Spatial genetic and morphologic structure of wolves and coyotes in relation to environmental heterogeneity in a Canis hybrid zone

Eastern wolves have hybridized extensively with coyotes and gray wolves and are listed as a ‘species of special concern’ in Canada. However, a distinct population of eastern wolves has been identified in Algonquin Provincial Park (APP) in Ontario. Previous studies of the diverse Canis hybrid zone adjacent to APP have not linked genetic analysis with field data to investigate genotype‐specific morphology or determine how resident animals of different ancestry are distributed across the landscape in relation to heterogeneous environmental conditions. Accordingly, we studied resident wolves and coyotes in and adjacent to APP to identify distinct Canis types, clarify the extent of the APP eastern wolf population beyond the park boundaries and investigate fine‐scale spatial genetic structure and landscape–genotype associations in the hybrid zone. We documented three genetically distinct Canis types within the APP region that also differed morphologically, corresponding to putative gray wolves, eastern wolves and coyotes. We also documented a substantial number of hybrid individuals (36%) that were admixed between 2 or 3 of the Canis types. Breeding eastern wolves were less common outside of APP, but occurred in some unprotected areas where they were sympatric with a diverse combination of coyotes, gray wolves and hybrids. We found significant spatial genetic structure and identified a steep cline extending west from APP where the dominant genotype shifted abruptly from eastern wolves to coyotes and hybrids. The genotypic pattern to the south and northwest was a more complex mosaic of alternating genotypes. We modelled genetic ancestry in response to prey availability and human disturbance and found that individuals with greater wolf ancestry occupied areas of higher moose density and fewer roads. Our results clarify the structure of the Canis hybrid zone adjacent to APP and provide unique insight into environmental conditions influencing hybridization dynamics between wolves and coyotes.     

Agouti sequence polymorphisms in coyotes, wolves and dogs suggest hybridization

Domestic dogs have been shown to have multiple alleles of the Agouti Signal Peptide (ASIP) in exon 4 and we wished to determine the level of polymorphism in the common wild canids of Canada, wolves and coyotes, in comparison. All Canadian coyotes and most wolves have banded hairs. The ASIP coding sequence of the wolf did not vary from the domestic dog but one variant was detected in exon 4 of coyotes that did not alter the arginine at this position. Two other differences were found in the sequence flanking exon 4 of coyotes compared with the 45 dogs and 1 wolf. The coyotes also demonstrated a relatively common polymorphism in the 3' UTR sequence that could be used for population studies. One of the ASIP alleles (R96C) in domestic dogs causes a solid black coat color in homozygotes. Although some wolves are melanistic, this phenotype does not appear to be caused by this same mutation. However, one wolf, potentially a dog-wolf hybrid or descendant thereof, was heterozygous for this allele. Likewise 2 coyotes, potentially dog-coyote or wolf-coyote hybrid descendants, were heterozygous for the several polymorphisms in and flanking exon 4. We could conclude that these were coyote-dog hybrids because both were heterozygous for 2 mutations causing fawn coat color in dogs.     

Y‐chromosome evidence supports asymmetric dog introgression into eastern coyotes

Hybridization has played an important role in the evolutionary history of Canis species in eastern North America. Genetic evidence of coyote–dog hybridization based on mitochondrial DNA (mtDNA) is lacking compared to that based on autosomal markers. This discordance suggests dog introgression into coyotes has potentially been male biased, but this hypothesis has not been formally tested. Therefore, we investigated biparentally, maternally, and paternally inherited genetic markers in a sample of coyotes and dogs from southeastern Ontario to assess potential asymmetric dog introgression into coyotes. Analysis of autosomal microsatellite genotypes revealed minimal historical and contemporary admixture between coyotes and dogs. We observed only mutually exclusive mtDNA haplotypes in coyotes and dogs, but we observed Y‐chromosome haplotypes (Y‐haplotypes) in both historical and contemporary coyotes that were also common in dogs. Species‐specific Zfy intron sequences of Y‐haplotypes shared between coyotes and dogs confirmed their homology and indicated a putative origin from dogs. We compared Y‐haplotypes observed in coyotes, wolves, and dogs profiled in multiple studies, and observed that the Y‐haplotypes shared between coyotes and dogs were either absent or rare in North American wolves, present in eastern coyotes, but absent in western coyotes. We suggest the eastern coyote has experienced asymmetric genetic introgression from dogs, resulting from predominantly historical hybridization with male dogs and subsequent backcrossing of hybrid offspring with coyotes. We discuss the temporal and spatial dynamics of coyote–dog hybridization and the conditions that may have facilitated the introgression of dog Y‐chromosomes into coyotes. Our findings clarify the evolutionary history of the eastern coyote.     

Sympatric wolf and coyote populations of the western Great Lakes region are reproductively isolated

Interpretation of the genetic composition and taxonomic history of wolves in the western Great Lakes region (WGLR) of the United States has long been debated and has become more important to their conservation given the recent changes in their status under the Endangered Species Act. Currently, the two competing hypotheses on WGLR wolves are that they resulted from hybridization between (i) grey wolves (Canis lupus) and western coyotes (C. latrans) or (ii) between grey wolves and eastern wolves (C. lycaon). We performed a genetic analysis of sympatric wolves and coyotes from the region to assess the degree of reproductive isolation between them and to clarify the taxonomic status of WGLR wolves. Based on data from maternal, paternal and bi‐parental genetic markers, we demonstrate a clear genetic distinction between sympatric wolves and coyotes and conclude that they are reproductively isolated and that wolf–coyote hybridization in the WGLR is uncommon. The data reject the hypothesis that wolves in the WGLR derive from hybridization between grey wolves and western coyotes, and we conclude that the extant WGLR wolf population is derived from hybridization between grey wolves and eastern wolves. Grey‐eastern wolf hybrids (C. lupus × lycaon) comprise a substantial population that extends across Michigan, Wisconsin, Minnesota and western Ontario. These findings have important implications for the conservation and management of wolves in North America, specifically concerning the overestimation of grey wolf numbers in the United States and the need to address policies for hybrids.     

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.     

Size‐assortative choice and mate availability influences hybridization between red wolves (Canis rufus) and coyotes (Canis latrans)

Anthropogenic hybridization of historically isolated taxa has become a primary conservation challenge for many imperiled species. Indeed, hybridization between red wolves (Canis rufus) and coyotes (Canis latrans) poses a significant challenge to red wolf recovery. We considered seven hypotheses to assess factors influencing hybridization between red wolves and coyotes via pair‐bonding between the two species. Because long‐term monogamy and defense of all‐purpose territories are core characteristics of both species, mate choice has long‐term consequences. Therefore, red wolves may choose similar‐sized mates to acquire partners that behave similarly to themselves in the use of space and diet. We observed multiple factors influencing breeding pair formation by red wolves and found that most wolves paired with similar‐sized conspecifics and wolves that formed congeneric pairs with nonwolves (coyotes and hybrids) were mostly female wolves, the smaller of the two sexes. Additionally, we observed that lower red wolf abundance relative to nonwolves and the absence of helpers increased the probability that wolves consorted with nonwolves. However, successful pairings between red wolves and nonwolves were associated with wolves that maintained small home ranges. Behaviors associated with territoriality are energetically demanding and behaviors (e.g., aggressive interactions, foraging, and space use) involved in maintaining territories are influenced by body size. Consequently, we propose the hypothesis that size disparities between consorting red wolves and coyotes influence positive assortative mating and may represent a reproductive barrier between the two species. We offer that it may be possible to maintain wild populations of red wolves in the presence of coyotes if management strategies increase red wolf abundance on the landscape by mitigating key threats, such as human‐caused mortality and hybridization with coyotes. Increasing red wolf abundance would likely restore selection pressures that increase mean body and home‐range sizes of red wolves and decrease hybridization rates via reduced occurrence of congeneric pairs.     

Degree of Behavioral Neoteny Differentiates Canid Polymorphs

As with juvenile wolves or coyotes, adult livestock conducting dogs displayed the first-half segment of a functional predatory system of motor patterns and did not express play or social bonding toward sheep; whereas, like wolf or coyote pups, adult livestock protecting dogs displayed sequences of mixed social, submissive, play and investigatory motor patterns and rarely expressed during ontogeny (even when fully adult) predatory behaviors. The most parsimonious explanation of our findings is that behavioral differences in the two types of livestock dogs are a case of selected differential retardation (neoteny) of ancestral motor pattern development.     

Inter-specific territoriality in a Canis hybrid zone: spatial segregation between wolves, coyotes, and hybrids

Gray wolves (Canis lupus) and coyotes (Canis latrans) generally exhibit intraspecific territoriality manifesting in spatial segregation between adjacent packs. However, previous studies have found a high degree of interspecific spatial overlap between sympatric wolves and coyotes. Eastern wolves (Canis lycaon) are the most common wolf in and around Algonquin Provincial Park (APP), Ontario, Canada and hybridize with sympatric gray wolves and coyotes. We hypothesized that all Canis types (wolves, coyotes, and hybrids) exhibit a high degree of spatial segregation due to greater genetic, morphologic, and ecological similarities between wolves and coyotes in this hybrid system compared with western North American ecosystems. We used global positioning system telemetry and probabilistic measures of spatial overlap to investigate spatial segregation between adjacent Canis packs. Our hypothesis was supported as: (1) the probability of locating wolves, coyotes, and hybrids within home ranges ( $\bar{x}$  = 0.05) or core areas ( $\bar{x}$  < 0.01) of adjacent packs was low; and (2) the amount of shared space use was negligible. Spatial segregation did not vary substantially in relation to genotypes of adjacent packs or local environmental conditions (i.e., harvest regulations or road densities). We provide the first telemetry-based demonstration of spatial segregation between wolves and coyotes, highlighting the novel relationships between Canis types in the Ontario hybrid zone relative to areas where wolves and coyotes are reproductively isolated. Territoriality among Canis may increase the likelihood of eastern wolves joining coyote and hybrid packs, facilitate hybridization, and could play a role in limiting expansion of the genetically distinct APP eastern wolf population.     

Genetic outcomes of wolf recovery in the western Great Lakes states

Conflicting interpretations of the influence of coyote hybridization on wolf recovery in the western Great Lakes (WGL) states have stemmed from disagreement over the systematics of North American wolves. Questions regarding their recovery status have resulted. We addressed these issues with phylogenetic and admixture analysis of DNA profiles of western wolves, WGL states wolves and Wisconsin coyotes developed from autosome and Y-chromosome microsatellites and mitochondrial DNA control region sequence. Hybridization was assessed by comparing the haplotypes exhibited by sympatric wolves and coyotes. Genetic variability and connectivity were also examined. These analyses support the recognition of Canis lycaon as a unique species of North American wolf present in the WGL states and found evidence of hybridization between C. lupus and C. lycaon but no evidence of recent hybridization with sympatric coyotes. The recolonized WGL states wolves are genetically similar to historical wolves from the region and should be considered restored.     

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.     

Sequence analysis of three pigmentation genes in the Newfoundland population of Canis latrans links the Golden Retriever Mc1r variant to white coat color in coyotes

Three genes, Mc1r, Agouti, and CBD103, interact in a type-switching process that controls much of the pigmentation variation observed in mammals. A deletion in the CBD103 gene is responsible for dominant black color in dogs, while the white-phased black bear (“spirit bear”) of British Columbia, Canada, is the lightest documented color variant caused by a mutation in Mc1r. Rare all-white animals have recently been discovered in a new northeastern population of the coyote in insular Newfoundland and Labrador, Canada. To investigate the causative gene and mutation of white coat in coyotes, we sequenced the three type-switching genes in white and dark-phased animals from Newfoundland. The only sequence variants unambiguously associated with white color were in Mc1r, and one of these variants causes the amino acid variant R306Ter, a premature stop codon also linked to coat color in Golden Retrievers and other dogs with yellow/red coats. The allele carrying R306Ter in coyotes matches that in the Golden Retriever at other variable amino acid sites and hence may have originated in these dogs. Coyotes experienced introgression with wolves and dogs as they colonized northeastern North America, and coyote/Golden Retriever interactions have been observed in Newfoundland. We speculate that natural selection, with or without a founder effect, may contribute to the observed frequency of white coyotes in Newfoundland, as it has contributed to the high frequency of white bears, and of a domestic dog-derived CBD allele in gray wolves.     

The influence of social and endocrine factors on urine-marking by captive wolves (Canis lupus)

Although serum hormones varied seasonally in all adult animals, only dominant male and female wolves urine-marked. Serum testosterone and urine-marking rates, which increased during the fall/winter breeding season, were positively correlated in both male and female dominant wolves. Estradiol, which increased in conjunction with proestrus and estrus, was not correlated with female urinemarking. These findings suggest that hormonal influence on urine-marking in the wolf is modulated by social factors and contrast with those for both domestic dogs and coyotes, two other members of the genus Canis.     

Optimal predator management for mountain sheep conservation depends on the strength of mesopredator release

Large predators often suppress ungulate population growth, but they may also suppress the abundance of smaller predators that prey on neonatal ungulates. Antagonistic interactions among predators may therefore need to be integrated into predator–prey models to effectively manage ungulate–predator systems. We present a modeling framework that examines the net impact of interacting predators on the population growth rate of shared prey, using interactions among wolves Canis lupus, coyotes Canis latrans and Dall sheep Ovis dalli dalli as a case study. Wolf control is currently employed on approximately 16 million ha in Alaska to increase the abundance of ungulates for human harvest. We hypothesized that the positive effects of wolf control on Dall sheep population growth could be counteracted by increased levels of predation by coyotes. Coyotes and Dall sheep adult females (ewes) and lambs were radiocollared in the Alaska Range from 1999–2005 to estimate fecundity, age‐specific survival rates, and causes of mortality in an area without wolf control. We used stage‐structured population models to simulate the net effect of wolf control on Dall sheep population growth (λ). Our models accounted for stage‐specific predation rates by wolves and coyotes, compensatory mortality, and the potential release of coyote populations due to wolf control. Wolves were the main predators of ewes, coyotes were the main predators of lambs, and wolves were the main source of mortality for coyotes. Population models predicted that wolf control could increase sheep λ by 4% per year in the absence of mesopredator release. However, if wolf control released coyote populations, our models predicted that sheep λ could decrease by up to 3% per year. These results highlight the importance of integrating antagonistic interactions among predators into predator–prey models, because the net effect of predator management on shared prey can depend critically on the strength of mesopredator release.     

Comparison of Mexican Wolf and Coyote Diets in Arizona and New Mexico

ABSTRACT Interactions between wolves (Canis lupus) and coyotes (C. latrans) can have significant impacts on their distribution and abundance. We compared diets of recently translocated Mexican wolves (C. l. baileyi) with diets of resident coyotes in Arizona and New Mexico, USA. We systematically collected scats during 2000 and 2001. Coyote diet was composed mostly of mammalian species, followed by vegetation and insects. Elk (Cervus elaphus) was the most common item in coyote scats. Mexican wolf diet had a higher proportion of large mammals and fewer small mammals than coyote diet; however, elk was also the most common food item in Mexican wolf scats. Our results suggest that Mexican wolf diet was more similar to coyote diet than previously reported, but coyotes had more seasonal variation. Considering results in other areas, we expect that Mexican wolves will have a negative impact on coyotes through direct mortality and possibly competition. Reintroduction of Mexican wolves may have great impacts on communities by changing relationships among other predators and their prey.     

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.     

Characterization of 59 canine single nucleotide polymorphisms in the Italian wolf (Canis lupus) population

We characterized 59 canine single nucleotide polymorphisms (SNPs) in the endangered Italian wolf (Canis lupus) population, which were discovered by resequencing sequence‐tagged‐site (STS) DNA sequences that are known to contain SNPs in domestic dogs. Dog SNPs were usually found also in wolves. Additional SNPs unique in dogs or wolves were discovered, which is important for detecting hybrids between dogs and wolves. We developed new primer sets and analysed 15 SNPs by Pyrosequencing. The characterized SNPs will provide an important addition to the genetic markers that are currently available for studying wild populations of canids.     

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.     

Polymorphism of transferrin and esterase in Alaskan wolves: evidence of close molecular homology with the dog

By the use of isoelectric focusing in polyacrylamide gels serum samples from 146 Alaskan wolves were studied with regard to transferrin (Tf) and esterase (ArE) polymorphism, comparing the phenotypic band patterns with those of selected Norwegian dogs. The study revealed Tf and ArE polymorphisms in the wolf with phenotypic band patterns being indistinguishable from the corresponding ones in dogs. This suggests the occurrence of the same two common Tf alleles in the wolf as in the dog. In the ArE system the results are consistent with the occurrence of three alleles which also occur in dogs whereas a fourth allele, so far not seen in dogs, is seen in Alaskan wolves.     

Bucking the Trend in Wolf-Dog Hybridization: First Evidence from Europe of Hybridization between Female Dogs and Male Wolves

Studies on hybridization have proved critical for understanding key evolutionary processes such as speciation and adaptation. However, from the perspective of conservation, hybridization poses a concern, as it can threaten the integrity and fitness of many wild species, including canids. As a result of habitat fragmentation and extensive hunting pressure, gray wolf (Canis lupus) populations have declined dramatically in Europe and elsewhere during recent centuries. Small and fragmented populations have persisted, but often only in the presence of large numbers of dogs, which increase the potential for hybridization and introgression to deleteriously affect wolf populations. Here, we demonstrate hybridization between wolf and dog populations in Estonia and Latvia, and the role of both genders in the hybridization process, using combined analysis of maternal, paternal and biparental genetic markers. Eight animals exhibiting unusual external characteristics for wolves - six from Estonia and two from Latvia - proved to be wolf-dog hybrids. However, one of the hybridization events was extraordinary. Previous field observations and genetic studies have indicated that mating between wolves and dogs is sexually asymmetrical, occurring predominantly between female wolves and male dogs. While this was also the case among the Estonian hybrids, our data revealed the existence of dog mitochondrial genomes in the Latvian hybrids and, together with Y chromosome and autosomal microsatellite data, thus provided the first evidence from Europe of mating between male wolves and female dogs. We discuss patterns of sexual asymmetry in wolf-dog hybridization.