If life ever existed on Mars, it may have developed survival strategies similar to those adopted by extremophiles living in terrestrial Martian analogs, such as the cryptoendolithic communities found in the rocky substrates of the McMurdo Dry Valleys or other ice-free areas of continental Antarctica. Nearly thirty years of research on these super-adapted organisms laid the foundation for the CRYPTOMARS project, which aims to disclose the genomic and phenotypic features allowing these microbial communities to withstand specific physico-chemical stresses that may be encountered on the Red Planet. This information will provide tools to outline, in terms of diversity and competences, a putative microbial community able to survive, adapt or even perpetuate under early or present Martian conditions. The project and the background information are here presented.

The Antarctic continent hosts highly specialized microbial ecosystems, particularly within endolithic habitats, where microorganisms colonize the interior of rocks in order to withstand conditions that otherwise cannot support life. Previous studies have characterized the composition and abundance of these communities, as well as their different degrees of stress power; furthermore, the effect of different lithic substrates in shaping their associated bacterial assemblages has been extensively investigated. By contrast, how rock typology exerts fungal endolithic colonization still remains unexplored. In this study, we have considered and compared fungal communities inhabiting granite and sandstone rocks collected across Victoria Land, Antarctica, using high-throughput sequencing of the Internal Transcribed Spacer (ITS) region. Our analyses revealed that both rock types were dominated by Ascomycota, with a marked prevalence of lichen-forming fungi, particularly within the class Lecanoromycetes. However, granite-supported communities exhibited significantly higher species richness, likely driven by the structural heterogeneity of the substrate and the presence of fissures enabling chasmoendolithic colonization. In contrast, sandstone communities were more specialized and dominated by strict cryptoendolithic taxa capable of surviving within the rock’s pore spaces. Differential abundance analysis identified key species associated with each substrate, including the lichen Buellia frigida in granite and the black fungus Friedmanniomyces endolithicus in sandstone, two endemic species in Antarctica. Moreover, the use of presence/absence- versus abundance-based diversity metrics revealed contrasting ecological patterns; substrate type had a stronger influence on species presence, whereas geographic location more significantly shaped abundance profiles, highlighting the complex interplay between both factors in determining fungal community composition. Additionally, alpha diversity analyses showed significantly higher species richness in granite compared to sandstone, suggesting that structural heterogeneity and chasmoendolithism may promote a more diverse fungal assemblage.

The present study has set up a pilot experiment to optimise the most promising assays for investigating the survival of Antarctic microbial cryptoendolithic communities – a natural astrobiological benchmark – when subjected to lethal/sub-lethal stresses testing viability, cell integrity and metabolic activity. Namely, the viability tests for culturable species are based on cultivation on a solid medium, while qPCR coupled to propidium monoazide (PMA) provides information of both culturable and non-culturable microorganisms. The fluorescein diacetate (FDA) and Adenosine 5’-TriPhosphate (ATP) assays, here optimised, consent to highlight the presence of metabolically active cells. The results revealed significant differences between the treated and untreated samples, proving the suitability of the selected tests for investigating the resilience of these astrobiological models.