Hydrogeological compartmentalization outweighs hydrochemistry in structuring microbial communities across contrasting subsurface habitats.

Deep groundwater ecosystems are energy-limited microbial habitats in which hydrochemical conditions, hydrogeological structure, and habitat isolation jointly influence community assembly. However, the relative importance of environmental filtering and hydrogeological compartmentalization in structuring subsurface microbial communities remains poorly understood. We characterized hydrochemistry, bacterial communities, and archaeal signatures across two contrasting groundwater systems in Québec, Canada: a shallow permeable groundwater system at pointe-du-lac (PDL) and deep fractured formation waters (1,460-1,690 m) at saint-Flavien (SF). Hydrochemical analyses, bacterial and archaeal amplicon sequencing, occupancy-based approaches, and multivariate statistical analyses were used to evaluate patterns of community composition, persistence, and environmental association. The two groundwater systems differed markedly in hydrochemical composition, with PDL enriched in major ions and SF characterized by elevated trace element concentrations. Despite these differences, bacterial communities were structured primarily at the scale of individual wells rather than by groundwater system. Well identity explained a substantially larger fraction of community variation than hydrochemistry, indicating strong hydrogeological compartmentalization. PDL supported a larger recurrent bacterial component, stronger taxon-environment relationships, and repeated methanogen-associated archaeal signatures, whereas SF contained a smaller persistent community fraction and a greater contribution from localized taxa. Across both systems, Acetobacterium was the dominant bacterial genus and was largely represented by a single recurrent lineage distributed across multiple wells and hydrochemically distinct habitats. Recurrent taxa in PDL were enriched in anaerobic and sulfur-associated lineages, while SF was characterized by a smaller set of persistent groundwater-associated taxa. Methanogen-associated archaea were largely restricted to PDL and included both methylotrophic and hydrogenotrophic lineages. These results demonstrate that hydrogeological compartmentalization outweighs hydrochemistry in structuring microbial communities across contrasting subsurface habitats. The persistence of recurrent bacterial lineages, together with localized archaeal assemblages, suggests that deep groundwater ecosystems are shaped by the combined effects of hydrogeological compartmentalization, environmental filtering, and adaptation to long-term energy limitation.