Background Lightless caves can harbour a wide range of living organisms. Cave animals have evolved a set of morphological, physiological, and behavioural adaptations known as troglomorphisms, enabling their survival in the perpetual darkness, narrow temperature and humidity ranges, and nutrient scarcity of the subterranean environment. In this study, we focused on adaptations of skull shape and sensory systems in the blind cave salamander, Proteus anguinus, also known as olm or simply proteus—the largest cave tetrapod and the only European amphibian living exclusively in subterranean environments. This extraordinary amphibian compensates for the loss of sight by enhanced non-visual sensory systems including mechanoreceptors, electroreceptors, and chemoreceptors. We compared developmental stages of P. anguinus with Ambystoma mexicanum, also known as axolotl, to make an exemplary comparison between cave- and surface-dwelling paedomorphic salamanders. Findings We used contrast-enhanced X-ray computed microtomography for the 3D segmentation of the soft tissues in the head of P. anguinus and A. mexicanum. Sensory organs were visualized to elucidate how the animal is adapted to living in complete darkness. X-ray microCT datasets were provided along with 3D models for larval, juvenile, and adult specimens, showing the cartilage of the chondrocranium and the position, shape, and size of the brain, eyes, and olfactory epithelium. Conclusions P. anguinus still keeps some of its secrets. Our high-resolution X-ray microCT scans together with 3D models of the anatomical structures in the head may help to elucidate the nature and origin of the mechanisms behind its adaptations to the subterranean environment, which led to a series of troglomorphisms.

Cave-dwelling animals might be regarded as dead-end points when it comes to their evolution and habitat exploitation. However, in the past different observations of the olms’ typical troglomorphic populations have been reported for springs of Venetia Giulia, along with a community rich in stygobionts (Bressi et al. 1999; Stoch 2017). The aim of this work is to point out the non-random active use of surface habitats by this community, providing a comparison with the occurrence observed in caves and performing an assessment of factors favouring ecotone habitats exploitation. Since 2020 we started multiple day and night surveys of olms in both 71 springs and 11 caves. Each spring and cave habitat monitored has been characterised by respect to abiotic and biotic features, including planktonic and benthic prey availability. Additionally, cave and spring populations of Troglocaris sp. were tested to determine whether they showed any behavioral difference regarding their response to light stimuli and predatory cues, as potential adaptations to the different conditions found in the two habitats. We used 34 individuals sampled from three springs and 24 from two caves of the Italian karst. We detected the olm at least once in 12 springs, with a maximum of 9 individuals occurring together. Detection probability in springs and caves was similar. Spring habitats provided higher density of potential prey available. Olms seem to prefer springs without predator fish and temporary hydroperiod. We recorded in one spring a larva of 3.5 cm which could be the smallest ever recorded in the field. Preliminary results of the behavioral experiments show no susceptibility to light neither for spring nor cave populations of Troglocaris sp., as for chemical cues we expect a difference in reactivity depending on the habitat of origin of the shrimp, consistently with the top predator present. We suggest that epigean habitats and borders with surface may have an underestimated importance for animals adapted to subterranean environments (Manenti and Piazza 2021), including the olm. Further studies focusing on the ipogean community which actively exploits spring habitats are underway, with the aim of determining the diel occupation of these complex ecotones.

The olm (Proteus anguinus) is considered the most representative example of all the stygobiont fauna. Nevertheless, it is also reported in some spring habitats of Friuli Venezia Giulia (North-eastern Italy). Springs are intriguing habitats that border two strongly distinct environments: surface and underground. They may provide distinct environmental pressures to stygobiont animals promoting new adaptations. The aim of this study is to verify if there are differences in the structural morphology of the head and in the presence of melanin in the skin between the olms that are found in caves compared to the individuals found in the external environment. During 2021, 28 olms, 18 from underground habitats and 10 from external sites, were photographed in standard conditions with a metric reference. For each individual, only the cephalic region has been considered. The pictures were processed using TPSdig software. For each picture we placed 2 landmark homologues, matching the emergence of the gills branches, and 48 semi-landmark to define the contour of the head. With regard to melanin 2 mm of skin were removed from the tail of the olm and analyzed through a spectrophotometer. The results indicate a significant effect of the environment on the shape of the olm’s head, in fact the shape of the head appears more elongated in the front and narrower in the parietal region in the olms coming from the hypogean environment. In addition, the olms that use the epigean environment show higher melanin levels in their skin. These findings provide a first evidence of differences occurring between olm populations/sub-populations living in caves and those exploiting surface habitats.