Microplastic pollution in karst systems is still poorly studied, despite the presence of protected species and habitats, and important water reserves. Vulnerable key species hosted in these habitats could consume or assimilate microplastics, which can irreversibly damage management efforts, and thus ecosystems functionality. This can be particularly true for subterranean water habitats where microplastic pollution effects on wildlife management programs are not considered. The aim of this study is to provide a case study from the Classical Karst Region, which hosts peculiar habitats and key species protected at European level, such as the olm Proteus anguinus. As this area has been deeply exploited and modified over time, and is adjacent to highways, roads and railways, which could contribute to pollution within the karst system, threatening the ecosystems, it provides a perfect model system. In this study we collected and investigated water and sediment samples from aquatic environments of surface and subterranean habitats hosting several subterranean environment-adapted organisms. Examined particles were counted and characterized by size, color and shape via visual identification under a microscope, with and without UV light. Furthermore, spectroscopic analyses were carried out in order to identify microplastics typology. Microplastics were found in all examined habitats. In water, microplastics concentration ranged from 37 to 86 items/L, in sediments from 776 to 2064 items/kg. Fibre-shape was the main present, followed by fragments and beads, suggesting multiple sources of pollution, especially textile products. Most of the particles were fluorescent under UV light and were mainly transparent, while not-fluorescent ones were especially black, blue or brown. Samples contained especially polyesters and copolymers. These results highlight intense MP pollution in karst areas, with significant impacts on water quality, and potential effects on subterranean environment-dwelling species. We stress the importance of monitoring pollution in these critical environments for biodiversity and habitat conservation: monitoring in karst areas must become a priority for habitat and species protection, and water resources management, improving analyses on a larger number of aquatic surface and subterranean habitats.

Stygobiont species show common, typical traits derived from their adaptation to subterranean life. Due to the general absence of light in cave environments, the majority of them are eyeless. Although the absence of eyes generally does not allow them to perceive luminous stimuli, some stygobionts still present phototaxis. Previous studies determined that different species of the eyeless amphipod crustaceans of the genus Niphargus are able to react to light; this has been interpreted as an adaptation to avoid dangerous surface habitats, even if recent studies suggest that this could also be an adaptation to exploit them when a situation is less dangerous (i.e., during the night). Niphargus thuringius is a stygobiont amphipod that can also be observed in spring environments despite possessing all the main morphological features of subterranean organisms, such as depigmentation and a lack of eyes. In the present study, we test how the species respond to light stimuli according to the light cycle and predation risk experienced during a conditioning period. We assessed the reactions to light stimuli of adult individuals of N. thuringius after 30 days of rearing in microcosms with different conditions of light occurrence (total darkness or a light/darkness daily cycle) and predation risk (without predators, with one predator, and with two predators). Both light stimuli during the test and rearing conditions affected the behavior of Niphargus thuringius. With light stimuli, individuals presented a strong photophobic response. Moreover, individuals reared in conditions of high predation risk preferred a more sheltered environment during behavioral tests than individuals reared in safe conditions. Our results add a new species to those of stygobiont amphipods known to display negative phototaxis, confirming that this pattern is widespread and conserved in the field. N. thuringius could be a good candidate model to perform further studies aiming to assess if differences occur between spring populations and populations present in deeper groundwater.

Springs are environments that can provide general insights into factors favouring diversity in ecotones, but they are often neglected in freshwater studies. One of the challenging processes acting in ecotones is the landscape of fear (LOF), the space–time variation of perceived predation risk. Spring exploitation often involves species that are mesopredators in surface fresh water and that can become apex predators in ground water, as in the case of the fire salamander larvae (Salamandra salamandra). Here, we aim to determine whether the activity and foraging patterns of the fire salamander in springs are affected by LOF. We surveyed the night‐ and daytime abundance of fire salamander larvae in 15 springs to assess predator occurrence. We also reared 48 salamander larvae with and without non‐lethal exposure to predators within tanks simulating groundwater or surface freshwater light features. Before and after a month of rearing, we tested larva efficiency in catching prey when exposed to predator chemical cues, both in light and dark conditions. In the field, the number of active fire salamander larvae was significantly higher during the night. At night, the number of active larvae across the transition area between ground water and surface water was higher in plots closer to the surface. Testing and rearing conditions significantly affected larva behaviour, and prey capture was significantly more effective in light conditions. It was less successful in larvae reared with predator chemical cues and in the presence of predators. Moreover, larvae reared with predators under light conditions were slower than those raised with predators in dark conditions. Our results show that LOF can interact with extant environmental features and constitute a significant behavioural pressure for mesopredator species living in freshwater ecotones.

Niche theory predicts that closely related and ecologically similar species with overlapping distribution ranges can coexist through resource partitioning, that limits interspecific competition. However, studies examining the mechanisms promoting coexistence of top predators at a large geographical scale are still scant. Here, we describe the foraging ecology of three sympatric owl species [Northern long-eared owl (Asio otus), Tawny owl (Strix aluco), Eurasian eagle owl (Bubo bubo)] in the Mediterranean Basin. We review 160 studies reporting diet information (212,236 vertebrate preys) and investigate among-species differences in diet metrics (diversity, evenness, prey size and proportion of mammals) and their variation along geographical and environmental gradients. Moreover, we test whether diet metrics differ in presence or absence of the other predators. All the three species mainly rely on small mammals, but they significantly differ in diet metrics. The smallest predator (i.e., long-eared owl) shows a higher level of specialism on small mammals (highest proportion but lowest diversity of mammals in the diet) compared to the larger ones. In addition, mean prey size significantly increases with predator body size (long-eared owl < tawny owl < eagle owl). Finally, interspecific competition results in an increase of diet diversity and evenness in the long-eared owl, and species’ diet also varies in response to environmental factors. The three species thus segregate along several dietary niche axes over a large spatial scale and according to both morphological characteristics (i.e., body size) and environmental variables. Such dietary niche segregation may adaptively buffer interspecific competition costs, ultimately allowing coexistence.

Benthic invertebrate communities are deeply influenced by hydrological variability in surface freshwater habitats. However, the responses of macroinvertebrates composing stygofauna in aquifers and unsaturated groundwater environments are not well understood nor specifically studied. Here, we present two new cases of resistance to drying in groundwater‐dwelling animals: planarians of the genus Dendrocoelum and the isopod crustacean Monolistra pavani. Multiple surveys were performed in 53 caves of the Italian Alps and Apennines, sampling different stable and unstable subterranean aquatic environments. Planarians and crustaceans were searched visually and by distressing the substrate. When we detected individuals in dry sectors, we photographed them, observed their features, and released them in pools or streams of the same cave. We rinsed dry individuals of M. pavani in small tanks and recorded the time of reactivation. In a dried subterranean stream, we observed a Dendrocoelum planarian encapsulated in a thick mucus layer, which after a total of 5 min started gliding slowly and reached a length of 18 mm. During the drought of January–April 2022, we detected several individuals of M. pavani in dry areas of two caves. In total, 72% of the tested individuals were able to reactivate. This percentage decreased to 42% after 20 days of dryness. Time to reactivate ranged between 0 and 30 s. Living individuals were observed even after 39 days of drought. Our observations provide new insights into the natural history of groundwater‐dwelling invertebrate taxa that are often neglected in the studies of freshwater ecology. Resistance of groundwater‐dwelling animals to droughts has several implications for global climatic changes and could be species‐ and site‐specific, as previously observed in surface macroinvertebrates.