Microbial diversity in Pyrenean ice caves: extreme cryogenic environments as analogues for extraterrestrial habitability

Abstract Pyrenean ice caves are the least studies cryogenic environments that preserve perennial ice. These caves host microbial communities adapted to extreme oligotrophy, low temperatures, and episodic water availability, making them valuable analogues for subsurface habitats on icy planetary bodies. This study investigated four ice caves in the Central Pyrenees (Devaux, Cotiella A294, Sarrios 1 and Somola SO-01) to (i) characterize bacterial and microeukaryotic assemblages, (ii) assess how ice origin, physicochemical gradients and cave geology structure these communities, and (iii) evaluate their relevance as terrestrial analogs for cold oligotrophic ecosystems. Amplicon sequencing of 16S and 18S rRNA genes, combined with detailed chemical profiling, revealed marked cave‑specific differences associated with pH, major ions, short‑chain organics and ice‑formation processes. Liquid water samples contained distinct assemblages dominated by ultra‑small Patesibacteria, whereas firn‑derived and congelation ice hosted stratified or hydrologically entrained communities. Microeukaryotic diversity was highest in light‑exposed ice from Cotiella A294 and lowest in Sarrios 1, where fungal taxa prevailed. Redundancy analyses identified acetate, nitrate, sulfate, pH and trace metals as the environmental variables most strongly aligned with microbial gradients. These findings provide a data‑driven characterization of microbial organization in Pyrenean ice caves and offer empirical baseline parameters to inform future studies of microbial persistence and biosignature formation in cold terrestrial and extraterrestrial environments.