Tracing Eastern Wolf Origins From Whole-Genome Data in Context of Extensive Hybridization.

Southeastern Canada is inhabited by an amalgam of hybridizing wolf-like canids, raising fundamental questions regarding their taxonomy, origins, and timing of hybridization events. Eastern wolves (Canis lycaon), specifically, have been the subject of significant controversy, being viewed as either a distinct taxonomic entity of conservation concern or a recent hybrid of coyotes (C. latrans) and grey wolves (C. lupus). Mitochondrial DNA analyses show some evidence of eastern wolves being North American evolved canids. In contrast, nuclear genome studies indicate eastern wolves are best described as a hybrid entity, but with unclear timing of hybridization events. To test hypotheses related to these competing findings we sequenced whole genomes of 25 individuals, representative of extant Canadian wolf-like canid types of known origin and levels of contemporary hybridization. Here we present data describing eastern wolves as a distinct taxonomic entity that evolved separately from grey wolves for the past ∼67,000 years with an admixture event with coyotes ∼37,000 years ago. We show that Great Lakes wolves originated as a product of admixture between grey wolves and eastern wolves after the last glaciation (∼8,000 years ago) while eastern coyotes originated as a product of admixture between "western" coyotes and eastern wolves during the last century. Eastern wolf nuclear genomes appear shaped by historical and contemporary gene flow with grey wolves and coyotes, yet evolutionary uniqueness remains among eastern wolves currently inhabiting a restricted range in southeastern Canada.

City divided: Unveiling family ties and genetic structuring of coyotes in Seattle.

Linear barriers pose significant challenges for wildlife gene flow, impacting species persistence, adaptation, and evolution. While numerous studies have examined the effects of linear barriers (e.g., fences and roadways) on partitioning urban and non-urban areas, understanding their influence on gene flow within cities remains limited. Here, we investigated the impact of linear barriers on coyote (Canis latrans) population structure in Seattle, Washington, where major barriers (i.e., interstate highways and bodies of water) divide the city into distinct quadrants. Just under 1000 scats were collected to obtain genetic data between January 2021 and December 2022, allowing us to identify 73 individual coyotes. Notably, private allele analysis underscored limited interbreeding among quadrants. When comparing one quadrant to each other, there were up to 16 private alleles within a single quadrant, representing nearly 22% of the population allelic diversity. Our analysis revealed weak isolation by distance, and despite being a highly mobile species, genetic structuring was apparent between quadrants even with extremely short geographic distance between individual coyotes, implying that Interstate 5 and the Ship Canal act as major barriers. This study uses coyotes as a model species for understanding urban gene flow and its consequences in cities, a crucial component for bolstering conservation of rarer species and developing wildlife friendly cities.

The value of hybrid genomes: Building two highly contiguous reference genome assemblies to advance Canis genomic studies.

Previous studies of canid population and evolutionary genetics have relied on high-quality domestic dog reference genomes that have been produced primarily for biomedical and trait mapping studies in dog breeds. However, the absence of highly contiguous genomes from other Canis species like the gray wolf and coyote, that represent additional distinct demographic histories, may bias inferences regarding interspecific genetic diversity and phylogenetic relationships. Here, we present single haplotype de novo genome assemblies for the gray wolf and coyote, generated by applying the trio-binning approach to long sequence reads generated from the genome of a female first-generation hybrid produced from a gray wolf and coyote mating. The assemblies were highly contiguous, with contig N50 sizes of 44.6 and 42.0 Mb for the wolf and coyote, respectively. Genome scaffolding and alignments between the two Canis assemblies and published dog reference genomes showed near complete collinearity, with one exception: a coyote-specific chromosome fission of chromosome 13 and fusion of the proximal portion of that chromosome with chromosome 8, retaining the Canis-typical haploid chromosome number of 2n = 78. We evaluated mapping quality for previous RADseq data from 334 canids and found nearly identical mapping quality and patterns among canid species and regional populations regardless of the genome used for alignment (dog, coyote, or gray wolf). These novel wolf and coyote genome reference assemblies will be important resources for proper and accurate inference of Canis demography, taxonomic evaluation, and conservation genetics.

Evolutionary legacy of the extirpated red wolf clings to life in gulf-coast canids.

Before Europeans colonized North America, a uniquely American wolf roamed the eastern forests of southern Canada to Florida and west to the Great Plains. Known today as "red wolf" (Canis rufus) in the south and "eastern wolf" (Canis lycaon) in the north, evidence suggests that these indigenous forest wolves shared a common evolutionary history until only a few centuries ago when they were extirpated from the intervening majority of their historical range. While the eastern wolf persists today primarily as a small population in Algonquin Provincial Park, Canada, the red wolf was ostensibly driven from its last stronghold in gulf-coastal Louisiana and Texas by 1980. The last-known red wolves were taken captive for propagation and reintroduction. Today, the red wolf exists as ~250 descendants of 12 founders and are distributed among 42 captive breeding facilities and one reintroduced population in coastal North Carolina. As red and eastern wolves declined in the 20th century, coyotes expanded from the west into the eastern forests, replacing them. Along with human persecution, coyote hybridization has been blamed for the late 20th century demise of the red wolf. However, rather than helping to drive the red wolf to extinction, coyote hybridization may have instead helped to preserve it. In this issue of Molecular Ecology, vonHoldt and colleagues provide the most comprehensive description yet of the substantial quantity and distribution of red wolf ancestry sequestered in southeastern coyote populations. They find the highest frequency of red wolf genes in coyotes from the gulf-coastal sites where the last known wild red wolves occurred, but also present evidence for a high prevalence of red wolf genes in coyotes throughout the southeastern expansion zone. Given the significant reduction in genetic diversity in extant red wolves owing to their late 20th century population bottleneck, this coyote-sequestered reservoir of red wolf genes could prove an important resource for red wolf conservation.

Persistence and expansion of cryptic endangered red wolf genomic ancestry along the American Gulf coast.

Admixture and introgression play a critical role in adaptation and genetic rescue that has only recently gained a deeper appreciation. Here, we explored the geographical and genomic landscape of cryptic ancestry of the endangered red wolf that persists within the genome of a ubiquitous sister taxon, the coyote, all while the red wolf has been extinct in the wild since the early 1980s. We assessed admixture across 120,621 single nucleotiode polymorphism (SNP) loci genotyped in 293 canid genomes. We found support for increased red wolf ancestry along a west-to-east gradient across the southern United States associated with historical admixture in the past 100 years. Southwestern Louisiana and southeastern Texas, the geographical zone where the last red wolves were known prior to extinction in the wild, contained the highest and oldest levels of red wolf ancestry. Further, given the paucity of inferences based on chromosome types, we compared patterns of ancestry on the X chromosome and autosomes. We additionally aimed to explore the relationship between admixture timing and recombination rate variation to investigate gene flow events. We found that X-linked regions of low recombination rates were depleted of introgression, relative to the autosomes, consistent with the large X effect and enrichment with loci involved in maintaining reproductive isolation. Recombination rate was positively correlated with red wolf ancestry across coyote genomes, consistent with theoretical predictions. The geographical and genomic extent of cryptic red wolf ancestry can provide novel genomic resources for recovery plans targeting the conservation of the endangered red wolf.

A sliver of the past: The decimation of the genetic diversity of the Mexican wolf.

The endangered Mexican wolf (Canis lupus baileyi) is known to carry exceedingly low levels of genetic diversity. This could be (i) the result of long-term evolutionary patterns as they exist at the southernmost limit of the species distribution at a relatively reduced effective size, or (ii) due to rapid population decline caused by human persecution over the last century. If the former, purifying selection is expected to have minimized the impact of inbreeding. If the latter, rapid and recent declines in genetic diversity may have resulted in severe fitness consequences. To differentiate these hypotheses, we conducted comparative whole-genome analyses of five historical Mexican wolves (1907-1917) and 18 contemporary Mexican and grey wolves from North America and Eurasia. Based on whole-genome data, historical and modern Mexican wolves together form a discrete unit. Moreover, we found that modern Mexican wolves have reduced genetic diversity and increased inbreeding relative to the historical population, which was widespread across the southwestern United States and not restricted to Mexico as previously assumed. Finally, although Mexican wolves have evolved in sympatry with coyotes (C. latrans), we observed lower introgression between historical Mexican wolves and coyotes than with modern Mexican wolves, despite similarities in body size. Taken together, our data show that recent population declines probably caused the reduced level of genetic diversity, but not the observed differentiation of the Mexican wolves from other North American wolves.

Pleistocene origins, western ghost lineages, and the emerging phylogeographic history of the red wolf and coyote.

The red wolf (Canis rufus) of the eastern US was driven to near-extinction by colonial-era persecution and habitat conversion, which facilitated coyote (C. latrans) range expansion and widespread hybridization with red wolves. The observation of some grey wolf (C. lupus) ancestry within red wolves sparked controversy over whether it was historically a subspecies of grey wolf with its predominant "coyote-like" ancestry obtained from post-colonial coyote hybridization (2-species hypothesis) versus a distinct species closely related to the coyote that hybridized with grey wolf (3-species hypothesis). We analysed mitogenomes sourced from before the 20th century bottleneck and coyote invasion, along with hundreds of modern amplicons, which led us to reject the 2-species model and to investigate a broader phylogeographic 3-species model suggested by the fossil record. Our findings broadly support this model, in which red wolves ranged the width of the American continent prior to arrival of the grey wolf to the mid-continent 60-80 ka; red wolves subsequently disappeared from the mid-continent, relegated to California and the eastern forests, which ushered in emergence of the coyote in their place (50-30 ka); by the early Holocene (12-10 ka), coyotes had expanded into California, where they admixed with and phenotypically replaced western red wolves in a process analogous to the 20th century coyote invasion of the eastern forests. Findings indicate that the red wolf pre-dated not only European colonization but human, and possibly coyote, presence in North America. These findings highlight the urgency of expanding conservation efforts for the red wolf.

Population genomics of grey wolves and wolf-like canids in North America.

North America is currently home to a number of grey wolf (Canis lupus) and wolf-like canid populations, including the coyote (Canis latrans) and the taxonomically controversial red, Eastern timber and Great Lakes wolves. We explored their population structure and regional gene flow using a dataset of 40 full genome sequences that represent the extant diversity of North American wolves and wolf-like canid populations. This included 15 new genomes (13 North American grey wolves, 1 red wolf and 1 Eastern timber/Great Lakes wolf), ranging from 0.4 to 15x coverage. In addition to providing full genome support for the previously proposed coyote-wolf admixture origin for the taxonomically controversial red, Eastern timber and Great Lakes wolves, the discriminatory power offered by our dataset suggests all North American grey wolves, including the Mexican form, are monophyletic, and thus share a common ancestor to the exclusion of all other wolves. Furthermore, we identify three distinct populations in the high arctic, one being a previously unidentified "Polar wolf" population endemic to Ellesmere Island and Greenland. Genetic diversity analyses reveal particularly high inbreeding and low heterozygosity in these Polar wolves, consistent with long-term isolation from the other North American wolves.

A Genome-Wide Perspective on the Persistence of Red Wolf Ancestry in Southeastern Canids.

The red wolf (Canis rufus), a legally recognized and critically endangered wolf, is known to interbreed with coyotes (Canis latrans). Declared extirpated in the wild in 1980, red wolves were reintroduced to northeastern North Carolina nearly a decade later. Interbreeding with coyotes was thought to be restricted to a narrow geographic region adjacent to the reintroduced population and largely believed to threaten red wolf recovery. However, red wolf ancestry was recently discovered in canids along the American Gulf Coast, igniting a broader survey of ancestry in southeastern canid populations. Here, we examine geographic and temporal patterns of genome-wide red wolf ancestry in 260 canids across the southeastern United States at over 164 000 SNP loci. We found that red wolf ancestry was most prevalent in canids sampled from Texas in the mid-1970s, although non-trivial amounts of red wolf ancestry persist in this region today. Further, red wolf ancestry was also observed in a subset of coyotes inhabiting North Carolina, despite management efforts to limit the occurrence of hybridization events. Lastly, we found no evidence of substantial red wolf ancestry in southeastern canids outside of these 2 admixture zones. Overall, this study provides a genome-wide survey of red wolf ancestry in canids across the southeastern United States, which may ultimately inform future red wolf restoration efforts.

Evaluation of an automated magnetic bead-based DNA extraction and real-time PCR in fecal samples as a pre-screening test for detection of Echinococcus multilocularis and Echinococcus canadensis in coyotes.

Efficient and sensitive diagnostic tools are essential for the study of the eco-epidemiology of Echinococcus species. We evaluated an automated magnetic bead-based DNA extraction commercial kit followed by qPCR (MB-qPCR), for the detection of Echinococcus multilocularis and Echinococcus canadensis in coyote (Canis latrans) fecal samples. The diagnostic sensitivity was determined by validating the method against the scraping, filtration, and counting technique (SFCT) for samples collected in Canada. From the 60 samples tested, 27 out of 31 SFCT positives samples for Echinococcus cestodes were positive in the MB-qPCR for E. multilocularis, with a sensitivity of 87.1% (95% CI 70.2 to 96.4%). Two samples were also positive for E. canadensis in the MB-qPCR and confirmed by morphological identification of adult worms. The agreement of the MB-qPCR and the SFCT was statistically significant with a kappa value of 0.67 (95% CI 0.48-0.85; p value < 0.001). The magnetic bead-based DNA extraction followed by qPCR proved to have a sensitivity comparable to the SFCT to detect E. multilocularis. Although the diagnostic sensitivity for E. canadensis was not estimated, MB-qPCR identified E. canadensis cases previously overlooked when using SFCT. We propose a combination of molecular and morphological identification using the MB-qPCR and the SFCT to detect both parasites, allowing for a more efficient large-scale surveillance, and detecting co-infections of Echinococcus species that can be difficult to identify when only based on morphology.

Analysis of the canid Y-chromosome phylogeny using short-read sequencing data reveals the presence of distinct haplogroups among Neolithic European dogs.

BACKGROUND: Most genetic analyses of ancient and modern dogs have focused on variation in the autosomes or on the mitochondria. Mitochondrial DNA is more easily obtained from ancient samples than nuclear DNA and mitochondrial analyses have revealed important insights into the evolutionary history of canids. Utilizing a recently published dog Y-chromosome reference, we analyzed Y-chromosome sequence across a diverse collection of canids and determined the Y haplogroup of three ancient European dogs. RESULTS: We identified 1121 biallelic Y-chromosome SNVs using whole-genome sequences from 118 canids and defined variants diagnostic to distinct dog Y haplogroups. Similar to that of the mitochondria and previous more limited studies of Y diversity, we observe several deep splits in the Y-chromosome tree which may be the result of retained Y-chromosome diversity which predates dog domestication or post-domestication admixture with wolves. We find that Y-chromosomes from three ancient European dogs (4700-7000 years old) belong to distinct clades. CONCLUSIONS: We estimate that the time to the most recent comment ancestor of dog Y haplogroups is 68-151 thousand years ago. Analysis of three Y-chromosomes from the Neolithic confirms long stranding population structure among European dogs.

Demographic history influences spatial patterns of genetic diversityin recently expanded coyote (Canis latrans) populations.

Human-mediated range expansions have increased in recent decades and represent unique opportunities to evaluate genetic outcomes of establishing peripheral populations across broad expansion fronts. Over the past century, coyotes (Canis latrans) have undergone a pervasive range expansion and now inhabit every state in the continental United States. Coyote expansion into eastern North America was facilitated by anthropogenic landscape changes and followed two broad expansion fronts. The northern expansion extended through the Great Lakes region and southern Canada, where hybridization with remnant wolf populations was common. The southern and more recent expansion front occurred approximately 40 years later and across territory where gray wolves have been historically absent and remnant red wolves were extirpated in the 1970s. We conducted a genetic survey at 10 microsatellite loci of 482 coyotes originating from 11 eastern U.S. states to address how divergent demographic histories influence geographic patterns of genetic diversity. We found that population structure corresponded to a north-south divide, which is consistent with the two known expansion routes. Additionally, we observed extremely high genetic diversity, which is atypical of recently expanded populations and is likely the result of multiple complex demographic processes, in addition to hybridization with other Canis species. Finally, we considered the transition of allele frequencies across geographic space and suggest the mid-Atlantic states of North Carolina and Virginia as an emerging contact zone between these two distinct coyote expansion fronts.

Ancestry-Specific Methylation Patterns in Admixed Offspring from an Experimental Coyote and Gray Wolf Cross.

Reduced fitness of admixed individuals is typically attributed to genetic incompatibilities. Although mismatched genomes can lead to fitness changes, in some cases the reduction in hybrid fitness is subtle. The potential role of transcriptional regulation in admixed genomes could provide a mechanistic explanation for these discrepancies, but evidence is lacking for nonmodel organisms. Here, we explored the intersection of genetics and gene regulation in admixed genomes derived from an experimental cross between a western gray wolf and western coyote. We found a significant positive association between methylation and wolf ancestry, and identified outlier genes that have been previously implicated in inbreeding-related, or otherwise deleterious, phenotypes. We describe a pattern of site-specific, rather than genome-wide, methylation driven by inter-specific hybridization. Epigenetic variation is thus suggested to play a nontrivial role in both maintaining and combating mismatched genotypes through putative transcriptional mechanisms. We conclude that the regulation of gene expression is an underappreciated key component of hybrid genome functioning, but could also act as a potential source of novel and beneficial adaptive variation in hybrid offspring.

Panmixia and Limited Interspecific Introgression in Coyotes (Canis latrans) from West Virginia and Virginia, USA.

The expansion of coyotes (Canis latrans) into the eastern United States has had major consequences for ecological communities and wildlife managers. Despite this, there has been little investigation of the genetics of coyotes across much of this region, especially outside of the northeast. Understanding patterns of genetic structure and interspecific introgression would provide insights into the colonization history of the species, its response to the modern environment, and interactions with other canids. We examined the genetic characteristics of 121 coyotes from the mid-Atlantic states of West Virginia and Virginia by genotyping 17 polymorphic nuclear DNA microsatellite loci. These genotypes were compared with those from other canid populations to evaluate the extent of genetic introgression. We conducted spatial clustering analyses and spatial autocorrelation to assess genetic structure among sampled coyotes. Coyotes across the 2 states had high genetic diversity, and we found no evidence of genetic structure. Six to sixteen percent of individuals displayed some evidence of genetic introgression from other species depending on the method and criteria used, but the population possessed predominantly coyote ancestry. Our findings suggested introgression from other canid populations has played less of a role in shaping the genetic character of coyotes in these states compared with populations closer to the Canadian border. Coyotes appear to display a panmictic population structure despite high habitat heterogeneity and heavy human influence in the spatial environment, underscoring the adaptability of the species.

Admixture mapping identifies introgressed genomic regions in North American canids.

Hybrid zones typically contain novel gene combinations that can be tested by natural selection in a unique genetic context. Parental haplotypes that increase fitness can introgress beyond the hybrid zone, into the range of parental species. We used the Affymetrix canine SNP genotyping array to identify genomic regions tagged by multiple ancestry informative markers that are more frequent in an admixed population than expected. We surveyed a hybrid zone formed in the last 100 years as coyotes expanded their range into eastern North America. Concomitant with expansion, coyotes hybridized with wolves and some populations became more wolflike, such that coyotes in the northeast have the largest body size of any coyote population. Using a set of 3102 ancestry informative markers, we identified 60 differentially introgressed regions in 44 canines across this admixture zone. These regions are characterized by an excess of exogenous ancestry and, in northeastern coyotes, are enriched for genes affecting body size and skeletal proportions. Further, introgressed wolf-derived alleles have penetrated into Southern US coyote populations. Because no wolves currently exist in this area, these alleles are unlikely to have originated from recent hybridization. Instead, they probably originated from intraspecific gene flow or ancient admixture. We show that grey wolf and coyote admixture has far-reaching effects and, in addition to phenotypically transforming admixed populations, allows for the differential movement of alleles from different parental species to be tested in new genomic backgrounds.

Ancient hybrid origin of the eastern wolf not yet off the table: a comment on Rutledge et al. (2015).

A recent study of North American canids by Rutledge et al. (Biol. Lett. 11, 20150303 (doi:10.1098/rsbl.2015.0303)) refutes the hypothesized hybrid origin of the eastern wolf (EW) based on genomic evidence against very recent hybridization. However, the analyses do not rule out the possibility of more ancient hybridization. Claims to have resolved the evolutionary origin of the EW are therefore inappropriate. Importantly, though, we plead that uncertainty about the ancient history of the taxon should not affect current conservation policy.

Mitochondrial DNA Variation in Southeastern Pre-Columbian Canids.

The taxonomic status of the red wolf (Canis rufus) is heavily debated, but could be clarified by examining historic specimens from the southeastern United States. We analyzed mitochondrial DNA (mtDNA) from 3 ancient (350-1900 year olds) putative wolf samples excavated from middens and sinkholes within the historic red wolf range. We detected 3 unique mtDNA haplotypes, which grouped with the coyote mtDNA clade, suggesting that the canids inhabiting southeastern North America prior to human colonization from Europe were either coyotes, which would vastly expand historic coyote distributions, an ancient coyote-wolf hybrid, or a North American evolved red wolf lineage related to coyotes. Should the red wolf prove to be a distinct species, our results support the idea of either an ancient hybrid origin for red wolves or a shared common ancestor between coyotes and red wolves.

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.

Single nucleotide polymorphism (SNP) variation of wolves (Canis lupus) in Southeast Alaska and comparison with wolves, dogs, and coyotes in North America.

There is considerable interest in the genetics of wolves (Canis lupus) because of their close relationship to domestic dogs (C. familiaris) and the need for informed conservation and management. This includes wolf populations in Southeast Alaska for which we determined genotypes of 305 wolves at 173662 single nucleotide polymorphism (SNP) loci. After removal of invariant and linked SNP, 123801 SNP were used to quantify genetic differentiation of wolves in Southeast Alaska and wolves, coyotes (C. latrans), and dogs from other areas in North America. There is differentiation of SNP allele frequencies between the species (wolves, coyotes, and dogs), although differentiation is relatively low between some wolf and coyote populations. There are varying levels of differentiation among populations of wolves, including low differentiation of wolves in interior Alaska, British Columbia, and the northern US Rocky Mountains. There is considerable differentiation of SNP allele frequencies of wolves in Southeast Alaska from wolves in other areas. However, wolves in Southeast Alaska are not a genetically homogeneous group and there are comparable levels of genetic differentiation among areas within Southeast Alaska and between Southeast Alaska and other geographic areas. SNP variation and other genetic data are discussed regarding taxonomy and management.

A genome-wide perspective on the evolutionary history of enigmatic wolf-like canids.

High-throughput genotyping technologies developed for model species can potentially increase the resolution of demographic history and ancestry in wild relatives. We use a SNP genotyping microarray developed for the domestic dog to assay variation in over 48K loci in wolf-like species worldwide. Despite the high mobility of these large carnivores, we find distinct hierarchical population units within gray wolves and coyotes that correspond with geographic and ecologic differences among populations. Further, we test controversial theories about the ancestry of the Great Lakes wolf and red wolf using an analysis of haplotype blocks across all 38 canid autosomes. We find that these enigmatic canids are highly admixed varieties derived from gray wolves and coyotes, respectively. This divergent genomic history suggests that they do not have a shared recent ancestry as proposed by previous researchers. Interspecific hybridization, as well as the process of evolutionary divergence, may be responsible for the observed phenotypic distinction of both forms. Such admixture complicates decisions regarding endangered species restoration and protection.