Red wolves (Canis rufus
) once inhabited the southeastern United States but were declared extinct in the wild by 1980 due to habitat loss, predator control programs, disease, and interbreeding with encroaching coyotes (Canis latrans
]. In 1967, the U.S. Fish and Wildlife Service (USFWS) listed red wolves as endangered under the U.S. Endangered Species Preservation Act due to their rapid population decline in the American south, and subsequently, red wolves were among the first species listed on the 1973 Endangered Species Act (ESA), the Unites States’ landmark environmental law [1
]. With red wolves on the brink of extinction, recovery was initiated through trapping what were believed to be the last wild red wolves along the Gulf Coast of Louisiana and Texas in the 1970s [1
]. Individuals were selected as founders for the captive breeding program based on morphology and behavioral traits considered to be species informative [6
]. Over 240 canids were trapped from coastal Louisiana and Texas between 1973 and 1977 [6
]. Forty individuals were selected for captive breeding, of which 17 were deemed 100% wolf. However, only 14 wolves successfully reproduced and became the founders from which all red wolves in the recovery program descend.
Due to the successful captive breeding program, red wolves were restored to the landscape in North Carolina less than a decade after being declared extinct in the wild [6
]. This historic event represented the first attempt to reintroduce a wild–extinct species in the United States and set a precedent for returning wild–extinct wildlife to the landscape. The success of the red wolf recovery program was the foundation upon which other wolf introductions were guided, including the gray wolf (C. lupus
) reintroduction to the northern Rocky Mountains in Yellowstone National Park, Wyoming, and central Idaho, and the ongoing restoration efforts for the Mexican wolves (C. lupus baileyi
) in the southwest [8
]. Although successful by many measures [7
], the North Carolina experimental population (NCEP) of red wolves was reduced by the USFWS in response to negative political pressure from the North Carolina Wildlife Resource Commission and a minority of private landowners [10
]. Further, gunshot-related mortalities have increased the probability that wolf packs deteriorate before the breeding season, which facilitates the establishment of coyote–wolf breeding pairs [11
]. Consequently, the NCEP has fewer than 40 surviving members [13
] and red wolves are once again on the brink of extinction in the wild.
Interbreeding between red wolves and coyotes is well documented and is viewed as a threat to red wolf recovery [14
]. When historic populations of red wolves along the Gulf Coast were surveyed, it was feared that these coastal populations were the last remnants of pre-recovery wild wolves and were likely to quickly become genetically extinct through introgressive swamping of coyote genetics [15
]. Yet, there continued to be reports of red wolves in rural regions of coastal Louisiana and Texas since the 1970s [5
]. Previous efforts to detect surviving red wolves or their hybrids in the region proved unsuccessful [17
]. However, the possibility remains that individuals with substantial red wolf ancestry have naturally persisted in isolated areas of the Gulf Coast. For example, body measurements of coyote-like canids in southwestern Louisiana were similar to those of confirmed red wolf–coyote hybrids in the NCEP [18
]. These individuals would harbor ghost alleles of the original red wolves, with these alleles lost in the contemporary red wolf population during the extreme population bottleneck, drift, and inbreeding.
For red wolf ghost alleles to persist, a remnant Gulf Coast population would need to be relatively isolated from frequent interbreeding with coyotes [14
]. Although red wolves that co-occur with coyotes in the NCEP exhibit assortative mating patterns [20
], a geographic island would promote genetic isolation and the persistence of red wolf alleles. We report evidence that Galveston Island, Texas (TX) may represent one such location. All contemporary red wolves descended from individuals trapped from Jefferson, Chambers, southern Orange, and eastern Galveston counties in Texas and Cameron and southern Calcasieu parishes in Louisiana [16
] (Figure 1
). Given Galveston Island’s location and isolation from the mainland, it is a probable region to harbor red wolf ghost alleles. Recent images captured of Galveston Island canids (Figure 2
) piqued interest of local naturalists and two genetic samples were taken from roadkill individuals. Accordingly, our objective was to conduct genomic analyses and determine if there was evidence of red wolf ancestry in modern-day Galveston Island canids.
We rediscovered red wolf ghost alleles present in the American southeast nearly 40 years after they were extinct in the region. Through interbreeding with coyotes, this endangered genetic variation has persisted and could represent a reservoir of previously lost red wolf ancestry. This unprecedented discovery opens new avenues for innovative conservation efforts, including the reintroduction of red wolf ghost alleles to the current captive and experimental populations. Consequently, these admixed individuals are of great conservation value, yet the ESA currently lacks any explicit policy providing protection for admixed individuals that serve as reservoirs for extinct genetic variation. An ‘intercross policy’ was introduced in 1996 to assist prioritizing protection efforts but was never fully adopted [40
]. Several commentaries have encouraged an updated implementation of the ESA and Species Status Assessments, especially as admixed genomes are increasingly being described and viewed as a source of potentially beneficial genetic variation in the face of rapid climate change (e.g., [41
]). Although red wolves represent one of the greatest species recovery stories in ESA history, debates regarding historical and ongoing interbreeding with coyotes highlight the ESA’s short-comings associated with admixed individuals and the difficulty in setting management objectives given our evolving understanding of admixed genomes across wild populations [42
Our analyses revealed a surprising amount of allele sharing with the captive breeding population of red wolves. This shared variation could be the consequence of two potential scenarios: (1) Surviving ancestral polymorphisms from the shared common ancestor of coyotes and red wolves that have drifted to a high frequency in the captive breeding red wolf population and in a small portion of Gulf Coast coyotes; or (2) coyotes in the Gulf Coast region are a reservoir of red wolf ghost alleles that have persisted into the 21st century. Neither of these potential explanations require adherence to a specific species concept. For instance, incomplete lineage sorting from a shared common ancestor could occur whether red wolves are a subspecies of the gray wolf, conspecific with Eastern wolves, or an independent lineage with a possible ancient hybrid origin [43
] (Figure S5
). Similarly, interbreeding with the ancestral red wolf population would have resulted in the introgression of red wolf alleles and associated phenotypes into Gulf Coast coyotes under each species concept. Our findings of admixture and composition of private alleles are most consistent with the second scenario, where the Galveston Island canids are admixed coyotes carrying red wolf ghost alleles. Further, Galveston Island is found within the historic red wolf range from where the original founders for the captive and reintroduced populations were captured in the 1970s (Figure S6
). This island population likely experienced reduced gene flow with southeastern coyotes. In further support that coyotes of the American Gulf Coast likely serve as a ghost allele reservoir of red wolf ancestry, we also identified two coyotes with red wolf admixture from Louisiana’s Gulf Coast, a second geographic region in which trapping efforts were conducted to build a captive red wolf population [16
]. These findings provide substantial support that ancestral red wolf genetic variation persists as ghost alleles in the regional coyotes of the southeastern United States.
While our primary objective was to determine the extent of red wolf allele sharing among the Galveston Island canids, our discovery warrants further genetic surveys of coyote populations in Louisiana and Texas to establish the level and extent to which remnant red wolf alleles are found exclusively in admixed coyotes. There are potentially admixed coyotes in the region that exhibit higher levels of red wolf ancestry, as exemplified by the two Louisiana coyotes that also exhibited partial assignment to the red wolf cluster. Broadly, admixture levels in southeastern coyotes could be impacted by variation in habitat, hunting, and dispersal barriers across the region. Given gunshot mortality is known to increase coyote–wolf hybridization [11
], there may be a need to regulate coyote hunting until we know more about the frequency of endangered wolf genetics in the American Gulf coast. With genetic surveys in place, conservation efforts then face the opportunity to consider the role of remnant genetic variation in the future of the red wolf. The NCEP of red wolves is a listable entity under the ESA in need of proactive conservation [43
]. However, in the age of an extinction crisis, innovative mechanisms to preserve and utilize adaptive potential are in great demand. Today, every federally recognized red wolf individual is a descendant from 14 founders, of which only 12 are genetically represented. These founders were removed from a single geographic location in the 1970s and vastly underrepresent the original genomic diversity present in southeastern wolves [5
]. Our discovery of red wolf ghost alleles in southeastern coyotes demonstrates the ability to uncover ancestral variation and establish a new component of biodiversity conservation. A minority of conservation priorities have considered a ‘de-introgression’ strategy in which admixed individuals are bred in a specific design to recover the extinct genotype [44
]. For instance, after identifying wild canids with red wolf ghost alleles, a breeding program could be established to prioritize individuals representing rare red wolf ancestry with the goal of recovering lost genomic variation, similar to Reference [45
While de-introgression may prove useful to recover extinct red wolf ancestry from admixed individuals, a new paradigm has been proposed to more broadly re-evaluate the role of admixed genomes [42
]. Red wolves face anthropogenically-mediated hybridization, but introgression is also likely a natural process in the evolution of Canis
lineages. As an important evolutionary process, introgression could protect adaptive potential and maintain processes that sustain ecosystems. Incorporating admixed entities into conservation policy and, here, red wolf restoration may be the next step in broader biodiversity conservation. Another pivotal step in red wolf restoration is the identification of a new reintroduction site for a wild population of red wolves. Our discovery of red wolf ghost alleles indicates there are geographic regions that can harbor endangered genetic variation and may guide future efforts for red wolf reintroduction. The foundation upon which that effort will be built rests exclusively on describing large-scale geographic patterns of red wolf ghost alleles in the American southeast.