Estimating distribution and abundance of wide-ranging species with integrated spatial models: Opportunities revealed by the first wolf assessment in south-central Italy.

Estimating demographic parameters for wide-ranging and elusive species living at low density is challenging, especially at the scale of an entire country. To produce wolf distribution and abundance estimates for the whole south-central portion of the Italian wolf population, we developed an integrated spatial model, based on the data collected during a 7-month sampling campaign in 2020-2021. Data collection comprised an extensive survey of wolf presence signs, and an intensive survey in 13 sampling areas, aimed at collecting non-invasive genetic samples (NGS). The model comprised (i) a single-season, multiple data-source, multi-event occupancy model and (ii) a spatially explicit capture-recapture model. The information about species' absence was used to inform local density estimates. We also performed a simulation-based assessment, to estimate the best conditions for optimizing sub-sampling and population modelling in the future. The integrated spatial model estimated that 74.2% of the study area in south-central Italy (95% CIs = 70.5% to 77.9%) was occupied by wolves, for a total extent of the wolf distribution of 108,534 km2 (95% CIs = 103,200 to 114,000). The estimate of total population size for the Apennine wolf population was of 2557 individuals (SD = 171.5; 95% CIs = 2127 to 2844). Simulations suggested that the integrated spatial model was associated with an average tendency to slightly underestimate population size. Also, the main contribution of the integrated approach was to increase precision in the abundance estimates, whereas it did not affect accuracy significantly. In the future, the area subject to NGS should be increased to at least 30%, while at least a similar proportion should be sampled for presence-absence data, to further improve the accuracy of population size estimates and avoid the risk of underestimation. This approach could be applied to other wide-ranging species and in other geographical areas, but specific a priori evaluations of model requirements and expected performance should be made.

Phylogenetic History and Phylogeographic Patterns of the European Wildcat (Felis silvestris) Populations.

Disentangling phylogenetic and phylogeographic patterns is fundamental to reconstruct the evolutionary histories of taxa and assess their actual conservation status. Therefore, in this study, for the first time, the most exhaustive biogeographic history of European wildcat (Felis silvestris) populations was reconstructed by typing 430 European wildcats, 213 domestic cats, and 72 putative admixed individuals, collected across the entire species' distribution range, at a highly diagnostic portion of the mitochondrial ND5 gene. Phylogenetic and phylogeographic analyses identified two main ND5 lineages (D and W) roughly associated with domestic and wild polymorphisms. Lineage D included all domestic cats, 83.3% of putative admixed individuals, and also 41.4% of wildcats; these latter mostly showed haplotypes belonging to sub-clade Ia, that diverged about 37,700 years ago, long pre-dating any evidence for cat domestication. Lineage W included all the remaining wildcats and putative admixed individuals, spatially clustered into four main geographic groups, which started to diverge about 64,200 years ago, corresponding to (i) the isolated Scottish population, (ii) the Iberian population, (iii) a South-Eastern European cluster, and (iv) a Central European cluster. Our results suggest that the last Pleistocene glacial isolation and subsequent re-expansion from Mediterranean and extra-Mediterranean glacial refugia were pivotal drivers in shaping the extant European wildcat phylogenetic and phylogeographic patterns, which were further modeled by both historical natural gene flow among wild lineages and more recent wild x domestic anthropogenic hybridization, as confirmed by the finding of F. catus/lybica shared haplotypes. The reconstructed evolutionary histories and the wild ancestry contents detected in this study could be used to identify adequate Conservation Units within European wildcat populations and help to design appropriate long-term management actions.

How to Choose? Comparing Different Methods to Count Wolf Packs in a Protected Area of the Northern Apennines.

Despite a natural rewilding process that caused wolf populations in Europe to increase and expand in the last years, human-wolf conflicts still persist, threatening the long-term wolf presence in both anthropic and natural areas. Conservation management strategies should be carefully designed on updated population data and planned on a wide scale. Unfortunately, reliable ecological data are difficult and expensive to obtain and often hardly comparable through time or among different areas, especially because of different sampling designs. In order to assess the performance of different methods to estimate wolf (Canis lupus L.) abundance and distribution in southern Europe, we simultaneously applied three techniques: wolf howling, camera trapping and non-invasive genetic sampling in a protected area of the northern Apennines. We aimed at counting the minimum number of packs during a single wolf biological year and evaluating the pros and cons for each technique, comparing results obtained from different combinations of these three methods and testing how sampling effort may affect results. We found that packs' identifications could be hardly comparable if methods were separately used with a low sampling effort: wolf howling identified nine, camera trapping 12 and non-invasive genetic sampling eight packs. However, increased sampling efforts produced more consistent and comparable results across all used methods, although results from different sampling designs should be carefully compared. The integration of the three techniques yielded the highest number of detected packs, 13, although with the highest effort and cost. A common standardised sampling strategy should be a priority approach to studying elusive large carnivores, such as the wolf, allowing for the comparison of key population parameters and developing shared and effective conservation management plans.

Possible origins and implications of atypical morphologies and domestication-like traits in wild golden jackals (Canis aureus).

Deciphering the origins of phenotypic variations in natural animal populations is a challenging topic for evolutionary and conservation biologists. Atypical morphologies in mammals are usually attributed to interspecific hybridisation or de-novo mutations. Here we report the case of four golden jackals (Canis aureus), that were observed during a camera-trapping wildlife survey in Northern Israel, displaying anomalous morphological traits, such as white patches, an upturned tail, and long thick fur which resemble features of domesticated mammals. Another individual was culled under permit and was genetically and morphologically examined. Paternal and nuclear genetic profiles, as well as geometric morphometric data, identified this individual as a golden jackal rather than a recent dog/wolf-jackal hybrid. Its maternal haplotype suggested past introgression of African wolf (Canis lupaster) mitochondrial DNA, as previously documented in other jackals from Israel. When viewed in the context of the jackal as an overabundant species in Israel, the rural nature of the surveyed area, the abundance of anthropogenic waste, and molecular and morphological findings, the possibility of an individual presenting incipient stages of domestication should also be considered.

"Guess Who's Coming to Dinner": Molecular Tools to Reconstruct multilocus Genetic Profiles from Wild Canid Consumption Remains.

Non-invasive genetic sampling is a practical tool to monitor pivotal ecological parameters and population dynamic patterns of endangered species. It can be particularly suitable when applied to elusive carnivores such as the Apennine wolf (Canis lupus italicus) and the European wildcat (Felis silvestris silvestris), which can live in overlapping ecological contexts and sometimes share their habitats with their domestic free-ranging relatives, increasing the risk of anthropogenic hybridisation. In this case study, we exploited all the ecological and genetic information contained in a single biological canid faecal sample, collected in a forested area of central Italy, to detect any sign of trophic interactions between wolves and European wildcats or their domestic counterparts. Firstly, the faecal finding was morphologically examined, showing the presence of felid hair and claw fragment remains. Subsequently, total genomic DNA contained in the hair and claw samples was extracted and genotyped, through a multiple-tube approach, at canid and felid diagnostic panels of microsatellite loci. Finally, the obtained individual multilocus genotypes were analysed with reference wild and domestic canid and felid populations to assess their correct taxonomic status using Bayesian clustering procedures. Assignment analyses classified the genotype obtained from the endothelial cells present on the hair sample as a wolf with slight signals of dog ancestry, showing a qi = 0.954 (C.I. 0.780-1.000) to the wolf cluster, and the genotype obtained from the claw as a domestic cat, showing a qi = 0.996 (95% C.I. = 0.982-1.000) to the domestic cat cluster. Our results clearly show how a non-invasive multidisciplinary approach allows the cost-effective identification of both prey and predator genetic profiles and their taxonomic status, contributing to the improvement of our knowledge about feeding habits, predatory dynamics, and anthropogenic hybridisation risk in threatened species.

A standardized approach to empirically define reliable assignment thresholds and appropriate management categories in deeply introgressed populations.

Anthropogenic hybridization is recognized as a major threat to the long-term survival of natural populations. While identifying F1 hybrids might be simple, the detection of older admixed individuals is far from trivial and it is still debated whether they should be targets of management. Examples of anthropogenic hybridization have been described between wolves and domestic dogs, with numerous cases detected in the Italian wolf population. After selecting appropriate wild and domestic reference populations, we used empirical and simulated 39-autosomal microsatellite genotypes, Bayesian assignment and performance analyses to develop a workflow to detect different levels of wolf x dog admixture. Membership proportions to the wild cluster (qiw) and performance indexes identified two q-thresholds which allowed to efficiently classify the analysed genotypes into three assignment classes: pure (with no or negligible domestic ancestry), older admixed (with a marginal domestic ancestry) and recent admixed (with a clearly detectable domestic ancestry) animals. Based on their potential to spread domestic variants, such classes were used to define three corresponding management categories: operational pure, introgressed and operational hybrid individuals. Our multiple-criteria approach can help wildlife managers and decision makers in more efficiently targeting the available resources for the long-term conservation of species threatened by anthropogenic hybridization.

Genomic approaches to identify hybrids and estimate admixture times in European wildcat populations.

The survival of indigenous European wildcat (Felis silvestris silvestris) populations can be locally threatened by introgressive hybridization with free-ranging domestic cats. Identifying pure wildcats and investigating the ancestry of admixed individuals becomes thus a conservation priority. We analyzed 63k cat Single Nucleotide Polymorphisms (SNPs) with multivariate, Bayesian and gene-search tools to better evaluate admixture levels between domestic and wild cats collected in Europe, timing and ancestry proportions of their hybrids and backcrosses, and track the origin (wild or domestic) of the genomic blocks carried by admixed cats, also looking for possible deviations from neutrality in their inheritance patterns. Small domestic ancestry blocks were detected in the genomes of most admixed cats, which likely originated from hybridization events occurring from 6 to 22 generations in the past. We identified about 1,900 outlier coding genes with excess of wild or domestic ancestry compared to random expectations in the admixed individuals. More than 600 outlier genes were significantly enriched for Gene Ontology (GO) categories mainly related to social behavior, functional and metabolic adaptive processes (wild-like genes), involved in cognition and neural crest development (domestic-like genes), or associated with immune system functions and lipid metabolism (parental-like genes). These kinds of genomic ancestry analyses could be reliably applied to unravel the admixture dynamics in European wildcats, as well as in other hybridizing populations, in order to design more efficient conservation plans.