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Statistical inferences about inbreeding depression are often derived from analyses with low power and a high risk of failing to detect inbreeding depression. That risk is widely appreciated by scientists familiar with the relevant statistical and genetical theory, but may be overlooked and underappreciated by decision-makers. Consequently, there is value in demonstrating this risk using a real example. We use data from the wolf population on Isle Royale to demonstrate the difficulty of making reliable statistical inferences about inbreeding depression. This wolf population is known-by other methods-to have gone effectively extinct due to deleterious genetic processes associated with inbreeding. Beyond that demonstration, we use two case-studies-wolves on Isle Royale and vaquita (porpoises) from the Gulf of California, Mexico-to show how statistical inferences about inbreeding depression can affect conservation decisions. According to most decision theory, decisions depend importantly on: 1) probabilities that certain states exist (e.g. inbreeding depression is present) and 2) the utility assigned to various outcomes (e.g. the value of acting to mitigate inbreeding when it is present). The probabilities are provided by statistical inference; whereas utilities are almost entirely determined by normative values and judgements. Our analysis suggests that decisions to mitigate inbreeding depression are often driven more by utilities (normative values) than probabilities (statistical inferences). As such, advocates for mitigating inbreeding depression will benefit from better communicating to decision-makers the value of populations persisting and the extent to which decisions should depend on normative values., (© The Author(s) 2023. Published by Oxford University Press on behalf of The American Genetic Association. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Previous studies of canid population and evolutionary genetics have relied on high-quality domestic dog reference genomes that have been produced primarily for biomedical and trait mapping studies in dog breeds. However, the absence of highly contiguous genomes from other Canis species like the gray wolf and coyote, that represent additional distinct demographic histories, may bias inferences regarding interspecific genetic diversity and phylogenetic relationships. Here, we present single haplotype de novo genome assemblies for the gray wolf and coyote, generated by applying the trio-binning approach to long sequence reads generated from the genome of a female first-generation hybrid produced from a gray wolf and coyote mating. The assemblies were highly contiguous, with contig N50 sizes of 44.6 and 42.0 Mb for the wolf and coyote, respectively. Genome scaffolding and alignments between the two Canis assemblies and published dog reference genomes showed near complete collinearity, with one exception: a coyote-specific chromosome fission of chromosome 13 and fusion of the proximal portion of that chromosome with chromosome 8, retaining the Canis-typical haploid chromosome number of 2n = 78. We evaluated mapping quality for previous RADseq data from 334 canids and found nearly identical mapping quality and patterns among canid species and regional populations regardless of the genome used for alignment (dog, coyote, or gray wolf). These novel wolf and coyote genome reference assemblies will be important resources for proper and accurate inference of Canis demography, taxonomic evaluation, and conservation genetics., (© The Author(s) 2024. Published by Oxford University Press on behalf of The American Genetic Association. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

RK Wayne has arguably been the most influential geneticist of canids, famously promoting the conservation of wolves in his homeland, the United States. His influence has been felt in other countries and regions outside the contiguous United States, where he inspired others, also including former graduate students and research fellows of his, to use modern molecular techniques to examine the evolutionary biology of canids to inform the conservation and management of wolves. In this review, we focus on the implications of Wayne's work on wolves outside the United States. He envisioned a clear future for wolf conservation research, involving the study of wolves' ecological and genetic diversity, and the description of ecotypes requiring conservation. He also documented widespread hybridization among canids and introgression of DNA from domestic dogs to wolves, a process that started dozens of thousands of years ago. His work therefore calls for innovative studies, such as examining the potential fitness benefits of introgression. Inspired by his results, for example, on the purging of deleterious alleles in small populations, wolf researchers should use novel molecular tools to challenge other conservation genetics paradigms. Overall, RK Wayne's work constitutes a call for answers, which as scientists or citizens concerned with conservation matters, we are obliged to address, as we contribute to monitoring and maintaining biodiversity during our period of dramatic transformations of the biosphere., (© The Author(s) 2024. Published by Oxford University Press on behalf of The American Genetic Association. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Among the three main divergent lineages of gray wolf (Canis lupus), the Holarctic lineage is the most widespread and best studied, particularly in North America and Europe. Less is known about Tibetan (also called Himalayan) and Indian wolf lineages in southern Asia, especially in areas surrounding Pakistan where all three lineages are thought to meet. Given the endangered status of the Indian wolf in neighboring India and unclear southwestern boundary of the Tibetan wolf range, we conducted mitochondrial and genome-wide sequencing of wolves from Pakistan and Kyrgyzstan. Sequences of the mitochondrial D-loop region of 81 wolves from Pakistan indicated contact zones between Holarctic and Indian lineages across the northern and western mountains of Pakistan. Reduced-representation genome sequencing of eight wolves indicated an east-to-west cline of Indian to Holarctic ancestry, consistent with a contact zone between these two lineages in Pakistan. The western boundary of the Tibetan lineage corresponded to the Ladakh region of India's Himalayas with a narrow zone of admixture spanning this boundary from the Karakoram Mountains of northern Pakistan into Ladakh, India. Our results highlight the conservation significance of Pakistan's wolf populations, especially the remaining populations in Sindh and Southern Punjab that represent the highly endangered Indian lineage., (© The Author(s) 2023. Published by Oxford University Press on behalf of The American Genetic Association. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The gray wolf (Canis lupus) population on the Iberian Peninsula was the largest in western and central Europe during most of the 20th century, with its size apparently never under a few hundred individuals. After partial legal protection in the 1970s in Spain, the northwest Iberian population increased to about 300 to 350 packs and then stabilized. In contrast to many current European wolf populations, which have been connected through gene flow, the Iberian wolf population has been isolated for decades. Here, we measured changes in genomic diversity and inbreeding through the last decades in a geographic context. We find that the level of genomic diversity in Iberian wolves is low compared with other Eurasian wolf populations. Despite population expansion in the last 50 years, some modern wolves had very high inbreeding, especially in the recently recolonized and historical edge areas. These individuals contrast with others with low inbreeding within the same population. The high variance in inbreeding despite population expansion seems associated with small-scale fragmentation of the range that is revealed by the genetic similarity between modern and historical samples from close localities despite being separated by decades, remaining differentiated from other individuals that are just over 100 km away, a small distance for a species with great dispersal capacity inhabiting a continuous range. This illustrates that, despite its demographically stable condition, the population would probably benefit from favoring connectivity within the population as well as genetic exchange with other European wolf populations to avoid excessive fragmentation and local inbreeding depression., (© The American Genetic Association. 2023.)

For species of management concern, accurate estimates of inbreeding and associated consequences on reproduction are crucial for predicting their future viability. However, few studies have partitioned this aspect of genetic viability with respect to reproduction in a group-living social mammal. We investigated the contributions of foundation stock lineages, putative fitness consequences of inbreeding, and genetic diversity of the breeding versus nonreproductive segment of the Yellowstone National Park gray wolf population. Our dataset spans 25 years and seven generations since reintroduction, encompassing 152 nuclear families and 329 litters. We found more than 87% of the pedigree foundation genomes persisted and report influxes of allelic diversity from two translocated wolves from a divergent source in Montana. As expected for group-living species, mean kinship significantly increased over time but with minimal loss of observed heterozygosity. Strikingly, the reproductive portion of the population carried a significantly lower genome-wide inbreeding coefficients, autozygosity, and more rapid decay for linkage disequilibrium relative to the nonbreeding population. Breeding wolves had significantly longer lifespans and lower inbreeding coefficients than nonbreeding wolves. Our model revealed that the number of litters was negatively significantly associated with heterozygosity (R = -0.11). Our findings highlight genetic contributions to fitness, and the importance of the reproductively active individuals in a population to counteract loss of genetic variation in a wild, free-ranging social carnivore. It is crucial for managers to mitigate factors that significantly reduce effective population size and genetic connectivity, which supports the dispersion of genetic variation that aids in rapid evolutionary responses to environmental challenges., (© The Author(s) 2023. Published by Oxford University Press on behalf of The American Genetic Association. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Despite periodic drops in popularity, Arctic sled dogs continue to play a vital role in northern societies, providing both freight transit and recreational race activities. In this study, we selected the Mackenzie River Husky, a freight dog of complex history, and the Chinook, an American Kennel Club recognized freight dog breed whose heritage reportedly overlaps that of the MKRH, for detailed population analysis. We tested each to determine their component breeds and used admixture analysis to ascertain their population structure. We utilized haplotype analysis to identify genomic regions shared between each population and their founding breeds. Our data show that the Alaskan Malamutes and modern Greenland sled dog contributed to both populations, but there are also unexpected contributions from the German Shepherd dog and Collie. We used haplotype analysis to identify genomic regions nearing fixation in population type and identify provocative genes in each region. Finally, in response to recent reports regarding the importance of dietary lipid genes in Arctic dogs, we analyzed 8 such genes in a targeted analysis observing signatures of selection in both populations at the MLXIPL gene loci. These data highlight the genetic routes that breeds of similar function have taken toward their occupation as successful sled dogs., (Published by Oxford University Press on behalf of The American Genetic Association 2022.)

In North American gray wolves, black coat color is dominantly inherited via a 3 base pair coding deletion in the canine beta defensin 3 (CBD103) gene. This 3 base pair deletion, called the KB allele, was introduced through hybridization with dogs and subsequently underwent a selective sweep that increased its frequency in wild wolves. Despite apparent positive selection, KBB wolves have lower fitness than wolves with the KyB genotype, even though the 2 genotypes show no observable differences in black coat color. Thus, the KB allele is thought to have pleiotropic effects on as-yet unknown phenotypes. Given the role of skin-expressed CBD103 in innate immunity, we hypothesized that the KB allele influences the keratinocyte gene expression response to TLR3 pathway stimulation and/or infection by canine distemper virus (CDV). To test this hypothesis, we developed a panel of primary epidermal keratinocyte cell cultures from 24 wild North American gray wolves of both Kyy and KyB genotypes. In addition, we generated an immortalized Kyy line and used CRISPR/Cas9 editing to produce a KyB line on the same genetic background. We assessed the transcriptome-wide responses of wolf keratinocytes to the TLR3 agonist polyinosinic:polycytidylic acid (polyI:C), and to live CDV. K locus genotype did not predict the transcriptional response to either challenge, suggesting that variation in the gene expression response does not explain pleiotropic effects of the KB allele on fitness. This study supports the feasibility of using cell culture methods to investigate the phenotypic effects of naturally occurring genetic variation in wild mammals., (© The American Genetic Association. 2021. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The red wolf (Canis rufus), a legally recognized and critically endangered wolf, is known to interbreed with coyotes (Canis latrans). Declared extirpated in the wild in 1980, red wolves were reintroduced to northeastern North Carolina nearly a decade later. Interbreeding with coyotes was thought to be restricted to a narrow geographic region adjacent to the reintroduced population and largely believed to threaten red wolf recovery. However, red wolf ancestry was recently discovered in canids along the American Gulf Coast, igniting a broader survey of ancestry in southeastern canid populations. Here, we examine geographic and temporal patterns of genome-wide red wolf ancestry in 260 canids across the southeastern United States at over 164 000 SNP loci. We found that red wolf ancestry was most prevalent in canids sampled from Texas in the mid-1970s, although non-trivial amounts of red wolf ancestry persist in this region today. Further, red wolf ancestry was also observed in a subset of coyotes inhabiting North Carolina, despite management efforts to limit the occurrence of hybridization events. Lastly, we found no evidence of substantial red wolf ancestry in southeastern canids outside of these 2 admixture zones. Overall, this study provides a genome-wide survey of red wolf ancestry in canids across the southeastern United States, which may ultimately inform future red wolf restoration efforts., (© The American Genetic Association 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The red fox (Vulpes vulpes) occurs on multiple continents in diverse habitats, making it an informative system for evolutionary genomic research. However, its phylogeography remains unclear. Previously, mitochondrial DNA and small numbers of nuclear loci provided discordant views. Both markers indicated deep divergence (~ 0.5 million years [MY]) between Eurasian and southern North American populations but differed in the apparent continental affinity of Alaskan red foxes, implying some degree of gene exchange during secondary contact (~0.1 MY). We assayed >173000 nuclear genomic sites in 52 red foxes, along with 2 Rueppell's foxes (Vulpes rueppellii) and a gray wolf (Canis lupus) using the Illumina CanineHD BeadChip. We obtained 5107 single nucleotide polymorphisms (SNPs) in the foxes. Consistent with the Afro-Eurasian origins of red foxes, genetic diversity was higher in Eurasian than North American samples. Phylogenetic trees indicated that Alaskan and southern North American red foxes formed a monophyletic group nested within the Eurasian clade. However, admixture models suggested Alaskan red foxes contained up to 40% Eurasian ancestry. We hypothesize that North American red foxes either hybridized with Eurasian foxes in Beringia at the start of the last glaciation or merged with a Beringian population after the last glaciation. Future work is needed to test between these scenarios and assess speciation.

Defining units that can be afforded legal protection is a crucial, albeit challenging, step in conservation planning. As we illustrate with a case study of the red wolf (Canis rufus) from the southeastern United States, this step is especially complex when the evolutionary history of the focal taxon is uncertain. The US Endangered Species Act (ESA) allows listing of species, subspecies, or Distinct Population Segments (DPSs) of vertebrates. Red wolves were listed as an endangered species in 1973, and their status remains precarious. However, some recent genetic studies suggest that red wolves are part of a small wolf species (C. lycaon) specialized for heavily forested habitats of eastern North America, whereas other authors suggest that red wolves arose, perhaps within the last ~400 years, through hybridization between gray wolves (C. lupus) and coyotes (C. latrans). Using published genetic, morphological, behavioral, and ecological data, we evaluated whether each evolutionary hypothesis would lead to a listable unit for red wolves. Although the potential hybrid origin of red wolves, combined with abundant evidence for recent hybridization with coyotes, raises questions about status as a separate species or subspecies, we conclude that under any proposed evolutionary scenario red wolves meet both criteria to be considered a DPS: they are Discrete compared with other conspecific populations, and they are Significant to the taxon to which they belong. As population-level units can qualify for legal protection under endangered-species legislation in many countries throughout the world, this general approach could potentially be applied more broadly.

The Mexican gray wolf (Canis lupus baileyi) was historically distributed throughout the southwestern United States and northern Mexico. Extensive predator removal campaigns during the early 20th century, however, resulted in its eventual extirpation by the mid 1980s. At this time, the Mexican wolf existed only in 3 separate captive lineages (McBride, Ghost Ranch, and Aragón) descended from 3, 2, and 2 founders, respectively. These lineages were merged in 1995 to increase the available genetic variation, and Mexican wolves were reintroduced into Arizona and New Mexico in 1998. Despite the ongoing management of the Mexican wolf population, it has been suggested that a proportion of the Mexican wolf ancestry may be recently derived from hybridization with domestic dogs. In this study, we genotyped 87 Mexican wolves, including individuals from all 3 captive lineages and cross-lineage wolves, for more than 172000 single nucleotide polymorphisms. We identified levels of genetic variation consistent with the pedigree record and effects of genetic rescue. To identify the potential to detect hybridization with domestic dogs, we compared our Mexican wolf genotypes with those from studies of domestic dogs and other gray wolves. The proportion of Mexican wolf ancestry assigned to domestic dogs was only between 0.06% (SD 0.23%) and 7.8% (SD 1.0%) for global and local ancestry estimates, respectively; and was consistent with simulated levels of incomplete lineage sorting. Overall, our results suggested that Mexican wolves lack biologically significant ancestry with dogs and have useful implications for the conservation and management of this endangered wolf subspecies.

Reduced fitness of admixed individuals is typically attributed to genetic incompatibilities. Although mismatched genomes can lead to fitness changes, in some cases the reduction in hybrid fitness is subtle. The potential role of transcriptional regulation in admixed genomes could provide a mechanistic explanation for these discrepancies, but evidence is lacking for nonmodel organisms. Here, we explored the intersection of genetics and gene regulation in admixed genomes derived from an experimental cross between a western gray wolf and western coyote. We found a significant positive association between methylation and wolf ancestry, and identified outlier genes that have been previously implicated in inbreeding-related, or otherwise deleterious, phenotypes. We describe a pattern of site-specific, rather than genome-wide, methylation driven by inter-specific hybridization. Epigenetic variation is thus suggested to play a nontrivial role in both maintaining and combating mismatched genotypes through putative transcriptional mechanisms. We conclude that the regulation of gene expression is an underappreciated key component of hybrid genome functioning, but could also act as a potential source of novel and beneficial adaptive variation in hybrid offspring., (© The American Genetic Association 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Inbreeding, relatedness, and ancestry have traditionally been estimated with pedigree information, however, molecular genomic data can provide more detailed examination of these properties. For example, pedigree information provides estimation of the expected value of these measures but molecular genomic data can estimate the realized values of these measures in individuals. Here, we generate the theoretical distribution of inbreeding, relatedness, and ancestry for the individuals in the pedigree of the Isle Royale wolves, the first examination of such variation in a wild population with a known pedigree. We use the 38 autosomes of the dog genome and their estimated map lengths in our genomic analysis. Although it is known that the remaining wolves are highly inbred, closely related, and descend from only 3 ancestors, our analyses suggest that there is significant variation in the realized inbreeding and relatedness around pedigree expectations. For example, the expected inbreeding in a hypothetical offspring from the 2 remaining wolves is 0.438 but the realized 95% genomic confidence interval is from 0.311 to 0.565. For individual chromosomes, a substantial proportion of the whole chromosomes are completely identical by descent. This examination provides a background to use when analyzing molecular genomic data for individual levels of inbreeding, relatedness, and ancestry. The level of variation in these measures is a function of the time to the common ancestor(s), the number of chromosomes, and the rate of recombination. In the Isle Royale wolf population, the few generations to a common ancestor results in the high variance in genomic inbreeding., (© The American Genetic Association 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The taxonomic status of the red wolf (Canis rufus) is heavily debated, but could be clarified by examining historic specimens from the southeastern United States. We analyzed mitochondrial DNA (mtDNA) from 3 ancient (350-1900 year olds) putative wolf samples excavated from middens and sinkholes within the historic red wolf range. We detected 3 unique mtDNA haplotypes, which grouped with the coyote mtDNA clade, suggesting that the canids inhabiting southeastern North America prior to human colonization from Europe were either coyotes, which would vastly expand historic coyote distributions, an ancient coyote-wolf hybrid, or a North American evolved red wolf lineage related to coyotes. Should the red wolf prove to be a distinct species, our results support the idea of either an ancient hybrid origin for red wolves or a shared common ancestor between coyotes and red wolves., (© The American Genetic Association. 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A variety of nonrandom mate choice strategies, including disassortative mating, are used by vertebrate species to avoid inbreeding, maintain heterozygosity and increase fitness. Disassortative mating may be mediated by the major histocompatibility complex (MHC), an important gene cluster controlling immune responses to pathogens. We investigated the patterns of mate choice in 26 wild-living breeding pairs of gray wolf (Canis lupus) that were identified through noninvasive genetic methods and genotyped at 3 MHC class II and 12 autosomal microsatellite (STR) loci. We tested for deviations from random mating and evaluated the covariance of genetic variables at functional and STR markers with fitness proxies deduced from pedigree reconstructions. Results did not show evidences of MHC-based disassortative mating. Rather we found a higher peptide similarity between mates at MHC loci as compared with random expectations. Fitness values were positively correlated with heterozygosity of the breeders at both MHC and STR loci, whereas they decreased with relatedness at STRs. These findings may indicate fitness advantages for breeders that, while avoiding highly related mates, are more similar at the MHC and have high levels of heterozygosity overall. Such a pattern of MHC-assortative mating may reflect local coadaptation of the breeders, while a reduction in genetic diversity may be balanced by heterozygote advantages., (© The American Genetic Association 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Genetic relatedness between individuals is an important measure in many areas of biology. However, some relatedness measures for use with molecular (allele) data assume that the individuals themselves are not inbred. Here, we present a new measure of relatedness based on the different modes of identity-by-descent for alleles that has an upper bound of 1 even when the individuals being compared are themselves inbred. This new measure is compared to several other measures of relatedness using several simple examples and pedigree data from the wolf population in Isle Royale National Park., (© The American Genetic Association 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

There is considerable interest in the genetics of wolves (Canis lupus) because of their close relationship to domestic dogs (C. familiaris) and the need for informed conservation and management. This includes wolf populations in Southeast Alaska for which we determined genotypes of 305 wolves at 173662 single nucleotide polymorphism (SNP) loci. After removal of invariant and linked SNP, 123801 SNP were used to quantify genetic differentiation of wolves in Southeast Alaska and wolves, coyotes (C. latrans), and dogs from other areas in North America. There is differentiation of SNP allele frequencies between the species (wolves, coyotes, and dogs), although differentiation is relatively low between some wolf and coyote populations. There are varying levels of differentiation among populations of wolves, including low differentiation of wolves in interior Alaska, British Columbia, and the northern US Rocky Mountains. There is considerable differentiation of SNP allele frequencies of wolves in Southeast Alaska from wolves in other areas. However, wolves in Southeast Alaska are not a genetically homogeneous group and there are comparable levels of genetic differentiation among areas within Southeast Alaska and between Southeast Alaska and other geographic areas. SNP variation and other genetic data are discussed regarding taxonomy and management., (© The American Genetic Association 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

There is a striking color polymorphism for wolves in the Yellowstone National Park where approximately half the wolves are black. The genetic basis for this polymorphism is known, and fitnesses of the genotypes are estimated. These estimates suggest that there is strong heterozygote advantage but substantial asymmetry in the fitness differences of the 2 homozygotes. Theoretically, such fitnesses in a finite population are thought to reduce genetic variation at least as fast as if there were no selection at all. Because the color polymorphism has remained at about the same frequency for 17 years, about 4 generations, we investigated whether this was consistent with the theoretical predictions. Counter to this general expectation of loss, given the initial frequency of black wolves, the theoretical expectation in this case was found to be that the frequency would only decline slowly over time. For example, if the effective population size is 20, then the expected black allele frequency after 4 generations would be 0.191, somewhat less than the observed value of 0.237. However, nearly 30% of the time the expected frequency is 0.25 or greater, consistent with the contemporary observed frequency. In other words and in contrast to general theoretical predictions, because of the short period of time in evolutionary terms and the relatively weak selection at low frequencies, the observed variation and the predicted theoretical variation are not inconsistent., (© The American Genetic Association 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

We studied the distribution of the mitochondrial DNA haplotypes and microsatellite genotypes at 8 loci in 102 gray wolves, 57 livestock guarding dogs, and 9 mongrel dogs from Georgia (Caucasus). Most of the studied dogs had mitochondrial haplotypes clustered with presumably East Asian dog lineages, and most of the studied wolves had the haplotypes clustered with European wolves, but 20% of wolves and 37% of dogs shared the same mitochondrial haplotypes. Bayesian inference with STRUCTURE software suggested that more than 13% of the studied wolves had detectable dog ancestry and more than 10% of the dogs had detectable wolf ancestry. About 2-3% of the sampled wolves and dogs were identified, with a high probability, as first-generation hybrids. These results were supported by the relatedness analysis, which showed that 10% of wolves and 20% of dogs had closest relatives from an opposite group. The results of the study suggest that wolf-dog hybridization is a common event in the areas where large livestock guarding dogs are held in a traditional way, and that gene flow between dogs and gray wolves was an important force influencing gene pool of dogs for millennia since early domestication events. This process may have been terminated 1) in areas outside the natural range of gray wolves and 2) since very recent time, when humans started to more tightly control contacts of purebred dogs.

Small, isolated populations may experience increased extinction risk due to reduced genetic variability at important functional genes, thus decreasing the population's adaptive potential. The major histocompatibility complex (MHC), a key immunological gene cluster, usually shows high variability maintained by positive or balancing selection in response to challenges by pathogens. Here we investigated for the first time, the variability of 3 MHC class II genes (DRB1, DQA1, and DQB1) in 94 samples collected from Italian wolves. The Italian wolf population has been long isolated south of the Alps and is presently recovering from a recent bottleneck that decreased the population to less than 100 individuals. Despite the bottleneck, Italian wolves show remarkable MHC variability with 6-9 alleles per locus, including 2 recently described alleles at DRB1. MHC sequences show signatures of historical selective pressures (high d N/d S ratio, ω > 1.74) but no evidence of ongoing selection. Variation at the MHC genes and 12 background microsatellite loci were not apparently affected by the recent bottleneck. Although MHC alleles of domestic dog origin were detected in 8 genetically admixed individuals, these alleles were rare or absent in nonadmixed wolves. Thus, despite known hybridization events between domestic dogs and Italian wolves, the Italian wolf population does not appear affected by deep introgression of domestic dog MHC alleles.

Domestic dogs have been shown to have multiple alleles of the Agouti Signal Peptide (ASIP) in exon 4 and we wished to determine the level of polymorphism in the common wild canids of Canada, wolves and coyotes, in comparison. All Canadian coyotes and most wolves have banded hairs. The ASIP coding sequence of the wolf did not vary from the domestic dog but one variant was detected in exon 4 of coyotes that did not alter the arginine at this position. Two other differences were found in the sequence flanking exon 4 of coyotes compared with the 45 dogs and 1 wolf. The coyotes also demonstrated a relatively common polymorphism in the 3' UTR sequence that could be used for population studies. One of the ASIP alleles (R96C) in domestic dogs causes a solid black coat color in homozygotes. Although some wolves are melanistic, this phenotype does not appear to be caused by this same mutation. However, one wolf, potentially a dog-wolf hybrid or descendant thereof, was heterozygous for this allele. Likewise 2 coyotes, potentially dog-coyote or wolf-coyote hybrid descendants, were heterozygous for the several polymorphisms in and flanking exon 4. We could conclude that these were coyote-dog hybrids because both were heterozygous for 2 mutations causing fawn coat color in dogs.

The canine major histocompatibility complex contains highly polymorphic genes, many of which are critical in regulating immune response. Since domestic dogs evolved from Gray Wolves (Canis lupus), common DLA class II alleles should exist. Sequencing was used to characterize 175 Gray Wolves for DLA class II alleles, and data from 1856 dogs, covering 85 different breeds of mostly European origin, were available for comparison. Within wolves, 28 new alleles were identified, all occurring in at least 2 individuals. Three DLA-DRB1, 8 DLA-DQA1, and 6 DLA-DQB1 alleles also identified in dogs were present. Twenty-eight haplotypes were identified, of which 2 three-locus haplotypes, and many DLA-DQA1/DQB1 haplotypes, are also found in dogs. The wolves studied had relatively few dog DLA alleles and may therefore represent a remnant population descended from Asian wolves. The single European wolf included carried a haplotype found in both these North American wolves and in many dog breeds. Furthermore, one wolf DQB1 allele has been found in Shih Tzu, a breed of Asian origin. These data suggest that the wolf ancestors of Asian and European dogs may have had different gene pools, currently reflected in the DLA alleles present in dog breeds.

X-linked retinitis pigmentosa (XLRP) is one of the most prevalent forms of a genetically heterogeneous group of inherited retinal disorders of man; more than 70% of XLRP families map to the RP2 or RP3 loci on the human X chromosome. Canine X-linked progressive retinal atrophy (XLPRA), observed in the Siberian husky, is the locus homologue of human RP3, but the gene responsible for XLPRA has not yet been identified. To develop polymorphic markers in the RP3 interval in dogs we have isolated microsatellites from canine BAC clones. Three tightly linked microsatellite loci, CUX20001, CUX30001, and CUX40002, have been investigated in 17 dog breeds or breed varieties. Calculated parameters of variability correspond with the number of repeats at each locus. Pedigree analyses showed tight linkage between the canine t-complex-associated testis-expressed 1-like gene (TCTE1l) and the gene ornithine carbamoyltransferase (OTC). Each microsatellite shows conservation within Canidae, and CUX20001 also amplified in Mustelidae and URSIDAE: These markers represent an important tool in the fine mapping process for the canine region homologous to the RP3 disease interval and are valuable for evaluation of conservation and homology of this region among related species.