<b>Differential DNA methylation is associated with coat color in Alaskan coastal wolves (</b><b><i>Canis lupus ligoni</i></b><b>)</b>

In North America, wolves generally have either gray or black coats, and the proportions of these colors vary across populations. Coat color is an important signal of individual condition and immunological status in wolves. Yet, the molecular mechanisms underpinning variation in this trait remain unclear. Epigenetic modifications, such as DNA methylation, play an important role in mediating environmental effects on melanin-based coloration in mammals, especially in relation to diet. Yet, how DNA methylation varies with coat color in wild animals has not been investigated, limiting our understanding of how variation in this trait emerges. Here, we used a population-level, reduced representation sequencing approach to survey DNA methylation among 147 black and gray Alexander Archipelago wolves (<i>Canis lupus ligoni</i>). We detected 880 differentially methylated regions (DMRs) between black and gray wolves. Of these DMRs, 93% of loci were hypermethylated in black compared to gray wolves. Across the entire genome however, black wolves had significantly lower levels of total DNA methylation compared to gray wolves. When we coupled our epigenomic data with information on wolves’ diet, we found that of the 880 DMRs detected between black and gray wolves, only 55 loci (6.5%) were also associated with diet. This finding suggests that the relationship between DNA methylation and coat color is not driven by dietary differences in our study. Together, our findings warrant future investigation into how coat color varies with DNA methylation at functionally relevant genetic loci (e.g., <i>MC1R</i>, <i>Agouti</i>, and <i>CBD103</i>). This will augment our understanding of how intraspecific coat color differences emerge among North American wolves, as well as its eco-evolutionary significance.