Protection of populations comprising admixed genomes is a challenge under the Endangered Species Act (ESA), which is regarded as the most powerful species protection legislation ever passed in the United States but lacks specific provisions for hybrids. The eastern wolf is a newly recognized wolf-like species that is highly admixed and inhabits the Great Lakes and eastern United States, a region previously thought to be included in the geographic range of only the gray wolf. The U.S. Fish and Wildlife Service has argued that the presence of the eastern wolf, rather than the gray wolf, in this area is grounds for removing ESA protection (delisting) from the gray wolf across its geographic range. In contrast, the red wolf from the southeastern United States was one of the first species protected under the ESA and was protected despite admixture with coyotes. We use whole-genome sequence data to demonstrate a lack of unique ancestry in eastern and red wolves that would not be expected if they represented long divergent North American lineages. These results suggest that arguments for delisting the gray wolf are not valid. Our findings demonstrate how a strict designation of a species under the ESA that does not consider admixture can threaten the protection of endangered entities. We argue for a more balanced approach that focuses on the ecological context of admixture and allows for evolutionary processes to potentially restore historical patterns of genetic variation.

Keywords

Although there is extensive literature on the red wolf and eastern wolf [for example, ( 2 , 11 , 18 , 19 )], only recently have genome-wide data been analyzed to support an admixed or ancient origin hypothesis. A previous study genotyped and analyzed more than 42,000 single-nucleotide polymorphisms (SNPs) in a large panel of North American and Eurasian wolf-like canids that supported an admixed origin for both red and eastern wolves ( 5 ). However, a reanalysis of these data found evidence for a genetic cluster in central Ontario representing the eastern wolf and concluded that the SNP array data may suffer from ascertainment bias ( 20 ). A more recent study presented new, ascertainment-free genome-wide SNP data from the eastern wolf and showed through simulation that admixture alone cannot explain the unique positions of the eastern wolf in a principal components analysis (PCA) ( 21 ). Here, we use a genome sequencing approach to directly search for regions of unique ancestry in the genomes of red and eastern wolves that cannot be explained by admixture between coyotes and gray wolves. We present 28 sequenced genomes from a diversity of large canids representing Eurasian and North American wolf populations, including North American regions where wolf/coyote admixture is currently absent and regions with suspected admixture. An exhaustive search of wolf genomes from the Great Lakes region including Algonquin Provincial Park, where pure eastern wolves are thought to exist, and from red wolves from the captive breeding colony reveals little unique ancestry and instead demonstrates a distinct geographic pattern of admixture between gray wolves and coyotes. We argue strongly for a less typologically oriented implementation of the ESA that allows interim protection of hybrids while encouraging the restoration of historic patterns of variation through habitat protection.

The U.S. Fish and Wildlife Service (USFWS) accepts the species status of both red and eastern wolves, with markedly divergent conservation implications. The red wolf is protected by the U.S. Endangered Species Act (ESA). However, the endangered eastern wolf, which was only recently recognized as a distinct species ( 8 – 14 ) and is currently restricted to a small portion of its historic range, would not be listed under the current political landscape. Instead, the acceptance of the eastern wolf species has led the USFWS to propose the delisting of the gray wolf. The reasoning for this action is that the historical range of the eastern wolf is hypothesized to include the Great Lakes region and 29 eastern states to the exclusion of the gray wolf ( 11 , 15 , 16 ). Because the geographic range of the gray wolf as originally listed in the 1975 ESA petition included these areas, the USFWS subsequently proposed that the entire original listing was invalid. Essentially, the presence of the eastern wolf, rather than the gray wolf, in the eastern United States would cause the original listing to be annulled. With the exception of the Mexican wolf, the gray wolf would be delisted (lose protection) from its entire North American range under the proposed USFWS rule change ( 17 ). These differing consequences of species listing, despite the possibility of similar admixed origin, provide a marked example of how taxonomy can both protect and threaten endangered species under the ESA.

Top: Previously proposed phylogenetic relationships among Canis lineages, with gray lines indicating putative admixture events ( 5 ). Bottom: Geographic distributions of Canis in North America. Sample locations are indicated by dots and abbreviations are described in Table 1 . Ancestry proportions from vonHoldt et al. ( 5 ) are indicated (proportion gray wolf/proportion coyote; see also new values in Table 3 ). IRNP, Isle Royale National Park; Ma, million years ago.

Two well-accepted species of wolf-like canids inhabit North America: the Holarctic gray wolf (Canis lupus) and the endemic coyote (Canis latrans). However, two other entities have been advanced as evolutionarily distinct species of North American origin: the red wolf (Canis rufus) of the southeastern United States and the eastern wolf (Canis lycaon), now found in the eastern Great Lakes (Algonquin Provincial Park and adjacent areas in Ontario) but were historically thought to inhabit a wider area, including the eastern United States ( 1 , 2 ) ( Fig. 1 ). However, an alternative hypothesis suggests that the red wolf is a hybrid between coyotes and gray wolves that historically inhabited the southeastern United States before gray wolves were eliminated through private and public bounty ( 3 – 5 ) ( Fig. 1 ). Similarly, the eastern wolf may have been generated through admixture between gray wolves and coyotes as they expanded eastward into the Great Lakes region at the end of the last century, concurrent with the near extirpation of wolves in the conterminous United States ( 6 – 8 ). Both red and eastern wolves are intermediate in body size between coyotes and gray wolves, which is consistent with an admixture scenario, and recent evidence has shown that gray wolves and coyotes can produce viable offspring in captivity ( 9 ).

RESULTS

Genome sequencing We sequenced 28 canid genomes to varying coverage ranging from 4 to 29-fold and mapped reads to the domestic dog reference genome (Table 1). After filtering for quality and minimum coverage, we retained 5,424,934 SNPs (referred to as 5.4 million SNPs) genotyped across all sequenced genomes. From these data, we estimated that heterozygosity (π) was highest in the Indian wolf (π = 1.71/kb) and lowest in the endangered Mexican wolf (π = 0.48/kb), which is consistent with previous observations of low diversity in the inbred captive Mexican wolf colony (22). We note that the fraction of missing data is negatively correlated with π, although we could not quantify the extent of this effect given the heterogeneous nature of the samples (Table 1). Table 1 Samples, origin, and genome code used in the article; average genome coverage; and ancestry proportions. If a population resides within the gray wolf and coyote hybrid zone, the location is indicated with “HZ”; reference populations are indicated by “REF.” When previously sequenced, the appropriate citation is provided. EuGW, Eurasian gray wolf; NAGW, North American gray wolf; RW, red wolf; GLW, Great Lakes region wolf; COY, coyote; DOG, dog; JACK, golden jackal. View this table: We quantified genetic differentiation using F ST , confining our coyote representatives to the three individuals (California, Alabama, and Quebec coyotes) most likely to be nonadmixed (see the “Estimating admixture by D and ” section). We found that the F ST between wolves of the Great Lakes region (which include putative eastern wolves from Algonquin Provincial Park) and gray wolves or coyotes is nearly half that between red wolves and gray wolves or coyotes (North American gray wolf–Great Lakes wolves, F ST = 0.057; coyote–Great Lakes region wolf, F ST = 0.045; North American gray wolf–red wolf, F ST = 0.177; coyote–red wolf, F ST = 0.108) (Table 2). The highest value of divergence was between the red wolf and the Eurasian gray wolf and may reflect, in part, the limited founding size and enhanced drift in the small population of captive red wolves. These estimates of interpopulation genetic differentiation (as measured by F ST ) are comparable to those found among human populations (23), suggesting that previously hypothesized divergence time estimates of hundreds of thousands of years between wolf-like canid lineages are overestimates and/or that these lineages have experienced a substantial amount of recent admixture. Using a simple isolation model and a summary likelihood approach, we estimated a Eurasian gray wolf–coyote divergence time of T = 0.38 N generations (95% confidence interval, 0.376 to 0.386 N), where N is the effective population size. If we assume a generation time of 3 years, and an effective population size of 45,000 (24, 25), then this corresponds to a divergence time of 50.8 to 52.1 thousand years ago (ka), roughly the same as previous estimates of the divergence time of extant gray wolves (26–28). Thus, the amount of genetic differentiation between gray wolves and coyotes is low and not much greater than the amount of differentiation within each species (for example, Eurasian versus North American gray wolf, F ST = 0.099; Table 2 and fig. S1). This result contradicts molecular clock calculations based on short mitochondrial control region sequences, which were calibrated using a 1-Ma (million years ago) divergence time between gray wolves and coyotes (10). Despite body size and other phenotypic differences between the two species [for example, (1)] and a long history of coyote- and wolf-like forms in North America (1, 29), the genomic data suggest that modern coyotes and gray wolves are very close relatives with a recent common ancestry. Table 2 Pairwise F ST estimates between canid lineages. Abbreviations are found in Table 1. View this table:

Cluster and ancestry analysis We first assessed the general pattern of sequence similarity across the observed 5.4 million SNPs using PCA and found distinct groups that corresponded to gray wolf, coyote, and putatively admixed populations, including the Algonquin wolf and red wolf (Fig. 2 and fig. S2). The overall PC space identified two clusters of wolves that can be explained by continental divergence (Eurasian and North American wolves) and identified the California coyote as the most distinct coyote and the Mexican wolf as a distinct North American wolf. The intermediate position on the first PC of Great Lakes region wolves and red wolves is consistent with a model of admixture between gray wolves and coyotes, although it is also consistent with the hypothesis that red and eastern wolves represent distinct conspecific populations [for example, Wilson et al. (10)]. The PCA also shows that coyotes in populations outside the present admixture zone are genetically distinct (5). These general patterns were found in a PCA of downsampled sequences that represented equivalent sampling of all the genomes (fig. S2). Fig. 2 PCA of 5.4 million unphased SNPs and 23 Canis genomes. The dashed line contains genomes that are considered admixed. We found 16,184 fixed differences between three nonadmixed coyotes (California, Alabama, and Quebec) and Eurasian gray wolves and used these to estimate wolf versus coyote ancestry proportions, scaled using simulations (Table 3). The Great Lakes region wolf genomes showed a majority of wolf-derived alleles (prop wolf = 0.61 to 0.67), unlike the eastern wolves from Algonquin Provincial Park (prop wolf = 0.39 to 0.47) and red wolves (prop wolf = 0.09 to 0.20). The three nonreference coyote genomes had no estimated wolf admixture (prop wolf = 0.00). We note that the results presented here are robust to changes in the specific genomes used for the wolf and coyote “reference” panels. For example, if we replace the three coyote genomes used with all six coyote genomes, the correlation in prop wolf estimates in the two analyses is r2 = 0.988. Table 3 Estimated fraction of wolf-like versus coyote-like alleles at the 16,184 fixed differences between wolves and putatively unadmixed coyotes. See Table 1 for sample abbreviations. View this table:

Estimating admixture by D and We tested for admixture among wolves, dogs, and coyotes using the D statistic (also known as the ABBA-BABA test) and quantified the proportion of ancestry using (30, 31). We performed all possible D statistic comparisons among our samples and used a San Nicolas Channel Islands fox (Urocyon littoralis dickeyi) [table S1, (32)] as the outgroup to identify derived alleles. Below, we report tests as D(P1, P2, candidate introgressor, outgroup). Because of the substantial controversy regarding the proper classification of eastern wolves and red wolves, we began by testing which North American canids, regardless of species assignment in the field, shared the most derived alleles with each Eurasian wolf using D(North America1, North America2, Eurasian wolf, fox), where “North America1, North America2” refers to all combinations of North American canids. We found that samples that were morphologically identified as coyotes tended to share the fewest derived alleles with Eurasian wolves (fig. S3), which is consistent with the expectation that gray wolves are a monophyletic species (33). The results of analyses with the Quebec wolf indicate that, like the California coyote and the Alabama coyote, it lacks detectable Eurasian wolf ancestry (Fig. 3 and fig. S3). This finding could reflect either an error in the field classification of the Quebec wolf specimen or a transcription error in the field or laboratory. Given the large variation in phenotype across the admixture zone [for example, Kolenosky et al. (34)] and the potential difficulty in making species-level assignments of hybrids under field conditions, we suggest that this genetic assignment of the “Quebec wolf” as a coyote is a more reliable guide to the ancestry of the sample than the field/laboratory assignment. Fig. 3 Estimates of ancestry proportions using thestatistic. Sequences grouped as coyotes are from Alabama, California, Illinois, Ohio, and Florida. (*Individual labeled as wolf but is likely to derive from a coyote; see discussion in the text.) To determine the proportion of coyote ancestry in North American canids, we next calculated D statistics using D(Eurasian wolf, North American wolf, California or Alabama coyote, fox) and quantified the result using the related statistic. In this test, we use the California and Alabama coyotes as potential introgressors because they had the fewest derived gray wolf alleles (they were the most coyote-like samples in the data set) and originated from outside of one or both admixture zones. Although the Alabama coyote is from the southeastern United States and may therefore be admixed with the red wolf, tests to detect red wolf ancestry in this sample were consistently nonsignificant (table S1). We found that all North American wolves and coyotes have significant amounts of coyote ancestry (table S1). In addition, we detect a strong geographic cline in the proportion of coyote ancestry across North American canids. Alaskan and Yellowstone wolves have 8 to 8.5% coyote ancestry, Great Lakes wolves have 21.7 to 23.9% coyote ancestry, Algonquin wolves have at least 32.5 to 35.5% coyote ancestry, and Quebec sequences have more than 50% coyote ancestry (Fig. 3). As expected, Eurasian wolves and dogs, which are allopatric to coyotes, do not have coyote ancestry (table S1). Finally, we estimated the amount of gray wolf introgression into other canids in our data set. We detected significant amounts of gray wolf ancestry in Illinois coyotes (prop wolf ≥ 0.06), Florida coyotes (prop wolf ≥ 0.09), Ohio coyotes (prop wolf ≥ 0.10), and red wolves (prop wolf ≥ 0.20). The two Quebec individuals have different amounts of gray wolf ancestry: The Quebec wolf has no detectable gray wolf ancestry, consistent with mislabeling (see above), and the Quebec coyote has at least 15.8% gray wolf ancestry. The highest inferred proportion of gray wolf ancestry among nonadmixed individuals was 61.1% in the Basenji, suggesting that these proportions may underestimate the amount of wolf contribution to the coyote gene pool (table S2). However, these proportions are similar in magnitude and ranking to those independently estimated using diagnostic SNPs from the canine genotyping array (Fig. 1 and see below). These results also highlight the mixed ancestry of red wolves and wolves from the Great Lakes region including Algonquin Provincial Park, with the latter having a substantial proportion of gray wolf ancestry.

Demographic analysis To better assess the demographic implications of a separate species origin, rather than one due entirely to admixture, we performed demographic inference by applying G-PhoCS (Generalized Phylogenetic Coalescent Sampler) to simple branching models (35). Notably, our models assume that red and eastern wolves have a phylogenetically distinct origin followed by admixture. Because of computational and coverage limitations, we focused on high-coverage genomes from nine individuals, each from a different population or species: a red wolf (Redwolf1), a Great Lakes region wolf with admixed history (Minnesota), a California coyote, two North American wolves (Yellowstone2 and Mexican wolf), two Eurasian wolves (Mongolia and Croatia), Basenji, and a golden jackal (Table 1). Our objective was to infer rates of gene flow into red and Great Lakes region wolves in the context of a complete demographic model that includes population divergence and changes in ancestral population sizes. We assumed four different plausible topologies for the population phylogeny (fig. S4); however, to capture the large contribution of genomic ancestry from gray wolves into Great Lakes region wolves and coyotes into red wolves, we focused on a model in which the eastern wolf branched from the population ancestral to North American wolves and the red wolf branched from the coyote lineage (Fig. 4). We analyzed 13,647 previously determined putative neutral loci of 1 kb in length (26). Fig. 4 Demographic history inferred using G-PhoCS. A schematic depiction of the population phylogeny assumed in the analysis. The phylogeny was augmented with migration bands from all canids to the red wolf and the Great Lakes region wolf. G-PhoCS infers significant rates of gene flow primarily from the gray wolf and the coyote to the red wolf and the Great Lakes region wolf (shaded box). Ninety-five percent Bayesian credible intervals are shown for the total rates transformed into proportions between 0 and 100% (see Materials and Methods). Similarly high rates were also inferred when assuming three alternative topologies for the population phylogeny (fig. S4). Under the assumed branching structure, we inferred high rates of gene flow from gray wolves and coyotes into the red wolf and the Great Lakes region wolf (fig. S4 and table S3). We converted these rates to admixture proportions and observed high proportions of coyote gene flow into the red wolf (48 to 88%) and high proportions of gene flow from the Yellowstone wolf into the Great Lakes region wolf (37 to 48%) (Fig. 4). High admixture proportions were also inferred from the coyote into the Great Lakes region wolf (25 to 34%) and between the red wolf and the Great Lakes region wolf (21 to 35%). We obtained similarly high estimated admixture proportions in the other three topologies examined (fig. S4), confirming that high rates of inferred gene flow were not a result of incorrect assumptions of the population phylogeny. Finally, the inferred divergence time under the model in Fig. 4 is relatively short for the Great Lakes region wolf (τ_GL_NA, 27 to 32 ka) (fig. S5). The estimated divergence time between the red wolf and the coyote is greater (τ_GL_NA, 55 to 117 ka), which may reflect the use of the high-coverage California coyote sequence, which is the most divergent sequence of our coyote samples (Fig. 2). This sequence is unlikely to represent the source of admixture for the red wolf in the American Southeast. Values of divergence time are even shorter in models without gene flow, as might be predicted (fig. S6). Consequently, these results show that even under the assumption of a distinct species origin, extensive gene flow into a recently diverged Great Lakes region wolf is needed to account for its genomic composition. Similarly, assuming a distinct origin for the red wolf still requires substantial gene flow from gray wolves and coyotes, although the inferred divergence time is greater. However, this divergence time might be inflated relative to that in which populations more directly ancestral to those comprising the red wolf admixture zone were used.