Fernando Martínez-Freiría’s research while affiliated with Research Centre in Biodiversity and Genetic Resources and other places

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 (<300 mm SVL), immature individuals feeding on lizards but start including mammals in their diet (between 300 and 440 mm), and mature individuals feeding on mammals and 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.

Since the last update of the list of the Spanish herpetofauna in 2018, recent studies have provided new evidence supporting the need to implement taxonomic changes in several groups. In this work, we present an updated reference list for the Spanish herpetofauna, which currently includes 132 native or historically introduced species, i.e. 37 amphibians and 95 reptiles, offering a standardized framework for amateurs, scientists, and environmental agencies. While updating the list, we had to undertake some nomenclatural acts. For instance, to solve the nomenclatural issues concerning Blanus species, we designate a neotype for Amphisbaena oxyura Wagler, 1824; thus, the valid name for the two recognized Iberian species are Blanus cinereus and Blanus oxyurus. We also provide an updated list for the 13 introduced species with documented breeding populations in Spain.

Snake genomes attract significant attention from multiple disciplines, including medicine, drug bioprospection, and evolutionary biology, due to the unique features found in snakes, especially, the evolution of venom. However, genomic research within the family Viperidae has mostly focused to date on the subfamily Crotalinae, while overlooking Viperinae, the Old World vipers. Among Viperinae, European vipers (Vipera) have been the subject of extensive research because of their venoms, phylogeographic, and ecological diversification. Nevertheless, venom research in this group has been conducted using mostly proteomes alone, while phylogeography and systematics in the genus have relied on biased information from mitochondrial phylogenies. Here, we generated chromosome-level genome assemblies for three Vipera species and whole-genome sequencing data for 94 samples representing 15 Vipera taxa. This comprehensive dataset has enabled us to disentangle the phylogenomic relationships of this genus, affected by mito-nuclear discordance and pervaded by ancestral introgression. Population-level analyses in the Iberian Peninsula, where the three oldest lineages within Vipera meet, revealed signals of recent adaptive introgression between ecologically dissimilar species, whereas chromosomal rearrangements isolate species occupying similar niches. Finally, using transcriptomic and proteomic data, we characterized the Vipera toxin-encoding genes, in which opposing selective forces were unveiled as common drivers of the evolution of venom as an integrated phenotype.

Speciation, i.e., the formation of new species, implies that diverging populations evolve genetic and phenotypic factors that promote reproductive isolation (RI), but the adaptive vs. neutral origin of these factors and their relative contributions across the speciation continuum remain elusive. Here we test which of genomic, bioacoustic, morphological and environmental differentiation best predicts RI across the midwife toads (genus Alytes), a diversification of Mediterranean amphibians. Hybrid zone analyses in the A. obstetricans complex support that without strong geographic barriers to dispersal, the extent of introgression (which should reflect the strength of RI) covaries with genomic divergence irrespective of other factors. Phenotypic divergence become important later along the continuum, namely between non-admixing species attributed to distinct subgenera. Our results suggest that by putatively causing intrinsic incompatibilities in hybrids, the genetic mutations accumulating randomly between allopatric populations act as the initial trigger of RI, while substantial ecological and behavioral differentiation is a long-term consequence of species divergence that ultimately promotes sympatry. Whereas speciation is usually claimed to be primarily adaptive, our study corroborates recent findings that new species may also be a neutral outcome of gradual phylogeographic divergence, which has practical implications for species delimitation.

Doñana (southern Spain), a region of notable biodiversity richness, is highly threatened by ongoing landscape transformation and climate change. We investigated the local effect of these anthropogenic factors on the temporal range dynamics of Lataste’s viper (Vipera latastei), an Iberian endemic Mediterranean reptile that has apparently become rare over the years in Doñana. Using ecological niche-based models, based on climatic and remote sensing variables, we analyzed historical (1959–1999) and contemporary (2000–2022) records of the species to assess range shifts and identify environmental factors that may influence them. Our results show that V. latastei is mostly restricted to the coastal region of Doñana and that one temperature variable is the most important factor explaining this distribution pattern in both periods. Additional climatic and vegetation variables play a role in its historical distribution, but they become less important in contemporary times, suggesting a niche simplification over time. We found 30.5% of reduction in the species suitable area from historical to contemporary conditions, a reduction that would be even greater (83.37%) in the absence of niche shift. These findings underscore the species’ heightened vulnerability to ongoing environmental changes and highlight the urgent need for targeted conservation strategies.

Understanding how hybridization influences the morphology and fitness of hybrids is essential for studying adaptive evolution and ecological speciation. Secondary contact zones, where separately evolving populations meet and hybridize, offer valuable insights into the evolutionary processes driving speciation and provide an excellent system to address these questions. In this study, we investigate patterns of morphological and genetic variation of two congeneric viper species, Vipera aspis and V. latastei, across a contact zone in northern Spain (Oja-Tirón), where vipers with mixed morphology are often detected, but genetic studies addressing hybridization and relating patterns of genetic and morphological admixture are lacking. Using nine morphological traits (scalation and colouration) and 18 microsatellite markers, we (1) estimated the extent of hybridization, (2) morphologically characterized parental species and hybrids, and (3) evaluated the correlation between patterns of genetic and morphological admixture. Analyses revealed a bimodal hybrid zone with high rate of hybridization (22%) and prevalence of late-generation hybrids (F2 and backcrosses). Morphological analyses differentiated the two parental species, and a positive correlation (r = 0.95) was found between morphological and genetic patterns. The hybrid group displayed on average an intermediate morphology between the parentals, yet morphologically intermediate hybrids were rare in our dataset. Instead, most hybrids resembled the parental species with whom they share most of the genetic background. Notably, the hybrid group exhibited greater morphological variation than the parental groups. Traits with adaptative value, such as ventral scales and dorsal marks, showed significant differences between hybrids and the two parental species. Introgression of these traits may confer ecological advantages to hybrids, enhancing local adaptation. Overall, this study reveals a positive correlation between patterns of morphological and genetic variation across a hybrid zone and provides insights into the phenotypic consequences of hybridization on these viper species.

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.