Watersheds are important suppliers of freshwater for human societies. Within mountainous watersheds, microbial communities impact water chemistry and element fluxes as water from precipitation events discharge through soils and underlying weathered rock, yet there is limited information regarding the structure and function of these communities. Within the East River, CO watershed, we conducted a depth‐resolved, hillslope to riparian zone transect study to identify factors that control how microorganisms are distributed and their functions. Metagenomic and geochemical analyses indicate that distance from the East River and proximity to groundwater and underlying weathered shale strongly impact microbial community structure and metabolic potential. Riparian zone microbial communities are compositionally distinct, from the phylum down to the species level, from all hillslope communities. Bacteria from phyla lacking isolated representatives consistently increase in abundance with increasing depth, but only in the riparian zone saturated sediments we found Candidate Phyla Radiation bacteria. Riparian zone microbial communities are functionally differentiated from hillslope communities based on their capacities for carbon and nitrogen fixation and sulfate reduction. Selenium reduction is prominent at depth in weathered shale and saturated riparian zone sediments and could impact water quality. We anticipate that the drivers of community composition and metabolic potential identified throughout the studied transect will predict patterns across the larger watershed hillslope system.

46 47 Watersheds are important suppliers of freshwater for human societies. Within mountainous 48 watersheds, microbial communities impact water chemistry and element fluxes as water from 49 precipitation events discharges through soils and underlying weathered rock, yet there is limited 50 information regarding the structure and function of these communities. Within the East River, CO 51 watershed, we conducted a depth-resolved, hillslope to riparian zone transect study to identify 52 factors that control how microorganisms are distributed and their functions. Metagenomic and 53 geochemical analyses indicate that distance from the East River and proximity to groundwater and 54 underlying weathered shale strongly impact microbial community structure and metabolic 55 potential. Riparian zone microbial communities are compositionally distinct from all hillslope 56 communities. Bacteria from phyla lacking isolated representatives consistently increase in 57 abundance with increasing depth, but only in the riparian zone saturated sediments did we find 58 Candidate Phyla Radiation bacteria. Riparian zone microbial communities are functionally 59 differentiated from hillslope communities based on their capacities for carbon and nitrogen 60 fixation and sulfate reduction. Selenium reduction is prominent at depth in weathered shale and 61 saturated riparian zone sediments. We anticipate that the drivers of community composition and 62 metabolic potential identified throughout the studied transect will predict patterns across the larger 63 watershed hillslope system. 64 Methods 129Site description and sample collection 130