Elena Fabbri

Noninvasive molecular tracking of colonizing wolf (Canis lupus) packs in the western Italian Alps.

We used noninvasive methods to obtain genetic and demographic data on the wolf packs (Canis lupus), which are now recolonizing the Alps, a century after their eradication. DNA samples, extracted from presumed wolf scats collected in the western Italian Alps (Piemonte), were genotyped to determine species and sex by sequencing parts of the mitochondrial DNA (mtDNA) control‐region and ZFX/ZFY genes. Individual genotypes were identified by multilocus microsatellite analyses using a multiple tubes polymerase chain reaction (PCR). The performance of the laboratory protocols was affected by the age of samples. The quality of excremental DNA extracts was higher in samples freshly collected on snow in winter than in samples that were older or collected during summer. Preliminary mtDNA screening of all samples allowed species identification and was a good predictor of further PCR performances. Wolf, and not prey, DNA targets were preferentially amplified. Allelic dropout occurred more frequently than false alleles, but the probability of false homozygote determinations was always < 0.001. A panel of six to nine microsatellites would allow identification of individual wolf genotypes, also whether related, with a probability of identity of < 0.015. Genealogical relationships among individuals could be determined reliably if the number of candidate parents was 6–8, and most of them had been sampled and correctly genotyped. Genetic data indicate that colonizing Alpine wolves originate exclusively from the Italian source population and retain a high proportion of its genetic diversity. Spatial and temporal locations of individual genotypes, and kinship analyses, suggest that two distinct packs of closely related wolves, plus some unrelated individuals, ranged in the study areas. This is in agreement with field observations.

From the Apennines to the Alps: colonization genetics of the naturally expanding Italian wolf ( Canis lupus ) population

Wolves in Italy strongly declined in the past and were confined south of the Alps since the turn of the last century, reduced in the 1970s to approximately 100 individuals surviving in two fragmented subpopulations in the central‐southern Apennines. The Italian wolves are presently expanding in the Apennines, and started to recolonize the western Alps in Italy, France and Switzerland about 16 years ago. In this study, we used a population genetic approach to elucidate some aspects of the wolf recolonization process. DNA extracted from 3068 tissue and scat samples collected in the Apennines (the source populations) and in the Alps (the colony), were genotyped at 12 microsatellite loci aiming to assess (i) the strength of the bottleneck and founder effects during the onset of colonization; (ii) the rates of gene flow between source and colony; and (iii) the minimum number of colonizers that are needed to explain the genetic variability observed in the colony. We identified a total of 435 distinct wolf genotypes, which showed that wolves in the Alps: (i) have significantly lower genetic diversity (heterozygosity, allelic richness, number of private alleles) than wolves in the Apennines; (ii) are genetically distinct using pairwise FST values, population assignment test and Bayesian clustering; (iii) are not in genetic equilibrium (significant bottleneck test). Spatial autocorrelations are significant among samples separated up to c. 230 km, roughly correspondent to the apparent gap in permanent wolf presence between the Alps and north Apennines. The estimated number of first‐generation migrants indicates that migration has been unidirectional and male‐biased, from the Apennines to the Alps, and that wolves in southern Italy did not contribute to the Alpine population. These results suggest that: (i) the Alps were colonized by a few long‐range migrating wolves originating in the north Apennine subpopulation; (ii) during the colonization process there has been a moderate bottleneck; and (iii) gene flow between sources and colonies was moderate (corresponding to 1.25–2.50 wolves per generation), despite high potential for dispersal. Bottleneck simulations showed that a total of c. 8–16 effective founders are needed to explain the genetic diversity observed in the Alps. Levels of genetic diversity in the expanding Alpine wolf population, and the permanence of genetic structuring, will depend on the future rates of gene flow among distinct wolf subpopulation fragments.

Who is who? Identification of livestock predators using forensic genetic approaches

Molecular identifications of salivary DNA are increasingly applied in wildlife forensic investigations, and are successfully used to identify unknown livestock predators, or elucidate cases of large carnivore attacks to humans. In Europe most of livestock predations are attributed to wolves (Canis lupus), thought free-ranging dogs are sometime the responsible, and false predations are declared by breeders to obtain compensations. In this study we analyzed 33 salivary DNA samples collected from the carcasses of 13 sheep and a horse presumably predated by wolves in seven farms in central Italy. Reliable individual genotypes were determined in 18 samples (corresponding to samples from nine sheep and the horse) using 12 unlinked autosomal microsatellites, mtDNA control-region sequences, a male-specific ZFX/ZFY restriction-site and four Y-linked microsatellites. Results indicate that eight animals were killed by five wolves (a male and four different females), the ninth by a female dog while the horse was post-mortem consumed by a male dog. The genotype of one female wolf matched with the genetic profile of a female wolf that was non-invasively sampled 4 years before in the same area near livestock predation remains. Genetic identifications always supported the results of veterinary reports. These findings show that salivary DNA genotyping, together with detailed veterinary field and necropsy reports, provides evidence which helps to correctly identify species, gender and individual genetic profiles of livestock predators, thus contributing to clarify attack dynamics and to evaluate the impact of wolf predations on husbandry.

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.

MHC Variability in an Isolated Wolf Population in Italy

Small, isolated populations may experience increased extinction risk due to reduced genetic variability at important functional genes, thus decreasing the populations 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.