Beyond the CaCl₂ Brine: Transient Microbial Communities at the Edge of Habitability in lifeless Don Juan Pond, McMurdo Dry Valleys, Antarctica

Abstract Background Don Juan Pond (DJP), located in the McMurdo Dry Valleys of Antarctica, is one of the most extreme aquatic environments on Earth due to its CaCl₂-rich brines, low water activity, exceptional chaotropicity, subzero temperatures, intense solar radiation, and recurrent desiccation. Although the central DJP brine has long been considered largely inhabitable, the surrounding basin seasonally harbors temporary streams, ponds, and sparse microbial mat. Here, we investigated the diversity, ecological organization, and environmental factor chaping the structure of microbial communities inhabiting these peripheral habitats by integrating hydrochemical analyses, in situ reflectance spectroscopy, DNA metabarcoding, and ecological network analysis. Results No DNA suitable for PCR amplification was recovered from the central CaCl₂ brine. In contrast, peripheral streams and ponds supported diverse prokaryotic, microeukaryotic, and fungal communities, with the highest alpha diversity consistently observed in the hydrologically heterogeneous S1 stream system. Prokaryotic assemblages were primarily associated with salinity-related variables, including chlorinity and Mg/Ca ratios, whereas eukaryotic and fungal communities were more strongly correlated with hyperspectral indices indicative of photosynthetic biomass. Transient habitats also hosted micro-metazoans, including rotifers, tardigrades, and nematodes, indicating ecological refugia capable of sustaining complex trophic interactions under polyextreme conditions. Hyperspectral indices successfully discriminated active microbial mats from inactive sedimentary crusts and were significantly associated with the distribution of multiple bacterial, fungal, and microeukaryotic taxa. Network analyses revealed highly interconnected multi-domain communities composed of bacteria, fungi, algae, protists, and microbial grazers. Conclusions The presence of life in the Don Juan Pond basin appears to be largely restricted to temporary habitats supported by seasonal meltwater inputs rather than the central CaCl₂ saturated brine itself, which is anathema to all life forms due to a number of strict physicochemical constraints imposed on life. The contrasting responses of prokaryotic and eukaryotic communities suggest that adaptation and persistence represent distinct ecological strategies in this polyextreme ecosystem. These findings indicate that environmental stability may be as important as salinity/chaotropicity in determining habitability limits and demonstrate the value of integrating molecular and hyperspectral approaches to investigate life in extreme terrestrial and exo-planetary analogous environments.