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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.

Large carnivores are making remarkable comebacks in Europe, but how this affects human-wildlife conflict remains unclear. Rebounding carnivore populations lead to increasing livestock depredation, which in turn leads to greater economic losses for farmers. However, returning carnivores could also influence the behavior of wild ungulates, which are themselves responsible for major crop damage and associated economic losses. Here, we exploit the natural experiment of a rebounding wolf population in the Italian Apennines to study how this affected both types of human-wildlife conflic. We used large datasets of wolf occurrences (n = 351), livestock depredation events (n = 165), and crop damage events by wild boar (n = 3442) to independently model the determinants of livestock depredation and crop damage distribution in relation to wolf habitat suitability over a ten-year period of increasing wolf numbers. These analyses yielded two major insights. First, livestock depredations were mainly related to insufficient prevention measures (e.g. lacking fencing) rather than landscape context, providing a clear pathway to conflict mitigation. Second, crop damage decreased in areas of higher wolf habitat suitability and became more likely in areas of lower wolf habitat suitability, closer to settlements. This suggests increasing predation pressure forces wild boars to avoid the most suitable wolf habitat, leading to a redistribution of crop damage in the landscape. More generally, our study highlights complex human-wildlife interactions as large carnivores recover in human-dominated landscapes, suggesting that multiple, co-occurring conflicts need to be assessed jointly and adaptively in order to foster coexistence between humans and wildlife.

In the European Union, all bat species are strictly protected and member states must ensure their conservation. However, if populations are genetically structured, conservation units that correspond to whole countries may be too large, putting small populations with specific conservation requirements at risk. Geoffroy’s bat (Myotis emarginatus) has undergone well-documented declines at its north-western European range edge between the 1960 and 1990s and is considered to be negatively affected by habitat fragmentation. Here we analysed the species’ genetic population structure and diversity to identify subpopulations with reduced genetic diversity and to scientifically inform conservation management. We generated 811 microsatellite-based genetic profiles obtained from 42 European nursery colonies and analysed a total of 932 sequences of the hypervariable region II of the mitochondrial control region sampled from across Europe. While two geographically widespread genetic populations were inferred to be present in north-western Europe, both nuclear and mitochondrial genetic diversity were lowest in the areas that had experienced a decline during the last century. A microsatellite-based analysis of demographic history did not permit, however, to unequivocally link that reduced genetic diversity to the population contraction event. Given the large geographic extent of the genetic populations, preserving the connectivity of mating sites requires concerted conservation efforts across multiple political jurisdictions. Genetic monitoring ought to be done on a regular basis to ensure that large-scale connectivity is maintained and further loss of genetic diversity is prevented.

The Middle Ages represented a crucial period for the evolutionary history of wolves (Canis lupus), marked by both significant ecosystem changes, especially through the degradation of wooded landscapes and heavy persecution, that drove this species to a dramatic demographic decline. In Europe, informative and well-documented wolf remains from the Medieval Ages are exceptionally rare and are mostly represented by teeth and postcranial elements. In this study, we describe a well-preserved wolf cranium dated to ca. 967–1157 AD from the Po Valley (northern Italy). The specimen was analysed through a multidisciplinary approach including CT-based, ancient DNA, and palaeopathological analyses. Morphological and genetic data supported the assignment of this sample to Canis lupus species. CT-based analyses indicated a typical wolf-like morphology falling into the extant variability of the medium-sized subspecies C. lupus italicus, whereas palaeopathological analyses indicated a severe periodontitis. Phylogenetic analyses showed that the Po valley wolf had a unique and never described mtDNA control region haplotype, testifying variability in the ancient Italian wolf, which has now been lost. This study provides the first comprehensive description of a wolf from the Middle Ages, adding useful information for a deeper knowledge about population dynamics, variability, and diseases of this species.

Background: Understanding the processes that lead to hybridization of wolves and dogs is of scientific and management importance, particularly over large geographical scales, as wolves can disperse great distances. However, a method to efficiently detect hybrids in routine wolf monitoring is lacking. Microsatellites offer only limited resolution due to the low number of markers showing distinctive allele frequencies between wolves and dogs. Moreover, calibration across laboratories is time-consuming and costly. In this study, we selected a panel of 96 ancestry informative markers for wolves and dogs, derived from the Illumina CanineHD Whole-Genome BeadChip (174 K). We designed very short amplicons for genotyping on a microfluidic array, thus making the method suitable also for non-invasively collected samples. Results: Genotypes based on 93 SNPs from wolves sampled throughout Europe, purebred and non-pedigree dogs, and suspected hybrids showed that the new panel accurately identifies parental individuals, first-generation hybrids and first-generation backcrosses to wolves, while second- and third-generation backcrosses to wolves were identified as advanced hybrids in almost all cases. Our results support the hybrid identity of suspect individuals and the non-hybrid status of individuals regarded as wolves. We also show the adequacy of these markers to assess hybridization at a European-wide scale and the importance of including samples from reference populations. Conclusions: We showed that the proposed SNP panel is an efficient tool for detecting hybrids up to the thirdgeneration backcrosses to wolves across Europe. Notably, the proposed genotyping method is suitable for a variety of samples, including non-invasive and museum samples, making this panel useful for wolf-dog hybrid assessments and wolf monitoring at both continental and different temporal scales. JH received funding from the Maj and Tor Nessling Foundation, the Finnish Cultural Foundation and Emil Aaltonen Foundation. Laboratory analyses were cofounded by the (LOEWE) program (Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz) of the German Federal State of Hessen. AvT received funding from the Karl und Marie Schack-Stiftung. RG was supported by the Portuguese Foundation for Science and Technology (FCT, DL57/2016). The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

This case report describes the rescue of an eight-month-old male Italian wolf (Canis lupus italicus), the victim of a car accident that caused it a pulmonary contusion, a fracture of the shaft of right femur, and a metaphyseal fracture of the left stifle. A lateral surgical approach was performed to treat the animal’s multiple contusions and fractures. Afterwards the wolf was transferred to a wild animal recovery center for its rehabilitation, where it fully recovered. After 35 days in captivity the wolf was thus released into the supposed home-range of its original pack, and its movements were monitored by a GPS satellite collar. The collar worked correctly for 479 days. During that period the collar acquired a total of 1202 locations, indicating that the wolf had traveled at least 1590 km, with an average monthly distance (± SD) of 102 ± 40 km, exploring an overall area of about 270 km2. During the first 10 days after its release, the wolf remained in the area of its supposed native pack, whereas at about the age of 10 months the wolf began to make wide extraterritorial movements. The wolf’s last localization was acquired on 13th May 2018, about 17 months after its release, at a linear distance of about 65 km from the release site. These preliminary data showed that the wolf was alive and travelled long distances after its release, and demonstrates how a multidisciplinary management approach can support the recovery and successful release into nature of a rescued wild animal belonging to a flagship species with a notable ecological role, such as the Italian wolf., Ovaj prikaz slučaja opisuje spašavanje osmomjesečnog mužjaka talijanskog vuka (Canis lupus italicus) koji je bio žrtva naleta vozila s posljedičnim nagnječenjem pluća, prijelomom vrata desne bedrene kosti prijelomom u području metafize lijevoga koljena. Primijenjen je lateralni kirurški pristup kako bi se sanirala višestruka nagnječenja i prijelomi u životinje. Nakon toga je vuk prevezen u centar za oporavak divljih životinja gdje se potpuno oporavio. Nakon 35 dana u zatočeništvu vuk je pušten u okoliš nalik na njegov prirodni te je praćen preko GPS ogrlice. Ogrlica je ispravno radila 479 dana. Za to je vrijeme pokazala 1202 lokacije, upućujući na to da je vuk prešao 1590 km, u prosjeku mjesečno 102 ± 40 km (± SD), istražujući područje od 270 km2. Prvih 10 dana nakon oslobađanja vuk je ostao u području nalik na njegov prirodni okoliš, no u dobi od 10 mjeseci počeo je odlaziti na šire područje. Posljednja je njegova lokacija zabilježena 13. svibnja 2018., oko 17 mjeseci nakon puštanja, na zračnoj udaljenosti od oko 65 km od mjesta ispuštanja. Ovaj preliminarni podatak pokazuje da je vuk bio živ i prelazio velike udaljenosti nakon oslobađanja. Također pokazuje da multidisciplinarni pristup spašavanju može pospješiti oporavak i uspješno vraćanje u prirodu divljih životinja koje pripadaju vodećim vrstama s velikom ekološkom važnošću, kao što je talijanski vuk.

International audience

Marmora’s Warbler (Curruca sarda) and Balearic Warbler (C. balearica) are allopatric sibling species and were recently split mostly based on morphological and ethological characteristics. Here we provide the first phylogenetic and phylogeographic analyses of this species complex to support the taxonomic status of C. sarda and C. balearica in light of integrative taxonomy. We sampled the two taxa in most of their breeding ranges and we sequenced three mitochondrial and one nuclear gene region. All C. balearica individuals had private haplotypes for the four markers and formed monophyletic clades. Genetic distances between the two taxa were comparable with those found between other species belonging to the Curruca genus. Furthermore, most of the genetic variance was expressed at the interspecific level, rather than between different populations within taxa or between individuals within populations. Our results strongly support the current taxonomic status of these two warblers as distinct species.

Over the last 40 years the gray wolf (Canis lupus) re-colonized its historical range in Italy increasing human-predator interactions. However, temporal and spatial trends in wolf mortality, including direct and indirect persecution, were never summarized. This study aims to fill this gap by focusing on the situation of Tuscany and Emilia-Romagna regions, hosting a significant proportion of the Italian wolf population, by: (i) identifying the prevalent causes of wolf mortality, (ii) summarizing their temporal and spatial patterns and (iii) applying spatially-explicit Generalized Linear Models to predict wolf persecution. Between October 2005 and February 2021, 212 wolf carcasses were collected and subjected to necropsy, being involved in collisions with vehicles (n = 104), poisoned (n = 45), wounded with gunshot (n = 24) or blunt objects (n = 4) and being hanged (n = 2). The proportion of illegally killed wolves did not increase through time. Most persecution events occurred between October and February. None of our candidate models outperformed a null model and covariates such as the density of sheep farms, number of predations on livestock, or human density were never associated to the probability of having illegally killed wolves, at the municipal scale. Our findings show that conventional correlates of wolf persecution, combined with a supposedly high proportion of non-retrieved carcasses, fail to predict illegal wolf killings in areas where the species have become ubiquitous. The widespread spatial distribution of illegal killings indicates that persecution probably arises from multiple kinds of conflicts with humans, beyond those with husbandry. Wolf conservation in Italy should thus address cryptic wolf killings with multi-disciplinary approaches, such as shared national protocols, socio-ecological studies, the support of experts’ experience and effective sampling schemes for the detection of carcasses.

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.

Hybridisation between wild and domestic taxa raises complex questions for conservation. Genetic advances offer new methods for hybrid identification, yet social and cultural factors can influence study design, and the interpretation, application, and communication of results. A relevant illustration is hybridisation between domestic dogs (Canis lupus familiaris) and wild canids, such as grey wolves (C. lupus). For regional European monitoring programs in areas with expanding wolf populations, priorities include shared genetic markers and inclusion of all relevant reference populations to ensure dispersing wolves are identified as such and not classified as wolf-dog hybrids, which may cause harmful management decisions. Beyond technical developments, hybrid research and conservation management can benefit from improved integration of legal and policy perspectives, recognition of phenotypic traits as broadly unreliable for identification, and attention to the drivers of, and responses to, evolution in human-dominated landscapes. Additionally, the proliferation of unsubstantiated reports about hybrids in popular and social media shows that communication based on verified findings of hybridisation is essential. Hybridisation requires more constructive discussion on how to balance potentially competing conservation objectives, and the integration of multidisciplinary perspectives. These encompass the welfare of individual animals and preservation of historical predator-prey relationships. Conservation measures centred on preserving the ecological function of wild canids likely offer the most sustainable prospects but require improved understanding of the extent to which their behavioural ecology might differ from that of hybrids. Accurate genetic identification is required to fill this critical knowledge gap, advance public discourse, and initiate relevant conservation actions.

The black-footed cat (Felis nigripes) is the smallest felid of Southern Africa, endemic to the arid steppe and savannah habitats. However, though threatened and characterized by decreasing sizes of its populations, a number of ecological, demographic, sanitary, and genetic aspects, essential for the long-term conservation of the species, still remain poorly known. Non-invasive genetic sampling may represent an appropriate and cost-effective tool to fill this lack of information. Thus, for the first time so far, we developed a protocol for species and individual identification of black-footed cats, starting from markers originally designed for the domestic cat and from 23 non-invasively collected samples of captive-bred individuals. We then tested its genotyping efficiency and reliability for future applications in non-invasive genetic monitoring programs of the wild populations. Most of the samples (65%), corresponding to 15 individuals, were successfully genotyped at 316 bp of the mtDNA ND5 and at nine autosomal microsatellites. We detected two black-footed cat mtDNA ND5 haplotypes that were clearly distinguishable from all the other wild and domestic felids. All microsatellites were polymorphic and showed low error rates, probabilities of identity < 0.001 and a mean observed heterozygosity H O = 0.68. Subsequent approximate Bayesian computation simulations confirmed that black-footed cats and African and European wildcats likely experienced sequential population splittings that started during the Late Pliocene and continued through the Early Pleistocene. Our study provided the first reliable and cost-effective molecular multilocus characterization of non-invasively collected samples of black-footed cats. Though solely tested on captive-bred individuals, our method could be applied to design and implement effective long-term monitoring and conservation plans of poorly investigated black-footed cat wild populations.

Through thousands of years of breeding and strong human selection, the dog (Canis lupus familiaris) exists today within hundreds of closed populations throughout the world, each with defined phenotypes. A singular geographic region with broad diversity in dog breeds presents an interesting opportunity to observe potential mechanisms of breed formation. Italy claims 14 internationally recognized dog breeds, with numerous additional local varieties. To determine the relationship among Italian dog populations, we integrated genetic data from 263 dogs representing 23 closed dog populations from Italy, seven Apennine gray wolves, and an established dataset of 161 globally recognized dog breeds, applying multiple genetic methods to characterize the modes by which breeds are formed within a single geographic region. Our consideration of each of five genetic analyses reveals a series of development events that mirror historical modes of breed formation, but with variations unique to the codevelopment of early dog and human populations. Using 142,840 genome‐wide SNPs and a dataset of 1,609 canines, representing 182 breeds and 16 wild canids, we identified breed development routes for the Italian breeds that included divergence from common populations for a specific purpose, admixture of regional stock with that from other regions, and isolated selection of local stock with specific attributes.

L’origine e l’unicità genetica del lupo italiano (Canis lupus italicus) sono state oggetto di diversi studi. Recenti pubbli- cazioni descrivono la distribuzione spazio-temporale di due ecomor , uno dei quali (HG2), associato alla megafauna pleistocenica, era dominante in Eurasia e in America durante l’ultimo massimo glaciale (LGM). In seguito al successivo cambiamento climatico un “nuovo” ecomorfo (HG1) ha sostituito i lupi preesistenti in Nord America, ed è divenuto progressivamente dominante in Eurasia. La popolazione italiana attuale è l’unica sopravvissuta interamente riferibile all’ecomorfo antico, ma le dinamiche che hanno portato a questa situazione non sono ben de nite. I reperti di ex Cava a Filo (16 campioni, datati da circa 25000 a circa 17400 anni fa) sono stati analizzati allo scopo di confrontare una regione del DNA mitocondriale con sequenze antiche e moderne di lupi e cani. Dai risultati emergono analogie con lupi antichi dell’Europa settentrionale e della Beringia, lupi moderni e anche cani primitivi e recenti. Alcuni campioni mostrano una notevole similitudine con i lupi italiani contemporanei. I lupi preistorici di ex Cava a Filo, associati a bisonti e megaceri, appartenevano solo alla popolazione riferita a HG2, caratterizzata da una maggiore variabilità e in continuità con la situazione attuale. Presumibilmente l’ondata migratoria dei lupi con HG1 non ha raggiunto l’Italia in numeri signi cativi, facendo della penisola italiana l’ultimo rifugio di un’antica popolazione di lupo un tempo di usa nell’intero emisfero nord.

Hybridization is a natural or anthropogenic process that can deeply affect the genetic make-up of populations, possibly decreasing individual fitness but sometimes favoring local adaptations. The population of Italian wolves (Canis lupus), after protracted demographic declines and isolation, is currently expanding in anthropic areas, with documented cases of hybridization with stray domestic dogs. However, identifying admixture patterns in deeply introgressed populations is far from trivial. In this study, we used a panel of 170,000 SNPs analyzed with multivariate, Bayesian and local ancestry reconstruction methods to identify hybrids, estimate their ancestry proportions and timing since admixture. Moreover, we carried out preliminary genotype–phenotype association analyses to identify the genetic bases of three phenotypic traits (black coat, white claws, and spur on the hind legs) putative indicators of hybridization. Results showed no sharp subdivisions between nonadmixed wolves and hybrids, indicating that recurrent hybridization and deep introgression might have started mostly at the beginning of the population reexpansion. In hybrids, we identified a number of genomic regions with excess of ancestry in one of the parental populations, and regions with excess or resistance to introgression compared with neutral expectations. The three morphological traits showed significant genotype–phenotype associations, with a single genomic region for black coats and white claws, and with multiple genomic regions for the spur. In all cases the associated haplotypes were likely derived from dogs. In conclusion, we show that the use of multiple genome-wide ancestry reconstructions allows clarifying the admixture dynamics even in highly introgressed populations, and supports their conservation management.

In the past century the Italian wolf has been repeatedly indicated as a distinct subspecies, Canis lupus italicus, due to its unique morphology and its distinctive mtDNA control region (CR) monomorphism. However, recent studies on wolf x dog hybridization in Italy documented the presence of a second mtDNA CR haplotype (W16), previously found only in wolves from Eastern Europe, casting doubts on the genetic uniqueness of the Italian wolves. To test whether this second haplotype belongs to the Italian wolf population, we genotyped 92 wolf DNA samples from Italy, Slovenia, Greece and Bulgaria at four mtDNA regions (control-region, ATP6, COIII and ND4 genes) and at 39 autosomal microsatellites. Results confirm the presence of two mtDNA multi-fragment haplotypes (WH14 and WH19) in the Italian wolves, distinct from all the other European wolves. Network analyses of the multi-fragment mtDNA haplotypes identified two strongly differentiated clades, with the Italian wolf WH14 and WH19 multi-fragment haplotypes rooted together. Finally, Bayesian clustering clearly assigned all the wolves sampled in Italy to the Italian population, regardless of the two different multi-fragment haplotypes. These results demonstrate that the W16 CR haplotype is part of the genetic pool of the Italian wolf population, reconfirming its distinctiveness from other European wolves. Overall, considering the presence of unique mtDNA and Y-linked haplotypes, the sharply different frequencies of genome-wide autosomal alleles and the distinct morphological features of Italian wolves, we believe that this population should be considered a distinct subspecies.

After centuries of massive decline, the recovery of the wolf (Canis lupus italicus) in Italy is a typical conservation success story. To learn more about the possible role of parasites in the wolves' individual and population health and conservation we used non-invasive molecular approaches on fecal samples to identify individual wolves, pack membership, and the taeniids present, some of which are zoonotic. A total of 130 specimens belonging to 54 wolves from eight packs were collected and examined. Taeniid eggs were isolated using a sieving/flotation technique, and the species level was identified by PCR (gene target: 12S rRNA and nad1). Taeniid prevalence was 40.7% for Taenia hydatigena, 22.2% for T. krabbei, 1.8% for T. polyachanta and 5.5% for Echinococcus granulosus. The prevalence of E. granulosus is discussed. Our results show that the taeniid fauna found in wolves from the Foreste Casentinesi National Park is comparable to that described for other domestic and wild Italian canids and provides insights into the wolves’ diet and their relationship with the environment.

Anthropogenic hybridization is widely perceived as a threat to the conservation of biodiversity. Nevertheless, to date, relevant policy and management interventions are unresolved and highly convoluted. While this is due to the inherent complexity of the issue, we hereby hypothesize that a lack of agreement concerning management goals and approaches, within the scientific community, may explain the lack of social awareness on this phenomenon, and the absence of effective pressure on decision-makers. By focusing on wolf x dog hybridization in Europe, we hereby (a) assess the state of the art of issues on wolf x dog hybridization within the scientific community, (b) assess the conceptual bases for different viewpoints, and (c) provide a conceptual framework aiming at reducing the disagreements. We adopted the Delphi technique, involving a three-round iterative survey addressed to a selected sample of experts who published at Web of Science listed journals, in the last 10 years on wolf x dog hybridization and related topics. Consensus was reached that admixed individuals should always be defined according to their genetic profile, and that a reference threshold for admixture (i.e., q-value in assignment tests) should be formally adopted for their identification. To mitigate hybridization, experts agreed on adopting preventive, proactive and, when concerning small and recovering wolf populations, reactive interventions. Overall, experts' consensus waned as the issues addressed became increasingly practical, including the adoption of lethal removal. We suggest three non-mutually exclusive explanations for this trend: (i) value-laden viewpoints increasingly emerge when addressing practical issues, and are particularly diverging between experts with different disciplinary backgrounds (e.g., ecologists, geneticists); (ii) some experts prefer avoiding the risk of potentially giving carte blanche to wolf opponents to (illegally) remove wolves, based on the wolf x dog hybridization issue; (iii) room for subjective interpretation and opinions result from the paucity of data on the effectiveness of different management interventions. These results have management implications and reveal gaps in the knowledge on a wide spectrum of issues related not only to the management of anthropogenic hybridization, but also to the role of ethical values and real-world management concerns in the scientific debate.

International audience; Estimating the relative abundance (prevalence) of different population segments is a key step in addressing fundamental research questions in ecology, evolution, and conservation. The raw percentage of individuals in the sample (naive prevalence) is generally used for this purpose, but it is likely to be subject to two main sources of bias. First, the detectability of individuals is ignored; second, classification errors may occur due to some inherent limits of the diagnostic methods. We developed a hidden Markov (also known as multievent) capture–recapture model to estimate prevalence in free-ranging populations accounting for imperfect detectability and uncertainty in individual's classification. We carried out a simulation study to compare naive and model-based estimates of prevalence and assess the performance of our model under different sampling scenarios. We then illustrate our method with a real-world case study of estimating the prevalence of wolf (Canis lupus) and dog (Canis lupus familiaris) hybrids in a wolf population in northern Italy. We showed that the prevalence of hybrids could be estimated while accounting for both detectability and classification uncertainty. Model-based prevalence consistently had better performance than naive prevalence in the presence of differential detectability and assignment probability and was unbiased for sampling scenarios with high detectability. We also showed that ignoring detectability and uncertainty in the wolf case study would lead to underestimating the prevalence of hybrids. Our results underline the importance of a model-based approach to obtain unbiased estimates of prevalence of different population segments. Our model can be adapted to any taxa, and it can be used to estimate absolute abundance and prevalence in a variety of cases involving imperfect detection and uncertainty in classification of individuals (e.g., sex ratio, proportion of breeders, and prevalence of infected individuals).

Thanks to protection by law and increasing habitat restoration, wolves (Canis lupus) are currently re-colonizing Europe from the surviving populations of Russia, the Balkan countries, Spain and Italy, raising the need to update conservation strategies. A major conservation issue is to restore connections and gene flow among fragmented populations, thus contrasting the deleterious consequences of isolation. Wolves in Italy are expanding from the Apennines towards the Alps, crossing the Ligurian Mountains (northern Italy) and establishing connections with the Dinaric populations. Wolf expansion is threatened by poaching and incidental killings, mainly due to livestock depredations and conflicts with shepherds, which could limit the establishment of stable populations. Aiming to find out the factors affecting the use of livestock by wolves, in this study we determined the composition of wolf diet in Liguria. We examined 1457 scats collected from 2008 to 2013. Individual scats were genotyped using a non-invasive genetic procedure, and their content was determined using microscopical analyses. Wolves in Liguria consumed mainly wild ungulates (64.4%; in particular wild boar Sus scrofa and roe deer Capreolus capreolus) and, to a lesser extent, livestock (26.3%; in particular goats Capra hircus). We modeled the consumption of livestock using environmental features, wild ungulate community diversity, husbandry characteristics and wolf social organization (stable packs or dispersing individuals). Wolf diet varied according to years and seasons with an overall decrease of livestock and an increase of wild ungulate consumption, but also between packs and dispersing individuals with greater livestock consumption for the latter. The presence of stable packs, instead of dispersing wolves, the adoption of prevention measures on pastures, roe deer abundance, and the percentage of deciduous woods, reduced predation on livestock. Thus, we suggest promoting wild ungulate expansion, the use of prevention tools in pastures, and supporting wolf pack establishment, avoiding lethal control and poaching, to mitigate conflicts between wolf conservation and husbandry.

Dog domestication is still largely unresolved due to time-gaps in the sampling of regions. Ancient Italian canids are particularly understudied, currently represented by only a few specimens. In the present study, we sampled 27 canid remains from Northern Italy dated between the Late Pleistocene and Bronze Age to assess their genetic variability, and thus add context to dog domestication dynamics. They were targeted at four DNA fragments of the hypervariable region 1 of mitochondrial DNA. A total of 11 samples had good DNA preservation and were used for phylogenetic analyses. The dog samples were assigned to dog haplogroups A, C and D, and a Late Pleistocene wolf was set into wolf haplogroup 2. We present our data in the landscape of ancient and modern dog genetic variability, with a particular focus on the ancient Italian samples published thus far. Our results suggest there is high genetic variability within ancient Italian canids, where close relationships were evident between both a similar to 24,700 years old Italian canid, and Iberian and Bulgarian ancient dogs. These findings emphasize that disentangling dog domestication dynamics benefits from the analysis of specimens from Southern European regions.

The grey wolf ( Canis lupus ) is one of the most widely distributed mammals in which a variety of distinct populations have been described. However, given their currently fragmented distribution and recent history of human-induced population decline, little is known about the events that led to their differentiation. Based on the analysis of whole canid genomes, we examined the divergence times between Southern European wolf populations and their ancient demographic history. We found that all present-day Eurasian wolves share a common ancestor ca 36 000 years ago, supporting the hypothesis that all extant wolves derive from a single population that subsequently expanded after the Last Glacial Maximum. We also estimated that the currently isolated European populations of the Iberian Peninsula, Italy and the Dinarics-Balkans diverged very closely in time, ca 10 500 years ago, and maintained negligible gene flow ever since. This indicates that the current genetic and morphological distinctiveness of Iberian and Italian wolves can be attributed to their isolation dating back to the end of the Pleistocene, predating the recent human-induced extinction of wolves in Central Europe by several millennia.

During historical times many local grey wolf (Canis lupus) populations underwent a substantial reduction of their sizes or became extinct. Among these, the wolf population once living in Sicily, the biggest island of the Mediterranean Sea, was completely eradicated by human persecution in the early decades of the XX century.In order to understand the genetic identity of the Sicilian wolf, we applied ancient DNA techniques to analyse the mitochondrial DNA of six specimens actually stored in Italian museums.We successfully amplified a diagnostic mtDNA fragment of the control region (CR) in four of the samples. Results showed that two samples shared the same haplotype, that differed by two substitutions from the currently most diffused Italian wolf haplotype (W14) and one substitution from the only other Italian haplotype (W16). The third sample showed a wolf-like haplotype never described before and the fourth a haplotype commonly found in dogs.Furthermore, all the wolf haplotypes detected in this study belonged to the mitochondrial haplogroup that includes haplotypes detected in all the known European Pleistocene wolves and in several modern southern European populations.Unfortunately, this endemic island population, bearing unique mtDNA variability, was definitively lost before it was possible to understand its taxonomic uniqueness and conservational value.

The distribution of intraspecific genetic variation and how it relates to environmental factors is of increasing interest to researchers in macroecology and biogeography. Recent studies investigated the relationships between the environment and patterns of intraspecific genetic variation across species ranges but only few rigorously tested the relation between genetic groups and their ecological niches. We quantified the relationship of genetic differentiation (FST) and the overlap of ecological niches (as measured by n‐dimensional hypervolumes) among genetic groups resulting from spatial Bayesian genetic clustering in the wolf (Canis lupus) in the Italian peninsula. Within the Italian wolf population, four genetic clusters were detected, and these clusters showed different ecological niches. Moreover, different wolf clusters were significantly related to differences in land cover and human disturbance features. Such differences in the ecological niches of genetic clusters should be interpreted in light of neutral processes that hinder movement, dispersal, and gene flow among the genetic clusters, in order to not prematurely assume any selective or adaptive processes. In the present study, we found that both the plasticity of wolves—a habitat generalist—to cope with different environmental conditions and the occurrence of barriers that limit gene flow lead to the formation of genetic intraspecific genetic clusters and their distinct ecological niches., Ecology and Evolution, 8 (22), ISSN:2045-7758

Wolf-dog hybridization is considered as one of the main threats for wolf conservation since the admixture and introgression of domestic genes may disrupt local adaptations and threaten the long term survival of wild wolf populations. We investigated the occurrence of wolf-dog hybridization in Croatia by analyzing a panel of 12 autosomal microsatellite markers using Bayesian admixture tests, and assessed its directionality by the use of maternally and paternally inherited markers in combination with morphometric data and morphological features. A systematic analysis of morphologic features and morphometric data was used to rank the studied individuals into either phenotypic wild-type wolves or suspected hybrids. By combining Bayesian assignment results with phenotypic features, we set three thresholds which differentiated wolves from hybrids with maximized hybrid detection and a minimized chance for false positive hybrid identification. On the basis of phenotype, out of 176 wild canids, 157 (89.2%) were categorized as wolves and 19 (10.8%) as suspected hybrids. On the basis of the Bayesian admixture tests and phenotype together, five (2.8 percent) animals were classified as wolf-dog hybrids, four of them as backcrosses with wolves, and one as a backcross with a dog. Mitochondrial DNA suggested that all hybrids originated from the mating of female wolves and male dogs. Two male hybrids had Y chromosome haplotypes common to both wolves and dogs, while the other two had wolf private Y chromosome haplotypes. One wolf had a dog Y-haplotype, indicating a past introgression of dog genes. All hybrids were found in Dalmatia, where wolves settled recently, and where they live close to humans, with a high rate of human-caused mortality. These conditions are considered as favorable for wolf-dog hybridization. However, we found a low hybridization prevalence in Croatia, which is nonetheless expected to persist as long as the conditions favoring its occurrence are met. The ecological, sociological, conservation and management implications of hybrid occurrence are yet to be determined., Križanje vukova i pasa jedna je od glavnih prijetnji očuvanju vukova jer se miješanjem i introgresijom gena domaćih životinja mogu narušiti lokalne prilagodbe te tako ugroziti dugoročni opstanak populacija vukova u prirodi. Istražili smo pojavu križanja vukova i pasa u Hrvatskoj analizom 12 autosomskih mikrosatelitskih markera Bayesian testom primjese te utvrdili smjer hibridizacije određivanjem markera nasljeđivanih i po majčinskoj i po očinskoj liniji, u kombinaciji s određivanjem morfometrijskih i morfoloških svojstava. Sustavna analiza morfoloških svojstava i morfometrijskih podataka korištena je za rangiranje istraživanih jedinki, bilo kao fenotipske divlje tipove vukova bilo kao sumnjive na križanost. Kombiniranjem rezultata Bayesian statistike s fenotipskim obilježjima postavili smo prag za razlikovanje vukova od križanaca, koji povećava vjerojatnost određivanja križanaca uz smanjivanje vjerojatnosti za pogrešno pozitivno određivanje. Temeljem fenotipa, od 176 divljih kanida 157 (89,2 %) bilo je kategorizirano kao vukovi, dok je 19 (10,8 %) bilo svrstano u sumnjive križance. Temeljem Bayesian testa primjese, pet (2,8 %) životinja bilo je svrstano u križance vuka i psa, četiri od njih određena su kao unatražno križani s vukovima, dok je jedan bio unatražno križan sa psom. Mitohondrijska DNK pokazala je da su svi križanci bili rezultat parenja vučice sa psom. Dva su muška križanca na Y-kromosomu imala haplotipove i od vukova i od pasa, dok su druga dva imala Y-kromosomske haplotipove dosad utvrđene samo kod vukova. Jedan je vuk imao pseći Y-kromosomski haplotip što je uputilo na introgresiju psećih gena. Svi su križanci pronađeni u Dalmaciji, gdje su se vukovi nedavno proširili te gdje žive blizu ljudi, s visokom stopom smrtnosti uzrokovane od ljudi. Te se okolnosti inače smatraju pogodujućima za nastanak križanaca vukova i pasa. Ipak, pronađena učestalost pojave križanaca bila je niska, ali i za očekivati je da će se zadržati sve dok postoje uvjeti koji pogoduju nastanku križanaca. Ekološke, sociološke te značajke hibridizacije za zaštitu i upravljanje populacijom vukova tek treba utvrditi.

Large carnivores in Italy and other European countries are protected by law to ensure their long-term conservation. Estimates of abundance and demographic trends of their populations are crucial for implementing effective conservation and management strategies. However, it is challenging to obtain basic demographic parameters for elusive species such as the wolf (Canis lupus). Monitoring wolf populations by standard field methods or non-invasive genetic approaches requires huge human efforts and may be exceedingly expensive on a nation-wide scale. Aiming to obtain a first approximate estimate of wolf distribution and abundance in Italy, we developed a systematic review procedure to analyze published data obtained from a variety of sources. We deduced relevant information on wolf presence and numbers from 20 peer-reviewed studies or official reports, and from 241 Standard Data Forms of Natura 2000 sites in Italy, referring to the period 2009–2013. We estimated the species abundance by combining the number of individuals reported in each study area with the values obtained by multiplying the estimated number of packs for the average pack size. Comparing our estimates with those previously reported, we evaluated the qualitative trend of the population for each of the two management units: Alps and Apennines. Results showed the occurrence of approximately 321 wolf packs in Italy, corresponding to 1269–1800 wolves, possibly still underestimated. The Apennine sub-population seems to be almost the double in size (with ca. 1212–1711 wolves in the period 2009–2013) compared to previous estimates (600–800 wolves between 2006 and 2011). The Alpine sub-population, despite its ongoing eastwards expansion, appears rather stable (with 57–89 wolves). Overall, the current wolf population size and trends seem favorable, although the species is still locally threatened by widespread poaching and accidents. These results represent the first estimate of abundance for the whole Italian wolf population in the last 40 years. Such information can be used to implement sound conservation strategies, especially in critical human-dominated landscapes, where conflicts with human activities and increasing rates of hybridization with free-ranging domestic dogs call for updated management plans.

Non-invasive genetic sampling has been used to reconstruct spatial patterns of carnivore distributions, identify regions where conflicts with human activities could threaten the survival of a species, and assess the effectiveness of conservation strategies. In this study, we used detailed information on wolf (Canis lupus) and livestock distributions to infer depredation risks in a wide area of the Italian Apennines. We carried out a General Niche Environment System Factor Analysis (GNESFA) to define the potential distribution of wolves genotyped from 8565 samples collected during 12 years of non-invasive genetic monitoring in 3622 locations. Habitat suitability models indicated that the proportion of meadows, altitude, slope, roughness, and distance from human settlements were the main factors positively related to the potential wolf distribution, in contrast with the extension of cultivated fields and human settlements. Results of GNESFA were used to infer the local depredation risk, which was high in 46.9 % of the pastures, and to rank the areas where prevention tools should be used with priority. In this way, the use of often-limited financial resources for prevention could be promoted in pastures with the highest depredation risk and conflicts between husbandry and wolf presence might be mitigated.