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.

Border habitats such as interfaces and ecotones are promising research targets being likely areas of high species richness and genetic and phenotypic diversity. Springs are intriguing habitats exploited by both subterranean and surface species. For subterranean species, springs can provide higher trophic resources but can be risky in terms of predation and UV radiation, while for surface species, springs can be safer but less productive environments. We coupled field surveys and laboratory experiments to understand how predation risk and physical constraints, like light occurrence, affect spring exploitation by both a subterranean (Niphargus thuringius) and a surface crustacean amphipod species (Echinogammarus stammeri). From March to May 2021, we surveyed multiple springs and evaluated the activity (both during day and night) of the amphipods and of their predators. Furthermore, in a subterranean laboratory, we reared 80 N. thuringius and 80 E. stammeri under safe and risky conditions with both constant darkness and diel light variation assessing their activity and survival. Risky conditions were represented by the occurrence of meso-predators alone or coupled with the presence of a top predator. In the field, N. thuringius activity was negatively related to the density of predators, while laboratory experiments revealed a main role played by light treatments and night period. E. stammeri activity in the field was higher close to surface while in laboratory conditions decreased during time. In laboratory conditions, predation risk negatively affected survival of both amphipods. Our findings reveal that physical constraints play a key role in affecting the exploitation of ecotones and can mediate antipredator responses, thus providing selective pressures for the exploitation of border environments. Significance statement Understanding environmental pressures acting on ecotones is a key point to verify if new adaptations may occur at the border between two distinct habitats. Using both field and laboratory approaches, we show that, in springs, the behaviour of subterranean invertebrates is affected by surface physical constraints which can mediate the effects of predation risk. Behavioural strategies to avoid predation, such as nocturnal activity, may promote spring ecotone exploitation by groundwater animals, such as amphipod crustaceans.

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.

Border habitats such as interfaces and ecotones promise research targets from an evolutionary and zoological point of view. Springs are typical ecotones that border two strongly distinct environments: surface and underground. They are exploited by both subterranean and surface species for which they may provide specific environmental pressures promoting phenotypic plasticity and local adaptations. The aim of this study is to understand how the landscape of fear (LOF) and physical constraints, like light occurrence, affect springs' exploitation by both a subterranean (Niphargus thuringius) and a surface crustacean amphipod species (Echinogammarus stammeri). From March to May 2021, we surveyed 15 springs, divided into 25 plots according to their distance to the border, and both day and night, we recorded amphipods activity and LOF levels for them. In a subterranean laboratory, we also reared 80 N. thuringius and 80 E. stammeri in safe and risky conditions with constant darkness and diel light variation assessing their activity and survival for 30 days. Risky conditions were represented by meso-predators (four fire salamander larvae) alone or with a top-predator (a dragonfly larva of the species Cordulegaster boltonii). While in field conditions, the activity of N. thuringius seemed negatively affected by the number of active predators, in laboratory experiments, the main role was played by the light treatment; activity was significantly higher in constant darkness conditions. E. stammeri activity in the field was higher in surface plots, while in laboratory conditions was affected by LOF. Predation risk negatively affected the survival of both amphipods. Our findings reveal that while light conditions seem to shape activity patterns of stygobionts strongly, predators have a lower effect on activity, even though predators have negative effects on survival. Moreover, physical constraints, such as light exposure, can affect antipredator responses of subterranean organisms, thus representing selective pressures for the exploitation of surface environments.