Kristine L. Pilgrim

Wolverine gene flow across a narrow climatic niche

Wolverines (Gulo gulo) are one of the rarest carnivores in the contiguous United States. Effective population sizes in Montana, Idaho, and Wyoming, where most of the wolverines in the contiguous United States exist, were calculated to be 35 (credible limits, 28 52) suggesting low abundance. Landscape features that influence wolverine population substructure and gene flow are largely unknown. Recent work has identified strong associations between areas with persistent spring snow and wolverine presence and range. We tested whether a dispersal model in which wolverines prefer to disperse through areas characterized by persistent spring snow cover produced least-cost paths among all individuals that correlated with genetic distance among individuals. Models simulating large preferences for dispersing within areas characterized by persistent spring snow explained the data better than a model based on Euclidean distance. Partial Mantel tests separating Euclidean distance from spring snow-cover-based effects indicated that Euclidean distance was not significant in describing patterns of genetic distance. Because these models indicated that successful dispersal paths followed areas characterized by spring snow cover, we used these understandings to derive empirically based least-cost corridor maps in the U.S. Rocky Mountains. These corridor maps largely explain previously published population subdivision patterns based on mitochondrial DNA and indicate that natural colonization of the southern Rocky Mountains by wolverines will be difficult but not impossible.

Identifying lynx and other North American felids based on MtDNA analysis

As part of a program to identify the distribution of Canada lynx (Lynx canadensis) across the U.S. using hair snags, we have developed a protocol to distinguish among all four felid species of northern North America (lynx, bobcat [Lynx rufus], cougar [Felis concolor], and domestic cat [Felis catus]) using mtDNA. Our tests were designed to be time and cost-efficient, and applicable to low-quantity or degraded DNA samples. Although it is possible to identify species using microsatellite DNA (e.g. Ernest et al. 2000), we favor mtDNA because of the greater copy number and because allele size constraints limit interspecific ranges of microsatellite DNA, potentially leading to allele frequency overlap (Nauta and Weissing 1996). Because mtDNA is highly conserved among tissue types within an individual, and because tissue samples amplify more consistently than hair, we developed and validated our protocol using both tissue and hair samples. For hair samples, 5–10 follicles with shafts were typically used in extraction, although in some cases we successfully used single hairs with or without follicles (see below). Genomic DNA was extracted using standard protocols for tissues (Dneasy tissue kit; Qiagen Inc.), with overnight incubation in lysis buffer and Proteinase K on a rocker at 60 • C. Elution of DNA was in 50 µl of buffer. To distinguish felid species from one another and from other species, we used the Polymerase Chain Reaction (PCR) to amplify two portions of the mito-chondrial genome (Figure 1). One region includes the control region, amplified using conserved, universal primers L16007 and H16498 (Kocher et al. 1989; Shields and Kocher 1991). Twenty-µl PCR reactions contained 50–100 ng DNA, 1× reaction buffer (Perkin-Elmer), 2.5 mM MgCl 2 , 200 µM each dNTP, 0.3 mg/ml BSA, 1 µM each primer, and 1 U Taq polymerase (Perkin-Elmer). After initial incubation at 94 • C for 5 min, the PCR profile was 35 cycles of 94 • C for 1 minute, 55 • C for 1 minute, and 72 • C for 1.5 minutes. PCR products were run in a 2.0% agarose gel (Asubel et al. 1989). The control region primers produced a PCR product of approximately 700 bp in all felid samples (Figure 1). This is the same region amplified by Foran et al. (1997a, b), except that primer L16007 results in a product 200 bp smaller, which we find amplifies with greater consistency. Felids have a 80–82 bp monomer within this region that …

Testing for wolf-coyote hybridization in the Rocky Mountains using mitochondrial DNA

Hybridization between gray wolves (Canis lupus) and coyotes (Canis latrans) has been documented in the Great Lakes region of the United States and Canada but has not been extensively studied in the Rocky Mountain region. We used mitochondrial DNA (mtDNA) to evaluate potential gray wolf-coyote hybridization in wolf populations in the western United States, Alberta, and British Columbia, including wolves reintroduced into Yellowstone National Park (YNP) and central Idaho. A restriction site and a length difference in the control region (D-loop) of mtDNA was used to differentiate wolf and coyote haplotypes. All 90 wolves tested had wolf haplotypes. We concluded that the wolf populations in the Rocky Mountain region have not hybridized with coyotes as they have in the Great Lakes region. This method could be used to test other wolf populations for wolf-coyote hybridization and monitor the translocated YNP and Idaho populations in the future.

Identification of mustelids using mitochondrial DNA and non-invasive sampling

, mitochondrial DNA, mtDNA, mustelids, non-invasivesampling,wolverineOne of the most fundamental issues in conservationbiology is the determination of species distributionand richness. Many species, including forest carni-vores, are elusive and secretive, making it difficult orimpossible to make even basic conservation decisionson listing, delisting, or threat status. Non-invasivesampling of hairs left on hair snares can substan-tially increase our detection of such species. In anextension of a nationwide project using hair snaresacross 12 states to identify lynx (

Integrating motion-detection cameras and hair snags for wolverine identification†

ABSTRACT We developed an integrated system for photographing a wolverines (Gulo gulo) ventral pattern while concurrently collecting hair for microsatellite DNA genotyping. Our objectives were to 1) test the system on a wild population of wolverines using an array of camera and hair-snag (C&H) stations in forested habitat where wolverines were known to occur, 2) validate our ability to determine identity (ID) and sex from photographs by comparing photographic data with that from DNA, and 3) encourage researchers and managers to test the system in different wolverine populations and habitats and improve the system design. Of the 18 individuals (10 M, 8 F) for which we obtained genotypes over the 2 years of our study, there was a 100% match between photographs and DNA for both ID and sex. The integrated system made it possible to reduce cost of DNA analysis by >74%. Integrating motion-detection cameras and hair snags provides a costeffective technique for wildlife managers to monitor wolverine populations in remote habitats and obtain information on important population parameters such as density, survival, productivity, and effective population size.

Wolverine Confirmation in California after Nearly a Century: Native or Long-Distance Immigrant?

Abstract We photo-verified the presence of a wolverine (Gulo gulo) in California for the first time in 86 years during February 2008. Herein we document the process of determining the origin of this wolverine using genetic, stable carbon (&dgr;13C) and stable nitrogen (&dgr;15N) isotope information. The wolverines origin was significant because it is a state-threatened species and California represents a historically unique genotype of wolverines in North America. We obtained both photographs and noninvasively-collected genetic evidence (scat and hair). DNA analysis revealed the animal was a male and not a remnant of a historical California population. Comparison with available data revealed the individual was most closely related to populations from the western edge of the Rocky Mountains. This represents the first evidence of connectivity between wolverine populations of the Rocky and Sierra Nevada Mountain Ranges.

DNA barcoding at riverscape scales: assessing biodiversity among fishes of the genus Cottus (Teleostei) in northern Rocky Mountain streams

There is growing interest in broad‐scale biodiversity assessments that can serve as benchmarks for identifying ecological change. Genetic tools have been used for such assessments for decades, but spatial sampling considerations have largely been ignored. Here, we demonstrate how intensive sampling efforts across a large geographical scale can influence identification of taxonomic units. We used sequences of mtDNA cytochrome c oxidase subunit 1 and cytochrome b, analysed with maximum parsimony networks, maximum‐likelihood trees and genetic distance thresholds, as indicators of biodiversity and species identity among the taxonomically challenging fishes of the genus Cottus in the northern Rocky Mountains, USA. Analyses of concatenated sequences from fish collected in all major watersheds of this area revealed eight groups with species‐level differences that were also geographically circumscribed. Only two of these groups, however, were assigned to recognized species, and these two assignments resulted in intraspecific genetic variation (>2.0%) regarded as atypical for individual species. An incomplete inventory of individuals from throughout the geographical ranges of many species represented in public databases, as well as sample misidentification and a poorly developed taxonomy, may have hampered species assignment and discovery. We suspect that genetic assessments based on spatially robust sampling designs will reveal previously unrecognized biodiversity in many other taxa.

Individual identification of Sitka black-tailed deer (Odocoileus hemionus sitkensis) using DNA from fecal pellets

We tested a protocol for extracting DNA from fecal pellets from Sitka black-tailed deer (Odocoileus hemionus sitkensis) and evaluated genotyping performance of previously developed microsatellite markers as well as a suite of new markers designed specifically for this study. We screened 30 microsatellites, and identified 7 (23%) loci including 4 new markers, that fit well into a single multiplex and consistently genotyped deer with low error rates. DNA was extracted from 2,408 fecal-pellet samples. Of those, 1,240 (52%) were genotyped successfully at all 7 markers allowing identification of 634 genetically unique deer. Using DNA from fecal pellets collected in the field was an effective technique for identifying and distinguishing among deer.

WHEN REINTRODUCTIONS ARE AUGMENTATIONS: THE GENETIC LEGACY OF FISHERS (MARTES PENNANTI) IN MONTANA

Abstract Fishers (Martes pennanti) were purportedly extirpated from Montana by 1930 and extant populations are assumed to be descended from translocated fishers. To determine the lineage of fisher populations, we sequenced 2 regions of the mitochondrial DNA genome from 207 tissue samples from British Columbia, Minnesota, Wisconsin, and Montana. In northwestern Montana, fishers share haplotypes with samples from the upper Midwest and British Columbia; in west-central Montana, we detected haplotypes found in British Columbia samples, but also detected a control region and cytochrome-b haplotype not found in source populations. Based on the unique haplotypes found in west-central Montana, we propose that individuals with these haplotypes are descended from a relic population. Fishers in northwestern Montana are likely descended from fishers from the Midwest and British Columbia.

Patterns of hybridization among cutthroat trout and rainbow trout in northern Rocky Mountain streams

Abstract Introgressive hybridization between native and introduced species is a growing conservation concern. For native cutthroat trout and introduced rainbow trout in western North America, this process is thought to lead to the formation of hybrid swarms and the loss of monophyletic evolutionary lineages. Previous studies of this phenomenon, however, indicated that hybrid swarms were rare except when native and introduced forms of cutthroat trout co‐occurred. We used a panel of 86 diagnostic, single nucleotide polymorphisms to evaluate the genetic composition of 3865 fish captured in 188 locations on 129 streams distributed across western Montana and northern Idaho. Although introgression was common and only 37% of the sites were occupied solely by parental westslope cutthroat trout, levels of hybridization were generally low. Of the 188 sites sampled, 73% contained ≤5% rainbow trout alleles and 58% had ≤1% rainbow trout alleles. Overall, 72% of specimens were nonadmixed westslope cutthroat trout, and an additional 3.5% were nonadmixed rainbow trout. Samples from seven sites met our criteria for hybrid swarms, that is, an absence of nonadmixed individuals and a random distribution of alleles within the sample; most (6/7) were associated with introgression by Yellowstone cutthroat trout. In streams with multiple sites, upstream locations exhibited less introgression than downstream locations. We conclude that although the widespread introduction of nonnative trout within the historical range of westslope cutthroat trout has increased the incidence of introgression, sites containing nonadmixed populations of this taxon are common and broadly distributed.