Wolves from the Great Lakes area were historically decimated due to habitat loss and predator control programmes. Under the protection of the US Endangered Species Act, the population has rebounded to approximately 3000 individuals. We show that the pre-recovery population was dominated by mitochondrial DNA haplotypes from an endemic American wolf referred to here as the Great Lakes wolf. In contrast, the recent population is admixed, and probably derives also from the grey wolf (Canis lupus) of Old World origin and the coyote (Canis latrans). Consequently, the pre-recovery population has not been restored, casting doubt on delisting actions.
Habitat modification can have both positive and negative effects on the abundance and distribution of species. A striking example in this regard is the distribution of two native American carnivorans: the grey wolf (Canis lupus) and the coyote (Canis latrans). Habitat loss and direct persecution led to the eradication of grey wolves over most of the contiguous USA and parts of southern Canada (Carbyn 1987). With the extinction of wolves and associated habitat changes, the smaller coyotes expanded their geographical range from the central plains and western North America north to Alaska, east to the Atlantic and south to Panama (Hilton 1978; Beckoff 1982; also see Sacks et al. 2004).
Owing to protection under the US Endangered Species Act, the restoration of wolves to the Great Lake states of Minnesota, Wisconsin and Michigan has been a remarkable success. The population was nearly exterminated approximately 100 years ago but now numbers over 3000 individuals, an increase which has led to a recent delisting action (Fish and Wildlife Service 2003). The delisting petition defined this wolf as Canis lupus lycaon, a subspecies of grey wolf. However, the petition also acknowledged controversy concerning the taxonomic status of Great Lakes wolves and the likelihood that hybridization between wolf-like taxa (the western grey wolf, C. lupus; the Great Lakes wolf, C. l. lycaon and the coyote, C. latrans) in the area had obscured genetic distinction. Consequently, a critical concern is the degree to which restoration has preserved the pre-recovery form of wolf that inhabited the US Great Lake states and whether delisting favours the persistence of hybrids at the expense of pure forms. These concerns are further complicated by the recent suggestion based on molecular genetic data that C. l. lycaon is a distinct wolf-like species unique to the region that evolved independently in North America from a coyote-like ancestor. Consequently, hybridization among three wolf-like species is a possibility (Lehman et al. 1991; Wilson et al. 2000; Kyle et al. 2006).
2. Material and methods
We genetically analysed 17 historic wolves approximately 100 years old and 68 recent wolves representing the diversity of coyote-like (n=54) and wolf-like (n=14) haplotypes identified in Canadian and US samples by Lehman et al. (1991; figure 1 and table 1). DNA was extracted from historic specimens with phenol–chloroform as in Leonard et al. (2005). Precautions for the analysis of low-copy DNA include nucleic acid isolation in separately designated ancient DNA facility, negative controls and replications. Two PCR and extraction blanks were run with each PCR to monitor for contamination. We used three different sets of primers to produce overlapping sequences for comparison to detect contamination (Leonard et al. 2002). Further, 26 fragments were replicated twice, 9 fragments three times and 1 fragment four times. One historic haplotype was also observed in modern specimens and two historic haplotypes were found in multiple specimens, suggesting that polymerase error was not the origin of these haplotypes. Recent samples were amplified as in Vilà et al. (1999).
Average sequence divergence within and between taxa and their standard errors, as well as bootstrap replicates, were assessed using Mega (Kumar et al. 2004). Population size of the historic population was estimated from nucleotide diversity (θ) using DnaSP v. 4.10.7 (Tajima 1996; Rozas & Rozas 1999). We estimated female effective population size from the expression θ=2 Nefμ, where Nef is the female effective population size and μ is the mutation rate. We used a value of μ based on a divergence time between coyotes and grey wolves of 2 million years (Nowak 2003) and a generation time of 3 years (Mech & Seal 1987), which yields a μ of 1.1×10−7 substitutions/site/generation. Consequently, a census size was estimated assuming equal reproduction of the sexes and that 30% of the population were reproductive adults (Vilà et al. 1999). A divergence rate of 2μ was used with the average HKY distances between Great Lakes area wolf haplotypes to estimate their age of origin.
Phylogenetic trees were based on 421–422 base pair (bp) sequences of the 5′ end of the mitochondrial control region from pre-recovery Great Lakes wolves, recent Great Lakes wolves and previously published North American grey wolf and coyote sequences (Vilà et al. 1999; Leonard et al. 2005). Three methods were used to construct the phylogeny. First, a neighbour-joining phylogeny was constructed in PAUP* v. 4.0.b10 (Swofford 2002) using a HKY model of sequence evolution with a gamma shape parameter a=0.0683, estimated in ModelTest v. 3.04 (Posada & Crandall 1998). The support for the internal nodes was estimated by 1000 bootstrap pseudoreplicates. Second, maximum parsimony trees using a heuristic search and indels as a fifth state were constructed using PAUP* v. 4.0.b10. Finally, Bayesian analyses using MrBayes v. 3.1.1 (Huelsenbeck & Ronquist 2001; Ronquist & Huelsenbeck 2003) were repeated twice for 3 500 000 steps, at which point the standard deviation of split frequencies was less than 0.01, and the first 25% of trees were discarded as burnin.
Departure from monophyly of the Great Lakes wolves was examined in MacClade v. 3.06 (Maddison & Maddison 2002) by determining the number of additional steps required to force monophyly of the pre-recovery Great Lakes wolf haplotypes (excluding GL4; see §3). The likelihood of the most parsimonious tree and one in which the pre-recovery Great Lakes wolves (except GL4) were monophyletic were calculated in PAUP* v. 4.0.b10.
Comparison of pre-recovery Great Lakes wolf sequences show that, with the exception of one Quebec wolf (haplotype GL4), all haplotypes are most closely related to each other and divergent from sequences in extant grey wolves and coyotes. Excluding haplotype GL4, pre-recovery sequences differ by 2–12 bps (0.5–4.5%) from each other and by an average of 13.6±3.8 (s.d.) (6.4±2.9%) and 24.7±2.5 bp (19.1±5.1%) from coyotes and grey wolves, respectively. There are 49 or 60 parsimony informative sites in the dataset used to construct the phylogeny, depending on inclusion of indels as informative sites.
Phylogenetic analysis with the grey wolf as out-group suggests that pre-recovery Great Lakes wolf sequences are basal to those in modern coyotes (figure 1). A tree that constrains all pre-recovery sequences to be monophyletic is only one step longer than the shortest tree and not less likely. We estimated female effective population size of historic Great Lakes wolves to be approximately 60 000 from the observed value of θ=0.013. This implies a census size of approximately 400 000 individuals, far greater than the current population size. Based on the average divergence among pre-recovery haplotypes excluding GL4, this diversity probably originated approximately 300 000 years ago.
We compared pre-recovery sequences with those in recent Great Lakes wolves and found that only haplotype GL2 persists (figure 1). Haplotype GL1 may also be extant as it is identical to a shorter sequence identified in recent Ontario wolves (haplotype C1; Wilson et al. 2000). Additionally, a haplotype more closely related to the pre-recovery haplotypes than to coyotes was identified in Minnesota (GL19). Haplotypes GL1 and GL19 were found in 21 of 68 (31%) of the recent Great Lakes wolves that we typed. The other 47 samples had grey wolf (n=14, 21%) or coyote haplotypes (n=33, 49%; figure 1). Consequently, the mitochondrial DNA data suggest that the dominant wolf currently existing in the Great Lakes area is derived principally from coyotes, grey wolves and their hybrid descendants, with a smaller contribution from the pre-recovery Great Lakes wolf. However, nuclear DNA analyses are needed to further confirm this conclusion.
Our results show that the wolves inhabiting the Great Lakes states prior to recovery were genetically distinct and had a large geographical range. Comparison of pre- and post-recovery Great Lakes wolves suggests that phylogenetically distinct wolf-like canids largely supplanted the pre-recovery form. These new invaders have a mixed ancestry including contributions from grey wolves, coyotes and Great Lakes wolves. Prior genetic evidence suggests that hybridization has been increasing in the Great Lakes region over the past 50 years and that hybrids now populate large areas of the Great Lakes states and New England (Lehman et al. 1991; Roy et al. 1994; Wilson et al. 2000; Kyle et al. 2006). Historical records indicate that hybridization between coyotes and wolf-like canids in the area did not begin until after 1900 when coyotes first entered the region (Hilton 1978; Lehman et al. 1991). The coyote invasion was probably a consequence of habitat alteration, changes in prey abundance, reduction in wolf numbers and other anthropogenic causes.
If hybridization is a result of natural factors, such as post-glacial range expansion, and involves entities listed by the Endangered Species Act, then hybrids can justifiably be protected (Jenks & Wayne 1992; Fish and Wildlife Service 1996). Indeed, hybrids may provide material for adaptation and evolution (Arnold 1997) and lead to increased population viability (Hedrick 1995). However, if hybridization is occurring in disturbed habitats and otherwise reflects human activities that can be mitigated, then delisting should be postponed to protect the listed entity until such contributing factors are ameliorated (Jenks & Wayne 1992). The US Endangered Species Act protects distinct populations (Pennock & Dimmick 1997) and hybrids (Fish and Wildlife Service 1996). Therefore, protection of the population in the US Great Lakes can be justified given that the historical conditions that have given rise to the current population can be sufficiently understood.
We thank Robert Fisher, National Museum of Natural History, Smithsonian Institution for historic samples, Carles Vilà for critical review and Genetics Laboratory, Smithsonian Institution for logistical support. Funding was provided by the National Science Foundation (OPP-0352634, OPP-9817937) and the Swedish Research Council.
- Received July 9, 2007.
- Accepted October 1, 2007.
- © 2007 The Royal Society