Assessing sexual dimorphism (SD) is an important step in evaluating divergence among cryptic species of Nannospalax leucodon superspecies, although evolution of SD remains poorly understood. We analysed sexual size dimorphism (SSD) and sexual shape dimorphism (SShD) in dorsal and ventral cranial views of four cryptic species from Serbia and one from North Macedonia. Significant SSD and SShD were found in both cranial views in all cryptic species except N. l. syrmiensis, where the absence of SD may be linked to urbanisation. Males and females of each cryptic species shared common allometric trajectories, and allometry significantly influenced cranial shape variation. After correcting for allometry, SSD in upper incisors width and SShD in the ventral cranium remained significant only in N. l. montanoserbicus. Shape differences between sexes were consistent across cryptic species and closely reflected allometric changes. The observed pattern of SD may represent an ancestrally inherited condition, as the most sexually dimorphic cryptic species, N. l. montanoserbicus, is also among the evolutionarily oldest. Because SSD and SShD were detected in most examined cryptic species, male and female size and overall shape data should not be pooled in future studies. Larger and more robust male crania, with broader upper incisors, may indicate greater bite force, while selection favouring SSD likely contributes to SShD. Although occupying similar ecological niches, males dig longer burrows, especially during mating, provide no parental care, and show higher aggression. These behavioural differences between sexes indicate that sexual, rather than natural, selection primarily drives SD in N. leucodon superspecies.

Cryptic biodiversity presents an ongoing challenge to the accuracy and effectiveness of biodiversity assessment and conservation. To address this, we developed a non-lethal genetic monitoring framework for the subterranean rodent, European lesser blind mole rat (BMR), Nannospalax leucodon species complex, which encompasses multiple chromosomally diversified and reproductively isolated cryptic-species and subspecies. Our workflow combines species and sex detection through: karyotyping, Inter-Simple Sequence Repeat (ISSR) PCR profiling, and Sry-based sex determination of 33 individual samples from five BMR cryptic-species collected at 25 localities in Serbia. As conventional karyotyping protocols for BMR required animal sacrifice, we developed the first non-lethal fibroblast culture-based karyotyping approach from BMR skin biopsy, producing high-quality metaphase chromosomes across five cryptic-species. Among twelve tested ISSR primers, three yielded reproducible, species-specific DNA profiles that resolved four of the five cryptic-species. The Sry assay accurately determined the sex of all examined individuals. Our findings demonstrate that integrating fibroblast culture-based karyotyping, with fast, cost-effective ISSR-PCR species identification, and Sry-based sex determination, provides a reliable approach for identifying and monitoring cryptic BMR species without sacrificing individuals. This framework has potential applications in conservation programmes and contributes to an integrative taxonomy approach essential for the study and protection of other cryptic taxa.

The evolutionary history and systematics of blind mole rats (BMR), subfamily Spalacinae (Gray, 1821), are complicated by uneven geographic sampling and insufficient phylogenetic resolution provided by a few individual gene sequences. We combined genome-wide SNPs (ddRAD-seq) and mitochondrial cytochrome b (cytb) sequences with cytogenetic and biogeographic context to resolve the key phylogenetic relationships in this diverse group, using a large set of samples from different parts of the subfamily's entire geographic range. The previously resolved phylogeny within the Spalax (G & uuml;ndenstaedt, 1770) genus is confirmed and refined. In the genus Nannospalax (Palmer, 1903), all sampled populations and chromosomal forms were grouped into four major clades: three of them had originated and diversified in Anatolia and the Northern Levant, while the fourth colonized the European subcontinent. Molecular dating suggested that intensive divergence in all branches of the subfamily occurred within the past 1 Myr, but there was a pause in the speciation process after the Middle Pleistocene Transition, between approximately 0.6 and 0.4 Mya. Presently, multiple and highly divergent subclades are found in small and fragmented geographic ranges, but a few occupy large continuous territories and demonstrate comparatively low levels of genetic diversity.

This study demonstrates the effectiveness of droplet digital PCR (ddPCR) for identifying vertebrate taxa from soil-derived environmental DNA (eDNA) a methodological combination not previously documented in literature. The protocol is optimized for detecting cryptic species of the lesser blind mole rat (BMR). A total of 38 soil samples were collected from five cryptic BMR species across 12 localities in Serbia. Species-specific primer/probe sets were designed based on the cytochrome b (cytb) gene. After optimization of ddPCR assays and verification of primer/ probe specificity, all samples were amplified in triplicates and the results' significance was tested using the RStudioV4.3.3 Environment. The total number of positive findings was detected in 33 out of 38 samples (86.84 %), highlighting the strength of ddPCR in detecting and quantifying rare and degraded target DNA from soil samples. Concentration values varied from 0.073 to 236 copies/mu L. The primer/probe sets were highly specific and the only difficulty emerged in distinguishing between two phylogenetically closest cryptic species, but with a discrepancy in fluorescence amplitudes in specific (target) and non-specific (non-target) cryptic species. We have confirmed that soil eDNA-based ddPCR species identification is a fast and highly sensitive noninvasive monitoring method. It allows quick and easy sampling of many locations across wide areas, providing essential data for conservation management. This protocol offers a valuable framework for conservation strategies targeting cryptic species, while also serving as a foundation for adaptation and broader implementation across other terrestrial vertebrates of concern-particularly those that are strictly protected or classified as endangered.--

Recent molecular analyses of the stone crayfish have revealed a high degree of genetic diversity. The greatest diversity is found in the western Balkans (Dinarides), where more than half of the known phylogroups exist in a relatively small geographical area, some of them having smaller distribution range than the others. While the Croatian and Slovenian parts of the Austropotamobius torrentium areal are well described, data from Bosnia and Herzegovina (BA) are lacking. Here we provide data from 13 different localities in the northwestern parts of BA. We analysed two mtDNA markers and the results revealed high genetic diversity with a total of 12 MT-COI and nine MT-16SrRNA haplotypes, with the majority of novel haplotypes. Both genes confirmed the presence of two known phylogroups and the discovery of a new group named VOJ. The CSE phylogroup was the most widespread and restricted to the Vrbas basin. The first detection of the BAN phylogroup in BA indicates its wider distribution and connects previously isolated findings from Croatia. The discovery of 18 unique haplotypes as well as a new phylogroup is of particular interest, but further studies are needed to clarify their exact relationship to other lineages.

Blind mole rats (genus Nannospalax) attract a great deal of attention because of their cancer resistance and longevity. Due to the high rate of chromosome rearrangements, 74 Nannospalax chromosomal forms have been discovered. The convergence of their external morphology complicates their taxonomy, and many cryptic species remain unrecognized. Thus, the European N. leucodon supersp. is listed in the IUCN Red List of Threatened Species with “Data Deficient” status. It is crucial for the conservation of biodiversity to clarify its taxonomy, to recognize each cryptic species, and assign to them the correct conservation status. Of the more than 20 chromosomal forms described within N. leucodon, five cryptic species occur in Serbia. The most threatened among them—N. l. syrmiensis, described and named 50 years ago in the regions of Srem, Belgrade and Mačva—has been declared extinct in the literature, which may have negative consequences for the conservation of wildlife genetic diversity. Through five years of fieldwork and comparison of 16SrRNA and MT-CYTB gene segments between old, archived teeth and recently collected material, we show that N. l. syrmiensis is not extinct. However, its habitat has been fragmented and reduced, owing primarily to anthropogenic impact. Therefore, detailed surveillance, population-structure studies, risk assessment, and appropriate conservation measures are needed.

Rana temporaria is one of the most widespread Palearctic brown frogs. We aimed to clarify distribution pattern of two main genetic clades in the understudied Balkan peninsula by using 16SrRNA and MT-CYTB sequences, already widely applied in analyses of populations from other parts of Europe, while focusing on the broad area along the Morava river (central Balkans) as a known gap in the species distribution. Additionally, we were interested in revealing the extent of haplotype diversity within the main genetic clades in the Balkans, particularly around the supposed suture zone. The results revealed a suture zone between the Western and Eastern Clades in the central part of the Balkan Peninsula. This indicated the existence of a historical barrier between the Balkan Mountain Belt and geographically close mountains surrounding the Vlasina Plateau (Rhodope/Serbian–Macedonian Massif). The overall observed haplotype diversity in populations of R. temporaria from the Balkan Peninsula seems high. Harboring both main genetic clades of R. temporaria qualifies the Balkan Peninsula as another important center of species’ genetic diversity, as well as rich in unique haplotypes. This points out the necessity of applying conservation measures focused on the common European frog populations and habitats in this part of the species’ distribution area.

This is a preliminary and exploratory study of cranial variation in European populations of grayling. We investigated the correspondence between size/shape variation of the dorsal ( dc ), ventral ( vc ) and occipital ( oc ) cranium and phylogenetic relationships (inferred from mitochondrial control region – mtDNA cr and microsatellite dna data) of six grayling populations: three from Balkan phylogenetic clade and two from Caspian phylogenetic clade of the European grayling Thymallus thymallus and one population of the Adriatic grayling Thymallus aeliani , which until recently was considered the Adriatic phylogenetic clade of T. thymallus . Significant size and shape differences were found between populations in all three cranial views. However, significant size-related shape variation (allometry) was found for dc and vc , but not for oc . The size variation of each cranial view does not contain phylogenetic signal, but size variation of oc is consistent with genetic variation inferred from microsatellite dna . Regarding shape variation, a significant phylogenetic signal was detected only for oc , and only the shape variation of oc is consistent with the genetic variation inferred from the mtDNA cr . Moreover, the Adriatic grayling T. aeliani (Soča population) was clearly separated from the three T. thymallus populations of the Balkan phylogenetic clade and the two T. thymallus populations of the Caspian phylogenetic clade only at the level of oc . Thus, our results suggest that different cranial regions differ in allometry, reflect phylo(genetic) relationships differently, and exhibit differences in ecophenotypic plasticity, with oc seeming best suited to represent the phylogenetic relationships of the grayling populations studied.

We explored the cryptic speciation of the Nannospalax leucodon species complex, characterised by intense karyotype evolution and reduced phenotypic variability that has produced different lineages, out of which 25 are described as chromosomal forms (CFs), so many cryptic species remain unnoticed. Although some of them should be classified as threatened, they lack the official nomenclature necessary to be involved in conservation strategies. Reproductive isolation between seven CFs has previously been demonstrated. To investigate the amount and dynamics of genetic discrepancy that follows chromosomal changes, infer speciation levels, and obtain phylogenetic patterns, we analysed mitochondrial 16S rRNA and MT-CYTB nucleotide polymorphism among 17 CFs-the highest number studied so far. Phylogenetic trees delineated 11 CFs as separate clades. Evolutionary divergence values overlapped with acknowledged higher taxonomic categories, or sometimes exceeded them. The fact that CFs with higher 2n are evolutionary older corresponds to the fusion hypothesis of Nannospalax karyotype evolution. To participate in conservation strategies, N. leucodon classification should follow the biological species concept, and proposed cryptic species should be formally named, despite a lack of classical morphometric discrepancy. We draw attention towards the syrmiensis and montanosyrmiensis CFs, estimated to be endangered/critically endangered, and emphasise the need for detailed monitoring and population survey for other cryptic species.

PURPOSE:The knowledge of heartworm disease in free ranging wild canid populations is limited. As it is very difficult to monitor this disease in live animals, sporadic findings are mostly obtained by examining culled individuals of game species.METHODS:As a part of a broader study on jackal (Canis aureus) ecology in Serbia, the necropsy of legally hunted animals was performed in 2020.RESULTS:Two cases of heartworm infection with unusually located adult heartworms were diagnosed. The first case is an adult female jackal harvested in the vicinity of the capital city of Belgrade. Three adult specimens of Dirofilaria immitis were found in the right atrium of the heart, and nine adult specimens were located at an atypical site, in the vena cava caudalis. Very similar, the second case was in an adult female jackal harvested in the vicinity of Bački Monoštor. In this case, six adult specimens of D. immitis were found in the right atrium of the heart and six more adult specimens in the vena cava caudalis.CONCLUSION:Although this nematode generally resides in the pulmonary arteries and the right ventricle of the heart, rare findings in other large blood vessels can occur. To the best of our knowledge, these are the first and only two confirmed cases of adult D. immitis found in vena cava caudalis in jackals in Europe.

The role of intraspecific karyotype variability in reproductive isolation and speciation has been widely studied. Among the 26 genera of Palaearctic mammals, the blind mole rats genus Nannospalax has the highest karyotype variability with 74 chromosomal forms (CFs). Although these CFs have been described in detail, taxonomic effects of chromosomal rearrangements are still lacking, especially among 25 recorded CFs of European N. leucodon superspecies. As genetic discrepancies for most of them are missing, we analyze nucleotide sequence polymorphism of the mitochondrial 16S rRNA gene between eight N. leucodon CFs. Here we provide for the first time nucleotide sequence data for three CFs: monticola , montanoserbicus and syrmiensis using 40–57-year-old archived samples from our mammalian collection and thus demonstrate the usefulness of archived/museum samples as starting material for DNA analysis. The topology of the phylogenetic tree is congruent with the traditional taxonomic separation of recent blind mole rats with high support. Diversification of N. leucodon cluster into discrete subclusters—CFs—and the extent of evolutionary divergence among them are in accordance with previous findings of complete reproductive isolation between six CFs analyzed here. Additionally, the level of evolutionary divergence among six N. leucodon CFs resembles those recorded among clearly distinct Spalax species and four proposed species of N. ehrenbergi. These facts suggest that they could be cryptic species and bring attention to their conservation and natural resource protection.

Thelazia callipaeda, originally known as an "Oriental eyeworm," is a small nematode parasitizing the conjunctival sacs of domestic and wild animals and humans. Previous studies conducted in Serbia have reported the eyeworm infections in dogs, cats, and foxes, as well as in a human patient. As the data regarding thelaziosis from wildlife is still scarce, the aim of this study was to investigate the presence of T. callipaeda in gray wolf (Canis lupus) from Serbia. All collected nematodes were morphologically identified as T. callipaeda males (n = 64) or females (n = 225). Molecular characterization, conducted by PCR amplification followed by sequence analysis of partial cytochrome c oxidase subunit 1 gene (cox1), revealed only haplotype 1 of T. callipaeda. The overall prevalence of thelaziosis was 38.1% (8/21). In all positive animals, both eyes were affected, with a total parasitic load ranging from four to 132 worms per animal. Our results indicate the important epidemiological role of wolves as wildlife reservoirs of T. callipaeda, expanding geographic range of infection, as well as intra- and interspecies contact rates, although the role of other wild carnivore species (i.e., foxes and jackals) should be investigated in future studies.

Mole rats are exclusively subterranean and highly specialized rodents. Their long lifespans, remarkable anti-cancer mechanisms, and various distinctive adaptive features make them a useful research model. Moreover, opposing convergence of morphological traits, they have developed extremely high karyotype variability. Thus, 74 chromosomal forms have been described so far and new ones are being revealed continuously. These evolved during the process of rapid radiation and occur in different biogeographical regions. During research into their reproductive biology we have already provided substantial evidence for species-level separation of these taxa. Here, we review diverse chromosomal forms of the lesser blind mole rat, Mediterranean Nannospalax leucodon, distributed in South-eastern Europe, their karyotype records, biogeography, origin, and phylogeny from our extensive research. In the light of new data from molecular genetic studies, we question some former valuations and propose a cryptospecies rank for seven reproductively isolated chromosomal forms with sympatric and parapatric distribution and clear ecogeographical discrepances in their habitats, as well as new experimental and theoretical methods for understanding the courses of speciation of these unique fossorial mammals.

Since the density of simple sequence repeats (SSRs) may vary between different chromosomes of the same species in eukaryotic genomes, we screened SSRs of the whole genome of the yellow necked mouse, Apodemus flavicollis, in order to reveal SSR profiles specific for animals carrying B chromosomes. We found that the 2200 bp band was amplified by primer (CAG)4AC to a highly increased level in samples with B chromosomes. This quantitative difference (B-marker) between animals with (+B) and without (0B) B chromosomes was used to screen 20 populations (387 animals). The presence/absence of Bs was confirmed in 96.5% of 342 non mosaic individuals, which recommends this method for noninvasive B-presence detection. A group of 45 animals with mosaic and micro B (μB) karyotypes was considered separately and showed 55.6% of overall congruence between karyotyping and molecular screening results. Relative quantification by qPCR of two different targeted sequences from B-marker indicated that these B-specific fragments are multiplied on B chromosomes. It also confirms our assumption that different types of Bs with variable molecular composition may exist in the same individual and between individuals of this species. Our results substantiate the origin of Bs from the standard chromosomal complement. The B-marker showed 98% sequence identity with the serine/threonine protein kinase VRK1 gene, similarly to findings reported for Bs from phylogenetically highly distant mammalian species. Evolutionarily conserved protein-coding genes found in Bs, including this one in A. flavicollis, could suggest a common evolutionary pathway.

Dobrava-Belgrade virus (DOBV) is a hantavirus species that causes the most severe form of haemorrhagic fever with renal syndrome (HFRS) in Europe. DOBV has been detected in three Apodemus rodents: A. flavicollis, A. agrarius and A. ponticus. These emerging viruses appear throughout the Balkan Peninsula including Serbia as its central part. In this study, we examined the seroprevalence, molecular epidemiology and phylogenetics of DOBV from A. flavicollis captured at six Serbian localities. Furthermore, we applied microsatellite typing of host animal genome to analyse the role of host kinship in DOBV animal transmission. The overall IgG seropositivity rate over 3 years (2008-2010) was 11.9% (22/185). All seropositive samples were subjected to RT-PCR and DNA sequencing for S and L genome segments (pos. 291-1079 nt and 2999-3316 nt, respectively). DOBV was genetically detected in three samples from mountain Tara in western Serbia, a newly detected DOBV focus in the Balkans. No sequence data from human cases from Serbia are available for the studied period. However, collected DOBV isolates in this work phylogenetically clustered together with isolates from Serbian human cases dating from 2002, with 1.9% nucleotide divergence. We determined the level of kinship between seropositive and seronegative animal groups and found no significant difference, suggesting that horizontal virus transmission in the studied population was the same within and among the hatches. Our findings are the first genetic detection of DOBV in rodents in Serbia. We confirm wide and continuous hantavirus presence in the examined parts of the Balkans, underlying the necessity of continual monitoring of hantavirus circulation in A. flavicollis.

Karyotyping and several molecular methods have allowed successful identification of two morphologically similar wide-ranging Western Palearctic species, the yellow-necked field mouse Apodemus flavicollis (Melchior, 1934) and the long-tailed wood mouse A. sylvaticus (Linnaeus, 1758), but reliable species diagnosis on the basis of morphometric characters is particularly problematic. Although they are easily morphologically distinguishable in Central and Northern Europe, this is not the case in southern parts of their distribution areas. Despite that, we have successfully discriminated A. flavicollis and A. sylvaticus from Serbia (Southern Europe) using geometric and traditional morphometric methods on a data set for ventral crania of specimens previously genotyped by the Inter Simple Sequence Repeat-PCR (ISSR-PCR). Discrimination power of applied approaches was more or less similar. The majority of our results were consistent with those obtained for specimens collected across the Czech Republic (Central Europe). Morphological differences observed herein, as well as those already reported between A. flavicollis and A. sylvaticus from the central and northern parts of their distribution areas, could be the outcome of their biology, i.e. ecological discrepancies, different assumed evolutionary scenarios considering biogeography, phylogeny, history and ontogeny.

The yellow-necked field mouse, Apodemus flavicollis (Melchior, 1934), and the long-tailed field mouse, Apodemus sylvaticus (Linnaeus, 1758) are morphologically similar species, with largely overlapping geographical areas and almost equal ecological requirements. This makes reliable species diagnosis based on external characters a real challenge. When advanced multivariate methods of skull and/or dental morphometrics are employed, specimens can be successfully distinguished in most cases, but not all, making single specimen identification impossible. Application of C-band karyotyping clearly distinguishes between these two species. However, it can be applied only to live animals. Several molecular methods have also allowed successful species diagnosis, but their universality has not been tested. Here we compare the diagnostic power of three molecular approaches and their applicability to populations from a species wide distributional area, as well as their simplicity of use, both for live animals and for samples stored in alcohol for a considerable period, without DNA sequencing. A total of 200 tissue samples from Morocco, France, Belgium, Serbia, Romania, Greece, Russia, Turkey and Kazakhstan, were analyzed by AP-PCR, ISSR-PCR and species-specific primers from the mitochondrial cytochrome b gene. The first two methods gave clear species-specific DNA profiles in complete agreement with previous C-band results. However, only partial agreement was observed between the third, species-specific primer method and the other two approaches and C-banding, as there were 4.5–10.5% false positive results. Hence we propose AP-PCR and ISSR-PCR techniques with particular chosen primers for quick and reliable diagnosis of these two species. A large number of samples can be assayed in a short period, with minimal cost and effort. Moreover, these techniques can be applied to a single specimen.

Bioaccumulation of heavy metals in the skulls of black-striped field mice (Apodemusagrarius) was compared for two localities in Serbia differing in the level of pollution. Eight heavy metals: Fe, Mn, Co, Cd, Zn, Ni, Pb and Cu, were quantitatively analyzed by atomic absorption spectrophotometry. Four of them (Mn, Cd, Fe and Ni) showed significantly higher concentrations at the polluted location than in the relatively unpolluted one. Concentrations of heavy metals in three age categories exhibited opposite patterns depending on pollution levels. At the unpolluted locality heavy metal concentrations were the highest in the youngest group and lower in older animals. In contrast, bone metal concentrations increased with age class at the more polluted site. At the same time, we found that young animals from the polluted location had a statistically significant lower relative body mass (RBM) than those from the unpolluted area(t = 7.155, p < 0.001), which may have been caused by heavy metals or other factors associated with pollution. In general, we found that age is a critical factor for estimating the level of heavy metal pollution, so proper investigations should account for the age structure of the samples.

Two alternative models are used to explain maintenance of polymorphism of B chromosomes (Bs) in populations of a great number of species. The parasitic model suggests deleterious effects of Bs on fitness of carriers, while the heterotic model assumes that, in the absence of drive, equilibrium is produced by beneficial effects of Bs at low numbers. In order to determine the potential contribution of Bs to genetic differentiation and diversity, four populations of Apodemus flavicollis , differing in frequency of Bs (from 0.23 to 0.38) and settled in ecologically different habitats, were analyzed by 471 AFLP markers. Although numerous loci were demonstrated to be population specific, none of them was associated with individuals with Bs. AMOVA showed that the presence of Bs does not affect population differentiation, pointing to greater genetic similarity of Bs to A chromosomes. The greatest genetic diversity (0.241) was found in the population settled in optimal conditions for this species featured by the lowest frequency of animals with Bs (0.23). We found that the majority of loci marked as loci under directional selection, are characteristic of either a population with lower or one with a higher frequency of Bs. Several loci detected as outliers were associated with environmental variables that could directly and/or indirectly influence population dynamics of A. flavicollis . Thus, we suggest that the different frequency of Bs carriers in populations is related to adaptive differentiation to diverse habitats, which is in accordance with the heterotic model of Bs maintenance.

The utility of the Inter Simple Sequence Repeat-Polymerase Chain Reaction (ISSR-PCR) was explored in order to determine genetic variation in six species of the genus Apodemus (A. flavicollis, A. sylvaticus, A. uralensis, A. agrarius, A. mystacinus and A. epimelas) at the individual level, population level, in separate geographic samples and in the species as a whole. Six optimized primers produced highly reproducible and polymorphic DNA markers with 98.3% polymorphic bands on a total sample of 91 individuals from 32 localities in Europe and Asia. Moreover, each primer allowed for an exact diagnosis of each of the six Apodemus species and thus provides a simple and reliable tool for the hitherto problematic discrimination of species from the subgenus Sylvaemus. Genetic distances between species ranged from 0.079, among the closely related A. flavicollis and A. sylvaticus, to 0.203 between A. mystacinus and A. agrarius. A. flavicollis, A. uralensis and A. sylvaticus display a strong population substructure. The range of genetic distances among geographic samples within last two species reaches the values obtained for closely related species. ISSR markers proved to be a simple and reliable tool for species diagnosis, as well as for estimating genetic diversity below the species level and for closely related species, but they showed questionable reliability for larger genetic distances.