by Bob Wayne*
Genetic evidence suggests that the red wolf is a
hybrid form,
not a distinct species. Should we continue to preserve it?
[Ed. - we plan to post the figures on-line soon!]
Conservationists today are doomed to struggle against an advancing tide of habitat destruction, human population growth, and persecution and exploitation of plant and animal species worldwide. They must face the unpleasant responsibility of prioritizing and allocating effort, in the certain knowledge that there are insufficient resources available to save and protect all of nature's diversity. That a triage-like system for prioritizing species' conservation is necessary is evidenced by one important failure of the Endangered Species Act (ESA) in the USA. Although nearly 1000 species are listed under the Act, only a few percent of them have received Federal support for research and conservation. The criteria for prioritizing species are multifarious, but one of considerable importance is the genetic uniqueness of candidates for conservation. The giant panda (Ailuropoda melanoleuca), for example, is the only surviving representative of a distinct subfamily of bears, and would thus seem to deserve the disproportionate attention and effort currently dedicated to its continued survival.
The genetic criterion is, however, only as reliable as the data available concerning the genetic uniqueness or value of an organism. Where the issue is unresolved conservation efforts may encounter problems of selection and justification of allocation of resources. A prime example of such a situation is the case of the North American red wolf, Canis rufus.
The red wolf was once fairly widespread in the southeastern USA, but predator control programmes and the conversion of mature woodland habitat to agriculture resulted in a reduction in both range and population size. By the 1970s the species had dwindled to a single population in eastern Texas, and this population was threatened by interbreeding with the red wolfµs close relative, the coyote (Canis latrans). The U.S. Fish and Wildlife Service (USFWS) initiated a last minute recovery plan to save the species from extinction. Fourteen animals, thought to be pure red wolves, were selected from a much larger sample of canids captured in east Texas and used to establish a captive breeding programme (Phillips & Parker, 1988).
The captive breeding and subsequent reintroductions to the wild have been successful beyond expectation, and can justifiably be viewed as a model for rescuing highly endangered species from the brink of extinction. Nevertheless, such success is not without price: the USFWS has a five year budget of around $4.5 million to cover field studies and captive breeding facilities. Does the genetic uniqueness of the red wolf warrant this level of resources? Since the recovery programme began the taxonomic status of the red wolf has been investigated using both morphological and genetic methods, giving rise to contradictory results. Due to the economic and political importance of the red wolf project, these contradictions have resulted in considerable controversy. I present here a summary of the findings of genetic research.
Efforts to use morphological criteria as a basis to classify the red wolf as a distinct species are problematic. Multivariate analysis of morphological measurements, such as done by Nowak (1979), express the overall similarity in cranial and dental form of red wolves to other canids. Indeed, his data indicate red wolves are distinct and intermediate in form between grey wolves (Canis lupus) and coyotes. Although acknowledging hybrid origin for the red wolf as a possibility, Nowak suggests that "the most reasonable explanation is that C.rufus represents a primitive line of wolves that has undergone less change than C.lupus, and has retained more characters found in the ancestral stock from which both wolves and coyotes arose".
The quantitative distinction of the red wolf has been used by Nowak to argue for separate species status. The use of quantitative differences alone to define a species is controversial (see O'Brien & Mayr, 1991; McKitrick & Zink, 1988; Avise & Ball, 1990; Mallet, 1995). For the red wolf, however, the phenotypic argument may be circular because hybrids between grey wolves and coyotes are expected to be intermediate in morphology. The phenotype of the red wolf may thus reflect either recent hybridization between grey wolves and coyotes in the southeast USA or, as Nowak suggests, an ancient origin and long distinct evolutionary heritage.
In fact, Nowak's multivariate morphological position of supposed grey wolf-coyote hybrids overlaps with the position of red wolves (Fig.1). Because of the possibility of a hybrid origin for the red wolf, discrete character state differences uniquely shared by red wolves, such as the shared presence of a specialized cusp or cranial foramen are needed to define them as a separate taxon. Molecular data provide such discrete character data, whose analysis using cladistic principles is consistent with modern systematic thinking. (Jenks & Wayne, 1992; Wayne, 1992). If the red wolf is a long distinct species, it should have unique genetic attributes, just as coyotes and grey wolves do today. The evolutionary heritage of the red wolf should be recorded in genes just a faithfully as it is in fossils. In reality, the fragmentary fossil remains of wolf-like canids and the conservative nature of canid dentition makes the interpretation of the sparse fossil record difficult.
The mitochondrial DNA (mtDNA) genome is a small circular loop of DNA only 16-18 thousand base pairs in length. It is a useful tool for the evolutionary geneticist since it has a rapid evolutionary rate, many times faster than the average nuclear gene. Closely related species whose nuclear DNA has diverged very little may thus be clearly distinguishable from differences in their mtDNA. In addition, mtDNA is inherited maternally and without recombination, so the evolution of sequences within a species defines a maternal phylogeny distinct from that in other species (see Avise, 1994). Given these properties, mtDNA analysis was a suitable tool to begin a study of the relationships between coyote, grey wolf and red wolf.
Initial investigations showed that Canis species are generally closely related, and form a distinct evolutionary group (Lehman et al., 1991; Wayne and Jenks, 1991; Girman et al., 1992; Gottelli et al., 1994). Within this group, restriction site and direct sequencing analysis of the mtDNA revealed appreciable differences between most species: jackals, for example, differed by 8% from wolves and coyotes. However, mtDNA from extant red wolves appeared to be identical to that found in coyotes from Louisiana (Wayne & Jenks, 1991).
Interpretation of this result was facilitated by earlier investigations of mtDNA from wolves and coyotes (Lehman et al., 1991). Wolves from a contiguous region in Minnesota, Ontario and Quebec had exhibited a high frequency (over 50%) of coyote type mtDNA, although mtDNA sequences of wolves from other more northern and western areas were grouped in a distinct cluster or clade (Fig. 2). For most of this century wolves declined in the Minnesota area due to predator control programmes and loss of prime habitat. Coyotes, meanwhile, expanded their range, and where they encountered wolves dispersing in the fragmented habitat, interspecific mating opportunities arose. Our data indicated that repeated hybridization between the two species resulted in the introgression of several coyote mtDNA genotypes into wolf populations. Mating between male wolves and female coyotes produced hybrid offspring carrying coyote mtDNA, which was then transferred into the wolf populations through backcrossing of female hybrids with male wolves. Such an event occurred at least six times. The analysis also revealed an asymmetry in the gene flow between the two species: although coyote mtDNA was found in many wolves, wolf mtDNA was not discovered in any coyotes.
Our results concerning the current captive bred red wolf population led us to propose that a similar explanation could be invoked, namely that coyote mtDNA found in red wolves reflected prior hybridization between the two species. Indeed, Ron Nowak's morphological analysis had indicated that as red wolves became rare in the southeast, they underwent hybridization with the abundant and expanding coyote population. Although great care was taken in choosing the 14 individuals for the captive breeding programme, our mtDNA showed that captive red wolves had a mtDNA genotype indistinguishable from that found in coyotes from Louisiana. This result indicated that an ancestor of captive red wolves paired with a female coyote, potentially some time before the captive breeding effort commenced.
A logical next step was to widen the search for diagnosable "red wolf" characters. We therefore analysed mtDNA from 77 canids captured between 1974-76 as part of the captive breeding programme. These canids had previously been classified on morphological grounds as either red wolves, coyotes, or hybrids of the two species. It thus came as rather a surprise when our mtDNA analysis revealed only genotypes otherwise found in southern coyotes or in grey wolves. We even found a grey wolf genotype characteristic of Mexican grey wolves that formerly inhabited parts of Texas (Wayne & Jenks, 1991).
Given the appreciably larger sample size of this investigation, the lack of a discernible "red wolf" genotype required some explanation. It was possible that our sample had still failed to include examples of red wolves with unique genotypes, but we proposed an alternative hypothesis. Previous studies had shown that coyotes can hybridize with both red and grey wolves. Grey wolves frequently disperse 50km or more, and theoretical models suggest that hybrid zones up to 50 times as large as an individual's dispersal distance are possible (Barton & Hewitt, 1989). Moreover the recent grey wolf/coyote hybrid zone in Minnesota and Eastern Canada comprises roughly half of the historical distribution of the red wolf. It would thus seem reasonable to propose that three centuries of human settlement in southeastern USA could have given rise to a grey wolf/coyote hybrid zone at least as large as the red wolf's range. Perhaps, then the red wolf might not be a distinct species, but rather represent a zone of various crosses or intergrades between grey wolf and coyote. Such an explanation would be in accordance with the recorded difficulties of classification using morphology alone.
This hypothesis could best be tested by a direct investigation of the evolution of the proposed hybrid zone, requiring analysis of historical specimens. The development of the polymerase chain reaction (PCR) made possible amplification of DNA from museum specimens, and this could then be sequenced directly, a technique which provides more information than restriction site analysis.
We analysed a sequence of 400 base pairs from the mitochondrial cytochrome b gene taken from six red wolf specimens in the Smithsonian Institution's fur vault. These specimens had been gathered from five US states between 1905-1930: before the hybridization with coyotes was thought to be widespread (Nowak, 1979). Our analysis revealed that all six individuals had genotypes which could be classified with either grey wolves of coyotes. No unique sequence was found, supporting our hypothesis of a hybrid swarm, and throwing considerable doubt on the accepted view of the red wolf as a long distinct taxon.
The hybrid hypothesis was not well received by the USFWS. The red wolf recovery programme was proving extremely successful but expensive, and thus politically sensitive. Furthermore the molecular data were admittedly incon-clusive. Our pre-1930 sample was limited, and although mtDNA has many advantages, its uniparental and clonal mode of inheritance promote rapid loss of diversity, and may provide a biased representation of gene flow. The molecular evidence would be far more convincing if distinct red wolf marker genes (alleles) could be shown to be absent in the nuclear genotype.
The majority of the nuclear genome evolves slowly, and differences between closely related species are usually too small to generate diagnostic nuclear markers. However, some parts of the genome consist of short sections of simple repeated sequences. These "microsatellite" loci have extremely high mutation rates, and can be amplified using PCR. Repeat loci are frequently so variable that a unique individual genetic "fingerprint" can be constructed often from typing just a few loci. Moreover, microsatellites can be amplified by PCR and scored using standard techniques. Thus microsatellites from both extant and historic samples can be sequenced and analysed and corresponding alleles compared between species.
Initial studies of over 300 coyotes and grey wolves confirmed earlier mtDNA results concerning the magnitude and asymmetry of the recent hybridization in Minnesota and eastern Canada (Roy et al., 1994a). In addition, the evidence from microsatellite analysis indicated that hybridization is only successful between male grey wolves and female coyotes, not visa versa. Considering the disparity in size between the two species, it seems unlikely that small male coyotes would be able to dominate the larger wolf females successfully in mating encounters.
Having shown that the microsatellite analysis appeared to correctly reflect the recent hybridization events, microsatellite polymorphisms in 40 red wolves from the captive population were evaluated (Roy et al., 1994a) The alleles found in this sample were compared with those from grey wolves and coyotes but no unique alleles characteristic of red wolves were discovered. All 53 alleles discovered in the red wolf sample were also found in coyotes.
Although not favourable to the ancient origin hypothesis, this result was not conclusive. The current red wolf stock was descended from so few founders that unique alleles could conceivably have been lost from the captive population, or indeed during the rapid decline of the species in its dwindling range. However, captive stocks of Mexican grey wolves, a subspecies that suffered a decline similar to the red wolf and were founded by only four individuals, had been shown to have a unique mtDNA genotype and unique microsatellite alleles (Wayne et al., 1992; García-Moreno et al., in press). The investigation therefore continued, as before, with a study of historic specimens. With the aid of Ron Nowak, 16 red wolf skins were selected from the Smithsonian vault, and their mtDNA and microsatellites amplified using the PCR. However, analysis of this additional sample also failed to reveal any unique mtDNA genotypes or microsatellite alleles when compared with those from grey wolves and coyotes (Roy et al., in review).
In an attempt to clarify the issue further, we then examined the genetic similarities between historic and recent red wolves, and between these and other wolf-like canids, using overall frequency similarities of microsatellite alleles as measured by genetic distance values among populations (Roy et al., 1994a; 1994b). Historic red wolves were shown to differ little from captive red wolves, indicating that the founder population was an excellent representative sample of the existing gene pool. This, however, was the extent of the genetic support for the species recovery programme; relationships between red wolves and other Canis species served only to reinforce the hybrid swarm hypothesis. Red wolves appeared to be closely related to populations of grey wolves that are known to hybridize with coyotes, and had allele frequencies generally intermediate between non-hybridizing populations of coyotes and grey wolves (Fig.3).
This analysis uncovered other important information. Both the current and historic red wolf samples showed surprisingly high levels of genetic variability. In the sample from the captive population, heterozygosity was over 60%, and several different alleles were discovered at most loci. Small populations tend to be characterized by low levels of variability, indeed two other endangered canids the Ethiopian wolf (Canis simensis) and the Mexican grey wolf, had previously been shown to have significantly lower heterozygosity (Gottelli et al., 1994, García-Moreno et al., in press). The high levels revealed in both red wolf samples are somewhat incongruous in a dwindling population reduced to only a few individuals from which 14 apparently pure-bred individuals were selected for captive breeding, but would be wholly consistent for a population representing a hybrid swarm containing a mixture of genes from both coyotes and grey wolves species.
The genetic evidence from both nuclear and mitochondrial DNA strongly supports the hypothesis that red wolves have hybridized with both grey wolves and coyotes in their recent past. This situation could have developed historically as a result of habitat changes that favoured hybridization of the two species such as is occurring today in eastern Canada. Alternatively, hybridization might have begun thousands of years ago (Dowling et al., 1992); the genetic data cannot easily discriminate between these two scenarios. In addition, our results are consistent with the red wolf possibly having been a distinct subspecies of grey wolf, such as the Mexican grey wolf is today. The Mexican wolf is the most distinct of surviving North American grey wolves and has a few unique genetic markers (García-Moreno et al., in press). Such markers may have existed historically in the red wolf but been lost due to hybridization with coyotes and small population size. However, our results are not consistent with the ancient origin and genetic isolation of the red wolf as envisioned by Nowak because the red wolf would be expected to have many unique genetic markers, more than coyotes and grey wolves have. We would be unlikely to miss so many genetic markers in our extensive recent and historical samples of the red wolf, even given hybridization and small population size.
The idea that the red wolf may have been a subspecies similar to the Mexican wolf is in fact quite appealing. Both the Mexican wolf and red wolf are smaller than other grey wolves (in accordance with Bergmann's rule) and some morphological studies have supported the idea that present-day red wolves are descendants of a now extinct unique southern subspecies of grey wolf (Lawrence & Bossert, 1967; 1975). If the red wolf was a southern subspecies of grey wolf, gene flow would have occurred with other grey wolf populations throughout the species' history in North America, but to a limited degree, allowing some morphological differentiation. The subspecies hypothesis would also explain the persistence of a red wolf morphology into the fossil record and the suggested "absence" of grey wolves in the historic range of red wolves. Otherwise, it is difficult to imagine why grey wolves, which have the largest distribution of any land mammal and are known from fossils in Virginia, Arkansas, Nebraska, Texas, and Georgia (Nowak, 1979) would have so carefully avoided entering the southeastern corner of the US where red wolves were hypothesized to exist in isolation.
The taxonomic status of the red wolf remains controversial. Although the genetic evidence is clear cut, morphological and behavioural evidence have been cited in support of its status as a unique species (Nowak, 1992; Phillips & Henry, 1992). Meanwhile the captive programme continues to flourish. If the hybrid hypothesis is correct, is the expense justifiable?
The protection of hybrid populations and issues of taxonomic distinction are not, in general, satisfactorily addressed by the US Endangered Species Act. Furthermore, the causes and consequences of hybridization are varied, and it would perhaps be best to evaluate each case separately. For example, many new plant species arise from a single cross between two species. Such ?hybrids' are of value in themselves and deserve protection as distinct species. Similarly, new hybrid forms of fish may form naturally as water drainages change and once distinct forms mix (DeMarais et al., 1992). In contrast, the genetic uniqueness of a number of rare species is at risk from hybridization. Conservation of the highly endangered Ethiopian wolf is beset by many problems, not least of which is interbreeding with free-ranging domestic dogs (Gottelli et al., 1994). In such situations the hybrid form is patently undesirable, and only merits protection if the pure-bred form becomes extinct or so reduced in numbers as to become unviable.
It can be hard to evaluate the conservation status of intermediate forms in hybrid zones whose geographical limits are determined by dynamic interaction between selection and dispersal. Nevertheless, there are certain generalities which should apply. Firstly, one should consider the causes of hybridization. Where hybrid zones develop as a result of disturbance or artificial introduction of non-native or domestic species, the hybrids should be accorded lower protection than distinct species. Hybridization may happen naturally, however, as two or more previously geographically isolated species shift or expand their range, perhaps due to climatic factors. The resulting hybrids may persist for some time and become an important part of the ecological landscape, and would thus merit preservation. Secondly, hybrids may develop unique genetic or phenotypic qualities over time, which may justify greater protection than hybrid forms which could be easily regenerated by future interbreeding of the parent species.
We can tentatively apply these criteria to the canids investigated. The hybrids resulting from the recent interbreeding of coyotes with grey wolves in the Minnesota region would not deserve protection as a distinct form. They arose as a result of habitat alterations and predator control measures which decimated the wolf population. Now, under protection of the ESA, grey wolves are once more flourishing in Minnesota, which currently supports over 2000 individuals. Previous analysis of grey wolves in this area suggests that hybridization with coyotes is no longer occurring, and grey wolves have been observed actively to exclude coyotes from their territories. Therefore not only did hybridization happen under unnatural conditions, it does not persist when the causal factors are reversed. Furthermore, the hybrids are of recent origin and do not appear to have developed any unique characteristics worthy of preservation.
The red wolf situation is more uncertain due to the time scale involved. Historical records and the genetic evidence would suggest that it is either an ancient, naturally formed hybrid which was in existence before human settlement of its range in the early 18th century, or a later hybrid which arose, at least in part, as a result of human settlement. It has nevertheless been in existence for quite some time and may have played an important role in the predator community of the area. Moreover, if we accept the possibility of the red wolf being a distinct subspecies of grey wolf (currently defined as populations that are generally allopatric and have a distinct evolutionary heritage (Ryder 1986; Avise & Ball 1990; O'Brien & Mayr 1991)), then although the red wolf may not now satisfy this definition because of hybridization, it may be the only living repository of characteristics once held by a valid subspecies of grey wolf. In order to justify curtailment of the captive breeding program, it should be demonstrated that the loss of the distinct red wolf phenotype (Nowak, 1979) and behaviour (Phillips & Henry, 1992) is reversible by simple interbreeding of extant coyotes and grey wolves. Ecological concerns need to be considered as well; red wolves, even if they are intergrades of grey wolf and coyote, may be extremely successful and have an important role in the predator community of disturbed habitats of the southcentral USA. The red wolf in its current form would therefore seem to merit some degree of protection.
The debate on the origin and conservation of the red wolf needs to be an open one and both sides should support their case with new data published in peer-reviewed journals. This should be an academic debate, removed from concerns about the importance and persistence of the red wolf reintroduction programme. Moreover, the facts on which both sides agree should be incorporated into the rationale and design underlying the reintroduction programme. For example, it is agreed by both sides that hybridization between coyotes and red wolves was common as the red wolf approached extinction in the wild. The same conditions that led to hybridization then (red wolves in small numbers relative to the abundant coyote) exist in areas where the red wolf is now being introduced. Reintroduced red wolves have been observed with coyotes and hybrid litters may have been produced in the wild (Mike Phillips, pers. comm.). Therefore, the reintroduction programme needs to confront the problems of hybridization, perhaps by introducing several packs of red wolves into areas where coyotes are rare in the hope that they may exclude invading coyotes and perhaps maintain their genetic integrity over time.
I am extremely grateful to Laura Handoca without whose collaboration and enormous editorial effort it would not have been possible to compile this article in time.
* Bob Wayne's training in molecular genetics began at the National Camcer Institute, followed by his tenure as Head of Conservation Genetics at London's Institute of Zoology. He is currently a professor at the University of California in Los Angeles. His molecular studies have made a major contribution to understanding canid taxonomy and shed light on many associated conservation issues.
© 1995 International Union for the Conservation of Nature and Natural Resources
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