Jennifer R. Adams

Using faecal DNA sampling and GIS to monitor hybridization between red wolves (Canis rufus) and coyotes (Canis latrans)

The US Fish and Wildlife Services (USFWS) Red Wolf Recovery Program recognizes hybridization with coyotes as the primary threat to red wolf recovery. Efforts to curb or stop hybridization are hampered in two ways. First, hybrid individuals are difficult to identify based solely on morphology. Second, managers need to effectively search 6000 km2 for the presence of coyotes and hybrids. We develop a noninvasive method to screen large geographical areas for coyotes and hybrids with maternal coyote ancestry by combining mitochondrial DNA sequence analysis of faeces (scat) and geographic information system (GIS) technology. This method was implemented on the Alligator River National Wildlife Refuge (1000 km2) in northeastern North Carolina. A total of 956 scats were collected in the spring of 2000 and 2001 and global positioning system (GPS) coordinates were recorded. Seventy‐five percent of the scats were assigned to species and five coyote/hybrid scats were detected. Placement of scat location coordinates on a map of the experimental population area revealed that four of the coyote/hybrid scats were detected within the home ranges of sterilized hybrids. The other coyote/hybrid scat indicated the presence of a previously unknown individual. We suggest this method be expanded to include more of the experimental population area and be optimized for use with nuclear markers to improve detection of hybrid and back‐crossed individuals.

Widespread occurrence of a domestic dog mitochondrial DNA haplotype in southeastern US coyotes

Sequence analysis of the mitochondrial DNA control region from 112 southeastern US coyotes (Canis latrans) revealed 12 individuals with a haplotype closely related to those in domestic dogs. Phylogenetic analyses grouped this new haplotype in the dog/grey wolf (Canis familiaris/Canis lupus) clade with 98% bootstrap support. These results demonstrate that a male coyote hybridized with a female dog, and female hybrid offspring successfully integrated into the coyote population. The widespread distribution of this haplotype from Florida to West Virginia suggests that the hybridization event occurred long ago before the southeastern USA was colonized by coyotes. However, it could have occurred in the southeastern USA before the main front of coyotes arrived in the area between male coyotes released for sport and a local domestic dog. The introgression of domestic dog genes into the southeastern coyote population does not appear to have substantially affected the coyotes genetic, morphological, or behavioural integrity. However, our results suggest that, contrary to previous reports, hybridization can occur between domestic and wild canids, even when the latter is relatively abundant. Therefore, hybridization may be a greater threat to the persistence of wild canid populations than previously thought.

Pedigree-based assignment tests for reversing coyote (Canis latrans) introgression into the wild red wolf (Canis rufus) population.

The principal threat to the persistence of the endangered red wolf (Canis rufus) in the wild is hybridization with the coyote (Canis latrans). To facilitate idengification and removal of hybrids, assignment tests are developed which use genotype data to estimate identity as coyote, 1/4, 1/2, 3/4 or full red wolf. The tests use genotypes from the red wolves that founded the surviving population and the resulting pedigree, rather than a contemporary red wolf sample. The tests are evaluated by analysing both captive red wolves at 18 microsatellite loci, and data simulated under a highly parameterized, biologically reasonable model. The accuracy of assignment rates are generally high, with over 95% of known red wolves idengified correctly. There are, however, tradeoffs between ambiguous assignments and misassignments, and between misidengifying red wolves as hybrids and hybrids as red wolves. These result in a compromise between limiting introgression and avoiding demographic losses. The management priorities and level of introgression determine the combination of test and removal strategy that best balances these tradeoffs. Ultimately, we conclude that the use of the assignment tests has the capacity to arrest and reverse introgression. To our knowledge, the presented approach is novel in that it accounts for genetic drift when the genotypes under analysis are temporally separated from the reference populations to which they are being assigned. These methods may be valuable in cases where reference databases for small populations have aged substantially, pedigree information is available or data are generated from historical samples.

Locating hybrid individuals in the red wolf (Canis rufus) experimental population area using a spatially targeted sampling strategy and faecal DNA genotyping

Hybridization with coyotes (Canis latrans) continues to threaten the recovery of endangered red wolves (Canis rufus) in North Carolina and requires the development of new strategies to detect and remove coyotes and hybrids. Here, we combine a spatially targeted faecal collection strategy with a previously published reference genotype data filtering method and a genetic test for coyote ancestry to screen portions of the red wolf experimental population area for the presence of nonred wolf canids. We also test the accuracy of our maximum‐likelihood assignment test for identifying hybrid individuals using eight microsatellite loci instead of the original 18 loci and compare its performance to the Bayesian approach implemented in newhybrids. We obtained faecal DNA genotypes for 89 samples, 73 of which were matched to 23 known individuals. The performance of two sampling strategies — comprehensive sweep and opportunistic spot‐check was evaluated. The opportunistic spot‐check sampling strategy required less effort than the comprehensive sweep sampling strategy but identified fewer individuals. Six hybrids or coyotes were detected and five of these individuals were subsequently captured and removed from the population. The accuracy and power of the genetic test for coyote ancestry is decreased when using eight loci; however, nonred wolf canids are identified with high frequency. This combination of molecular and traditional field‐based approaches has great potential for addressing the challenge of hybridization in other species and ecosystems.

Genetic rescue in Isle Royale wolves: genetic analysis and the collapse of the population

While genetic rescue is known to benefit population viability, the duration of that benefit is poorly understood. We document what appears to be the waning benefit of genetic rescue after approximately 2–3 generations for the wolf population in Isle Royale National Park. The fitness benefit of genetic rescue declined because of inbreeding and population abundance declined when the inbred individuals exhibited low reproduction and survival. Only detailed studies of other cases will reveal what aspects of these dynamics represent general features of genetic rescue. We also present evidence indicating that numerous past immigration events have likely gone undetected. This finding is of particular significance because the Isle Royale wolf population has maintained good population viability for decades even though it was small and thought to be isolated from the mainland population of wolves. Past gene flow also suggests that human-assisted gene flow is necessary to conserve the ecosystem services associated with predation, since climate warming has reduced the frequency of ice bridges and with it the only opportunity for unassisted gene flow.

Helper effects on pup lifetime fitness in the cooperatively breeding red wolf (Canis rufus)

The evolutionary maintenance of cooperative breeding systems is thought to be a function of relative costs and benefits to breeders, helpers and juveniles. Beneficial effects of helpers on early-life survivorship and performance have been established in several species, but lifetime fitness benefits and/or costs of being helped remain unclear, particularly for long-lived species. We tested for effects of helpers on early- and late-life traits in a population of reintroduced red wolves (Canis rufus), while controlling for ecological variables such as home-range size and population density. We found that the presence of helpers in family groups was positively correlated with pup mass and survival at low population density, but negatively correlated with mass/size at high density, with no relation to survival. Interestingly, mass/size differences persisted into adulthood for both sexes. While the presence of helpers did not advance age at first reproduction for pups of either sex, females appeared to garner long-term fitness benefits from helpers through later age at last reproduction, longer reproductive lifespan and a greater number of lifetime reproductive events, which translated to higher lifetime reproductive success. In contrast, males with helpers exhibited diminished lifetime reproductive performance. Our findings suggest that while helper presence may have beneficial short-term effects in some ecological contexts, it may also incur long-term sex-dependent costs with critical ramifications for lifetime fitness.

Molecular species identification for multiple carnivores

Species identification is crucial for carnivore conservation and ecological studies. We present a simple molecular genetic test that amplifies DNA of 16 wild carnivore species from three continents. The test is based on co-amplification of two mitochondrial DNA fragments and scoring of the resulting species-specific size patterns. We evaluated the performance of this method using 332 known tissue, blood, hair and fecal samples from 23 carnivore and 11 potential prey species. Results demonstrate that this test can distinguish many Caniform species but not members of Felidae. The test can be performed with a single PCR and capillary sequencer run for cost-effective processing of large sample numbers typical of non-invasive genetic projects.

Evaluating the ability of Bayesian clustering methods to detect hybridization and introgression using an empirical red wolf data set

Bayesian clustering methods have emerged as a popular tool for assessing hybridization using genetic markers. Simulation studies have shown these methods perform well under certain conditions; however, these methods have not been evaluated using empirical data sets with individuals of known ancestry. We evaluated the performance of two clustering programs, baps and structure, with genetic data from a reintroduced red wolf (Canis rufus) population in North Carolina, USA. Red wolves hybridize with coyotes (C. latrans), and a single hybridization event resulted in introgression of coyote genes into the red wolf population. A detailed pedigree has been reconstructed for the wild red wolf population that includes individuals of 50–100% red wolf ancestry, providing an ideal case study for evaluating the ability of these methods to estimate admixture. Using 17 microsatellite loci, we tested the programs using different training set compositions and varying numbers of loci. structure was more likely than baps to detect an admixed genotype and correctly estimate an individuals true ancestry composition. However, structure was more likely to misclassify a pure individual as a hybrid. Both programs were outperformed by a maximum‐likelihood‐based test designed specifically for this system, which never misclassified a hybrid (50–75% red wolf) as a red wolf or vice versa. Training set composition and the number of loci both had an impact on accuracy but their relative importance varied depending on the program. Our findings demonstrate the importance of evaluating methods used for detecting admixture in the context of endangered species management.

Kin encounter rate and inbreeding avoidance in canids

Mating with close kin can lead to inbreeding depression through the expression of recessive deleterious alleles and loss of heterozygosity. Mate selection may be affected by kin encounter rate, and inbreeding avoidance may not be uniform but associated with age and social system. Specifically, selection for kin recognition and inbreeding avoidance may be more developed in species that live in family groups or breed cooperatively. To test this hypothesis, we compared kin encounter rate and the proportion of related breeding pairs in noninbred and highly inbred canid populations. The chance of randomly encountering a full sib ranged between 1–8% and 20–22% in noninbred and inbred canid populations, respectively. We show that regardless of encounter rate, outside natal groups mates were selected independent of relatedness. Within natal groups, there was a significant avoidance of mating with a relative. Lack of discrimination against mating with close relatives outside packs suggests that the rate of inbreeding in canids is related to the proximity of close relatives, which could explain the high degree of inbreeding depression observed in some populations. The idea that kin encounter rate and social organization can explain the lack of inbreeding avoidance in some species is intriguing and may have implications for the management of populations at risk.

Reevaluating and Broadening the Definition of Genetic Rescue

A widely accepted definition of genetic rescue from the classic review by Tallmon et al. (2004) is: “when population fitness, inferred from some demographic vital rate or phenotypic trait, increases by more than can be attributed to the demographic contribution of immigrants.” However, this definition limits assessment of the success of genetic rescue, decreases the probability of detecting benefits of genetic rescue, and thus restricts application of genetic rescue in increasing the viability of endangered species and populations. More specifically, there is reason to expect the demographic benefits of genetic rescue may be masked if environmental conditions are limiting or deteriorating, a situation that may have been previously overlooked because instances of genetic rescue receiving the most attention involve environmental conditions that permitted population expansion (Bouzat et al. 2009; Hedrick & Fredrickson 2010). As a result, one needs to consider more carefully the definition of, and standards for, assessing genetic rescue and include documentation of genetic change in ancestry following genetic rescue. The loss of genetic viability is a critical and widespread threat to threatened and endangered species and populations. Genetic rescue, which entails the introduction of unrelated individuals, is a potentially important tool to increase the viability of such populations (Tallmon et al. 2004). Despite its potential, <10 cases of genetic rescue have been well documented (Adams et al. 2011). Obstacles to the collection of adequate data to assess genetic rescue are widely appreciated; however, there is likely an additional, unappreciated obstacle to assessing genetic rescue. That impediment is the very definition of genetic rescue and the standards used to judge whether genetic rescue has occurred. The need to reconsider the definition of genetic rescue is revealed when one compares how several well-studied populations have responded to the introduction of unrelated individuals. First, when Texas pumas were artificially introduced into the endangered Florida panther population (both are Puma concolor), the population expanded into new areas as it increased from around 25 to over 100 individuals in just over a decade (Johnson et al. 2010). Similarly, the Swedish wolf population (Canis lupus) naturally reestablished itself in the early 1980s with 2 migrants from Finland. The population consisted of a single pack until another male wolf immigrated into the population around 1990 (Vilà et al. 2003). As a result of this natural genetic-rescue event, the population quickly increased to over 200 individuals by populating previously unoccupied areas. In contrast, the immigration of an individual into the wolf (Canis lupus) population on Isle Royale (U.S.A.), an island in Lake Superior (Adams et al. 2011), was not followed by an increase in abundance, a demographic measure of population fitness. Prior to the immigration event, every portion of Isle Royale had been occupied by a wolf pack for approximately 50 years; thus, there was no opportunity for the population to increase by expanding into available habitat. Second, deteriorating environmental conditions may be common to many populations of conservation concern and may obscure the demographic benefits of genetic rescue. For example, the immigration of the male wolf to Isle Royale coincided with a substantial decline in moose (Alces alces), their main prey. In contrast, prey was plentiful after the genetic-rescue events for Florida panthers and Swedish wolves. Third, for both Florida panthers and Swedish wolves genetic changes associated with genetic rescue were confirmed by detailed pedigree analysis with molecular genetic markers. The ancestral contribution of the 5 successfully contributing Texas pumas in the total Florida panther population was approximately 41% (Johnson et al. 2010), and the contribution from the Swedish wolf immigrant was around 30% (Liberg et al. 2005). The 1997 immigration of the male wolf to Isle Royale was only discovered in 2009 in an examination of molecular data. Subsequent pedigree analysis revealed the fitness of this immigrant so exceeded that of native wolves that