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.     

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.     

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 individual's 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.     

Mitochondrial DNA restriction-fragment-length monomorphism in the Italian wolf (Canis lupus) population

Mitochondrial-DNA (mtDNA) restriction patterns were studied in 22 wolves (Canis lupus) sampled in central-northern Italy. A total of 60 restriction sites were detected, encompassing about 2 % of the mitochondrial genome of canids. All wolves showed the same restriction pattern. Therefore, a single mtDNA haplotype was detected in the Italian wolf population. Historical information on peninsular isolation and demographic decline suggest that low genetically effective population size and random drift may have strongly reduced the mtDNA variability of wolves in Italy over the last 100–200 years. A different mtDNA restriction pattern in feral dogs sampled from a wolf range in central Italy was detected. These findings suggest that the hybridization and introgression of female dog genomes into the Italian wolf population may be rare or absent.     

Y‐chromosome evidence supports widespread signatures of three‐species Canis hybridization in eastern North America

There has been considerable discussion on the origin of the red wolf and eastern wolf and their evolution independent of the gray wolf. We analyzed mitochondrial DNA (mtDNA) and a Y‐chromosome intron sequence in combination with Y‐chromosome microsatellites from wolves and coyotes within the range of extensive wolf–coyote hybridization, that is, eastern North America. The detection of divergent Y‐chromosome haplotypes in the historic range of the eastern wolf is concordant with earlier mtDNA findings, and the absence of these haplotypes in western coyotes supports the existence of the North American evolved eastern wolf (Canis lycaon). Having haplotypes observed exclusively in eastern North America as a result of insufficient sampling in the historic range of the coyote or that these lineages subsequently went extinct in western geographies is unlikely given that eastern‐specific mtDNA and Y‐chromosome haplotypes represent lineages divergent from those observed in extant western coyotes. By combining Y‐chromosome and mtDNA distributional patterns, we identified hybrid genomes of eastern wolf, coyote, gray wolf, and potentially dog origin in Canis populations of central and eastern North America. The natural contemporary eastern Canis populations represent an important example of widespread introgression resulting in hybrid genomes across the original C. lycaon range that appears to be facilitated by the eastern wolf acting as a conduit for hybridization. Applying conventional taxonomic nomenclature and species‐based conservation initiatives, particularly in human‐modified landscapes, may be counterproductive to the effective management of these hybrids and fails to consider their evolutionary potential.     

Origin and status of the Great Lakes wolf

An extensive debate concerning the origin and taxonomic status of wolf-like canids in the North American Great Lakes region and the consequences for conservation politics regarding these enigmatic predators is ongoing. Using maternally, paternally and biparentally inherited molecular markers, we demonstrate that the Great Lakes wolves are a unique population or ecotype of gray wolves. Furthermore, we show that the Great Lakes wolves experienced high degrees of ancient and recent introgression of coyote and western gray wolf mtDNA and Y-chromosome haplotypes, and that the recent demographic bottleneck caused by persecution and habitat depletion in the early 1900s is not reflected in the genetic data.     

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

The US Fish and Wildlife Service's (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 km(2) 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 km(2)) 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.     

Detecting introgressive hybridization between free-ranging domestic dogs and wild wolves (Canis lupus) by admixture linkage disequilibrium analysis

Occasional crossbreeding between free-ranging domestic dogs and wild wolves (Canis lupus) has been detected in some European countries by mitochondrial DNA sequencing and genotyping unlinked microsatellite loci. Maternal and unlinked genomic markers, however, might underestimate the extent of introgressive hybridization, and their impacts on the preservation of wild wolf gene pools. In this study, we genotyped 220 presumed Italian wolves, 85 dogs and 7 known hybrids at 16 microsatellites belonging to four different linkage groups (plus four unlinked microsatellites). Population clustering and individual assignments were performed using a Bayesian procedure implemented in structure 2.1, which models the gametic disequilibrium arising between linked loci during admixtures, aiming to trace hybridization events further back in time and infer the population of origin of chromosomal blocks. Results indicate that (i) linkage disequilibrium was higher in wolves than in dogs; (ii) 11 out of 220 wolves (5.0%) were likely admixed, a proportion that is significantly higher than one admixed genotype in 107 wolves found previously in a study using unlinked markers; (iii) posterior maximum-likelihood estimates of the recombination parameter r revealed that introgression in Italian wolves is not recent, but could have continued for the last 70 (+/- 20) generations, corresponding to approximately 140-210 years. Bayesian clustering showed that, despite some admixture, wolf and dog gene pools remain sharply distinct (the average proportions of membership to wolf and dog clusters were Q(w) = 0.95 and Q(d) = 0.98, respectively), suggesting that hybridization was not frequent, and that introgression in nature is counteracted by behavioural or selective constraints.     

Molecular genetics of the most endangered canid: the Ethiopian wolf Canis simensis

The world's most endangered canid is the Ethiopian wolf Canis simensis , which is found in six isolated areas of the Ethiopian highlands with a total population of no more than 500 individuals. Ethiopian wolf populations are declining due to habitat loss and extermination by humans. Moreover, in at least one population, Ethiopian wolves are sympatric with domestic dogs, which may hybridize with them, compete for food, and act as disease vectors. Using molecular techniques, we address four questions concerning Ethiopian wolves that have conservation implications. First, we determine the relationships of Ethiopian wolves to other wolf-like canids by phylogenetic analysis of 2001 base pairs of mitochondrial DNA (mtDNA) sequence. Our results suggest that the Ethiopian wolf is a distinct species more closely related to gray wolves and coyotes than to any African canid. The mtDNA sequence similarity with gray wolves implies that the Ethiopian wolf may hybridize with domestic dogs, a recent derivative of the gray wolf. We examine this possibility through mtDNA restriction fragment analysis and analysis of nine microsatellite loci in populations of Ethiopian wolves. The results imply that hybridization has occurred between female Ethiopian wolves and male domestic dogs in one population. Finally, we assess levels of variability within and between two Ethiopian wolf populations. Although these closely situated populations are not differentiated, the level of variability in both is low, suggesting long-term effective population sizes of less than a few hundred individuals. We recommend immediate captive breeding of Ethiopian wolves to protect their gene pool from dilution and further loss of genetic variability.     

A new mitochondrial haplotype confirms the distinctiveness of the Italian wolf (Canis lupus) population

In the past century the Italian wolf has been repeatedly indicated as a distinct subspecies, Canis lupus italicus, due to its unique morphology and its distinctive mtDNA control region (CR) monomorphism. However, recent studies on wolf x dog hybridization in Italy documented the presence of a second mtDNA CR haplotype (W16), previously found only in wolves from Eastern Europe, casting doubts on the genetic uniqueness of the Italian wolves. To test whether this second haplotype belongs to the Italian wolf population, we genotyped 92 wolf DNA samples from Italy, Slovenia, Greece and Bulgaria at four mtDNA regions (control-region, ATP6, COIII and ND4 genes) and at 39 autosomal microsatellites. Results confirm the presence of two mtDNA multi-fragment haplotypes (WH14 and WH19) in the Italian wolves, distinct from all the other European wolves. Network analyses of the multi-fragment mtDNA haplotypes identified two strongly differentiated clades, with the Italian wolf WH14 and WH19 multi-fragment haplotypes rooted together. Finally, Bayesian clustering clearly assigned all the wolves sampled in Italy to the Italian population, regardless of the two different multi-fragment haplotypes. These results demonstrate that the W16 CR haplotype is part of the genetic pool of the Italian wolf population, reconfirming its distinctiveness from other European wolves. Overall, considering the presence of unique mtDNA and Y-linked haplotypes, the sharply different frequencies of genome-wide autosomal alleles and the distinct morphological features of Italian wolves, we believe that this population should be considered a distinct subspecies.     

Assessment of coyote–wolf–dog admixture using ancestry‐informative diagnostic SNPs

The evolutionary importance of hybridization as a source of new adaptive genetic variation is rapidly gaining recognition. Hybridization between coyotes and wolves may have introduced adaptive alleles into the coyote gene pool that facilitated an expansion in their geographic range and dietary niche. Furthermore, hybridization between coyotes and domestic dogs may facilitate adaptation to human‐dominated environments. We genotyped 63 ancestry‐informative single‐nucleotide polymorphisms in 427 canids to examine the prevalence, spatial distribution and the ecology of admixture in eastern coyotes. Using multivariate methods and Bayesian clustering analyses, we estimated the relative contributions of western coyotes, western and eastern wolves, and domestic dogs to the admixed ancestry of Ohio and eastern coyotes. We found that eastern coyotes form an extensive hybrid swarm, with all our samples having varying levels of admixture. Ohio coyotes, previously thought to be free of admixture, are also highly admixed with wolves and dogs. Coyotes in areas of high deer density are genetically more wolf‐like, suggesting that natural selection for wolf‐like traits may result in local adaptation at a fine geographic scale. Our results, in light of other previously published studies of admixture in Canis, revealed a pattern of sex‐biased hybridization, presumably generated by male wolves and dogs mating with female coyotes. This study is the most comprehensive genetic survey of admixture in eastern coyotes and demonstrates that the frequency and scope of hybridization can be quantified with relatively few ancestry‐informative markers.     

Detection of an East European wolf haplotype puzzles mitochondrial DNA monomorphism of the Italian wolf population

Southern European wolves suffered from reiterated population declines during glacial periods and historically due to human persecution. Differently from other European wolf populations, a single mitochondrial DNA (mtDNA) control region haplotype (W14) has been so far described in the Italian wolves, although no intensive genetic sampling has ever been conducted in historical source populations from central and southern Italy. Using non-invasive genetic techniques, we report the occurrence of an unexpected mtDNA haplotype (W16) in the wolf population of the Abruzzo, Lazio and Molise National Park (PNALM), central Italy. This haplotype, detected in three out of 90 faecal samples from the PNALM, was previously reported in wolves from the North Carpathians, Slovakia and the Balkans only. Microsatellite analysis and molecular sex determination confirmed that the W16 samples belonged to three distinct wolves. Although alternative explanations can be formulated for the origin of this mtDNA haplotype in the otherwise monomorphic Italian wolf population, assignment procedures indicated the likely admixed ancestry of one W16 sample with East European wolves. Anthropogenic introgression with dogs has been detected in the Italian wolf population using nuclear DNA microsatellites, but no population-wide genetic survey had previously reported a mtDNA control region variant in Italian wolves. Our findings strongly suggest that, in addition to wolf × dog hybridization, captive-released wolves or wolf × dog hybrids may successfully interbreed with wolves in the wild, and that human-mediated introgression may occur even in well established protected areas.     

Major histocompatibility complex variation in red wolves: evidence for common ancestry with coyotes and balancing selection

We examined variation at a class II major histocompatibility complex (MHC) gene (DRB1) in the captive red wolf population and samples of coyotes from Texas and North Carolina. We found 4 alleles in the 48 red wolves, 8 alleles in the 10 coyotes from Texas and 15 alleles in the 29 coyotes from North Carolina. Two of the four alleles found in red wolves, Caru-2 and Caru-4, were found in both the Texas and North Carolina coyote samples. Allele Caru-1, previously found in gray wolves, was also found in the North Carolina sample. The most frequent red wolf allele, Caru-3, was not found in any of the coyote samples. However, an allele found in both the Texas and North Carolina coyote samples is only one nucleotide (one amino acid) different from this red wolf allele. Overall, it appears from examination of this MHC gene that red wolves are more closely related to coyotes than to gray wolves. There were a number of different types of evidence supporting the action of balancing selection in red wolves. Namely, there was: (i) an excess of heterozygotes compared with expectations; (ii) a higher rate of nonsynonymous than synonymous substitution for the functionally important antigen-binding site positions; (iii) an eight times higher average heterozygosity of individual amino acids at the positions identified as part of the antigen-binding site than those not associated with it; (iv) the amino acid divergence of four red wolf alleles was greater than that expected from a simulation of genetic drift; and (v) the distribution of alleles, and the distributions of amino acids at many positions were more even than expected from neutrality. Examination of the level and pattern of linkage disequilibria between pairs of sites suggest that the heterozygosity, substitution and frequencies at individual amino acids are not highly dependent upon each other.     

Combined use of maternal,paternal and bi-parental genetic markers for the identification of wolf-dog hybrids

The identification of hybrids is often a subject of primary concern for the development of conservation and management strategies, but can be difficult when the hybridizing species are closely related and do not possess diagnostic genetic markers. However, the combined use of mitochondrial DNA (mtDNA), autosomal and Y chromosome genetic markers may allow the identification of hybrids and of the direction of hybridization. We used these three types of markers to genetically characterize one possible wolf-dog hybrid in the endangered Scandinavian wolf population. We first characterized the variability of mtDNA and Y chromosome markers in Scandinavian wolves as well as in neighboring wolf populations and in dogs. While the mtDNA data suggested that the target sample could correspond to a wolf, its Y chromosome type had not been observed before in Scandinavian wolves. We compared the genotype of the target sample at 18 autosomal microsatellite markers with those expected in pure specimens and in hybrids using assignment tests. The combined results led to the conclusion that the animal was a hybrid between a Scandinavian female wolf and a male dog. This finding confirms that inter-specific hybridization between wolves and dogs can occur in natural wolf populations. A possible correlation between hybridization and wolf population density and disturbance deserves further research.     

Hybridization among three native North American Canis species in a region of natural sympatry

Background

Population densities of many species throughout the world are changing due to direct persecution as well as anthropogenic habitat modification. These changes may induce or increase the frequency of hybridization among taxa. If extensive, hybridization can threaten the genetic integrity or survival of endangered species. Three native species of the genus Canis, coyote (C. latrans), Mexican wolf (C. lupus baileyi) and red wolf (C. rufus), were historically sympatric in Texas, United States. Human impacts caused the latter two to go extinct in the wild, although they survived in captive breeding programs. Morphological data demonstrate historic reproductive isolation between all three taxa. While the red wolf population was impacted by introgressive hybridization with coyotes as it went extinct in the wild, the impact of hybridization on the Texas populations of the other species is not clear.

Methodology/ Principal Findings

We surveyed variation at maternally and paternally inherited genetic markers (mitochondrial control region sequence and Y chromosome microsatellites) in coyotes from Texas, Mexican wolves and red wolves from the captive breeding programs, and a reference population of coyotes from outside the historic red wolf range. Levels of variation and phylogenetic analyses suggest that hybridization has occasionally taken place between all three species, but that the impact on the coyote population is very small.

Conclusion/Significance

Our results demonstrate that the factors driving introgressive hybridization in sympatric Texan Canis are multiple and complex. Hybridization is not solely determined by body size or sex, and density-dependent effects do not fully explain the observed pattern either. No evidence of hybridization was identified in the Mexican wolf captive breeding program, but introgression appears to have had a greater impact on the captive red wolves.     

Detecting rare introgression of domestic dog genes into wild wolf (Canis lupus) populations by Bayesian admixture analyses of microsatellite variation

Hybridization with free-ranging dogs isthought to threat the genetic integrity ofwolves in Europe, although available mtDNA dataevidenced only sporadic cases of crossbreeding.Here we report results of population assignmentand genetic admixture analyses in 107wild-living Italian wolves, 95 dogs including30 different breeds and feral dogs, andcaptive-reared wolves of unknown or hybridorigins, which were genotyped at 18microsatellites. Two Italian wolves showedunusually dark coats (``black wolves'), and oneshowed a spur in both hindlegs (``fifth fingerwolf'), suggesting hybridization. Italianwolves showed significant deficit ofheterozygotes, positive F_IS values anddeviations from Hardy-Weinberg equilibrium.Genetic variability was significantlypartitioned between groups, suggesting thatwolves and dogs represent distinct gene pools.Multivariate ordination of individual genotypesand clustering of inter-individual geneticdistances split wolves and dogs into twodifferent clusters congruent with the priorphenotypic classification, but hybrids andwolves of unknown origin were not identifiedfrom genetic information alone. By contrast, aBayesian admixture analysis assigned all theItalian wolves and dogs to two differentclusters, independent of any prior phenotypicinformation, and simultaneously detected theadmixed gene composition of the hybrids, whichwere assigned to more than one cluster.Captive-reared wolves of unknown origin wereprevalently assigned to the Italian wolfpopulation. Admixture analyses showed that one``black wolf' had mixed ancestry in the dog genepool and could be a hybrid, while the other twowolves with unusual phenotypes were assigned tothe Italian wolf population.     

Estimating Admixture at the Population Scale: Taking Imperfect Detectability and Uncertainty in Hybrid Classification Seriously

Introgressive hybridization between domestic dogs and wolves (Canis lupus) represents an emblematic case of anthropogenic hybridization and is increasingly threatening the genomic integrity of wolf populations expanding into human-modified landscapes. But studies formally estimating prevalence and accounting for imperfect detectability and uncertainty in hybrid classification are lacking. Our goal was to present an approach to formally estimate the proportion of admixture by using a capture-recapture (CR) framework applied to individual multilocus genotypes detected from non-invasive samples collected from a protected wolf population in Italy. We scored individual multilocus genotypes using a panel of 12 microsatellites and assigned genotypes to reference wolf and dog populations through Bayesian clustering procedures. Based on 152 samples, our dataset comprised the capture histories of 39 individuals sampled in 7 wolf packs and was organized in bi-monthly sampling occasions (Aug 2015−May 2016). We fitted CR models using a multievent formulation to explicitly handle uncertainty in individual classification, and accordingly examined 2 model scenarios: one reflecting a traditional approach to classifying individuals (i.e., minimizing the misclassification of wolves as hybrids; Type 1 error), and the other using a more stringent criterion aimed to balance Type 1 and Type 2 error rates (i.e., the misclassification of hybrids as wolves). Compared to the sample proportion of admixed individuals in the dataset (43.6%), formally estimated prevalence was 50% under the first and 70% under the second scenario, with 71.4% and 85.7% of admixed packs, respectively. At the individual level, the proportion of dog ancestry in the wolf population averaged 7.8% (95% CI = 4.4−11%). Balancing between Type 1 and 2 error rates in assignment tests, our second scenario produced an estimate of prevalence 40% higher compared to the alternative scenario, corresponding to a 65% decrease in Type 2 and no increase in Type 1 error rates. Providing a formal and innovative estimation approach to assess prevalence in admixed wild populations, our study confirms previous population modeling indicating that reproductive barriers between wolves and dogs, or dilution of dog genes through backcrossing, should not be expected per se to prevent the spread of introgression. As anthropogenic hybridization is increasingly affecting animal species globally, our approach is of interest to a broader audience of wildlife conservationists and practitioners. © 2021 The Authors. The Journal of Wildlife Management published by Wiley Periodicals LLC on behalf of The Wildlife Society.     

An efficient method for screening faecal DNA genotypes and detecting new individuals and hybrids in the red wolf (<Emphasis Type="Italic">Canis rufus</Emphasis>) experimental population area

Previously, sequencing of mitochondrial DNA (mtDNA) from non-invasively collected faecal material (scat) has been used to help manage hybridization in the wild red wolf (Canis rufus) population. This method is limited by the maternal inheritance of mtDNA and the inability to obtain individual identification. Here, we optimize the use of nuclear DNA microsatellite markers on red wolf scat DNA to distinguish between individuals and detect hybrids. We develop a data filtering method in which scat genotypes are compared to known blood genotypes to reduce the number of PCR amplifications needed. We apply our data filtering method and the more conservative maximum likelihood ratio method (MLR) of Miller et al. (2002 Genetics 160:357–366) to a scat dataset previously screened for hybrids by sequencing of mtDNA. Using seven microsatellite loci, we obtained genotypes for 105 scats, which were matched to 17 individuals. The PCR amplification success rate was 50% and genotyping error rates ranged from 6.6% to 52.1% per locus. Our data filtering method produced comparable results to the MLR method, and decreased the time and cost of analysis by 25%. Analysis of this dataset using our data filtering method verified that no hybrid individuals were present in the Alligator River National Wildlife Refuge, North Carolina in 2000. Our results demonstrate that nuclear DNA microsatellite analysis of red wolf scats provides an efficient and accurate approach to screen for new individuals and hybrids.     

INTROGRESSION OF COYOTE MITOCHONDRIAL DNA INTO SYMPATRIC NORTH AMERICAN GRAY WOLF POPULATIONS

Mitochondrial DNA (mtDNA) genotypes of gray wolves and coyotes from localities throughout North America were determined using restriction fragment length polymorphisms. Of the 13 genotypes found among the wolves, 7 are clearly of coyote origin, indicating that genetic transfer of coyote mtDNA into wolf populations has occurred through hybridization. The transfer of mtDNA appears unidirectional from coyotes into wolves because no coyotes sampled have a wolf-derived mtDNA genotype. Wolves possessing coyote-derived genotypes are confined to a contiguous geographic region in Minnesota, Ontario, and Quebec, and the frequency of coyote-type mtDNA in these wolf populations is high (>50%). The ecological history of the hybrid zone suggests that hybridization is taking place in regions where coyotes have only recently become abundant following conversion of forests to farmlands. Dispersing male wolves unable to find conspecific mates may be pairing with female coyotes in deforested areas bordering wolf territories. Our results demonstrate that closely related species of mobile terrestrial vertebrates have the potential for extensive genetic exchange when ecological conditions change suddenly.