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.

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.--

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.

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.

A new species of eriophyoid mite, Aculodes marcelli sp. nov., was discovered on cheatgrass, Anisantha tectorum (L.) Nevski (syn. Bromus tectorum L.), an annual grass that is native to Eurasia and Northern Africa. This grass was introduced to North America near the end of the 19th century and now is widespread and associated with the observed increases in the size, frequency, and intensity of wildfires in western N. America. In this paper, A. marcelli sp. nov., is morphologically described and illustrated. Compared with other Aculodes spp., it differs based on morphology and the sequence of the mitochondrial cytochrome oxidase gene, subunit I (MT-CO1). Results of morphometric analysis showed clear differentiation between A. marcelli sp. nov., and the most similar congener, A. altamurgiensis from Taeniatherum caput-medusae. Analysis of MT-CO1 sequence divergence revealed significant levels of genetic variation (17.7%) and supported the results from the morphometric analysis; therefore, it is determined that they are two different species. Aculodes marcelli sp. nov., is a new candidate agent for classical biological control of A. tectorum.

Background Mammalian mandible and cranium are well-established model systems for studying canalization and developmental stability (DS) as two elements of developmental homeostasis. Nematode infections are usually acquired in early life and increase in intensity with age, while canalization and DS of rodent skulls could vary through late postnatal ontogeny. We aimed to estimate magnitudes and describe patterns of mandibular and cranial canalization and DS related to age and parasite intensity (diversity) in adult yellow-necked mice ( Apodemus flavicollis ). Results We found the absence of age-related changes in the levels of canalization for mandibular and cranial size and DS for mandibular size. However, individual measures of mandibular and cranial shape variance increased, while individual measures of mandibular shape fluctuating asymmetry (FA) decreased with age. We detected mandibular and cranial shape changes during postnatal ontogeny, but revealed no age-related dynamics of their covariance structure among and within individuals. Categories regarding parasitism differed in the level of canalization for cranial size and the level of DS for cranial shape. We observed differences in age-related dynamics of the level of canalization between non-parasitized and parasitized animals, as well as between yellow-necked mice parasitized by different number of nematode species. Likewise, individual measures of mandibular and cranial shape FA decreased with age for the mandible in the less parasitized category and increased for the cranium in the most parasitized category. Conclusions Our age-related results partly agree with previous findings. However, no rodent study so far has explored age-related changes in the magnitude of FA for mandibular size or mandibular and cranial FA covariance structure. This is the first study dealing with the nematode parasitism-related canalization and DS in rodents. We showed that nematode parasitism does not affect mandibular and cranial shape variation and covariance structure among and within individuals. However, parasite intensity (diversity) is related to ontogenetic dynamics of the levels of canalization and DS. Overall, additional studies on animals from natural populations are required before drawing some general conclusions.

We conducted a comparative (2D landmark-based geometric and traditional) morphometric analysis on tadpoles at early developmental stages. Two species of brown frog (Rana dalmatina and R. temporaria) and the common toad (Bufo bufo) were involved, all raised in the laboratory from fertilized eggs collected in their natural habitat. Taxonomic identification was confirmed by the DNA barcoding method with the 16S rRNA sequence as the gene marker. Interested to compare the methodologies for quantification and description of morphological differences among tadpoles of mentioned species, we aimed to: 1) calculate interspecies genetic distances as the most relevant measurement for species differentiation, 2) determine and describe size and shape variation, 3) identify relationships among the analyzed species at the morphological level and 4) assess their classification accuracy. Within the framework of the specified aims, both methodologies produced very similar results, i.e., the smallest divergence was between R. dalmatina and R. temporaria, while the most discriminative were B. bufo and R. temporaria. However, we observed subtle shape variation of the distal region of the tail that was detected only by the geometric morphometrics. Our findings support the following. Geometric morphometric method captures more subtle shape differences that were unable to be recovered from linear measurements. It performs slightly better in classification rate. Although it was not quantified, it stands to reason that there is no difference in time investment between the two approaches. Geometric morphometrics provides more information that can be leveraged to answer further questions and it has a clear advantage in visualizing.

We employed geometric morphometric techniques to investigate external (body) morphology of European grayling (Thymallus thymallus) populations from the wild and hatchery facilities. Wild graylings were representative of Balkan and Caspian phylogenetic clades, whereas hatchery-reared specimens originated from Balkan and Adriatic phylogenetic clades. Individuals of T. thymallus from the Adriatic phylogenetic clade were the largest, followed by those from the Balkan phylogenetic clade, while graylings from the Caspian phylogenetic clade were the smallest. Graylings from hatchery facilities were larger than graylings from the wild. Body shape variation in T. thymallus coincides with genetic differentiation of the analyzed populations, whereas it is less influenced by difference in environment they experience in wild and captive habitats. Although hatcheries can generate large numbers of individuals, some of which will have an extreme phenotype, the variance in body shape was similar in captive and wild populations. Allometric relations were different between specimens from the wild and from hatchery facilities, as well as among those belonging to different phylogenetic clades. Allometric analyses performed separately for the wild and hatchery-reared populations revealed significant effect of allometry and similar trends in size-related shape variation among populations from different phylogenetic clades. We found that phenetic relationships among the studied wild grayling populations inferred from non-allometric body shape variation better reflected their phylogenetic relationships than equivalent data from hatchery populations.

We examined morphological and genetic differences among Aceria spp. inhabiting six Brassicaceae species in Serbia. Five of them have been already mentioned in the literature as original or alternate hosts of Aceria spp. (Berteroa incana (L.) DC., Capsella bursa-pastoris (L.) Medik., Cardamine hirsuta L., Lepidium draba L. and Sisymbrium orientale L.), whereas Aurinia petraea (Ard.) Schur was registered here for the first time as a host for Aceria sp. Results of morphometric analyses indicated clear differentiation of Aceria spp. from B. incana, L. draba, A. petraea and S. orientale, whereas the other two entities were less diverse and clustered together. Molecular analyses indicated that the average mean divergence over all sequence pairs was 18.3% (11.8–25.9%) and disclosed that the observed mtCOI distances between the six host-associated Aceria entities are large enough to represent differences between species. The Aceria sp. hosted by B. incana was morphologically and genetically most distinctive, whereas discrepancy between phenetic and phylogenetic relationships was found in the position of Aceria sp. hosted by L. draba when compared with those inhabiting C. hirsuta, S. orientale and C. bursa-pastoris.

We used geometric morphometric techniques to examine variability in Serbian red fox (Vulpes vulpes) crania. Male crania are about 5% larger than female ones and sexual size dimorphism (SSD) was significant. Also, significant cranial shape differences were found between the sexes. Males are characterized by reduced basicranial, but broader rostral and zygomatic regions. Although a slight and significant allometric effect was detected and larger specimens are smaller in the region of basicranium, cranial sexual shape dimorphism (SShD) pattern is not influenced by SSD. As small to moderate SSD in canid species is a general characteristics of the family, additional studies are needed to determine whether basicranial reduction in males, i.e. its enlargement in females, could be an important feature in characterizing cranial SShD patterns in other Canidae members. While uniform considering size, the shape of Serbian red fox crania varies geographically, as well as depending on proportion of agricultural habitats. In comparison to those from central and eastern Serbia, specimens from the northern Serbian region Vojvodina (with higher proportions of agricultural areas) have more robust crania with shorter snouts and maxillae, larger palatine bones accompanied with anteriorly moved posterior edges of the canine alveolus and laterally expanded zygomatic arches. Encompassing mostly facial and temporal cranial regions these shape changes are probably related to diet differences, although genetic diversification cannot be excluded as a possible contributing factor.

Geometric morphometric approaches were employed for the first time to explore skull size and shape changes in golden jackal (Canis aureus) from Serbia. We examined three different, but connected aspects of its cranial variability: geographic variation, sexual dimorphism and allometry. To elucidate the pattern of cranial size and shape variation, we analyzed ventral crania of subadults and adults from two geographic regions of Serbia. For both age groups there were no significant size, but significant shape differences between golden jackals from northeastern and central Serbia. The observed shape differences were subtle, probably due to recent range expansion of this species in the Balkans and a strong founder effect in the newly established populations. The detected significant sexual size and shape dimorphisms (SSD and SShD) were slight and more evident in adult crania. Although the SShD observed in adult crania is influenced by SSD, factors responsible for the non-allometric component of shape variation, and not pure size differences between sexes, are those that are most important for generation of SShD in this species. A monogamous reproductive system, male parental care, omnivorous and opportunistic feeding behavior and the absence of division of labor between males and females in activities other than parental care, could all be related to the driving of morphological similarity between the sexes.

We explored modularity and morphological integration of the ventral cranium during postnatal ontogeny in Martino's vole (Dinaromys bogdanovi). Two closely related phylogenetic groups, originating from the Central and Southeastern part of the species range in the western Balkans, were considered. As expected, both phylogroups had similar patterns of ontogenetic changes in cranial size and shape variation, modularity and integration. At the level of within individual variation, the hypothesis that the viscerocranial and neurocranial regions are separate modules was rejected, indicating that the hypothesized modules are not developmental, but rather functional. At the level of among individual variation, the viscerocranium and the neurocranium could not be recognized as separate modules at the juvenile stage. The strength of association between the hypothesized modules becomes lower with age which finally results in a clear 2-module organization of the ventral cranium at the adult stage. On the other hand, patterns of morphological integration for the cranium as a whole, the viscerocranium and the neurocranium stay consistent across ontogenetic stages. The developmental mechanism producing integration of the cranium as a whole, as well as integration of the neurocranium, varies throughout postnatal ontogeny. In contrast, we detected the ontogenetic stability of the mechanism responsible for covariation of viscerocranial traits which could provide ongoing flexibility of the viscerocranial covariance structure for high functional demands during lifetime. Findings from our study most likely support the idea of the 'palimpsest-like' model of covariance structure. Moreover, similarity or dissimilarity in the patterns of within and among individual variation in different sets of analyzed traits and comparisons across ontogenetic stages demonstrate how studies on small mammals other than mice can give new insights into postnatal cranial development.

As a basis for future conservation activities, the genetic and external body morphology variability of the European mudminnow Umbra krameri, a highly endangered fish species in Serbia and in Bosnia and Herzegovina, was determined for existing populations with the use of molecular markers (mitochondrial and microsatellite DNA) and geometric morphometric methods. Mitochondrial DNA cytochrome b gene analysis revealed two previously undescribed haplotypes: Da1 (the Lugomir population from the Danube River basin) and Sa1 (the Bakreni Batar and the Gromiželj populations from the Sava River system), with a corresponding genetic distance of 0·7%. Paired values of FST and DAS distances for microsatellite marker data show that the difference between the Danube and the Sava populations is seven to nine times higher than the difference between the populations within the Sava River system. Geometric morphometric analyses also support a clear separation of the Lugomir population from the Bakreni Batar and the Gromiželj populations. The analysis of the body shape variation, however, indicates a significant difference between the two genetically indistinguishable Sava populations. The observed genetic and phenetic relationships of the analysed mudminnow populations most probably represent a consequence of historical, geographical and ecological factors. These results will offer guidelines for future protection, conservation and sustainable management of this species in the region.

We report the first evaluation of mandibular and cranial modularity in the greater horseshoe bat (Rhinolophus ferrumequinum). Although some authors found no modular pattern of these morphological structures in mammals, we discovered that traits integration in R. ferrumequinum is not uniform throughout the mandible and cranium, but structured into two distinct modules. Allometry does not affect mandibular and cranial modularity in R. ferrumequinum probably as a result of the low fraction of shape variation explained by size. Observed at the static level, mandible and cranium modularity in this species could be either functional or developmental or both, but we cannot rule out a genetic nature for underlying mandibular and cranial trait interactions. Evolutionary modularity among related taxa is influenced by genetic and functional modularity. Therefore, future studies on cranial integration and modularity at multiple levels of variation may shed more light on these important features of morphological variability in bats, including R. ferrumequinum.

Cranial size and shape variation of the greater horseshoe bat Rhinolophus ferrumequinum from territories in Serbia and Montenegro was examined using geometric morphometric methods. Statistically significant size and shape differences among specimens from distinct geographic regions (the Carpatho–Balkanides, the Internal and External Dinarides) were observed. Bats from the Carpatho–Balkanides have the smallest crania, while those from the External Dinarides have the largest ones. Compared to specimens from the Carpatho–Balkanides, bats from the other two regions have crania wider in the temporal and elongated in the facial region, while the basicranial region is smaller. Our analysis of sexual size dimorphism revealed no statistically significant differences between males and females. Even though significant sexual shape dimorphism was observed, cranial shape differences among bats from different geographic regions exceeded those between sexes. We also found that size and shape vary with climatic factors. Allometry has statistically significant effect on cranial shape variation and somehow contributes to covariation between cranial shape and environmental variables. Although the examined bats were from a relatively small territory, we have provided new insights into important issues like geographic variation, sexual dimorphism and allometry in this species.

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 russet mite, Aceria anthocoptes (Nalepa), is the only eriophyoid that has been recorded on Cirsium arvense (L.) Scop. It has been noted in several European countries and recently in the USA. In this study we explored the geographic and host-related variability of Aceria spp. inhabiting different Cirsium spp. We applied landmark-based geometric morphometric methods to study morphological variability of three body regions (ventral, coxigenital and prodorsal) of 13 Aceria spp. populations inhabiting five Cirsium spp. in Serbia (Europe) and four Cirsium spp. in Colorado (North America). Analyses of size and shape variation revealed statistically significant differences between Aceria spp. living on European native and North American native Cirsium spp., as well as between A. anthocoptes s.s. inhabiting European C. arvense and North American C. arvense. The coxigenital region was the most informative when considering inter-population shape differences. European Aceria spp. dwelling on Cirsium spp., including A. anthocoptes s.s. from C. arvense, are characterized by higher inter-population size and shape variability than their North American counterparts. This finding supports a Eurasian origin of A. anthocoptes, presumed to consist of a complex of cryptic taxa probably coevolved with host plants in the native environment. Morphological similarity among Aceria spp. inhabiting North American native Cirsium spp. may indicate that speciation of A. anthocoptes started relatively soon after the host shift to plants different from C. arvense in the invaded region.

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.