Northeastern Naturalist 598 J.W.B. Power, N. LeBlanc, S. Bondrup-Nielsen, M.J. Boudreau, M.S. O’Brien, and D.T. Stewart 22001155 NORTHEASTERN NATURALIST 2V2(o3l). :2529,8 N–6o1. 23 Spatial Genetic and Body-Size Trends in Atlantic Canada Canis latrans (Coyote) Populations Jason W.B. Power1, Nathalie LeBlanc1, Søren Bondrup-Nielsen1, Mike J. Boudreau2, Mike S. O’Brien2, and Donald T. Stewart1,* Abstract - Eastern Canis latrans (Coyote) dispersed into northeastern North America during the last century and into Nova Scotia in the 1970s. En route, Coyotes hybridized extensively with C. lycaon (Eastern Wolf). Coyote populations in northeastern North America contain mitochondrial and nuclear DNA characteristics of both species. In samples collected from Nova Scotia, Prince Edward Island, New Brunswick, and Newfoundland, we found mitochondrial DNA haplotypes characteristic of Coyote and/or Eastern Wolf with decreasing haplotype diversity consistent with sequential founder events/bottlenecks moving from west to east generally and on islands. Principal components analysis of a suite of morphological characters indicated that male eastern Coyotes from Nova Scotia with Eastern Wolf mitochondrial DNA are significantly larger than male eastern Coyotes from the same region with Coyote mitochondrial DNA. Introduction Canis latrans Say (Coyote), was historically restricted to the Great Plains region of North America west of the Mississippi River (Banfield 1974, Stains 1975) but Coyotes now inhabit almost all regions between Alaska and Costa Rica (Fener et al. 2005, Harrison 1992, Hidalgo-Mihart et al. 2004, O’Brien 1983, Parker 1995). During the latter part of the 19th century, the Coyotes’ range expanded throughout southern Canada and the Great Lakes region, eventually reaching New Brunswick by 1958, mainland Nova Scotia by 1977, Cape Breton Island by 1980, Prince Edward Island by 1983, and Newfoundland by 1985 (Parker 1995). Nova Scotia is connected to New Brunswick (and the rest of North America) via the small Isthmus of Chignecto, which has served as a dispersal corridor for several large mammals including Coyotes into the province (Scott and Hebda 2004). Coyotes have demonstrated phenomenal dispersal ability at several biogeographic scales (Bekoff 1977, Parker 1995, Patterson 1995) and have been photographed on ice flows in the Northumberland Strait and the Gulf of St. Lawrence (McGrath 2004). Cape Breton Island, Prince Edward Island, and Newfoundland are isolated islands historically inaccessible by bridges. It is thought that Coyotes arrived on these islands via ice flows from the mainland (i.e., Prince Edward Island and Cape Breton Island were colonized from a mainland source) or via ice flows from another island (e.g., Newfoundland may have been colonized from Cape Breton Island). Coyotes may also have 1Department of Biology, Acadia University, Wolfville, NS, Canada B4P 2R6. 2Wildlife Division, Nova Scotia Department of Natural Resources, Kentville, NS, Canada B4N 2E5. *Corresponding author - don.stewart@acadiau.ca. Manuscript Editor: Thomas W. French Northeastern Naturalist Vol. 22, No. 3 J.W.B. Power, N. LeBlanc, S. Bondrup-Nielsen, M.J. Boudreau, M.S. O’Brien, and D.T. Stewart 2015 599 colonized Cape Breton Island via the Canso Causeway, which is ~1 km in length and was constructed in 1955. Extensive range expansion of Coyotes into eastern North America occurred after a decrease in the Canis lupus L. (Gray Wolf) population resulting from their local extirpation by over-harvesting and habitat destruction (Thurber and Peterson 1991). Although the phylogenetic and phylogeographic history of the genus Canis in North America is complex, it appears that Western Coyotes dispersed east, where they hybridized extensively with Canis lycaon Schreber, commonly referred to as the Eastern Wolf (e.g., Monzón et al. 2014, Wheeldon et al. 2010), the Great Lakes Wolf (e.g., Kays et al. 2010, Koblmuller et al. 2009, vonHoldt et al. 2011), or the Algonquin Wolf (e.g., Chambers et al. 2012). In this paper, we use the name Eastern Wolf for this taxon. Coyote colonization occurred at a particularly fast rate in Ontario, where they were exposed to Eastern Wolf populations. Their more southerly route through Ohio has been extensively studied (e.g., Benson et al. 2012, Kays et al. 2010, Koblmuller et al. 2009, Leonard and Wayne 2008, Schwartz and Vucetich 2009, Wheeldon and White 2009, Wilson et al. 2009). In contrast to earlier hypotheses that eastern Coyotes in Ohio did not hybridize with Eastern Wolves or with Gray Wolves, recent assessment of eastern Coyote genomes by Monzon et al. (2014) indicated widespread admixture of Eastern Wolf, Gray Wolf, and even Canis lupus familiaris L. (Domestic Dog). Monzon et al. (2014) have proposed several plausible yet presently untested hypotheses to account for these patterns of admixture of the nuclear genomes of these various canid species. In terms of mitochondrial DNA, there is an asymmetric pattern of matings among canid species resulting in a discordant pattern of mtDNA introgression compared to the nuclear genome (Monzon et al. 2014). Eastern Coyote populations possess primarily western Coyote mitochondrial DNA (mtDNA) with varying degrees of introgressed Eastern Wolf mtDNA (e.g., Way et al. 2010). Perhaps not surprisingly, eastern Coyotes are morphologically distinct from their ancestors in the west. Eastern Coyotes are the largest morphotype within C. latrans (Gompper 2002, Way 2007, Way and Proietto 2005), weighing ~15–20% more than typical western Coyotes (Blake 2006). Body size in eastern Coyotes may reflect selection for Eastern Wolf alleles or mtDNA haplotypes that contribute to larger body size, possibly in response to an increase in predation on larger prey—e.g., Odocoileus virginianus (Zimmermann) (White-tailed Deer)—compared to the diet of western Coyotes (Kays et al. 2010, Monzón et al. 2014, Wheeldon et al. 2010). Indeed, Benson and Patterson (2013) recently demonstrated 4 definitive cases of eastern Coyote and/or eastern Coyote x Eastern Wolf hybrids killing Alces alces (L.) (Moose). Although hybridization is a natural evolutionary process, it may also be an indicator of environmental change over time (e.g., Rutledge et al. 2010). Mitochondrial DNA, which is inherited through the maternal lineage, can be used to track such hybridization events (e.g., Nunes et al. 2010). Distinct mtDNA sequences, called haplotypes, can be used as one genetic marker in the analysis of other traits such as morphology, behavior, health, etc. For example, Kays et al. (2010) showed that Northeastern Naturalist 600 J.W.B. Power, N. LeBlanc, S. Bondrup-Nielsen, M.J. Boudreau, M.S. O’Brien, and D.T. Stewart 2015 Vol. 22, No. 3 male eastern Coyotes with Eastern Wolf mtDNA haplotypes exhibited significantly wider skulls than eastern Coyotes with western Coyote mtDNA haplotypes in northeastern North America. With this background in mind, the objectives of this study were to: (1) determine the diversity and frequency of mtDNA control-region haplotypes for Coyotes in Atlantic Canada, particularly Cape Breton Island and mainland Nova Scotia and (2) investigate morphological differences between groups of eastern Coyotes in Nova Scotia defined by mtDNA haplotype sequences. Methods Sample collection and study area The majority of samples collected and analyzed in this study were from mainland Nova Scotia (n = 94) and Cape Breton Island (n = 77), Canada. We obtained samples from fur harvesters in Nova Scotia who took part in a legalized harvest and voluntary Coyote carcass-collection program. These carcasses were brought to regional offices of the Nova Scotia Department of Natural Resources (NSDNR) during the 2010–2013 harvest seasons. We obtained additional tissue samples for New Brunswick (n = 11), Prince Edward Island (n = 2), and Newfoundland (n = 14) from the North American Fur Auction depot in Truro, NS, Canada with collection data recorded from trappers upon pick up. No Canadian Council of Animal Care protocol review was required for this work because we collected all samples from specimens that had already been killed for reasons other than this research program. We obtained detailed information on sex, geographic location, date, and method of collection for as many animals as possible. We mapped sampling locations on a 1-km2 grid using the Nova Scotia Atlas (Service Nova Scotia 2006). We collected a tissue sample for genetic analysis from each carcass and stored carcasses at -20 ºC until shipping them to the NSDNR-Wildlife Division in Kentville, NS, for processing. For carcasses that had front and back foot pads intact, we measured left foot length (length from the back edge of the heel pad to the outer tip-toe pad) and width (distance from the outer edges of outer toe pad); the right foot was measured in cases where the left foot was missing. We weighed carcasses turned in with intact pelts with the pelt on before skinning. For each skinned carcass, we weighed and measured the body length (length from tip of nose to base of tail along the dorsal midline contour), chest girth (circumference around torso behind shoulders), skull length (length from the incisors to the tip of sagittal crest), and skull width (distance from the outer edges of the zygomatic arch). Carcass masses predominately reflected winter weight based on harvest dates (trapping season). We omitted incomplete carcasses from weight comparisons. We simmered the skulls in water for ~15 min to facilitate extraction of the lower jaw and 2 lower canines. We classified Coyotes as adults if radiographs of canine teeth indicated a pulp cavity–tooth-width ratio of ≤0.45 as measured ~15 mm from the tip of the root (Knowlton and Whittemore 2001) and by submitting a lower canine to Matson’s Laboratory (Missoula, MT) for cementum analysis to confirm age (Ballard et al. 1995). We measured lower canine Northeastern Naturalist Vol. 22, No. 3 J.W.B. Power, N. LeBlanc, S. Bondrup-Nielsen, M.J. Boudreau, M.S. O’Brien, and D.T. Stewart 2015 601 total length and maximum width prior to submitting specimens for analysis. We identified 56 adults in our sample (25 females and 31 males). Genetic analysis We extracted DNA from tissue samples from 199 carcasses using a DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA). For each sample, we amplified and sequenced a 343–347-base pair (bp) fragment of the mitochondrial DNA (mtDNA) control region using published primers (AB13279 [Pilgrim et al. 1998], AB13280 [Wilson et al. 2000]). We also sent polymerase chain reaction (PCR) products to the McGill University and Génome Québec Innovation Centre (Montreal, PQ, Canada) for sample sequencing. We edited mtDNA sequences to 223–228 bp in length using Jalview version 2.8 (2012). We employed the Akaike information criterion in Modeltest (Posada and Crandall 1998) to determine that the HKY substitution model was the best-fitting model of DNA evolution for these samples. We used this model to construct a neighbor-joining tree in MEGA5 version 2.2 (Tamura et al. 2011), and assigned haplotypes, denoted as cla28, cla29 and GL20, corresponding to previously described sequences (Kays et al. 2010). We assessed genetic diversity by measuring relative polymorphism levels using nucleotide diversity, haplotypic diversity (π), and θ as implemented in dnaSP 5 (Librado and Rozas 2009). We used Arlequin v. 3.5 (Excoffier and Lischer 2010) to calculate overall genetic variance within and among populations using an analysis of molecular variance (AMOVA), as well as to calculate pairwise FST values between all populations. Morphological comparisons We compared body measurements of the 56 adult eastern Coyotes obtained from mainland Nova Scotia and Cape Breton Island. We determined differences in body measurements between groups defined by haplotypes using Student’s t-tests and analysis of variance (ANOVA) as implemented in Excel and the R package aov (R Development Core Team 2008), respectively, and principal component analysis (PCA) followed by ANOVA and Tukey’s honest significant differences, as implemented in the R packages prcomp, aov, and TukeyHSD, respectively. For each haplogroup and sex combination, we replaced missing data (which comprised 19% of total male data and 4% of total female data) with the variable mean (e.g., Pigott 2001). We scaled variables to have unit variance. Results and Discussion Genetic analyses The sequences obtained in our study are consistent with previously named haplotypes (Fig. 1; Kays et al. 2010) and show that eastern Coyote populations in Atlantic Canada have both western Coyote and Eastern Wolf mtDNA. The Atlantic Canada population of eastern Coyotes carries 1 of 2 Coyote haplotypes (cla28 or cla29) or an Eastern Wolf haplotype (GL20) (Fig. 1) consistent with the haplotype distribution found in northeastern US populations (Kays et al. 2010). The presence Northeastern Naturalist 602 J.W.B. Power, N. LeBlanc, S. Bondrup-Nielsen, M.J. Boudreau, M.S. O’Brien, and D.T. Stewart 2015 Vol. 22, No. 3 of GL20 is the product of recent hybridization between Coyote and Eastern Wolf and introgression of Eastern Wolf mtDNA into eastwardly dispersing populations of eastern Coyotes (Fig. 1). Several generalizations about mitochondrial diversity in Atlantic Canada eastern Coyotes can be drawn from our analysis, although in some cases these observations must be considered preliminary due to small sample size (e.g., n = 2 for Prince Edward Island). Nucleotide diversity (Table 1) in New Brunswick was comparable to levels found previously in Ohio populations, while diversity in Nova Scotia was similar to that of northeast populations (Kays et al. 2010). Diversity was reduced in Cape Breton samples, and Newfoundland samples had no haplotype variation. Observed diversity levels east of New Brunswick are consistent with populations founded by a small number of migrants. Pairwise FST values revealed significant differences between Newfoundland and each of the following regions: Cape Breton Island, Nova Scotia, Prince Edward Island, and New Brunswick, indicating historical bottlenecks and/or restricted gene-flow between Newfoundland and the southern provinces (Table 2). Similarly, there is evidence of genetic differentiation between the New Brunswick and the Cape Breton Island populations (FST = 0.31, P < 0.001). The FST value between the Nova Scotia and Cape Breton Island populations is lower, though still significantly different from zero (FST = 0.05, P < 0.05). There is likely some on-going Figure 1. Mitochondrial haplotype frequencies of eastern Coyotes from Atlantic Canada (NB, mainland NS, Cape Breton Island, PE, and NL). Northeastern Naturalist Vol. 22, No. 3 J.W.B. Power, N. LeBlanc, S. Bondrup-Nielsen, M.J. Boudreau, M.S. O’Brien, and D.T. Stewart 2015 603 Table 2. Pairwise FST values of Coyote mtDNA CR sequences for 5 locations in Atlantic Canada, calculated in Arlequin, and their associated P-values. FST values are above the diagonal; P-values are below the diagonal. The total FST value for all samples was 0.275 (P < 0.001). * indicates that the FST value is significantly different from 0 with the associated P-value. New Brunswick Nova Scotia Newfoundland Prince Edward Island Cape Breton Island New Brunswick - 0.09402 0.49083* 0.04661 0.31424* Nova Scotia 0.06306 ± 0.0237 - 0.51736* -0.06738 0.04661* Newfoundland 0.00901 ± 0.0091 0.00000 ± 0.0000 - 1 0.75539* Prince Edward Island 0.39640 ± 0.0528 0.65766 ± 0.0526 0.01802 ± 0.0121 - -0.19644 Cape Breton Island 0.00000 ± 0.0000 0.03604 ± 0.0148 0.00000 ± 0.0000 0.99099 ± 0.0030 - Table 1. Summary of genetic diversity observed in the total sample and the 5 sampled locations in Atlantic Canada. Total New Brunswick Nova Scotia Newfoundland Prince Edward Island Cape Breton Island Sample size 197 10 94 14 2 77 # haplotypes 3 3 3 1 1 2 Haplotype diversity 0.465 0.733 0.480 0 0 0.267 Nucleotide diversity, π (per site) 0.01189 0.02259 0.01231 0 0 0.00661 θ (per site) 0.01189 0.01607 0.00889 0 0 0.00504 Average pairwise number of 2.87683 5.467 2.98 0 0 1.599 nucleotide differences, k Northeastern Naturalist 604 J.W.B. Power, N. LeBlanc, S. Bondrup-Nielsen, M.J. Boudreau, M.S. O’Brien, and D.T. Stewart 2015 Vol. 22, No. 3 genetic exchange between mainland Nova Scotia and Cape Breton Island facilitated by the Canso Causeway that connects mainland Nova Scotia to Cape Breton Island. As a consequence of the causeway, currents through the Strait of Canso are severely impeded leading to a build-up of ice across the strait in winter. The causeway has directly or indirectly facilitated the invasion of Cape Breton Island by several meso-carnivores including Procyon lotor L. (Raccoon), Mephitis mephitis (Schreber) (Striped Skunk), Lynx rufus (Schreber) (Bobcat ), and Coyote (Scott and Hebda 2004). Although the FST value between Nova Scotia and New Brunswick is fairly large, it is not significantly different from zero (FST 0.09, P = 0.06). Populations in these provinces likely exchange some genes via the Isthmus of Chignecto, which is apparently not a significant barrier to Coyote dispersal (Scott and Hebda 2004). Other population pairs show no statistically significant genetic structure (Table 2). The particularly small sample size for the Prince Edward Island population makes it difficult to draw inferences about population structure in that province. The reduced nucleotide diversity observed in populations located further east, as well as the haplotypes present, support previous hypotheses proposed for this species; i.e., that eastern Coyote populations in these regions were founded by relatively few individuals who migrated from the southwest and that gene flow i s likely impeded by water (e.g., Kays et al. 2010, Parker 1995). New Brunswick samples did not exhibit any of the rare haplotypes that Kays et al. (2010) detected in their northeastern samples. The absence of rare alleles in our study is possibly due, in part, to sample size; we analyzed only 11 samples from New Brunswick, whereas Kays et al. (2010) determined haplotypes for 453 samples from northeastern North America. The contact zone reported by Kays et al. (2010) contains considerably more rare haplotypes compared to the more northeasterly samples, suggesting that rare haplotypes were lost as Coyotes dispersed farther to the east. This loss of genetic diversity is typical during dispersal, and especially dispersal with limited migration between the new population and the source population (Boileau et al. 1992). Continued sampling efforts will help to further elucidate the genetic connectivity between Coyote populations in the northeastern US and New Brunswick. The mainland Nova Scotia population of eastern Coyotes has the same 3 haplotypes as the New Brunswick population, although the frequencies of occurrence of cla28 and cla29 are quite different. One factor contributing to this discrepancy is likely a degree of bottlenecking in the establishment of the Nova Scotia population, which is connected to New Brunswick by the small Isthmus of Chignecto. This type of sequential bottlenecking has been found previously in feral Mink (Neovision vision (Schreber) in Poland (Zalewski et al. 2011) and island-colonizing birds (Clegg et al. 2002). Presumably a small number of individuals initially traveled this route into mainland Nova Scotia; in particular, it appears that only Coyotes with the cla28 and GL20 haplotypes colonized this province. The relatively low frequency of cla29 suggests few Coyotes with this haplotype made it into the province. Interestingly, individuals with the cla29 haplotype were all collected near the Isthmus of Northeastern Naturalist Vol. 22, No. 3 J.W.B. Power, N. LeBlanc, S. Bondrup-Nielsen, M.J. Boudreau, M.S. O’Brien, and D.T. Stewart 2015 605 Chignecto, in the counties of Colchester, Cumberland, and Pictou, in west-central Nova Scotia. Therefore, it is possible that the cla29 haplotype has only recently entered Nova Scotia. Further evidence supporting the slow spread of cla29 into Nova Scotia is the fact that the Cape Breton Island population only has 2 haplotypes in our sample of 77 individuals, cla28 and GL20. The path Coyotes took to get to Newfoundland is not yet understood (McGrath 2004), but it is suspected that they crossed over from the mainland or from Cape Breton Island on ice flows. Cape Breton Island is the closest likely source of Coyotes for Newfoundland (~105 km across the Cabot Strait). It is possible, however, that Coyotes colonized Newfoundland from New Brunswick, Prince Edward Island, or Quebec. The first known report of Coyotes on Newfoundland was from the western portion of the province in 1985 (Parker 1995). Only one or a few females carrying the GL20 haplotype may have made it across on an ice flow possibly from Cape Breton. Coyotes also likely dispersed onto Prince Edward Island on ice flows with the first confirmed record in 1983 (Parker 1995). The Confederation Bridge, which was completed in 1997, now links New Brunswick to Prince Edward Island. Although Coyotes have been documented crossing bridges (e.g., the Golden Gate Bridge, San Francisco, CA; Sacks et al. 2006), it is unlikely that many individuals would cross the Confederation Bridge given its length (~11 km) and high level of traffic. Again, the most likely scenario involves crossing on ice flows, limiting the number of individuals that make the crossing. Perhaps only those carrying cla28 made it to Prince Edward Island. A larger sample size will be necessary to confirm the absence of other haplotypes in these areas. Morphological analyses We recorded skinned body-weight averages for adult female and male eastern Coyotes in all of our samples collected through the voluntary Coyote carcasscollection program. Averages for adult female and male eastern Coyotes were 12.17 kg for females (n = 195) and 14.57 kg for males (n = 230). Using the formula of Nelson and Lloyd (2005) to convert skinned-carcass mass to non-skinned weight gives values of 14.95 kg and 17.83 kg for females and males, respectively. In our univariate analyses, there were no statistically significant differences in morphological variables between groups of adult male or adult female Coyotes from Nova Scotia as defined by the Coyote or Eastern Wolf haplotype group when we analyzed each measurement separately (Tables 3, 4). In contrast, a composite measure of overall body size based on PCA demonstrated a statistically significant difference between groups of adult male Coyotes defined by haplotype. Principal component 1 (PC1) which is generally interpreted as the size axis (e.g., Rising and Somers 1989), comprised 59% of total variation in males (Fig. 2) and 44% of total variation in females (Fig. 3). Male eastern Coyotes that possessed GL20 (i.e., the Eastern Wolf haplotype) were significantly larger than male eastern Coyotes that possessed western Coyote haplotypes (Tukey’s difference = 3.8 ± 2.9, P = 0.015). There was no significant difference in body size between groups of females defined by haplotype (Tukey’s difference = -1.5 ± 3.0, P = 0.285). Northeastern Naturalist 606 J.W.B. Power, N. LeBlanc, S. Bondrup-Nielsen, M.J. Boudreau, M.S. O’Brien, and D.T. Stewart 2015 Vol. 22, No. 3 Table 4. Adult male morphological characteristics ± 1 SD grouped by mtDNA haplotype (hap). All samples are from Nova Scotia. There were no statistically significant differences between groups defined by haplotype (i.e., cla28 vs. GL 20). Skinned Chest Body Tail Front-paw Back-paw Shoulder Canine-tooth Skull weight girth length length width length width length height length width length width Hap (kg) (cm) (cm) (cm) (mm) (mm) (mm) (mm) (cm) (mm) (mm) (cm) (cm) cla28 14.7 ± 2.8 52.8 ± 3.6 87.6 ± 21.1 34.4 ± 9.3 48.5 ± 4.6 62.4 ± 5.6 42.8 ± 6.7 56.8 ± 3.6 53.6 ± 3.7 40.0 ± 2.5 9.5 ± 0.8 21.2 ± 1.6 11.6 ± 1.3 n = 20 n = 20 n =20 n = 20 n = 19 n = 19 n = 20 n = 20 n = 19 n = 18 n = 20 n =19 n = 19 GL20 15.1 ± 2.1 54.4 ± 6.3 95.9 ± 5.9 39.0 ± 3.9 49.0 ± 4.6 63.2 ± 4.9 42.3 ± 1.6 56.9 ± 4.8 53.6 ± 3.6 39.0 ± 3.3 9.4 ± 1.0 21.3 ± 1.4 11.9 ± 0.9 n = 9 n = 10 n = 10 n = 10 n = 9 n = 9 n = 10 n = 10 n = 8 n = 6 n = 8 n = 8 n = 8 Table 3. Adult female morphological characteristics ± 1 SD grouped by mtDNA haplotype . All samples are from Nova Scotia. There were no statistically significant differences between groups defined by haplotype (i.e., cla28 vs. GL 20). Skinned Chest Body Tail Front-paw Back-paw Shoulder Canine-tooth Skull weight girth length length width length width length height length width length width Hap (kg) (cm) (cm) (cm) (mm) (mm) (mm) (mm) (cm) (mm) (mm) (cm) (cm) cla28 11.9 ± 1.7 48.7 ± 2.9 89.3 ± 4.4 34.9 ± 2.4 44.3 ± 3.9 59.9 ± 4.9 40.1 ± 4.0 54.9 ± 3.1 50.0 ± 4.7 37.6 ± 1.6 8.8 ± 0.8 20.5 ± 1.1 11.1 ± 0.9 n = 19 n = 19 n = 19 n = 19 n = 17 n = 17 n = 18 n = 18 n = 16 n = 14 n = 17 n = 17 n = 17 GL20 11.5 ± 1.8 47.3 ± 2.3 87.2 ± 2.9 34.0 ± 2.8 43.2 ± 3.4 57.2 ± 4.7 37.7 ± 5.0 51.8 ± 4.4 51.5 ± 1.9 37.0 ± 1.0 8.9 ± 0.47 19.3 ± 1.3 10.1 ± 0.9 n = 5 n = 5 n = 5 n = 5 n = 4 n = 4 n = 5 n = 5 n = 5 n = 3 n = 4 n = 3 n = 3 Northeastern Naturalist Vol. 22, No. 3 J.W.B. Power, N. LeBlanc, S. Bondrup-Nielsen, M.J. Boudreau, M.S. O’Brien, and D.T. Stewart 2015 607 The observation that body size was correlated with mitochondrial DNA haplotype warrants further exploration. As mentioned, eastern Coyotes from the northeastern US and southeastern Canada have been shown to be fairly well genetically admixed in terms of nuclear genes from western Coyote (61.73%), Eastern Wolf (13.58%), Gray Wolf (13.62%), and Domestic Dog (11.07%) (Monzón et al. 2014). Although we plan to pursue additional nuclear genetic analysis (sensu Monzón et al. 2014) of our samples to determine if the morphological differences correlate with degree of Eastern Wolf nuclear background, it is possible that the mtDNA genome confers an effect on morphology. For example, Pichaud et al. (2012) recently demonstrated that mtDNA, and not nuclear background or mito-nuclear interactions, was responsible for metabolic activity in experimental Drosophila (fruit fly) populations. Zhang et al. (2008) showed that mitochondrial mutations per se have been implicated in growth rates (and body size) in Bos taurus L. (Nanyang Cattle). Toews et al. (2013) showed that Figure 2. First principal component (PC1) values for male Coyotes with 1 of 2 mtDNA haplotypes, Cla28 or GL20, using 11 morphological measurements and representing 59% of total variation. PC1 has the following loadings: 0.31 * weight (lbs), 0.30 * chest (cm), 0.34 * body length (cm), 0.31 * tail length (cm), 0.22 * front-paw width (mm), 0.32 * front-paw length (mm), 0.17 * back-paw width (mm), 0.29 * back-paw length (mm), 0.30 * shoulder (cm), 0.36 * skull width (cm), 0.36 * skull length (cm). This component can be interpreted as representing general size differences because all measurements load in the same direction. Boxes on this plot represent 25th and 75th percentiles, and the line represents the 50th percentile. Whiskers extend to the highest and lowest values that fall within 1.5 * the interquartile range. Data beyond the ends of whiskers are outliers represented as points. Northeastern Naturalist 608 J.W.B. Power, N. LeBlanc, S. Bondrup-Nielsen, M.J. Boudreau, M.S. O’Brien, and D.T. Stewart 2015 Vol. 22, No. 3 patterns of introgression of mtDNA in the Setophaga coronata spp. (L.) (Yellowrumped Warbler) complex could be explained by selection for mitochondrially encoded differences in respiration rate and ATP production efficiency. More generally, da Fonseca et al. (2008) compared 12 mitochondrial protein-coding genes from 41 mammalian species and found evidence that adaptive evolution of mitochondrial mutations was associated with metabolic requirements such as adaptations to the energy available in the diet and the body size of the species. As summarized by Galtier et al. (2009), there is growing evidence that mtDNA is not always a simple neutral marker of molecular diversity and it may be an important driver of functional evolutionary change. As Kays et al. (2010) and Monzón et al. (2014) have suggested, the recent range expansion of Coyotes into eastern North America and the concomitant hybridization with Eastern Wolves presents a fascinating case study in rapid adaptive evolution; feeding on large prey such as White-tailed Deer and Moose Figure 3. First principal component (PC1) values for female Coyotes with 1 of 2 mtDNA haplotypes, Cla28 or GL20, using 11 morphological measurements and representing 44% of total variation. PC1 has the following loadings: 0.28 * weight (lbs), 0.30 * chest (cm), 0.17 * body length (cm), 0.25 * tail length (cm), 0.34 * front-paw width (mm), 0.39 * front-paw length (mm), 0.37 * back-paw width (mm), 0.38 * back-paw length (mm), -0.02 * shoulder (cm), 0.21 * skull width (cm), 0.39 * skull length (cm). This component can be interpreted as representing general size differences because all measurements load in the same direction. Boxes on this plot represent 25th and 75th percentiles, and the line represents the 50th percentile. Whiskers extend to the highest and lowest values that fall within 1.5 times the inter-quartile range. Northeastern Naturalist Vol. 22, No. 3 J.W.B. Power, N. LeBlanc, S. Bondrup-Nielsen, M.J. Boudreau, M.S. O’Brien, and D.T. Stewart 2015 609 may select for wolf-like traits in eastern Coyotes, leading to larger body size. In the future, we plan to pursue whether there is localized selection for behavioral changes in eastern Coyotes in Nova Scotia compared to their western ancestors, possibly associated with introgression of Eastern Wolf mitochondrial genes and selection for wolf-like behaviors. Acknowledgments We thank the trappers of Nova Scotia, especially J. Camus, and members of the Trappers Association of Nova Scotia for sample submission. We thank B. Ettinger (Furafee Trading, Truro, NS), E. Tichenor, G. Bourgeois, P. Sanderson, W. Pitts, C. Englehart, E. Muntz, B. Galaviz, C. Bossi, S. Nairn, D. Keizer, and F. 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