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Abstract Background Mosquitoes (Culicidae), as disease vectors, represent a risk for human health worldwide. Repeated introductions of alien mosquito species and the spread of invasive species have been recorded in different countries. Traditionally, identification of mosquitoes relies on morphological observation. However, morphology-based identification is associated with a number of potential disadvantages, such as the high level of specialisation of the operator and its limited applicability to damaged samples. In these cases, species identification is achieved through molecular methods based on DNA amplification. Molecular-based taxonomy has also enabled the development of techniques for the study of environmental DNA (eDNA). Previous studies indicated the 16S mitochondrial ribosomal RNA (rRNA) gene as a promising target for this application; however, 16S rRNA sequences are available for only a limited number of mosquito species. In addition, although primers for the 16S rRNA gene were designed years ago, they are based on limited numbers of mosquito sequences. Thus, the aims of this study were to: (i) design pan-mosquito 16S rRNA gene primers; (ii) using these primers, generate a 16S rRNA gene mosquito reference library (with a focus on mosquitoes present in Italy); and (iii) compare the discriminatory power of the 16S rRNA gene with two widely used molecular markers, cytochrome c oxidase subunit 1 mitochondrial gene (COI) and internal transcribed spacer 2 (ITS2). Methods A total of six mosquito genera (28 mosquito species) were included in this study: Aedes (n = 16 species), Anopheles (5 species), Coquillettidia (1 species), Culex (3 species), Culiseta (2 species) and Uranotaenia (1 species). DNA was extracted from the whole mosquito body, and more than one specimen for each species was included in the analysis. Sanger sequencing was used to generate DNA sequences that were then analysed through the Barcode of Life Data Systems (BOLD). Phylogenetic analyses were also performed. Results Novel 16S rDNA gene, COI and ITS2 sequences were generated. The 16S rRNA gene was shown to possess sufficient informativeness for the identification of mosquito species, with a discriminatory power equivalent to that of COI. Conclusions This study contributes to the generation of DNA barcode libraries, focussed on Italian mosquitoes, with a significant increase in the number of 16S rRNA gene sequences. We hope that these novel sequences will provide a resource for studies on the biodiversity, monitoring and metabarcoding of mosquitoes, including eDNA-based approaches. Graphical abstract

Abstract A pattern of increasing species richness from the poles to the equator is frequently observed in many animal taxa. Ecological limits, determined by the abiotic conditions and biotic interactions within an environment, are one of the major factors influencing the geographical distribution of species diversity. Energy availability is often considered a crucial limiting factor, with temperature and productivity serving as empirical measures. However, these measures may not fully explain the observed species richness, particularly in marine ecosystems. Here, through a global comparative approach and standardised methodologies, such as Autonomous Reef Monitoring Structures (ARMS) and DNA metabarcoding, we show that the seasonality of primary production explains sessile animal richness comparatively or better than surface temperature or primary productivity alone. A Hierarchical Generalised Additive Model (HGAM) is validated, after a model selection procedure, and the prediction error is compared, following a cross-validation approach, with HGAMs including environmental variables commonly used to explain animal richness. Moreover, the linear effect of production magnitude on species richness becomes apparent only when considered jointly with seasonality, and, by identifying world coastal areas characterized by extreme values of both, we postulate that this effect may result in a positive relationship in environments with lower seasonality.

Abstract The ongoing biodiversity crisis is strongly threatening amphibians, mostly because of their peculiar physiology, their sensitivity to climate change and the spread of diseases. Effective monitoring involving assessments of pressure effects across time and estimation of population trends play a key role in mitigating amphibian decline. To improve implementation of standardized protocols and conservation efforts, we present here a dataset related to one of the amphibian genera whose onservation status is considered the most declining according to the IUCN. We report information on 66 populations of the endangered European cave salamanders, genus Speleomantes, that was collected through a standardized monitoring along a four-year period (2021–2024). Demographics data of the populations and fitness-related data of single individuals are reported. Furthermore, we include 3,836 high quality images of individuals that can allow to perform studies aiming to assess the phenotypic variability within the genus, and to perform long-term capture-mark-recaptured studies.

Competition between native and alien species is often described as a main driver of biodiversity loss. Nevertheless, there is limited evidence of animal declines and extinctions actually determined by competition. The Aeolian lizard, Podarcis raffonei, is critically endangered because it suffered dramatic declines and extinctions throughout its range. Competition and hybridization with invasive Italian lizards, Podarcis siculus, have been proposed as a driver of the shrinkage of Aeolian lizards, still the mechanisms underlying their decline remain poorly resolved. We used observations of behavioral encounters, combined with morphological data and robust species identification based on genomics, to test whether agonistic interactions with the invasive Italian lizard can explain the competitive exclusion and rapid decline of the native Aeolian lizard while accounting for hybridization. Invasive lizards were larger, with larger heads, and showed higher bite tendency against neutral items. In agonistic encounters between males, Aeolian lizards received more attacks and escaped more frequently than invasive males. The performance of Aeolian males was particularly poor in interspecific encounters. Genomic data verified that tested individuals were all pure P. raffonei or P. siculus, with a single hybrid individual detected. The strong competitive advantage of invasive males can allow them monopolizing territories, potentially hampering the reproduction of both native males and females, thus resulting in a mechanism of sterilizing interference. Reproductive interference competition mediated by spatial exclusion might be an unappreciated process determining rapid decline in endemic species. Safeguarding areas devoid of invasive species should be the priority strategy to avoid the extinction of the Aeolian lizards.

Summary: Interspecific hybridization can be consequential for insular species. The Critically Endangered Aeolian wall lizard, Podarcis raffonei, severely declined due to interactions with the invasive Italian wall lizard, Podarcis siculus. The largest population of P. raffonei survives on a narrow peninsula (Capo Grosso) that is mildly connected to the island of Vulcano, which has been entirely invaded by P. siculus. Recent observation of individuals with an intermediate phenotype raised concern over the risk that hybridization might swamp this last stronghold. We genetically characterized lizards from Vulcano, considering individuals showing multiple phenotypes (native, invasive, and “intermediate”). Hybridization rate was low (∼3%), with just two F1 hybrids and two backcrosses, suggesting that hybridization does not currently represent a major threat. However, we identified low genetic diversity, a small effective population size, and a low Ne/Nc ratio. Management strategies are urgently needed to control invasive species and maintain the genetic diversity of P. raffonei.

Individual diet specialization is a key behavioral trait, with multifaceted impacts on population dynamics. Long-term studies are fundamental to properly understand such dynamics and their main causes. Indeed, individuals may vary from specialism to generalism in response to multiple biological and environmental factors, thus snapshot studies can have limited relevance for the overall biology of species. Yet, so far longitudinal studies testing the potential variation of diet specialization through time are lacking. Here we present the results of a study on individual diet specialization performed over a four-year period. Using three populations of the Ambrosis’ cave salamander Speleomantes ambrosii we tested: i) whether seasonal variation in the occurrence of specialized individuals is maintained through different years, and ii) if the degree of specialization observed in a single season is constant through different years. Our results showed that the species maintains a high proportion of specialized individuals in spring and more generalist in autumn during different years, confirming a consistent seasonal variation of trophic specialization through time. On the other hand, the proportion of specialized individuals of the same period can vary up to ten-fold across years, suggesting that a single study may be not enough to properly understand the dynamics of a species’ trophic niche. This study highlights the need of repeated surveys to accurately understand this behavioral trait, especially for species that are strongly dependent on local environmental conditions.

Abstract Landscapes nearby glaciers are disproportionally affected by climate change, but we lack detailed information on microclimate variations that can modulate the impacts of global warming on proglacial ecosystems and their biodiversity. Here, we use near-subsurface soil temperatures in 175 stations from polar, equatorial and alpine glacier forelands to generate high-resolution temperature reconstructions, assess spatial variability in microclimate change from 2001 to 2020, and estimate whether microclimate heterogeneity might buffer the severity of warming trends. Temporal changes in microclimate are tightly linked to broad-scale conditions, but the rate of local warming shows great spatial heterogeneity, with faster warming nearby glaciers and during the warm season, and an extension of the snow-free season. Still, most of the fine-scale spatial variability of microclimate is one-to-ten times larger than the temporal change experienced during the past 20 years, indicating the potential for microclimate to buffer climate change, possibly allowing organisms to withstand, at least temporarily, the effects of warming.

Nematodes are keystone actors of soil, freshwater and marine ecosystems, but the complexity of morphological identification has limited broad-scale monitoring of nematode biodiversity. DNA metabarcoding is increasingly used to assess nematode diversity but requires universal primers with high taxonomic coverage and high taxonomic resolution. Several primers have been proposed for the metabarcoding of nematode diversity, many of which target the 18S rRNA gene. In silico analyses have a great potential to assess key parameters of primers, including taxonomic coverage, resolution and specificity. Based on a recently-available reference database, we tested in silico the performance of fourteen commonly used and one newly optimized primer for nematode metabarcoding. Most primers showed very good coverage, amplifying most of the sequences in the reference database, while four markers showed limited coverage. All primers showed good taxonomic resolution. Resolution was particularly good if the aim was the identification of higher-level taxa, such as genera or families. Overall, species-level resolution was higher for primers amplifying long fragments. None of the primers was highly specific for nematodes as, despite some variation, they all amplified a large number of other eukaryotes. Differences in performance across primers highlight the complexity of the choice of markers appropriate for the metabarcoding of nematodes, which depends on a trade-off between taxonomic resolution and the length of amplified fragments. Our in silico analyses provide new insights for the identification of the most appropriate primers, depending on the study goals and the origin of DNA samples. This represents an essential step to design and optimize metabarcoding studies assessing nematode diversity.