et al. (in litt.), who indicated high density of cougars visiting or residing within the park. It is our opinion that management efforts should not include reintroduction of naive bighorn sheep unfamiliar with either cougars or the terrain. A meta-population of bighorn sheep, consisting of >800 sheep distributed throughout the Sierra Diablo, Baylor, and Beach mountain ranges, occurs south and southeast of Guadalupe Mountains National Park (Richardson, 2007). Current reports indicate movements of sheep between these ranges and the Delaware Mountains, which may provide a potential migration corridor for sheep to enter Guadalupe Mountains National Park (Gilad, 2006). We recommend that more investigation of this potential corridor be conducted. Natural recolonization of the park, when and if it occurs, will prevent the inherent problems associated with reintroduction programs of animals unfamiliar with terrain and local predators. In addition, such an action might eliminate the need to kill cougars within the park.
We thank personnel at Guadalupe Mountains National Park for collecting samples and providing continuous support of this project. This study was funded by the Guadalupe Mountains-Carlsbad Cavern Association. We acknowledge M. Culver for providing primers and T. Evens for providing the experimental DNA-repair kit. We thank P. Thomason for translating our abstract into Spanish.
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Submitted 18 August 200009. Accepted 8 May 2011.
Associate Editor was Marlis R. Douglas.
Oranit Gilad, * Jan E. Janecka, Fred Armstrong, Michael E. Tewes, and Rodney L. Honeycutt
Department of Biology, Texas A&M University, TAMU 2258, College Station, TX 77843-2258 (OG)
Department of Veterinary Integrative Biosciences, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843 (JEJ)
Resource Management, Guadalupe Mountains National Park, Salt Flat, TX 79847 (FA)
Feline Research Center, Caesar Kleberg Wildlife Research Institute, Texas A&M University-Kingsville, MSC 218, 700 University Boulevard, Kingsville, TX 78363 (MET)
Division of Natural Science, Pepperdine University, 24255 Pacific Coast Highway, Malibu, CA 90263 (RLH)
* Correspondent: firstname.lastname@example.org
TABLE 1--Fecal samples of cougars (Puma concolor) collected in Guadalupe Mountains National Park, Texas, by year and location. Year Transect n 1997 Middle McKittrick 2 Upper south McKittrick 3 1998 Upper south McKittrick 1 Middle McKittrick 2 Dog Canyon 1 1999 Middle McKittrick 1 Dog Canyon 7 Frijole Ridge 1 Bush Mountain 1 2000 Cox 2 Upper south McKittrick 1 Dog Canyon 5 Bush Mountain 1 2001 Frijole Ridge 4 Middle McKittrick 2 Dog Canyon 2 2002 Bush Mountain 11 West Dog Canyon 14 Upper south McKittrick 4 Middle McKittrick 2 Frijole Ridge 4 Cox 3 2003 Upper south McKittrick 3 Dog Canyon 4 Frijole Ridge 4 Cox 1 Bush Mountain 1 El Capitan 1 2004 El Capitan 2 West Dog Canyon 2 Bush Mountain 5 Shumard Canyon 1 TABLE 2--Genetic estimates for cougars (Puma concolor) in Guadalupe Mountains National Park, Texas, based on six microsatellite loci and data from 31 unique genotypes. Name of locus Expected and number Number of heterozygosity Observed of base pairs n alleles ([+ or -] SE) heterozygosity FCA23 32 5 0.690 (0.040) 0.742 FCA26 31 7 0.523 (0.070) 0.469 FCA35 32 6 0.672 (0.043) 0.563 FCA43 30 7 0.753 (0.029) 0.875 FCA82 30 5 0.690 (0.031) 0.813 FCA96 30 9 0.785 (0.037) 0.742 Population 6.5 0.672 (0.042) 0.684 Name of locus and number Rate of allelic False rate of of base pairs dropout alleles FCA23 0.019 0.000 FCA26 0.000 0.000 FCA35 0.016 0.000 FCA43 0.061 0.028 FCA82 0.180 0.147 FCA96 0.065 0.031 Population 0.089 0.034
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|Author:||Gilad, Oranit; Janecka, Jan E.; Armstrong, Fred; Tewes, Michael E.; Honeycutt, Rodney L.|
|Date:||Sep 1, 2011|
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