Linda Y. Rutledge

Genetic differentiation of eastern wolves in Algonquin Park despite bridging gene flow between coyotes and grey wolves.

Distinguishing genetically differentiated populations within hybrid zones and determining the mechanisms by which introgression occurs are crucial for setting effective conservation policy. Extensive hybridization among grey wolves (Canis lupus), eastern wolves (C. lycaon) and coyotes (C. latrans) in eastern North America has blurred species distinctions, creating a Canis hybrid swarm. Using complementary genetic markers, we tested the hypotheses that eastern wolves have acted as a conduit of sex-biased gene flow between grey wolves and coyotes, and that eastern wolves in Algonquin Provincial Park (APP) have differentiated following a history of introgression. Mitochondrial, Y chromosome and autosomal microsatellite genetic data provided genotypes for 217 canids from three geographic regions in Ontario, Canada: northeastern Ontario, APP and southern Ontario. Coyote mitochondrial DNA (mtDNA) haplotypes were common across regions but coyote-specific Y chromosome haplotypes were absent; grey wolf mtDNA was absent from southern regions, whereas grey wolf Y chromosome haplotypes were present in all three regions. Genetic structuring analyses revealed three distinct clusters within a genetic cline, suggesting some gene flow among species. In APP, however, 78.4% of all breeders and 11 of 15 known breeding pairs had assignment probability of Q⩾0.8 to the Algonquin cluster, and the proportion of eastern wolf Y chromosome haplotypes in APP breeding males was higher than expected from random mating within the park (P<0.02). The data indicate that Algonquin wolves remain genetically distinct despite providing a sex-biased genetic bridge between coyotes and grey wolves. We speculate that ongoing hybridization within the park is limited by pre-mating reproductive barriers.

Genetic Characterization of Eastern “Coyotes” in Eastern Massachusetts

Abstract This study examined the genetic nature and relatedness of Canis latrans (Coyotes) in eastern Massachusetts (i.e., eastern Coyotes). We characterized 67 animals at the mitochondrial DNA control region, and 55 of those at 8 microsatellite loci. Structure analysis and factorial correspondence analysis of the microsatellite genotypes indicated that the eastern Coyotes in Massachusetts clustered with other northeastern Canis populations and away from western Coyotes, C. lycaon (Eastern Wolves), and C. lupus (Gray Wolves). They contained mitochondrial haplotypes from both western Coyotes and Eastern Wolves, consistent with their hybrid origin from these two species. There was no evidence of either C. lupus familiaris (Domestic Dog) or Gray Wolf mitochondrial DNA in the animals. These results indicate that the eastern Coyote should more appropriately be termed “Coywolf” to reflect their hybrid (C. latrans x lycaon) origin. Genetic data were also used to assess parental and kinship relationships, and confirmed that family units typically contain an unrelated breeding pair and their offspring. Lastly, a synthesis of knowledge of the eastern Coyote as well as implications for Wolf recovery in the northeast US is provided.

Intense harvesting of eastern wolves facilitated hybridization with coyotes

Despite ethical arguments against lethal control of wildlife populations, culling is routinely used for the management of predators, invasive or pest species, and infectious diseases. Here, we demonstrate that culling of wildlife can have unforeseen impacts that can be detrimental to future conservation efforts. Specifically, we analyzed genetic data from eastern wolves (Canis lycaon) sampled in Algonquin Provincial Park (APP), Ontario, Canada from 1964 to 2007. Research culls in 1964 and 1965 killed the majority of wolves within a study region of APP, accounting for approximately 36% of the parks wolf population at a time when coyotes were colonizing the region. The culls were followed by a significant decrease in an eastern wolf mitochondrial DNA (mtDNA) haplotype (C1) in the Parks wolf population, as well as an increase in coyote mitochondrial and nuclear DNA. The introgression of nuclear DNA from coyotes, however, appears to have been curtailed by legislation that extended wolf protection outside park boundaries in 2001, although eastern wolf mtDNA haplotype C1 continued to decline and is now rare within the park population. We conclude that the wolf culls transformed the genetic composition of this unique eastern wolf population by facilitating coyote introgression. These results demonstrate that intense localized harvest of a seemingly abundant species can lead to unexpected hybridization events that encumber future conservation efforts. Ultimately, researchers need to contemplate not only the ethics of research methods, but also that future implications may be obscured by gaps in our current scientific understanding.

Genetic and morphometric analysis of sixteenth century Canis skull fragments: implications for historic eastern and gray wolf distribution in North America

Resolving the taxonomy and historic ranges of species are essential to recovery plans for species at risk and conservation programs that aim to restore extirpated populations. In eastern North America, planning for wolf population restoration is complicated by the disputed historic distributions of two wolf species: the Old World-evolved gray wolf (Canis lupus) and the New World-evolved eastern wolf (C. lycaon). We used genetic and morphometric data from 4- to 500-year-old Canis samples excavated in London, Ontario, Canada to help clarify the historic range of these two wolf species in the eastern temperate forests of North America. We isolated DNA and sequenced the mitochondrial control region and found that none of the samples were of gray wolf origin. Two of the DNA sequences corresponded to those found in present day coyotes (C. latrans), but morphometric comparisons show an eastern wolf, not coyote, origin. The remaining two sequences matched ancient domestic dog haplotypes. These results suggest that the New World-evolved eastern wolf, not the gray wolf, occupied this region prior to the arrival of European settlers, although eastern-gray wolf hybrids cannot be ruled out. Furthermore, our data support the idea of a shared common ancestry between eastern wolves and western coyotes, and that the distribution of gray wolves at this time probably did not include the eastern temperate forests of North America.

An Improved Field Method to Obtain DNA for Individual Identification From Wolf Scat

Abstract Sampling of feces for genetic studies of wild populations can be problematic because of the low quality and quantity of template DNA obtained. We used cotton swabs in the field to isolate the mucous layer on the surface of fresh wolf (Canis lupus, C. lycaon, and their hybrids) scats followed by immediate preservation, and compared microsatellite genotyping of DNA from these fresh field swabs (FS) to that of previously frozen laboratory swabs (LS). In single polymerase chain reactions (PCRs) of 2 multiplexes, amplification at 8 loci was higher in the FS samples (FS  =  50%, LS  =  15%; P  =  0.02) because proportion, quantity, and quality of large fragment wolf nuclear DNA from these samples was greater (2.5–25%, 6.25–62.5 ng/swab, 35% amplified at 1,000 base pairs [bp]) than from the LS samples (1.9%–10%, 4.7–25 ng/swab, 10% amplified at 1,000 bp). Paired blood and fresh field-swabbed samples had identical genotypes. In 84 multiplex PCRs we found no evidence of allelic dropout associated with low template quality or quantity. We conclude that field swabbing of fresh wolf scat facilitates field storage and reduces the need for multiple amplifications at single microsatellite loci, thereby reducing the genotyping costs for wildlife projects that use noninvasive samples.

Analysis of Canis mitochondrial DNA demonstrates high concordance between the control region and ATPase genes

BackgroundPhylogenetic studies of wild Canis species have relied heavily on the mitochondrial DNA control region (mtDNA CR) to infer species relationships and evolutionary lineages. Previous analyses of the CR provided evidence for a North American evolved eastern wolf (C. lycaon), that is more closely related to red wolves (C. rufus) and coyotes (C. latrans) than grey wolves (C. lupus). Eastern wolf origins, however, continue to be questioned. Therefore, we analyzed mtDNA from 89 wolves and coyotes across North America and Eurasia at 347 base pairs (bp) of the CR and 1067 bp that included the ATPase6 and ATPase8 genes. Phylogenies and divergence estimates were used to clarify the evolutionary history of eastern wolves, and regional comparisons of nonsynonomous to synonomous substitutions (dN/dS) at the ATPase6 and ATPase8 genes were used to elucidate the potential role of selection in shaping mtDNA geographic distribution.ResultsWe found high concordance across analyses between the mtDNA regions studied. Both had a high percentage of variable sites (CR = 14.6%; ATP = 9.7%) and both phylogenies clustered eastern wolf haplotypes monophyletically within a North American evolved lineage apart from coyotes. Divergence estimates suggest the putative red wolf sequence is more closely related to coyotes (DxyCR = 0.01982 ± 0.00494 SD; DxyATP = 0.00332 ± 0.00097 SD) than the eastern wolf sequences (DxyCR = 0.03047 ± 0.00664 SD; DxyATP = 0.00931 ± 0.00205 SD). Neutrality tests on both genes were indicative of the population expansion of coyotes across eastern North America, and dN/dS ratios suggest a possible role for purifying selection in the evolution of North American lineages. dN/dS ratios were higher in European evolved lineages from northern climates compared to North American evolved lineages from temperate regions, but these differences were not statistically significant.ConclusionsThese results demonstrate high concordance between coding and non-coding regions of mtDNA, and provide further evidence that the eastern wolf possessed distinct mtDNA lineages prior to recent coyote introgression. Purifying selection may have influenced North American evolved Canis lineages, but detection of adaptive selection in response to climate is limited by the power of current statistical tests. Increased sampling and development of alternative analytical tools will be necessary to disentangle demographic history from processes of natural selection.

RAD sequencing and genomic simulations resolve hybrid origins within North American Canis

Top predators are disappearing worldwide, significantly changing ecosystems that depend on top-down regulation. Conflict with humans remains the primary roadblock for large carnivore conservation, but for the eastern wolf (Canis lycaon), disagreement over its evolutionary origins presents a significant barrier to conservation in Canada and has impeded protection for grey wolves (Canis lupus) in the USA. Here, we use 127 235 single-nucleotide polymorphisms (SNPs) identified from restriction-site associated DNA sequencing (RAD-seq) of wolves and coyotes, in combination with genomic simulations, to test hypotheses of hybrid origins of Canis types in eastern North America. A principal components analysis revealed no evidence to support eastern wolves, or any other Canis type, as the product of grey wolf × western coyote hybridization. In contrast, simulations that included eastern wolves as a distinct taxon clarified the hybrid origins of Great Lakes-boreal wolves and eastern coyotes. Our results support the eastern wolf as a distinct genomic cluster in North America and help resolve hybrid origins of Great Lakes wolves and eastern coyotes. The data provide timely information that will shed new light on the debate over wolf conservation in eastern North America.

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.

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.

Comment on “Whole-genome sequence analysis shows two endemic species of North American wolf are admixtures of the coyote and gray wolf”

Whole-genome data do not support a recent hybrid origin for red and eastern wolves.