Melanism and Wolves

Animal models of aggression are an important tool for understanding what is correlated with differential aggression within a species. Epigenetics and its correlation to aggression is not an area that has thoroughly explored for gray wolves, Canis lupus. Through methylation data of 59 wolves, this project aimed to determine whether melanism has an effect on methylation, and if differential methylation is a significant factor in predicting aggression in wolves. I synthesized and collected data that included aggressiveness ratings, wolf pedigree, sex, age, and pack information for predictor and covariate factors. Reduced Representation Bisulfite Sequencing (RRBS) was used to obtain the methylation data. Instead of focusing on the entire genome, I looked specifically for regions that are differentially methylated with respect to aggression. To accomplish this, I used linear mixed models to analyze the relationship between methylation and other external and environmental factors. After regions were identified, I determined their function, if they were silenced by melanism, and whether or not they play a significant role in regulating aggression. This method allowed me to assess the relative importance of factors such as sex, age, coat color, and methylation with respect to aggressive behavior. The linear mixed model produced several hundred methylated sites, which were then ranked by p-value and grouped by position and beta values. This left three main region candidates to examine. Two of the groups were on chromosome 7, and both were associated with immune responses. A third group was on chromosome 10, and although non-coding was near protein coding regions. Although unable to determine a genetic mechanistic effect melanism has on a wolf’s genome, this project leads to further research on how methylated sites effect gray wolf aggression.

Aggression is a quantitative trait deeply entwined with individual fitness. Mapping the genomic architecture underlying such traits is complicated by complex inheritance patterns, social structure, pedigree information and gene pleiotropy. Here, we leveraged the pedigree of a reintroduced population of grey wolves (Canis lupus) in Yellowstone National Park, Wyoming, USA, to examine the heritability of and the genetic variation associated with aggression. Since their reintroduction, many ecological and behavioural aspects have been documented, providing unmatched records of aggressive behaviour across multiple generations of a wild population of wolves. Using a linear mixed model, a robust genetic relationship matrix, 12,288 single nucleotide polymorphisms (SNPs) and 111 wolves, we estimated the SNP‐based heritability of aggression to be 37% and an additional 14% of the phenotypic variation explained by shared environmental exposures. We identified 598 SNP genotypes from 425 grey wolves to resolve a consensus pedigree that was included in a heritability analysis of 141 individuals with SNP genotype, metadata and aggression data. The pedigree‐based heritability estimate for aggression is 14%, and an additional 16% of the phenotypic variation was explained by shared environmental exposures. We find strong effects of breeding status and relative pack size on aggression. Through an integrative approach, these results provide a framework for understanding the genetic architecture of a complex trait that influences individual fitness, with linkages to reproduction, in a social carnivore. Along with a few other studies, we show here the incredible utility of a pedigreed natural population for dissecting a complex, fitness‐related behavioural trait.

Pigmentation is often used to understand how natural selection affects genetic variation in wild populations since it can have a simple genetic basis, and can affect a variety of fitness-related traits (e.g., camouflage, thermoregulation, and sexual display). In gray wolves, the K locus, a β-defensin gene, causes black coat color via a dominantly inherited K^B allele. The allele is derived from dog-wolf hybridization and is at high frequency in North American wolf populations. We designed a DNA capture array to probe the geographic origin, age, and number of introgression events of the K^B allele in a panel of 331 wolves and 20 dogs. We found low diversity in K^B, but not ancestral k^y, wolf haplotypes consistent with a selective sweep of the black haplotype across North America. Further, North American wolf K^B haplotypes are monophyletic, suggesting that a single adaptive introgression from dogs to wolves most likely occurred in the Northwest Territories or Yukon. We use a new analytical approach to date the origin of the K^B allele in Yukon wolves to between 1,598 and 7,248 years ago, suggesting that introgression with early Native American dogs was the source. Using population genetic simulations, we show that the K locus is undergoing natural selection in four wolf populations. We find evidence for balancing selection, specifically in Yellowstone wolves, which could be a result of selection for enhanced immunity in response to distemper. With these data, we demonstrate how the spread of an adaptive variant may have occurred across a species’ geographic range.

The use of functional mutations, in addition to standard noncoding molecular markers, can help to detect hybridization and gene introgression in wild canid populations. We analyzed ancestry of a canid pack breeding in Central Italy that showed black coats and other unusual morphological traits suggesting wolf × dog hybrid origins. Individuals were identified by genotyping excremental DNA at 13 autosomal microsatellites, mtDNA control region sequences, a male-specific restriction site on the ZFX/Y gene to determine the gender of the individuals, four Y-linked microsatellites to determine male haplotypes, and two melanistic mutations: a SNP at exon 4 of the Agouti locus and a 3-bp deletion at a β-Defensin gene, the K locus. Results showed that: (1) the pack was founded by a single breeding pair of related individuals, probably brother and sister, and no immigrant was detected; (2) parents and offspring showed signals of admixture at autosomal microsatellites; and (3) the melanistic K locus deletion was present in the black-coated female parent and in 8/14 offspring, but it was absent in the wild type male parent. This deletion was found also in 17/40 village dogs randomly sampled from nearby areas, but it was absent in a random sample of 40 Italian wolves. These findings suggest that the pack received the K locus deletion from dogs. Admixture analyses of empirical and simulated genotypes indicate the parents of the pack originated through a single hybridization event at least two generations back. Genetic and phenotypic assessments of coat color mutations can contribute to investigation of the origin and dynamics of functional polymorphisms in hybridizing wolf populations and to develop appropriate guidelines to contrast hybridization with their domesticated relatives.

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Morphological diversity within closely related species is an essential aspect of evolution and adaptation. Mutations in the Melanocortin 1 receptor (Mc1r) gene contribute to pigmentary diversity in natural populations of fish, birds, and many mammals. However, melanism in the gray wolf, Canis lupus, is caused by a different melanocortin pathway component, the K locus, that encodes a beta-defensin protein that acts as an alternative ligand for Mc1r. We show that the melanistic K locus mutation in North American wolves derives from past hybridization with domestic dogs, has risen to high frequency in forested habitats, and exhibits a molecular signature of positive selection. The same mutation also causes melanism in the coyote, Canis latrans, and in Italian gray wolves, and hence our results demonstrate how traits selected in domesticated species can influence the morphological diversity of their wild relatives.