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Abstract A comprehensive understanding of the dietary habits of carnivores is essential to get ecological insights into their role in the ecosystem, potential competition with other carnivorous species, and their effect on prey populations. Genetic analysis of non-invasive samples, such as scats, can supplement behavioural or microscopic diet investigations. The objective of this study was to employ DNA metabarcoding to accurately determine the prey species in grey wolf (Canis lupus) and Eurasian lynx (Lynx lynx) scat samples collected in the Julian Alps and the Dinaric Mountains, Slovenia. The primary prey of wolves were red deer (Cervus elaphus) (detected in 96% scat samples), European roe deer (Capreolus capreolus) (68%), and wild boar (Sus scrofa) (45%). A smaller portion of their diet consisted of mesocarnivores, small mammals, and domestic animals. In contrast, the lynx diet mostly consisted of European roe deer (82%) and red deer (64%). However, small mammals and domestic animals were also present in lynx diet, albeit to a lesser extent. Our findings indicate that the dietary habits of wolves and lynx are influenced by geographical location. Snapshot dietary analyses using metabarcoding are valuable for comprehending the behaviour and ecology of predators, and for devising conservation measures aimed at sustainable management of both their natural habitats and prey populations. However, to gain a more detailed understanding of wolf and lynx dietary habits and ecological impact, it would be essential to conduct long-term genetic monitoring of their diet.

Abstract The rapid expansion of wild pigs (Sus scrofa) throughout the United States has been fueled by unlawful introductions, with invasive populations causing extensive crop losses, damaging native ecosystems, and serving as a reservoir for disease. Multiple states have passed laws prohibiting the possession or transport of wild pigs. However, genetic and phenotypic similarities between domestic pigs and invasive wild pigs—which overwhelmingly represent domestic pig and wild boar hybrids—pose a challenge for the enforcement of such regulations. We sought to exploit wild boar ancestry as a common attribute among the vast majority of invasive wild pigs as a means of genetically differentiating wild pigs from breeds of domestic pig found within the United States. We organized reference high‐density single nucleotide polymorphism genotypes (1039 samples from 33 domestic breeds and 382 samples from 16 wild boar populations) into five genetically cohesive reference groups: mixed‐commercial breeds, Durocs, heritage breeds, primitive breeds, and wild boar. Building upon well‐established genetic clustering approaches, we structured the test statistic to describe the difference in the likelihood of a given genotype's ancestry vectors (sensu genetic clustering analysis) if derived strictly from the four described domestic pig reference groups versus allowing for admixture from the wild boar group. By fitting statistical distributions to test statistics of reference domestic pigs, we characterized the distribution of the null hypothesis that a given genotype descends strictly from domestic pig reference groups. We tested the approach with simulated genotypes and empirical data from an additional 29 breeds of domestic pig represented by 435 unique genotypes; all associated test statistics for simulated and empirical domestic pig challenge sets fell within the distribution of reference domestic pigs. We then evaluated 6566 invasive wild pigs sampled across the contiguous United States, of which 63% exceeded the maximum threshold for domestic pigs and could be statistically classified as possessing wild boar ancestry. This approach provides a scientific foundation to enforce regulations prohibiting the possession of this destructive invasive species. Further, this computationally efficient and generalizable approach could be readily adapted to quantify gene flow among ecological systems of conservation or management concern.

The translocation of wild animal species became a common practice worldwide to re-establish local populations threatened with extinction. Archaeological data confirm that chamois once lived in the Biokovo Mountain but, prior to their reintroduction in the 1960s, there was no written evidence of their recent existence in the area. The population was reintroduced in the period 1964–1969, when 48 individuals of Balkan chamois from the neighbouring mountains in Bosnia and Herzegovina were released. The main objective of this study was to determine the accuracy of the existing historical data on the origin of the Balkan chamois population from the Biokovo Mountain and to assess the genetic diversity and population structure of the source and translocated populations 56 years after reintroduction. Sixteen microsatellite loci were used to analyse the genetic structure of three source chamois populations from Prenj, Čvrsnica and Čabulja Mountains and from Biokovo Mountain. Both STRUCTURE and GENELAND analyses showed a clear separation of the reintroduced population on Biokovo from Prenj’s chamois and considerable genetic similarity between the Biokovo population and the Čvrsnica-Čabulja population. This suggests that the current genetic composition of the Biokovo population does not derive exclusively from Prenj, as suggested by the available literature and personal interviews, but also from Čvrsnica and Čabulja. GENELAND analysis recognised the Balkan chamois from Prenj as a separate cluster, distinct from the populations of Čvrsnica and Čabulja. Our results thus highlight the need to implement genetic monitoring of both reintroduced and source populations of endangered Balkan chamois to inform sustainable management and conservation strategies in order to maximise the chances of population persistence.

Abstract With an accelerating negative impact of anthropogenic actions on natural ecosystems, non-invasive biodiversity assessments are becoming increasingly crucial. As a consequence, the interest in the application of environmental DNA (eDNA) survey techniques has increased. The use of eDNA extracted from faeces from generalist predators, have recently been described as “biodiversity capsules” and suggested as a complementary tool for improving current biodiversity assessments. In this study, using faecal samples from two generalist omnivore species, the Eurasian badger and the red fox, we evaluated the applicability of eDNA metabarcoding in determining dietary composition, compared to macroscopic diet identification techniques. Subsequently, we used the dietary information obtained to assess its contribution to biodiversity assessments. Compared to classic macroscopic techniques, we found that eDNA metabarcoding detected more taxa, at higher taxonomic resolution, and proved to be an important technique to verify the species identification of the predator from field collected faeces. Furthermore, we showed how dietary analyses complemented field observations in describing biodiversity by identifying consumed flora and fauna that went unnoticed during field observations. While diet analysis approaches could not substitute field observations entirely, we suggest that their integration with other methods might overcome intrinsic limitations of single techniques in future biodiversity surveys.

Abstract Patterns of genetic differentiation within and among animal populations might vary due to the simple effect of distance or landscape features hindering gene flow. An assessment of how landscape connectivity affects gene flow can help guide management, especially in fragmented landscapes. Our objective was to analyze population genetic structure and landscape genetics of the native wild boar (Sus scrofa meridionalis) population inhabiting the island of Sardinia (Italy), and test for the existence of Isolation‐by‐Distance (IBD), Isolation‐by‐Barrier (IBB), and Isolation‐by‐Resistance (IBR). A total of 393 Sardinian wild boar samples were analyzed using a set of 16 microsatellite loci. Signals of genetic introgression from introduced non‐native wild boars or from domestic pigs were revealed by a Bayesian cluster analysis including 250 reference individuals belonging to European wild populations and domestic breeds. After removal of introgressed individuals, genetic structure in the population was investigated by different statistical approaches, supporting a partition into five discrete subpopulations, corresponding to five geographic areas on the island: north‐west (NW), central west (CW), south‐west (SW), north‐central east (NCE), and south‐east (SE). To test the IBD, IBB, and IBR hypotheses, we optimized resistance surfaces using genetic algorithms and linear mixed‐effects models with a maximum likelihood population effects parameterization. Landscape genetics analyses revealed that genetic discontinuities between subpopulations can be explained by landscape elements, suggesting that main roads, urban settings, and intensively cultivated areas are hampering gene flow (and thus individual movements) within the Sardinian wild boar population. Our results reveal how human‐transformed landscapes can affect genetic connectivity even in a large‐sized and highly mobile mammal such as the wild boar, and provide crucial information to manage the spread of pathogens, including the African Swine Fever virus, endemic in Sardinia.

Human activities can globally modify natural ecosystems determining ecological, demographic and range perturbations for several animal species. These changes can jeopardize native gene pools in different ways, leading either to genetic homogenization, or conversely, to the split into genetically divergent demes. In the past decades, most European wild boar (Sus scrofa) populations were heavily managed by humans. Anthropic manipulations have strongly affected also Italian populations through heavy hunting, translocations and reintroductions that might have deeply modified their original gene pools. In this study, exploiting the availability of the well-mapped porcine genome, we applied genomic tools to explore genome-wide variability in Italian wild boar populations, investigate their genetic structure and detect signatures of possible introgression from domestic pigs and non-native wild boar. Genomic data from 134 wild boar sampled in six areas of peninsular Italy and in Sardinia were gathered using the Illumina Porcine SNP60 BeadChip (60k Single Nucleotide Polymorphisms – SNPs) and compared with reference genotypes from European specimens and from domestic pigs (both commercial and Italian local breeds), using multivariate and maximum-likelihood approaches. Pairwise FST values, multivariate analysis and assignment procedures indicated that Italian populations were highly differentiated from all the other analyzed European wild boar populations. Overall, a lower heterozygosity was found in the Italian population than in the other European regions. The most diverging populations in Castelporziano Presidential Estate and Maremma Regional Park can be the result of long-lasting isolation, reduced population size and genetic drift. Conversely, an unexpected similarity was found among Apennine populations, even at high distances. Signatures of introgression from both non-Italian wild boar and domestic breeds were very limited. To summarize, we successfully applied genome-wide procedures to explore, for the first time, the genomic diversity of Italian wild boar, demonstrating that they represent a strongly heterogeneous assemblage of demes with different demographic and manipulation histories. Nonetheless, our results suggest that a native component of genomic variation is predominant over exogenous ones in most populations.

Abstract Native domestic breeds represent important cultural heritage and genetic diversity relevant for production traits, environmental adaptation and food security. However, risks associated with low effective population size, such as inbreeding and genetic drift, have elevated concerns over whether unique within‐breed lineages should be kept separate or managed as one population. As a conservation genomic case study of the genetic diversity represented by native breeds, we examined native and commercial cattle (Bos taurus) breeds including the threatened Danish Jutland cattle. We examined population structure and genetic diversity within breeds and lineages genotyped across 770K single nucleotide polymorphism loci to determine (a) the amount and distribution of genetic diversity in native breeds, and (b) the role of genetic drift versus selection. We further investigated the presence of outlier loci to detect (c) signatures of environmental selection in native versus commercial breeds, and (d) native breed adaptation to various landscapes. Moreover, we included older cryopreserved samples to determine (e) whether cryopreservation allows (re)introduction of original genetic diversity. We investigated a final set of 195 individuals and 677K autosomal loci for genetic diversity within and among breeds, examined population structure with principal component analyses and a maximum‐likelihood approach and searched for outlier loci suggesting artificial or natural selection. Our findings demonstrate the potential of genomics for identifying the uniqueness of native domestic breeds, and for maintaining their genetic diversity and long‐term evolutionary potential through conservation plans balancing inbreeding with carefully designed outcrossing. One promising opportunity is the use of cryopreserved samples, which can provide important genetic diversity for populations with few individuals, while helping to preserve their traditional genetic characteristics. Outlier tests for native versus commercial breeds identified genes associated with climate adaptation, immunity and metabolism, and native breeds may carry genetic variation important for animal health and robustness in a changing climate.

The Sardinian population of wild boar (WB, Sus scrofa meridionalis) has evolved on this Mediterranean island since its arrival in Neolithic age. Climate and land use vary across the island; high temperatures and dryness represent limiting factors for the development and reproduction of the species. Hence, the environment can have contributed to create the morphological differences we observe today across the island and could sustain the genetic structure that has been previously observed using neutral molecular markers. We therefore searched for genomic signatures of local adaptation in a sample of Sardinian WB genotyped at almost 50 K single nucleotide polymorphisms (SNPs). Genetic structure was observed in the population separating the northwest and southwest from the east of the island, where internal substructure also emerged. We identified 49 SNPs as candidate loci involved in adaptation and 61 genes. Gene ontology enrichment analysis revealed over-representation of terms related to cell localization, motility, and adhesion, but also related to anatomical development and immunity. According to our results, the environment seems to have played a role in shaping the genetic differentiation of the Sardinian wild boar in a limited evolutionary timescale.

Many ungulate populations have a complex history of isolation and translocation. Consequently, ungulate populations may have experienced substantial reductions in the level of overall gene flow, yet simultaneously have augmented levels of long-distance gene flow. To investigate the effect of this dual anthropogenic effect on the genetic landscape of ungulates, we genotyped 35K SNPs in 47 red deer (Cervus elaphus) of Netherlands, including putative autochthonous relic populations as well as allochthonous populations established in private estates and rewilding areas. We applied FST and ordination analyses to determine the meta-population genetic structure and thereby the occurrence of hybridization. At population level, we investigated levels of inbreeding through individual-based diversity measures, including Runs of Homozygosity. We documented that both spatial genetic structure and within-population genetic variation differed markedly from patterns assumed from present-day abundance and distribution. Notwithstanding the small spatial scale, red deer populations formed distinct genetic clusters, and some had higher genetic similarity to distant than to nearby populations. Moreover, the putative autochthonous relic deer populations had much reduced levels of polymorphism and multi-locus heterozygosity, despite relatively large current population sizes. Accordingly, genomes of these deer contained a high proportion of long (>5 Mb) Runs of Homozygosity. Whereas the observed high levels of inbreeding warrant defragmentation measures, the presence of adjacent autochthonous and allochthonous genetic stocks imply that facilitation of gene flow would cause genetic homogenization. Such distortions of the genetic landscape of ungulates creates management dilemmas that cannot be properly anticipated without baseline genetic monitoring.

Abstract Background Pigs were domesticated independently in Eastern and Western Eurasia early during the agricultural revolution, and have since been transported and traded across the globe. Here, we present a worldwide survey on 60K genome-wide single nucleotide polymorphism (SNP) data for 2093 pigs, including 1839 domestic pigs representing 122 local and commercial breeds, 215 wild boars, and 39 out-group suids, from Asia, Europe, America, Oceania and Africa. The aim of this study was to infer global patterns in pig domestication and diversity related to demography, migration, and selection. Results A deep phylogeographic division reflects the dichotomy between early domestication centers. In the core Eastern and Western domestication regions, Chinese pigs show differentiation between breeds due to geographic isolation, whereas this is less pronounced in European pigs. The inferred European origin of pigs in the Americas, Africa, and Australia reflects European expansion during the sixteenth to nineteenth centuries. Human-mediated introgression, which is due, in particular, to importing Chinese pigs into the UK during the eighteenth and nineteenth centuries, played an important role in the formation of modern pig breeds. Inbreeding levels vary markedly between populations, from almost no runs of homozygosity (ROH) in a number of Asian wild boar populations, to up to 20% of the genome covered by ROH in a number of Southern European breeds. Commercial populations show moderate ROH statistics. For domesticated pigs and wild boars in Asia and Europe, we identified highly differentiated loci that include candidate genes related to muscle and body development, central nervous system, reproduction, and energy balance, which are putatively under artificial selection. Conclusions Key events related to domestication, dispersal, and mixing of pigs from different regions are reflected in the 60K SNP data, including the globalization that has recently become full circle since Chinese pig breeders in the past decades started selecting Western breeds to improve local Chinese pigs. Furthermore, signatures of ongoing and past selection, acting at different times and on different genetic backgrounds, enhance our insight in the mechanism of domestication and selection. The global diversity statistics presented here highlight concerns for maintaining agrodiversity, but also provide a necessary framework for directing genetic conservation.

The majority of the nearly 400 existing local pig breeds are adapted to specific environments and human needs. The demand for large production quantities and the industrialized pig production have caused a rapid decline of many local pig breeds in recent decades. Black Slavonian pig and Turopolje pig, the latter highly threatened, are the two Croatian local indigenous breeds typically grown in extensive or semi-intensive systems. In order to guide a long-term breeding program to prevent the disappearance of these breeds, we analyzed their genetic diversity, inbreeding level and relationship with other local breeds across the world, as well as modern breeds and several wild populations, using high throughput genomic data obtained using the Illumina Infinium PorcineSNP60 v2 BeadChip. Multidimensional scaling analysis positioned Black Slavonian pigs close to the UK/North American breeds, while the Turopolje pig clustered within the Mediterranean breeds. Turopolje pig showed a very high inbreeding level (FROH>4Mb = 0.400 and FROH>8Mb = 0.332) that considerably exceeded the level of full-sib mating, while Black Slavonian pig showed much lower inbreeding (FROH>4Mb = 0.098 and FROH>8Mb = 0.074), indicating a planned mating strategy. In Croatian local breeds we identified several genome regions showing adaptive selection signals that were not present in commercial breeds. The results obtained in this study reflect the current genetic status and breeding management of the two Croatian indigenous local breeds. Given the small populations of both breeds, a controlled management activity has been implemented in Black Slavonian pigs since their commercial value has been recognized. In contrast, the extremely high inbreeding level observed in Turopolje pig argues for an urgent conservation plan with a long-term, diversity-oriented breeding program.

Although the two species of chamois (Rupicapra rupicapra and R. pyrenaica) are currently classified as least-concern by the IUCN (International Union for Conservation of Nature), inconsistencies on the subspecies classification reported in literature make it challenging to assess the conservation status of the single subspecies. Previous studies relying on mitochondrial genes, sometimes in combination with nuclear or Y-chromosome markers, reported the presence of clusters corresponding to the geographic distribution but highlighting ambiguities in the genus phylogeny. Here we report novel de novo assembled sequences of the mitochondrial genome from nine individuals, including previously unpublished R. r. balcanica and R. r. tatrica subspecies, and use them to untangle the genus phylogeny. Our results based on the full mitogenome inferred phylogeny confirm the previously reported genus subdivision in three clades and its monophyletic positioning within the Caprinae. Phylogeny and taxonomy of Rupicapra species thus remain controversial prompting for the inclusion of archeological remains to solve the controversy.

Genes of the major histocompatibility complex (MHC) code for cell surface proteins essential for adaptive immunity. They show the most outstanding genetic diversity in vertebrates, which has been connected with various fitness traits and thus with the long-term persistence of populations. In this study, polymorphism of the MHC class II DRB locus was investigated in chamois with Single-Strand Conformation Polymorphism (SSCP)/Sanger genotyping and Ion Torrent S5 next-generation sequencing (NGS). From eight identified DRB variants in 28 individuals, five had already been described, and three were new, undescribed alleles. With conventional SSCP/Sanger sequencing, we were able to detect seven alleles, all of which were also detected with NGS. We found inconsistencies in the individual genotypes between the two methods, which were mainly caused by allelic dropout in the SSCP/Sanger method. Six out of 28 individuals were falsely classified as homozygous with SSCP/Sanger analysis. Overall, 25% of the individuals were identified as genotyping discrepancies between the two methods. Our results show that NGS technologies are better performing in sequencing highly variable regions such as the MHC, and they also have a higher detection capacity, thus allowing a more accurate description of the genetic composition, which is crucial for evolutionary and population genetic studies.

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

A ‘rewilding’ process is occurring in Europe and beyond, centered on landscapes reverting from agricultural use to a more natural state. Wild species are arriving by natural or human-mediated range expansion, at times reclaiming ranges from which they were lost centuries ago. Recent colonizers include alien invasives and species expanding their historical distribution without direct human assistance. These events can deepen our understanding of contemporary evolution, including wildlife responses to changing climatic conditions. Rewilding can advance conservation genomics by encouraging study of wildlife in environments where these species represent novel arrivals or return after prolonged absences. Such efforts could facilitate more experimental approaches to research design than that typically feasible for landscape-scale ecological and evolutionary studies. Correspondingly, developments in conservation genomics offer unparalleled means for testing predictions about rewilding, and advancing evolutionary enlightened and proactive conservation planning. In this perspective article, we examine four European carnivore species relevant as case studies of (re)colonization and/or rewilding, and discuss examples of emerging research opportunities in genomics, evolutionary ecology, and human-wildlife relationships. Keywords: Conservation planning, Dispersal, Evolutionary ecology, Human-modified landscapes, Local adaptation

Runs of homozygosity (ROH), uninterrupted stretches of homozygous genotypes resulting from parents transmitting identical haplotypes to their offspring, have emerged as informative genome-wide estimates of autozygosity (inbreeding). We used genomic profiles based on 698 K single nucleotide polymorphisms (SNPs) from nine breeds of domestic cattle (Bos taurus) and the European bison (Bison bonasus) to investigate how ROH distributions can be compared within and among species. We focused on two length classes: 0.5–15 Mb to investigate ancient events and >15 Mb to address recent events (approximately three generations). For each length class, we chose a few chromosomes with a high number of ROH, calculated the percentage of times a SNP appeared in a ROH, and plotted the results. We selected areas with distinct patterns including regions where (1) all groups revealed an increase or decrease of ROH, (2) bison differed from cattle, (3) one cattle breed or groups of breeds differed (e.g., dairy versus meat cattle). Examination of these regions in the cattle genome showed genes potentially important for natural and human-induced selection, concerning, for example, meat and milk quality, metabolism, growth, and immune function. The comparative methodology presented here permits visual identification of regions of interest for selection, breeding programs, and conservation.

Among the European fauna, the Sardinian hare (Lepussp.) is peculiar in that it differs from all other hares inhabiting the continent. Here, we report on the variation of a 461 bp sequence of hypervariable domain 1 of the mitochondrial control region, examined in 42 hares collected throughout Sardinia and compared to the corresponding sequences of differentLepustaxa. Seventeen novel haplotypes were found in the Sardinian population, resulting in a haplotype diversity of 0.840 and a nucleotide diversity of 0.012. As a result of Bayesian and principal coordinates analyses, Sardinian hares were grouped with North African hares, constituting a monophyletic clade that diverges from all other Old World hares, including Cape hares from South Africa and East Asia. Hence, our data agree that populations inhabiting North Africa and Sardinia form a distinct taxon, which could possibly be included in theL. capensissuperspecies. Moreover, two corresponding lineages can be found in Sardinia and Tunisia, providing evidence of a common origin of the two populations and thus supporting the hypothesis that North African hares were introduced into the island in historical times. Our data show that the two lineages differ in their geographic distribution throughout the island and that the wild Sardinian population also shows the signature of a postintroduction demographic expansion. [ABSTRACT FROM AUTHOR]