Marco Galaverni

Genome Sequencing Highlights the Dynamic Early History of Dogs

To identify genetic changes underlying dog domestication and reconstruct their early evolutionary history, we generated high-quality genome sequences from three gray wolves, one from each of the three putative centers of dog domestication, two basal dog lineages (Basenji and Dingo) and a golden jackal as an outgroup. Analysis of these sequences supports a demographic model in which dogs and wolves diverged through a dynamic process involving population bottlenecks in both lineages and post-divergence gene flow. In dogs, the domestication bottleneck involved at least a 16-fold reduction in population size, a much more severe bottleneck than estimated previously. A sharp bottleneck in wolves occurred soon after their divergence from dogs, implying that the pool of diversity from which dogs arose was substantially larger than represented by modern wolf populations. We narrow the plausible range for the date of initial dog domestication to an interval spanning 11–16 thousand years ago, predating the rise of agriculture. In light of this finding, we expand upon previous work regarding the increase in copy number of the amylase gene (AMY2B) in dogs, which is believed to have aided digestion of starch in agricultural refuse. We find standing variation for amylase copy number variation in wolves and little or no copy number increase in the Dingo and Husky lineages. In conjunction with the estimated timing of dog origins, these results provide additional support to archaeological finds, suggesting the earliest dogs arose alongside hunter-gathers rather than agriculturists. Regarding the geographic origin of dogs, we find that, surprisingly, none of the extant wolf lineages from putative domestication centers is more closely related to dogs, and, instead, the sampled wolves form a sister monophyletic clade. This result, in combination with dog-wolf admixture during the process of domestication, suggests that a re-evaluation of past hypotheses regarding dog origins is necessary.

Worldwide patterns of genomic variation and admixture in gray wolves

The gray wolf (Canis lupus) is a widely distributed top predator and ancestor of the domestic dog. To address questions about wolf relationships to each other and dogs, we assembled and analyzed a data set of 34 canine genomes. The divergence between New and Old World wolves is the earliest branching event and is followed by the divergence of Old World wolves and dogs, confirming that the dog was domesticated in the Old World. However, no single wolf population is more closely related to dogs, supporting the hypothesis that dogs were derived from an extinct wolf population. All extant wolves have a surprisingly recent common ancestry and experienced a dramatic population decline beginning at least ∼30 thousand years ago (kya). We suggest this crisis was related to the colonization of Eurasia by modern human hunter-gatherers, who competed with wolves for limited prey but also domesticated them, leading to a compensatory population expansion of dogs. We found extensive admixture between dogs and wolves, with up to 25% of Eurasian wolf genomes showing signs of dog ancestry. Dogs have influenced the recent history of wolves through admixture and vice versa, potentially enhancing adaptation. Simple scenarios of dog domestication are confounded by admixture, and studies that do not take admixture into account with specific demographic models are problematic.

Hypoxia adaptations in the grey wolf (Canis lupus chanco) from Qinghai-Tibet Plateau.

The Tibetan grey wolf (Canis lupus chanco) occupies habitats on the Qinghai-Tibet Plateau, a high altitude (>3000 m) environment where low oxygen tension exerts unique selection pressure on individuals to adapt to hypoxic conditions. To identify genes involved in hypoxia adaptation, we generated complete genome sequences of nine Chinese wolves from high and low altitude populations at an average coverage of 25× coverage. We found that, beginning about 55,000 years ago, the highland Tibetan grey wolf suffered a more substantial population decline than lowland wolves. Positively selected hypoxia-related genes in highland wolves are enriched in the HIF signaling pathway (P = 1.57E-6), ATP binding (P = 5.62E-5), and response to an oxygen-containing compound (P≤5.30E-4). Of these positively selected hypoxia-related genes, three genes (EPAS1, ANGPT1, and RYR2) had at least one specific fixed non-synonymous SNP in highland wolves based on the nine genome data. Our re-sequencing studies on a large panel of individuals showed a frequency difference greater than 58% between highland and lowland wolves for these specific fixed non-synonymous SNPs and a high degree of LD surrounding the three genes, which imply strong selection. Past studies have shown that EPAS1 and ANGPT1 are important in the response to hypoxic stress, and RYR2 is involved in heart function. These three genes also exhibited significant signals of natural selection in high altitude human populations, which suggest similar evolutionary constraints on natural selection in wolves and humans of the Qinghai-Tibet Plateau.

Noninvasive sampling and genetic variability, pack structure, and dynamics in an expanding wolf population

Abstract After centuries of population decline and range contraction, gray wolves (Canis lupus) are now expanding in Europe. Understanding wolf social structure and population dynamics and predicting their future range expansion is mandatory to design sound conservation strategies, but field monitoring methods are difficult or exceedingly expensive. Noninvasive genetic sampling offers unique opportunities for the reliable monitoring of wolf populations. We conducted a 9-year-long monitoring program in a large area (approximately 19,171 km2) in northern Italy, aiming to identify individuals, estimate kinship, reconstruct packs, and describe their dynamics. Of 5,065 biological samples (99% scats), we genotyped and sexed 44% reliably using 12 unlinked autosomal microsatellites, 4 Y-linked microsatellites, and a diagnostic mitochondrial DNA control-region sequence. We identified 414 wolves, 88 dogs, and 16 wolf × dog hybrids. Wolves in the study area belonged to at least 42 packs. We reconstructed the genealogy of 26 packs. The mean pack size was 5.6 ± 2.4 SD, including adoptees, with a mean minimum pack home range of 74 km2 ± 52 SD. We detected turnovers of breeding pairs in 19% of the packs. Reproductive wolves were unrelated and unrelated dispersers founded new packs, except for 1 pack founded by a brother–sister pair. We did not detect multiple breeding females in any packs. Overall, the population was not inbred. We found significant isolation by distance and spatial autocorrelation, with nonrandom genetic structure up to a distance of approximately 17 km. We detected 37 dispersers, 14 of which became breeders in new or already existing packs. Our results can be used to model habitat use by wolves, to estimate survival rates, to predict future expansion of the wolf population, and to build risk maps of wolf–human conflicts.

Multilocus detection of wolf x dog hybridization in Italy, and guidelines for marker selection

Hybridization and introgression can impact the evolution of natural populations. Several wild canid species hybridize in nature, sometimes originating new taxa. However, hybridization with free-ranging dogs is threatening the genetic integrity of grey wolf populations (Canis lupus), or even the survival of endangered species (e.g., the Ethiopian wolf C. simensis). Efficient molecular tools to assess hybridization rates are essential in wolf conservation strategies. We evaluated the power of biparental and uniparental markers (39 autosomal and 4 Y-linked microsatellites, a melanistic deletion at the β-defensin CBD103 gene, the hypervariable domain of the mtDNA control-region) to identify the multilocus admixture patterns in wolf x dog hybrids. We used empirical data from 2 hybrid groups with different histories: 30 presumptive natural hybrids from Italy and 73 Czechoslovakian wolfdogs of known hybrid origin, as well as simulated data. We assessed the efficiency of various marker combinations and reference samples in admixture analyses using 69 dogs of different breeds and 99 wolves from Italy, Balkans and Carpathian Mountains. Results confirmed the occurrence of hybrids in Italy, some of them showing anomalous phenotypic traits and exogenous mtDNA or Y-chromosome introgression. Hybridization was mostly attributable to village dogs and not strictly patrilineal. The melanistic β-defensin deletion was found only in Italian dogs and in putative hybrids. The 24 most divergent microsatellites (largest wolf-dog FST values) were equally or more informative than the entire panel of 39 loci. A smaller panel of 12 microsatellites increased risks to identify false admixed individuals. The frequency of F1 and F2 was lower than backcrosses or introgressed individuals, suggesting hybridization already occurred some generations in the past, during early phases of wolf expansion from their historical core areas. Empirical and simulated data indicated the identification of the past generation backcrosses is always uncertain, and a larger number of ancestry-informative markers is needed.

Forensic DNA against wildlife poaching: Identification of a serial wolf killing in Italy

The recent expansion of the Italian wolf population through the Apennine and western Alps, after centuries of contractions, is causing conflicts with human activities leading to a rise in poaching or illegal killings. Here we show how molecular population genetics has been used to identify a suspect serial wolf killer. We analysed DNA extracted from a necklace made of ten presumed wolf canine teeth, confiscated in 2008 to a man living in the northern Italian Apennine (Liguria Region). Individual genotypes were determined using 12 unlinked autosomal microsatellites (STRs), mtDNA control-region sequences, a male-specific ZFX/ZFY restriction-site and three Y-linked STRs. Results indicate that the teeth belonged to six different individuals (three males and three females), which were assigned to the Italian wolf population with p > 0.90 by Bayesian procedures. One of these genotypes matched with the genetic profile of a male wolf previously found-dead and already non-invasively sampled in the same area. Another genotype matched with that of a female wolf non-invasively sampled twice in the same area 1 year before. These data are being used as forensic genetic evidence in the ongoing criminal trial against the suspect serial wolf killer.

Black coats in an admixed wolf × dog pack is melanism an indicator of hybridization in wolves?

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.

Monitoring wolves (Canis lupus) by non-invasive genetics and camera trapping: a small-scale pilot study

Monitoring populations of elusive large carnivores like wolves (Canis lupus), which are often distributed at low density in widespread forested areas, is difficult or exceedingly expensive. Aiming to assess the power of two indirect monitoring methods, non-invasive genetic sampling and camera trapping, we designed a small-scale pilot study that was carried out from 2006 to 2008 in and around the Corno alle Scale Regional Park, Bologna, northern Italian Apennine. We collected 103 non-invasive samples (mainly scats) that were genotyped at 12 microsatellite loci and sexed using the ZFX gene. We identified 11 distinct wolf genotypes within the park and four wolf genotypes outside. Spatial locations and kinship analyses showed that the wolves belong to three different packs. The breeding pair of the ‘Park’ pack showed a complete turnover in the two sampling seasons. Two dogs, but no hybrids, were identified in the area. Up to five unbaited camera traps were activated (for 1,250 trapping-nights) close to recent wolf presence marks. We obtained 103 photos of wolves, documenting the reproduction events, the minimum number of adult and young wolves, and phenotype information each year. We obtained information on health conditions detecting probable sarcoptic mange in three individuals. Camera trapping also showed that the presence of wolves in a chase area during wild boar (Sus scrofa) hunting sessions was significantly higher in the nights just after a chase (P < 0.01, χ2 test; P < 0.07¸ exact Fisher test). The data obtained from genetics and camera trapping were consistent with one other, and in part complementary. The total cost of the study (c. 28,000 €) was moderate, suggesting that this integrated approach can be successfully used to monitor the structure and dynamics of local wolf packs.

Demographically-Based Evaluation of Genomic Regions under Selection in Domestic Dogs.

Controlling for background demographic effects is important for accurately identifying loci that have recently undergone positive selection. To date, the effects of demography have not yet been explicitly considered when identifying loci under selection during dog domestication. To investigate positive selection on the dog lineage early in the domestication, we examined patterns of polymorphism in six canid genomes that were previously used to infer a demographic model of dog domestication. Using an inferred demographic model, we computed false discovery rates (FDR) and identified 349 outlier regions consistent with positive selection at a low FDR. The signals in the top 100 regions were frequently centered on candidate genes related to brain function and behavior, including LHFPL3, CADM2, GRIK3, SH3GL2, MBP, PDE7B, NTAN1, and GLRA1. These regions contained significant enrichments in behavioral ontology categories. The 3rd top hit, CCRN4L, plays a major role in lipid metabolism, that is supported by additional metabolism related candidates revealed in our scan, including SCP2D1 and PDXC1. Comparing our method to an empirical outlier approach that does not directly account for demography, we found only modest overlaps between the two methods, with 60% of empirical outliers having no overlap with our demography-based outlier detection approach. Demography-aware approaches have lower-rates of false discovery. Our top candidates for selection, in addition to expanding the set of neurobehavioral candidate genes, include genes related to lipid metabolism, suggesting a dietary target of selection that was important during the period when proto-dogs hunted and fed alongside hunter-gatherers.

First evidence of hybridization between golden jackal (Canis aureus) and domestic dog (Canis familiaris) as revealed by genetic markers

Interspecific hybridization is relatively frequent in nature and numerous cases of hybridization between wild canids and domestic dogs have been recorded. However, hybrids between golden jackals (Canis aureus) and other canids have not been described before. In this study, we combined the use of biparental (15 autosomal microsatellites and three major histocompatibility complex (MHC) loci) and uniparental (mtDNA control region and a Y-linked Zfy intron) genetic markers to assess the admixed origin of three wild-living canids showing anomalous phenotypic traits. Results indicated that these canids were hybrids between golden jackals and domestic dogs. One of them was a backcross to jackal and another one was a backcross to dog, confirming that golden jackal–domestic dog hybrids are fertile. The uniparental markers showed that the direction of hybridization, namely females of the wild species hybridizing with male domestic dogs, was common to most cases of canid hybridization. A melanistic 3bp-deletion at the K locus (β-defensin CDB103 gene), that was absent in reference golden jackal samples, but was found in a backcross to jackal with anomalous black coat, suggested its introgression from dogs via hybridization. Moreover, we demonstrated that MHC sequences, although rarely used as markers of hybridization, can be also suitable for the identification of hybrids, as long as haplotypes are exclusive for the parental species.