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The International Symposium on Wild Boar and Other Suids (IWBS 2022), which took place in Montseny Biosphere Reserve (Catalonia, Spain) in September 2022, provided to ENETWILD with the opportunity to meet in‐person for the first time after 2.5 years, and meet the international scientific community with expertise on wild suids and other ungulates. Twelve members of ENETWILD consortium representing 6 partners were present. Bringing together international experts, stakeholders and ENETWILD collaborators was a perfect occasion to present the European Observatory of Wildlife (EOW). Two hundred and twenty‐five wildlife experts from 25 countries were present at symposium, and at presentation of the EOW. Overall, 3 'Plenary Talks' and 118 presentations (62 oral and 56 posters) were made. The meeting has gone through all the possible topics regarding wild suids, from genetics to monitoring and management. This was the optimal context to introduce the EOW to an ideal target audience, both in terms of interest and in terms of potential new member of the Network. From our presentation, it emerged the importance of comparable data on geographical distribution and abundance of wildlife hosts in Europe, fundamental to develop the best management policies and to perform effective risk assessments for shared emergent diseases. The adoption of a common and effective protocol adopted throughout the continent would ensure such comparability. Moreover, the discussion highlighted the need of extending the network to as many European countries as possible and, when feasible, of having multiple sites within each country. A number of participants manifested their interest to join the EOW during the 2023 campaign. Such a capillary distribution of observation points would provide solid and comparable density estimates as well as effective feedback about the field protocol implemented by the EOW. A number of questions were raised by the audience during the presentation of the EOW. [ABSTRACT FROM AUTHOR]

During summer 2022, the European Wildlife Observatory (EOW2), involving 50 study areas in at least 30 European countries, has applied similar camera‐trapping‐based population estimation protocols and data collection standards to facilitate harmonization and interoperability. For this purpose, continuous training of the network of wildlife professionals in Europe is a key activity of the EOW. In this context, in May 2022 the ENETWILD consortium organized a first online training course to update information on methodology to process with next steps in the field. The course here reported took place in two different dates, 28th September, and 21st November 2022, online. The first part of the course (28th September) presented the progress of the EOW and focused on the analytical tools incorporated to Agouti app, on the use of information technology tools and artificial intelligence to process and analyse photo‐trappings to finally obtain a standardized database, ready for analysis. The main objective of the course offered on 21st November was providing detailed instructions on running the random encounter model (REM) on camera trap data packages from Agouti and incorporating some recommendations to improve the calibration of images for distance estimation. We also presented SMART app adapted to the EOW, which facilitates the collection of information in the field using stablished density estimation protocols to make these data available in the destock in real time (cloud‐based solution). SMART is flexible enough to incorporate new protocols and species, as methods (such as camera traps‐based) and needs continuously evolve. The integration of SMART tools on EOW was successfully done for (i) distance sampling, (ii) hunting data and (iii) camera trap protocols. This course was successfully attended by 46 wildlife biologists, animal health professionals and wildlife experts from national hunting and forestry authorities. Detailed explanations, protocols, and examples for applying such protocols were provided. [ABSTRACT FROM AUTHOR]

One of the main objectives of ENETWILD consortium is to collect data on density, hunting statistics and wildlife occurrence in order to model the geographical distribution and abundance of wildlife species across Europe as a tool to support the assessment of risks associated, for example, with disease transmission. Created in the framework of the ENETwild project, the European Wildlife Observatory (EOW2) provides the backbone for an integrated, interdisciplinary, multi‐sectoral and multi‐institutional approach to wildlife monitoring, initially focusing on terrestrial mammals in Europe. The EOW applies similar camera‐trapping‐based protocols for population estimation and data collection standards to facilitate harmonization and interoperability. For this purpose, continuous training of the network of wildlife professionals in Europe is a key activity of the EOW. In this context, during the last few years the ENETWILD consortium has organized different online training courses and workshops on the use of camera traps, addressing different approaches from the design and handling of camera traps to the processing of the collected data. Many of the participants in our previous courses are now part of the EOW and require updated information on methodology to process with next steps in the field. The course here reported presented improvements and refinements in the sampling protocols, aimed specially at new collaborators to be incorporated in the network. Therefore, the objectives of this introductory online course held on 5th May 2022 were: (i) to present milestones and achievements of the ENETWILD project and the EOW, and (ii) to review scientific methods for determining wildlife abundance and density, providing specific training on camera trapping methods and protocols, specifically the random encounter method (REM) and other methods which do not require identification of individuals. This course was attended by 46 wildlife biologists, animal health professionals and wildlife experts from national hunting and forestry authorities. Detailed explanations, protocols, and examples for applying such protocols were provided. [ABSTRACT FROM AUTHOR]

The 2nd ENETWILD Annual General Meeting took place on 5‐6th October 2021, bringing together experts, stakeholders and ENETWILD collaborators in online workshop discussions. First, workshop discussions contributed to the analysis and proposal of approaches for a harmonized European‐wide wildlife monitoring framework able of sustaining coordinated decision‐making. Secondly, participants identified the key challenges that managers face in making decisions for wildlife in Europe and data needs for policies. Finally, we illustrated these challenges with the case of wild boar as a model species widely distributed across Europe. Inputs from the participants were collated into a plan of proposed steps and objectives for the mid‐term (5‐year time frame) to achieve progress on harmonised, coordinated, and integrated wildlife monitoring at the European level, which requires the contribution of experts from the early stages.. Specific proposed actions include the creation of a trans‐disciplinary authority at the European level, effective points of reference for data collection and sharing at different administrative levels and countries, a standing committee to coordinate and exchange experience and capacities on data collection between countries, and expert groups for problem solving, with proper EU financial support, establishing regular policy meetings.. To provide useful results, wildlife monitoring must ensure proper design and data analysis for subsequent science‐based management and best allocation of management resources. The 'Observatory' approach (a representative network of intensively monitored sites) can provide long‐term systematic and representative insights, normally more feasible for comparative studies, providing less biases and support for decision‐making. For international decision‐making by wildlife managers and politicians based on scientific knowledge and interdisciplinary research, experts should define the foundations of a common European wildlife decision‐making framework (inter‐institutional and inter‐sectorial). The development of a European legislation on wildlife management may represent an opportunity for addressing the abovementioned steps, identifying data priorities matching the needs of the various European Directorates, Agencies, and monitoring frameworks. [ABSTRACT FROM AUTHOR]

The ENETWILD consortium (www.enetwild.com) aims at progressively defining the spatial interface between wild ungulates and livestock in Europe, which is essential to evaluate the risk for shared diseases. This is to provide preliminary risk maps of possible wild‐domestic interfaces at European scale using relatively similar sized regions by compiling, for the first time, comprehensive data for both groups, wild and domestic ungulates in the continent. We spatially represented (i) the richness of species (livestock and wild ungulates), (ii) their specific occupancy and abundance (the latter for livestock), and finally, (iii) their spatial overlapping over Europe. Species richness in animal communities, including wildlife and domestic hosts, may moderate pathogen transmission and disease outcome.. As a first step, we should characterize the diverse assemblages of animal communities at large scale to better understand possible scenarios for further assessment of shared infection dynamics. About 90% of Europe land area hosts from one to five species of wild native ungulates. Therefore, the interface between livestock and wildlife is wide spread over the European continent. Native wild boar, roe deer and reed deer are widely distributed species, present in most possible assemblages of wild/domestic communities over Europe. The richness of ungulate species is high in Central Europe, from West to East, from the Alps (where the presence of mountain ungulates adds richness), extending to countries with important big game tradition and presence of introduced species, and finally, to Eastern Europe (where also typically northern species such as bisons appear)... To sum, we described by pair of species a wide diversity of potential interfaces, which had variable distribution areas.. While the analysis presented herein is purely spatial and at administrative level, the interface between wild and domestic ungulates is influenced by livestock husbandry (e.g., enclosed, herded or free‐ranging, level of biosecurity), landscape and land uses, and wildlife management practices, among other factors, operating locally. Therefore, there is need for a more detailed picture of the interface at European scale. [ABSTRACT FROM AUTHOR]

Reproductive synchrony among gregarious mammals has a strong adaptive value and may lead to cooperative behaviors aimed at maximizing offspring survival. Additionally, temporal clustering of estrus has important implications on individual mating tactics and ultimately affects the degree of polygamy in a population. Although several studies have examined the reproductive biology of wild boar (Sus scrofa), much remains to be understood about the patterns of timing and synchrony of reproduction in natural populations. We analyzed the spatiotemporal distribution of conception dates in an Italian wild boar population taking into account the effects of environmental and individual factors, in order to determine the main variables influencing the timing of reproduction and to detect the signs of a socially-driven reproductive synchrony. Specifically, for each litter belonging to 354 pregnant sows culled between 2006 and 2013 in a mountain area of Tuscany, we determined the conception date (CD) from an estimate of the mean fetal age and the culling date. We then investigated which factors drove the variation in CD, by implementing linear mixed models, Mantel tests and spatial autocorrelation analyses. The selected model showed significant effects of rainfall, temperatures, and previous and current productivity on CD, as well as a strong correlation of CDs among sows culled in close spatial and temporal proximity (i.e., in the same hunting ground and hunting season). Likewise, autocorrelation analyses and Mantel tests consistently indicated that closer sows had similar conception dates. Overall, our results confirm the effect of resource availability and climate on wild boar reproductive phenology, and suggest socially-driven reproductive patterns, in spite of a high turn-over in social groups due to hunting. Finally, possible advantages and evolutionary implications of reproductive synchrony in wild boar are discussed. [ABSTRACT FROM AUTHOR]

Dispersal patterns can have vital consequences for the transfer of genes in a population, thus shaping its genetic make-up and structure. Genetic relationships between individuals can, in turn, affect their social behaviour and the emergent social organization of the population. Using combination of behavioural and genetic data from the wild boar S us scrofa population in Białowieża Primeval Forest (eastern Poland), we evaluated the socio-genetic structure of wild boar groups, the spatial genetic structure of the population and dispersal patterns. We found that wild boar post-weaning movements were largely spatially limited to the vicinity of maternal range, with female boars showing a tendency to settle in the direct neighbourhood of the kin and male boars dispersing further away from the natal area. Consequently, such dispersal patterns were reflected in the kin-based social organization and the spatial genetic structure of the population, which was manifested at a spatial scale corresponding to the size of a few home ranges (<5 km). A negative relationship between geographic distance and genetic relatedness, which was particularly strong in female boars, indicated a presence of local kin clusters dominated by female boars and the importance of female philopatry in shaping the structure of wild boar population. This was confirmed by the genetic profile and composition of social groups. This study showed the role dispersal decisions can play in the emergence of the kin-based and matrilineal social system of wild boars. [ABSTRACT FROM AUTHOR]

Game species are often manipulated by human beings, whose activities can deeply affect their genetic make-up and population structure. We focused on a geographically isolated wild boar population (Sardinia, Italy), which is classified, together with the Corsican population, as a separate subspecies (Sus scrofa meridionalis). Two hundred and ten wild boars collected across Sardinia were analysed with a set of 10 microsatellites and compared with 296 reference genotypes from continental wild populations and to a sample of domestic pigs. The Sardinian population showed remarkable diversity and a high proportion of private alleles, and strongly deviated from the equilibrium. A Bayesian cluster analysis of only the Sardinian sample revealed a partition into five subpopulations. However, two different Bayesian approaches to the assignment of individuals, accounting for different possible source populations, produced consistent results and proved the admixed nature of the Sardinian population. Indeed, introgressive hybridization with boars from multiple sources (Italian peninsula, central Europe, domestic stocks) was detected, although poor evidence of crossbreeding with free-ranging domestic pigs was unexpectedly found. After excluding individuals who carried exotic genes, the population re-entered Hardy-Weinberg proportions and a clear population structure with three subpopulations emerged. Therefore, the inclusion of introgressed animals in the Bayesian analysis implied an overestimation of the number of clusters. Nonetheless, two of them were consistent between analyses and corresponded to highly pure stocks, located, respectively, in north-west and south-west Sardinia. This work shows the critical importance of including adequate reference samples when studying the genetic structure of managed wild populations. [ABSTRACT FROM AUTHOR]

The wild boar (Sus scrofa meridionalis) arrived in Sardinia with the first human settlers in the early Neolithic with the potential to hybridize with the domestic pig (S. s. domesticus) throughout its evolution on the island. In this paper, we investigated the possible microevolutionary effects of such introgressive hybridization on the present wild boar population, comparing Sardinian wild specimens with several commercial pig breeds and Sardinian local pigs, along with a putatively unadmixed wild boar population from Central Italy, all genotyped with a medium density SNP chip. We first aimed at identifying hybrids in the population using different approaches, then examined genomic regions enriched for domestic alleles in the hybrid group, and finally we applied two methods to find regions under positive selection to possibly highlight instances of domestic adaptive introgression into a wild population. We found three hybrids within the Sardinian sample (3.1% out of the whole dataset). We reported 11 significant windows under positive selection with a method that looks for overly differentiated loci in the target population, compared with other two populations. We also identified 82 genomic regions with signs of selection in the domestic pig but not in the wild boar, two of which overlapped with genomic regions enriched for domestic alleles in the hybrid pool. Genes in these regions can be linked with reproductive success. Given our results, domestic introgression does not seem to be pervasive in the Sardinian wild boar. Nevertheless, we suggest monitoring the possible spread of advantageous domestic alleles in the coming years., (© 2023. The Author(s).)

We estimated the current size and dynamics of the wolf population in Tuscany and investigated the trends and demographic drivers of population changes. Estimates were obtained by two different approaches: (i) mixed-technique field monitoring (from 2014 to 2016) that found the minimum observed pack number and estimated population size, and (ii) an individual-based model (run by Vortex software v. 10.3.8.0) with demographic inputs derived from a local intensive study area and historic data on population size. Field monitoring showed a minimum population size of 558 wolves (SE = 12.005) in 2016, with a density of 2.74 individuals/100 km 2 . The population model described an increasing trend with an average annual rate of increase λ = 1.075 (SE = 0.014), an estimated population size of about 882 individuals (SE = 9.397) in 2016, and a density of 4.29 wolves/100 km 2 . Previously published estimates of wolf population were as low as 56.2% compared to our field monitoring estimation and 34.6% in comparison to our model estimation. We conducted sensitivity tests to analyze the key parameters driving population changes based on juvenile and adult mortality rates, female breeding success, and litter size. Mortality rates played a major role in determining intrinsic growth rate changes, with adult mortality accounting for 62.5% of the total variance explained by the four parameters. Juvenile mortality was responsible for 35.8% of the variance, while female breeding success and litter size had weak or negligible effects. We concluded that reliable estimates of population abundance and a deeper understanding of the role of different demographic parameters in determining population dynamics are crucial to define and carry out appropriate conservation and management strategies to address human-wildlife conflicts.

Lectin-like molecules play a key role in mammalian sperm functionality. These multifunctional proteins have been proven to be involved in sperm capacitation, sperm motility, and viability, formation of the oviductal sperm reservoir, and in sperm-oocyte interaction. In a previous study, we reported the presence of a novel seminal plasma lectin, sperm lectin 15 kDa (SL15), adsorbed to the llama sperm. In order to gain knowledge in the understanding of SL15 and its functions, the aims of this study were to (a) elucidate the presence and localization of SL15 in the llama male reproductive tract and sperm, and (b) determine whether the sperm cryopreservation process of cooling and freeze-thawing affects the SL15 levels and distribution on llama sperm. We found that SL15 protein was expressed along the male reproductive system: testis, epididymis, prostate, and bulbourethral glands, being the prostate the main site of SL15 secretion. SL15 was localized on the sperm head, following different localization patterns. In order to understand if sperm cryopreservation induces modifications in the SL15 adsorption pattern, immunocytochemistry and flow cytometry analysis were carried out on fresh, 24 h cooled, and frozen-thawed sperm. Both cooled and frozen sperm showed particular SL15 patterns, that were not observed in the freshly ejaculated, indicating loss of SL15. Flow cytometry analysis also exhibited a decrease of SL15 in the cooled sperm (P < 0.05), whereas a tendency to decrease was found in frozen-thawed sperm (P < 0.1) when compared with freshly ejaculated sperm. This study extends the knowledge about the SL15 in the llama male physiology and provides evidence that cryopreservation-related techniques disrupt SL15 adsorption to the sperm membrane, possibly affecting sperm functionality and fertility., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The aim of the study was to assess which kill site characteristics were selected by a lone wolf living in a protected Mediterranean coastal area near the city of Pisa, Italy, where both wild and domestic ungulates were available as potential prey. Between 2017 and 2019, we monitored the wolf's predatory behaviour through a combination of camera trapping and active search for kill sites and prey carcasses. The main prey found was the fallow deer (n = 82); only two wild boars and no domestic ungulates were found preyed upon. The features and habitat of kill sites were modelled to test for selection by the wolf. The habitat type of kill site was composed of meadows and pastures (89.3%), woods (7.3%), degraded coastal areas (1.9%), roads and rivers (1.1%), and marshes (0.5%). We calculated their distance from landscape features and ran a binomial generalised linear model to test the influence of such landscape variables. The distance of kill sites from landscape elements was significantly different from random control sites, and a positive selection for fences was found. In fact, the wolf pushed fallow deer towards a fence to constrain them and prevent them from escaping. We also analysed the body condition of predated fallow deer as a percentage of fat content in the bone marrow of the hind legs. Our results revealed the selection of the lone wolf for deer in good body condition. This is a possible outcome of the habitat selection shown by fallow deer in the study area, where fenced open pastures are the richest in trophic resources; therefore, our findings suggest a high efficacy for the lone wolf hunting strategy, but also the adoption of a high risk feeding strategy by deer. This study suggests that a lone predator can take advantage of human infrastructures to maximise its predatory effectiveness.

An amendment to this paper has been published and can be accessed via the original article.

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 (q iw ) 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.

The phylogeography of the European wild boar was mainly determined by postglacial recolonization patterns from Mediterranean refugia after the last ice age. Here we present the first analysis of SNP polymorphism within the complete mtDNA genome of West Russian ( n  = 8), European ( n  = 64), and North African ( n  = 5) wild boar. Our analyses provided evidence of unique lineages in the East-Caucasian (Dagestan) region and in Central Italy. A phylogenetic analysis revealed that these lineages are basal to the other European mtDNA sequences. We also show close connection between the Western Siberian and Eastern European populations. Also, the North African samples were clustered with the Iberian population. Phylogenetic trees and migration modeling revealed a high proximity of Dagestan sequences to those of Central Italy and suggested possible gene flow between Western Asia and Southern Europe which was not directly related to Northern and Central European lineages. Our results support the presence of old maternal lineages in two Southern glacial refugia (i.e., Caucasus and the Italian peninsula), as a legacy of an ancient wave of colonization of Southern Europe from an Eastern origin., Competing Interests: The authors declare no conflict of interests.

A new sequence of eruptions occurred at Mt. Etna volcano during the first half of 2017, after almost 8 months of quiescence. These episodes had low-to-mild intensity and markedly differ from the violent paroxysms occurred at the Voragine Crater (VOR) during December 2015 and May 2016. Despite the general weak explosive nature of the eruptions, the activity during 2017 revealed unusually complex dynamics of magma ascent and interaction. Detection and investigation of such dynamics required a multidisciplinary approach in which bulk rock compositions, crystal chemical zoning, diffusion chronometry and ground deformation data have been combined. Bulk rock major and trace elements suggest that the 2017 magmas followed a differentiation path similar to that experienced by magmas erupted at Mt. Etna during the 2015-16 eruptions at VOR. Olivine core compositions and zoning patterns indicate the presence of multiple magmatic environments at depth that strictly interacted each other through some episodes of intrusion and mixing before and during the 2017 eruptive events. Timescales retrieved from diffusion chronometry on olivine normal and reverse zoning correlate well with the ground deformation stages detected through geodetic data and associated models, thus allowing to track the evolution through time of the 2017 volcanic activity. Combination of all petrological and geodetic observations supports the idea that dynamics of magma transfer driving the eruptive episodes of 2017 have been a direct consequence of the violent eruptions occurred at VOR on May 2016, which boosted the ascent of new magma from depth and improved the efficiency of the plumbing system to transfer it upward to the surface. We propose a mechanism of self-feeding replenishment of the volcano plumbing system during 2017, where magma recharge from depth is triggered by sudden unloading of the magma column consequential to the violent paroxysmal activity occurred on May 2016 at VOR.

After a strong demographic decline before World War II, wild boar populations are expanding and the species is now the second-most abundant ungulate in Europe. This increase raises concerns due to wild boar impact on crops and natural ecosystems and as potential vector of diseases. Additionally, wild boar can hybridize with domestic pigs, which could increase health risks and alter wild boar adaptive potential. We analysed 47,148 Single Nucleotide Polymorphisms in wild boar from Europe (292) and the Near East (16), and commercial (44) and local (255) pig breeds, to discern patterns of hybridization across Europe. We identified 33 wild boars with more than 10% domestic ancestry in their genome, mostly concentrated in Austria, Bosnia and Herzegovina, Bulgaria and Serbia. This difference is probably due to contrasting practices, with free-ranging vs. industrial farming but more samples would be needed to investigate larger geographic patterns. Our results suggest hybridization has occurred over a long period and is still ongoing, as we observed recent hybrids. Although wild and domestic populations have maintained their genetic distinctiveness, potential health threats raise concerns and require implementation of management actions and farming practices aimed at reducing contact between wild and domestic pigs.

European brown hare, Lepus europaeus, from Central and Eastern European countries (Hungary, Poland, Serbia, Lithuania, Romania, Georgia and Italy) were sampled, and phylogenetic analyses were carried out on two datasets: 1.) 137 sequences (358 bp) of control region mtDNA; and 2.) 105 sequences of a concatenated fragment (916 bp), including the cytochrome b, tRNA-Thr, tRNA-Pro and control region mitochondrial DNA. Our sequences were aligned with additional brown hare sequences from GenBank. A total of 52 and 51 haplotypes were detected within the two datasets, respectively, and assigned to two previously described major lineages: Anatolian/Middle Eastern (AME) and European (EUR). Furthermore, the European lineage was divided into two subclades including South Eastern European (SEE) and Central European (CE). Sympatric distribution of the lineages of the brown hare in South-Eastern and Eastern Europe revealed contact zones there. BAPS analysis assigned sequences from L. europaeus to five genetic clusters, whereas CE individuals were assigned to only one cluster, and AME and SEE sequences were each assigned to two clusters. Our findings uncover numerous novel haplotypes of Anatolian/Middle Eastern brown hare outside their main range, as evidence for the combined influence of Late Pleistocene climatic fluctuations and anthropogenic activities in shaping the phylogeographic structure of the species. Our results support the hypothesis of a postglacial brown hare expansion from Anatolia and the Balkan Peninsula to Central and Eastern Europe, and suggest some slight introgression of individual haplotypes from L. timidus to L. europaeus., Competing Interests: The authors have declared that no competing interests exist.

Background: Density estimation is a key issue in wildlife management but is particularly challenging and labour-intensive for elusive species. Recently developed approaches based on remotely collected data and capture-recapture models, though representing a valid alternative to more traditional methods, have found little application to species with limited morphological variation. We implemented a camera trap capture-recapture study to survey wolf packs in a 560-km 2 area of Central Italy. Individual recognition of focal animals (alpha) in the packs was possible by relying on morphological and behavioural traits and was validated by non-invasive genotyping and inter-observer agreement tests. Two types (Bayesian and likelihood-based) of spatially explicit capture-recapture (SCR) models were fitted on wolf pack capture histories, thus obtaining an estimation of pack density in the area., Results: In two sessions of camera trapping surveys (2014 and 2015), we detected a maximum of 12 wolf packs. A Bayesian model implementing a half-normal detection function without a trap-specific response provided the most robust result, corresponding to a density of 1.21 ± 0.27 packs/100 km 2 in 2015. Average pack size varied from 3.40 (summer 2014, excluding pups and lone-transient wolves) to 4.17 (late winter-spring 2015, excluding lone-transient wolves)., Conclusions: We applied for the first time a camera-based SCR approach in wolves, providing the first robust estimate of wolf pack density for an area of Italy. We showed that this method is applicable to wolves under the following conditions: i ) the existence of sufficient phenotypic/behavioural variation and the recognition of focal individuals (i.e. alpha, verified by non-invasive genotyping); ii ) the investigated area is sufficiently large to include a minimum number of packs (ideally 10); iii ) a pilot study is carried out to pursue an adequate sampling design and to train operators on individual wolf recognition. We believe that replicating this approach in other areas can allow for an assessment of density variation across the wolf range and would provide a reliable reference parameter for ecological studies., Competing Interests: Not applicable.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.