The Deep Darkness microbiome: functional and taxonomic diversity in an oligotrophic temperate cave

Many subterranean ecosystems are chronically energy limited, yet the mechanisms governing microbial community assembly and metabolic function under extreme carbon scarcity remain poorly resolved. We combined 16S rRNA gene sequencing with community-level physiological profiling (CLPP) to examine how physicochemical gradients regulate microbial diversity and carbon-use strategies across a surface-subsurface transition into the Deep Darkness zone of Indiana Caverns. Progressive isolation from surface inputs resulted in sharp declines in microbial biomass, total organic carbon, and nitrogen, accompanied by increased water content and C:N ratios, indicating strong attenuation of allochthonous organic matter and intensified resource filtering. Microbial communities spanned 42 bacterial phyla, but exhibited compositional shifts consistent with selection for carbon-efficient and non-heterotrophic metabolisms. Cave sediments were dominated by Proteobacteria (47.6%), Acidobacteria (13.6%), Chloroflexi (9.1%), and Nitrospirae (8.3%). In contrast, Actinobacteria, typically abundant in oligotrophic caves, were exceptionally rare (1.2%), suggesting that extreme depletion of refractory organic substrates constrains decomposer-based energy acquisition and favors taxa adapted to chemolithotrophy or resource-conserving strategies. Despite severe carbon limitation, cave communities retained high phylogenetic richness, but were strongly structured by sediment chemistry and moisture availability, indicating niche differentiation driven by environmental filtering rather than passive dispersal. Only 19.8% of operational taxonomic units (OTUs) were shared across all sites, consistent with a small, persistent core microbiome. CLPP analyses further revealed functional reorganization: surface reference soil preferentially oxidized labile substrates, whereas cave communities relied more heavily on recalcitrant and polymeric carbon sources. Together, these results demonstrate that extreme oligotrophy restructures cave microbiomes around resource-efficient metabolic guilds shaped by hydrological and geochemical constraints. IMPORTANCE: Caves provide natural laboratories for understanding how microbial communities persist under extreme energy limitation. Yet, the mechanisms linking subterranean physicochemistry with microbial functional capacity remain largely unresolved. By integrating culture-independent sequencing with metabolic profiling across spatial and hydrological gradients, this study shows how carbon scarcity, sediment stoichiometry, and microhabitat structure filter microbial taxa and select for specialized metabolic guilds. The work highlights that subterranean environments can harbor high microbial diversity despite chronic oligotrophy, and that functional potential shifts predictably toward degradation of complex substrates under nutrient scarcity. The unusually low abundance of Actinobacteria, typically dominant in oligotrophic caves, highlights a distinct subterranean energy regime that favors slow-growing, resource-efficient taxa. Our findings provide new insight into how environmental filtering, hydrologic connectivity, and metabolic specialization structure microbiomes in deep karst systems, informing broader models of microbial survival in low-energy environments.