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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.

Autochthonous transmissions of mosquito-borne diseases were described in close proximity to Slovenia where the knowledge of mosquito species and the viruses they transmit remains very scarce. Therefore we aimed to assess the burden mosquitoes bring to the local community and to test mosquitoes for the presence of the selected zoonotic arboviruses, i. e. Dengue (DENV), West Nile (WNV) and Chikungunya (CHIKV). A pilot mosquito monitoring was conducted in 2012 within two municipalities based on adult mosquito trapping. Altogether 2007 adult mosquitoes were caught. Females of Aedes albopictus and Culex pipiens represented the majority of the total mosquito catch. In sites with more households and nearby greenery, catches of Ae. albopictus were more abundant. Highest numbers of Cx. pipiens females were caught close to a nature reserve, where many bird species stop during their migrations. Trapping results are consistent with both species’ feeding behaviour. Female mosquitoes were grouped into 58 pools according to species, location and date of catch. Real time RT-PCRs for WNV, DENV and CHIKV were performed, but no arboviral genomes were detected in any of the tested mosquito pools. However, amplicons were obtained in three pools when using generic RT-PCR markers for flaviviruses. The viruses detected were propagated and isolated in mosquito cell line C3/36 and identified with sequencing as mosquito-only flaviviruses (MOFs). The important contributions of this study are a successful first isolation of MOFs and confirmation of the presence of the invasive mosquito Ae. albopictus in Slovenia.

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.

The Bukovina blind mole rat Spalax graecus is the westernmost representative of the genus and one of the least known European mammals. As currently under- stood, the species contains three isolates on both sides of the south-eastern Carpathian Mountains. Our focus was on Bukovina blind mole rats from north- eastern Romania and adjacent Ukraine, i.e. on the nominal subspecies Spalax graecus graecus Topachevskii 1976. 2. Phylogenetic reconstruction based on 1140-bp-long cytochrome b sequence revealed a sister position of Spalax graecus against the genus Nannospalax. Pair- wise Kimura two-parameter genetic divergences were evidently higher between Spalax graecus and the three species of Nannospalax (mean distances standard errors between 0.177 0.014 and 0.197 0.016) than between the three species of Nannospalax (up to 0.128 0.010). The two genera were separated by 178 mutational steps. 3. The Bukovina blind mole rat is so far known from 13 localities in north- eastern Romania and adjacent Ukraine. 4. The most influential environmental factors in the Spalax graecus habitat model were seasonality in precipitation, type of soil and altitude. The elevational range of suitable habitats is 39-848 m. The area occupied is estimated at 15581 km 2 , but patches with high habitat suitability cover only 1604 km 2 . 5. The main habitat for the Bukovina blind mole rat is likely to degrade and the conservation needs of the species will soon have to be taken into account. We rec- ommend the inclusion of the species on Annex II or IV of the European Union's Habitats Directive on grounds of its (i) narrow endemism, (ii) small range of occupancy, (iii) small overall population size and (iv) vulnerability to changes in agricultural land use. bs_bs_banner

Mole rats (genus Nannospalax) display prolific chromosomal variation with more than 50 distinct cytotypes. These cytotypes are largely indistinguishable morphologically, are mainly allopatric and their taxonomic ranking is contradictory. We established a cytochrome b phylogeny for 15 cytotypes belonging to all three species recognized on morphological grounds (morphospecies): N. leucodon, N. xanthodon and N. ehrenbergi. Phylogenetic reconstructions yielded two highly divergent groups which are in agreement with the current division into two subgenera (Nannospalax and Mesospalax). The former comprised samples from south-eastern Turkey, Israel and Egypt (the morphospecies N. ehrenbergi). Basal dichotomy within Mesospalax remained unresolved and the putative sister position of N. leucodon against the two lineages of N. xanthodon was not supported in our analysis. Net divergences between sister cytotypes were low (

Mesotriton alpestris lacusnigri is regarded as being an extinct subspecies of the Alpine newt endemic to a small fishless alpine lake called Črno jezero (Black Lake) in the Julian Alps in Slovenia. To re-assess its taxonomic position we sequenced fragments of two mitochondrial genes (309 bp of cytochrome b and 315 bp of 16S rRNA) of two museum specimens collected in 1953. Specimens of the ssp. lacusnigri yielded two new haplotypes which formed a monophyletic group within a cluster of other Slovenian and central European haplotypes of ssp. alpestis. The name lacusnigri is therefore merely a junior synonym of the nominotypical subspecies and should be removed from red data listing. Our study highlights the outstanding value of natural history collections as baselines for contemporary biodiversity assessments.

The phylogeographic architecture of the common vole, Microtus arvalis, has been well-studied using mitochondrial DNA and used to test hypotheses relating to glacial refugia. The distribution of the five described cytochrome b (cyt b) lineages in Europe west of Russia has been interpreted as a consequence of postglacial expansion from both southern and central European refugia. A recently proposed competing model suggests that the 'cradle' of the M. arvalis lineages is in western central Europe from where they dispersed in different directions after the Last Glacial Maximum. In the present study, we report a new cyt b lineage of the common vole from the Balkans that is not closely related to any other lineage and whose presence might help resolve these issues of glacial refugia. The Balkan phylogroup occurs along the southern distributional border of M. arvalis in central and eastern Bosnia and Herzegovina, Montenegro, and eastern Serbia. Further north and west in Slovenia, Bosnia and Herzegovina, and Serbia, common voles belong to the previously-described Eastern lineage, whereas both lineages are sympatric in one site in Bosnia and Herzegovina. The Balkan phylogroup most reasonably occupied a glacial refugium already known for various Balkan endemic species, in contrast to the recently proposed model. South-east Europe is an absolutely crucial area for understanding the postglacial colonization history of small mammals in Europe and the present study adds to the very few previous detailed phylogeographic studies of this region.

Microtus irani Thomas, 1921, is known with certainty from two localities which are separated by a gap of almost 2000 km. In this paper we describe a population from Balkusan in Turkey as a new subspecies Microtus irani karamani. In a complete sequence for cytochrome b gene (1140 bp), the new subspecies has unique mutations when compared with a sequence of M. i. irani from the type locality (Shiraz, Iran) at 32 positions, and differs in 35 mutations and a mean nucleotide divergence of 3.19% ±0.50. It has 60 acrocentric chromosomes in the diploid complement. Morphologically, M. i. karamani ssp. n. is smaller than M. i. irani, with a shallower brain-case, shallower rostrum, and shorter bullae. The new subspecies can be reliably separated from social voles occupying Turkey, Iran and adjacent regions of the Near East, by a combination of morphological, chromosomal and molecular data.

The Blind Mole Rats of Anatolia (Nannospalax xanthodon (Nordmann, 1840)) are characterised by prolific chromosomal diversification. While the geographic distribution of various cytotypes is well documented, opinions on their taxonomic ranking varies amongst authorities. A partial sequence (630 bp) of mitochondrial cytochrome b gene in 13 Blind Mole Rats from the Konya basin, central Anatolia, which represented three distinct cytotypes (2n=40, 58, and 60) yielded nine cyt b haplotypes. Phylogenetic reconstructions recognized three well supported lineages which matched diploid number counts. Genetic divergences between cytotypes were high (K2P between 8.16% ± 1.19 and 11.33% ± 1.42) and application of the 2% divergence rate to the net divergence estimates suggests their divergence about 3.84 and 5.43 Mya (95% confidence interval=1.53–8.19 Mya). If one would rely on genetic operational criteria in species delimitation, there would be little doubt that the three Nannospalax cytotypes analysed in this...

We established a cytochrome b (cyt b) phylogeny for six species of social voles. A Bayesian approach to phylogenetic reconstruction (BI) and a maximum likelihood (ML) tree revealed a dichotomy into two major clusters, namely a Microtus guentheri cluster and a M. socialis cluster. The three main lineages that emerged within each of these two clusters were separated by the K2P divergences which are above the intraspecific variation in Microtus. All six species were also retrieved in the minimum spanning network. Within its present taxonomic scope, M. guentheri is paraphyletic and consists of two allopatric sibling species: M. guentheri (Syria, Israel) and M. hartingi (Anatolia and the Balkans). The closest relative to these two species is M. dogramacii, which is possibly a sister species to M. hartingi. The two geographic samples were identified as M. irani, one from Shiraz (Iran) and the other from Balkusan (Turkey). The cyt b sequence confirmed the specific status of M. anatolicus within the M. socialis cluster. Although five species of social voles occur within a radius of

Martino's vole Dinaromys bogdanovi is the only living member of the Tertiary genus Dinaromys, and probably also the only surviving member of the Pliomys lineage. The range of the genus Dinaromys has historically been small and its rate of evolution has been low. 2. Martino's vole shows all three attributes of rarity in accordance with Rabinowitz's 'seven forms of rarity' model: (i) its range is estimated at 43 545 km 2 but the area of occupancy is

Taxonomy of thicket rats (Grammomys) is highly provisional and the genus is in a critical need of a thorough revision. We compared G. cometes from Eastern Cape Province (n = 150) with G. ibeanus, G. macmillani, and the southern African G. dolichurus, applying analyses of a partial cytochrome-b (Cytb) sequence (375 base pairs), karyotypes, and cranial morphology. Genetically, G. cometes appeared to be very close to G. dolichurus (mean sequence divergence of 3.4% ± 0.8% SE), whereas G. ibeanus and G. macmillani were separated by a mean sequence divergence of 5.4% ± 1.2%. Nucleotide diversity among haplotypes was higher in G. dolichurus (π = 0.0080 ± 0.0010 SD) than in G. cometes (π = 0.0040 ± 0.0009). G. cometes and G. dolichurus showed the same diploid chromosome number (2N = 52) of mostly acrocentric autosomes. None of the karyotypes reported so far for various Grammomys species match the chromosomal sets we found in Eastern Cape Province. Discriminant function analysis on 5 cranial measurements that are not affected by age variation was successful in separating G. cometes and G. ibeanus, but G. dolichurus appeared very similar to the former. In spite of their close genetic and morphological proximity, G. cometes and G. dolichurus tend toward ecological segregation and behave as distinct biological species. G. cometes is endemic to the southern African subregion and the 4 Eastern Cape Province localities are possibly isolates. Specimens were caught in the Afromontane forest above 1,000 m elevation and the lowland riverine forests dominated by Combretum caffra.

We examined, in the context of phylogeny, variations in ventral cranial shape in Martino’s vole (Dinaromys bogdanovi), a rare rodent endemic to the western Balkans. Our analysis was based on 138 complete adult skulls, which were pooled into 3 phylogeographic groups (Northwestern, Central, and Southeastern). These groups were retrieved in an earlier study based on a 555‐base-pair fragment of mitochondrial cytochrome b gene, which suggested a stepping-stone pattern of southward expansion followed by allopatry. Ventral skull shape was analyzed with geometric morphometrics using 23 two-dimensional landmarks. The primary shape differences across groups are the sizes of the auditory bulla and the foramen magnum, the length of the incisive foramen, and the width of the rostrum. Consistent geographic trends in shape changes were rare, and size was stable across phylogeographic groups. Morphological relationships among groups closely resemble genetic distances, implying their neutral evolution rate. We suggest that the adaptive zone occupied by Martino’s vole imposes narrow limits to its phenotypic variation. Under the strong pressures of stabilizing selection for the normative (intermediate) phenotype, random drift in isolated populations produced only minor deviations in the ventral cranium.