Allopatric populations living under distinct ecological conditions are excellent systems to infer factors underlying intraspecific venom variation. The venom composition of two populations of Vipera ammodytes, insular with a diet based on ectotherms and mainland with a diet based on ectotherms and endotherms, was compared considering the sex and age of individuals. Ten toxin families, dominated by PLA₂, svMP, svSP, and DI, were identified through a bottom-up approach. The venom profiles of adult females and males were similar. Results from 58 individual SDS-PAGE profiles and venom pool analysis revealed significant differences between juveniles compared to subadults and adults. Two venom phenotypes were identified: a juvenile svMP-dominated and KUN-lacking phenotype and an adult PLA₂/svMP-balanced and KUN-containing phenotype. Despite differences in prey availability (and, therefore, diet) between populations, no significant differences in venom composition were found. As the populations are geographically isolated, the lack of venom diversification could be explained by insufficient time for natural selection and/or genetic drift to act on the venom composition of island vipers. However, substantial differences in proteomes were observed when compared to venoms from geographically distant populations inhabiting different conditions. These findings highlight the need to consider ecological and evolutionary processes when studying venom variability. SIGNIFICANCE: This study provides the first comprehensive analysis of the venom composition of two allopatric populations of Vipera ammodytes, living under similar abiotic (climate) but distinct biotic (prey availability) conditions. The ontogenetic changes in venom composition, coupled with the lack of differences between sex and between populations, shed light on the main determinants of venom evolution in this medically important snake. Seven new proteomes may facilitate future comparative studies of snake venom evolution. This study highlights the importance of considering ecological and evolutionary factors to understand snake venom variation.

In the most recent comprehensive distribution of vipers in the western and central Balkans, 210 precise records (146 UTM cells) for all three species of vipers (Vipera ammodytes, V. berus, and V. ursinii) were presented for Serbia. During the past eight years, extensive field investigations within the ongoing conservation projects in our country (e.g. Ecological Networks and Natura 2000), significantly increased the number of faunistic records, which urged the need for publishing the updated distribution of all three species of vipers in Serbia. We collected a total of 770 records of vipers in Serbia, of which literature data constitute 49.4% (n = 380) and unpublished field data made as much as 50.6% (n = 390) of the records. As expected, V. ammodytes was the best-represented species, accounting for 614 records (79.8%), followed by V. berus with 145 records (18.8%), and V. ursinii with 11 records (1.4%). Results of the present study showed that in our country there are three areas crucially important for the diversity of vipers, with all three species present: Koritnik Mt. and Žljeb Mt. in Metohija, as well as Mokra Gora Mt. in south-western Serbia. We also found 25 10×10 km UTM squares with sympatry of two species of vipers. The designation of areas of special importance for vipers (e.g. Mokra Gora Mt., Dukat Mt., Besna kobila Mt., Kamena Gora Mt.) should be one of the priority actions for improvement of their conservation statuses in our country.

Identifying individual age and size is crucial in venom research to understand potential phenotypic changes. When studying venom ontogeny, juveniles and adults are often determined by size at sexual maturity. However, in gape-limited predators such as snakes, venom shifts may occur earlier, coinciding with an increase in jaw size that allows the intake of larger prey. This study explored venom variation in Vipera ammodytes along the snout-vent length (SVL), linking these changes to dietary shifts and reproductive status. A total of 57 venom samples from two populations were analysed using chip electrophoresis (CE) and MALDI-TOF mass spectrometry. Individuals were categorised into three groups: immature individuals feeding on lizards (440 mm). Significant venom composition changes around 300 mm SVL were observed, aligning with a dietary shift marked by increased mammalian prey intake and preceding sexual maturity. This finding highlights the need to use SVL as a metric for accurate venom analysis along ontogeny. Our results indicate that when pooling venom samples is necessary, allocating individuals to age categories should be based on dietary shifts rather than sexual maturity. Additionally, CE and MALDI-TOF MS proved effective in detecting ontogenetic venom changes, offering a fast and affordable approach to venom profiling. This study emphasises the importance of integrating natural history data in venom studies to better understand the ecological and evolutionary drivers of venom adaptation.

We present the updated distribution of batracho-and herpetofauna in southern and south-eastern Serbia, based on literature and new field records for 16 amphibian and 22 reptile species. In these two biogeographic regions, already recognized as biodiversity hotspots, we discovered two new amphibian species with limited distribution: Hyla orientalis and Pelobates balcanicus. Also, one species (Triturus ivanbureshi) has restricted distribution, while one (Bombina bombina) is rare. Four reptile species have limited distributions and six are rare in these regions. The presence of hybrid zones and the influence of the Mediterranean climate make southern and south-eastern Serbia areas of great importance and the target for future ecological and conservation studies in Serbia.