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Multi-proxy analysis of the coprolites which were found during excavations at two Late Neolithic (fourth millennium bc) pile-dwelling sites (Crnelnik and Stare gmajne) in Slovenia yielded some new insights into human–dog relations and behaviour. The digested content is presented in a multidisciplinary approach, in which palynological, palaeoparasitological, archaeobotanical and archaeozoological features are studied and genetic signs are tested. Beside the origin of the coprolites, the size of an animal and the diet, the faeces provided some additional information, such as health, status, nutrition habits, environment and season.

Maintenance of traditional cultural landscapes largely depends on traditional agricultural practices, which are nowadays in decline as a result of increasingly intensive and mechanised land use. Losing traditional practices may result in impoverishing of picturesque mosaic landscape and biodiversity. This research focuses on land-use changes in two time periods (2002–2008; 2013–2016) and effects of changes reflecting on populations of critically endangered butterfly. False Ringlet, Coenonympha oedippus (Lepidoptera: Nymphalidae), is a habitat specialist, which in Slovenia inhabits two geographically distinct contrasting habitats – dry meadows in south-western and wetlands in central Slovenia. We compared nine environmental parameters to assess environmental differences, which shape species habitat; seven parameters significantly differ among the four geographical regions and five among the two habitat types. Four parameters significantly differ (i.e. at least in two regions) when tested for homogeneity, while in dry habitat type all (except slope) were significant and none in wet habitat. Changes in land use in two studied periods lit up two processes: transformation of meadows into agricultural land and overgrowing of the meadows, both processes affecting species severely. We believe that maintaining of traditional landscapes in future could serve as a good conservation practice for this endangered species.

The development of new diagnostic methods resulted in the discovery of novel hepaciviruses in wild populations of the bank vole (Myodes glareolus, syn. Clethrionomys glareolus). The naturally infected voles demonstrate signs of hepatitis similar to those induced by hepatitis C virus (HCV) in humans. The aim of the present research was to investigate the geographical distribution of bank vole-associated hepaciviruses (BvHVs) and their genetic diversity in Europe. Real-time reverse transcription polymerase chain reaction (RT-qPCR) screening revealed BvHV RNA in 442 out of 1838 (24.0%) bank voles from nine European countries and in one of seven northern red-backed voles (Myodes rutilus, syn. Clethrionomys rutilus). BvHV RNA was not found in any other small mammal species (n = 23) tested here. Phylogenetic and isolation-by-distance analyses confirmed the occurrence of both BvHV species (Hepacivirus F and Hepacivirus J) and their sympatric occurrence at several trapping sites in two countries. The broad geographical distribution of BvHVs across Europe was associated with their presence in bank voles of different evolutionary lineages. The extensive geographical distribution and high levels of genetic diversity of BvHVs, as well as the high population fluctuations of bank voles and occasional commensalism in some parts of Europe warrant future studies on the zoonotic potential of BvHVs.

Simple Summary The major histocompatibility complex is a family of genes of central importance in vertebrate adaptive immunity. Genes of the major histocompatibility complex are among the most polymorphic genes ever described in vertebrates, with their number differing greatly among species. Due to their functional significance and exceptional diversity, they represent excellent markers in evolutionary ecology and conservation. Traditional methods were commonly used for their genotyping, but the introduction of next-generation sequencing facilitated more accurate and reproducible genotyping of such polymorphic gene families. Nevertheless, due to the high polymorphism of the major histocompatibility complex genes, genotyping is inherently difficult. Here, we compared the performance of traditional and next-generation sequencing on genotyping of the major histocompatibility complex genes in chamois. Although the major histocompatibility complex system in chamois is quite simple, we found genotyping discrepancies between the two methods in 25% of individuals. Our results show that next-generation sequencing has a higher detection capacity and thus allows for more accurate genotyping of highly polymorphic genes. Abstract 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.

At the southern limit of its range the endangered butterfly Coenonympha oedippus inhabits grasslands (wet, dry) that differ significantly in the abundance of its larval hostplants (wet > dry) and mean annual air temperature (wet < dry). We determined the difference in the wing morphology of individuals in the two contrasting habitats to test whether and how traits associated with wing size, shape and eye like spots vary in the sexes and two ecotypes. We show that sexual dimorphism follows the same (wing size and shape, number of eyespots on forewing) or different (relative area of eyespots on hindwings) patterns in the two contrasting habitats. Irrespective of ecotype, females had larger, longer and narrower wings, and more forewing eyespots than males. Sexual dimorphism in the relative area of eyespots on hindwing was female-biased in the wet, but male-biased in the dry ecotype. Ecotype dimorphism in wing size and the relative area of eyespots on the hindwing is best explained by mean annual air temperature and abundance of host-plants. While ecotype dimorphism in wing size did not differ between sexes, neither in direction (wet > dry) or in degree, in the two sexes the relative area of eyespots on hindwing had opposite patterns (males: dry > wet; females: wet > dry) and was more pronounced in males than in females. The differences in wing shape between ecotypes were detected only in the hindwings of males, with more rounded apex in the dry than in the wet ecotype. We discuss the life-history traits, behavioural strategies and selection mechanisms, which largely account for the sex- and ecotype-specific variation in wing morphology.

Spiny dogfish Squalus acanthias is a widely distributed, highly migratory mesopredatory shark that is extremely sensitive to overexploitation. Because of unregulated targeted and incidental capture and a lack of enforceable management in the Mediterranean Sea, the spiny dogfish subpopulation has declined by at least 50% in the Mediterranean Sea over the past 75–105 years, and is regionally classified as endangered. In this study we sampled 124 spiny dogfish in the northern-most part of the Mediterranean (i.e. the Adriatic Sea) to: (1) assess levels of genetic diversity using 13 microsatellite loci and mitochondrial (mt)DNA (NADH dehydrogenase subunit 2 (ND2) gene); and (2) infer its evolutionary placement while assessing further possible genetic divergence across the Atlantic and Pacific Ocean basins based on ND2. Analysis of mtDNA revealed 12 unique haplotypes and a high level of genetic variation in the Mediterranean region, whereas results from microsatellite markers showed significant genetic heterogeneity and a fine-scale stock structuring involving both sexes. The findings support an Atlantic–South Pacific origin for the spiny dogfish in the Adriatic Sea, with little or no present-day connectivity with the Atlantic population. Consequently, this commercially exploited and regionally endangered coastal shark in the Adriatic Sea should be considered as a separate management unit, with implementation of regional protective management plans.

Migration is one of several marine vertebrate traits increasingly affected by human encroachment. The Adriatic Sea is an important foraging and wintering site for loggerhead turtles (Caretta caretta), and one of the Mediterranean regions where they are most heavily impacted, particularly by fisheries bycatch. Conservation measures concern foraging and wintering areas and nesting grounds, and must, therefore, be informed by the natal origin of individuals in these mixed aggregates. Genetic diversity was investigated among 488 loggerheads found stranded or incidentally captured in fishing gear across the Adriatic and the origin of individuals was assessed through mixed-stock analysis based on comparison of 755 bp of mitochondrial DNA control region sequence. In addition, we highlight the importance of the Gulf of Manfredonia (Apulia, Italy), and of the northeastern Adriatic--a previously genetically undescribed foraging ground. No significant genetic divergence was recorded among sampling areas, between turtles sampled in cold and warm months or between adults and juveniles. The distribution of turtles across the Adriatic Sea appeared not to depend on individual origin. Rookeries in western Greece and Crete provided the most important contributions to the Adriatic mixed stock. In particular, the Greek populations were the most abundant locally in the Gulf of Manfredonia, so they are likely to experience an even greater impact than previously thought because of the severe fishery bycatch levels in this area. This study also provides an example of how substantial increases in sample sizes permit a relatively comprehensive testing of genetic structure across groupings in foraging aggregations.