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.

Central European Institute of Technology Brno University of Technology Purkyňova 123 Brno 61200 Czech Republic

Department of Information Technology Uppsala University Box 337 Uppsala 755 01 Sweden

Department of Integrative Biology University of Texas at Austin Austin 78712 Texas USA

Elettra Sincrotrone Trieste S C p A S S 14 km 163 5 in Area Science Park Basovizza Trieste 34149 Italy

Institute Tular Cave Laboratory Oldhamska 8a Kranj 4000 Slovenia

Karolinska Institutet Department of Physiology and Pharmacology Solnavagen 9 17165 Solna Sweden

Laboratory of Functional Morphology University of Antwerp Universiteitsplein 1 Wilrijk 2610 Belgium

Max Planck Institute for Evolutionary Biology August Thienemann Str 2 Plon 24306 Germany

Medical University of Vienna Center for Brain Research Department of Neuroimmunology Spitalgasse 4 1090 Vienna Austria

Research Centre of the Slovenian Academy of Sciences and Arts Karst Research Institute Titov trg 2 Postojna 6230 Slovenia

Speleovivarium Erwin Pichl Adriatic Speleology Society Via Guido Reni 2 C Trieste 34123 Italy

University of Ljubljana Biotechnical Faculty Department of Biology Večna pot 111 Ljubljana 1000 Slovenia

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Living in darkness: Exploring adaptation of Proteus anguinus in 3 dimensions by X-ray imaging