Originality-Significance Statement. Subsurface zones of groundwater and surface water 21 mixing (hyporheic zones) are hotspots of biogeochemical activity and strongly influence carbon, 22 nutrient and contaminant dynamics within riverine ecosystems. Hyporheic zone microbiomes are 23 responsible for up to 95% of riverine ecosystem respiration, yet the ecology of these 24 microbiomes remains poorly understood. While significant progress is being made in the 25 development of microbially-explicit ecosystem models, poor understanding of hyporheic zone 26 microbial ecology impedes development of such models in this critical zone. To fill the 27 knowledge gap, we present a comprehensive analysis of biogeographical patterns in hyporheic 28 microbiomes as well as the ecological processes that govern their composition and function 29 through space and time. Despite pronounced hydrologic connectivity throughout the hyporheic 30 zone-and thus a strong potential for dispersal-we find that ecological selection 31 deterministically governs microbiome composition within local environments, and we identify 32 specific groups of organisms that correspond to seasonal changes in hydrology. Based on our 33 results, we propose a conceptual model for hyporheic zone metabolism in which comparatively 34 high-organic C conditions during surface water intrusion into the hyporheic zone support 35 heterotrophic metabolisms that succumb to autotrophy during time periods of groundwater 36 discharge. These results provide new opportunities to develop microbially-explicit ecosystem 37 models that incorporate the hyporheic zone and its influence over riverine ecosystem function. 38 39 Keywords: hyporheic zone, selection, subsurface, microbial community structure, community 40 assembly, freshwater biology, microbial ecology, stochastic assembly 41 42 2
Summary. 43Subsurface groundwater-surface water mixing zones (hyporheic zones) have enhanced 44 biogeochemical activity, but assembly processes governing subsurface microbiomes remain a 45 critical uncertainty in understanding hyporheic biogeochemistry. To address this obstacle, we 46 investigated (a) biogeographical patterns in attached and waterborne microbiomes across three 47 hydrologically-connected, physicochemically-distinct zones (inland hyporheic, nearshore 48 hyporheic, and river); (b) assembly processes that generated these patterns; (c) groups of 49 organisms that corresponded to deterministic changes in the environment; and (d) correlations 50 between these groups and hyporheic metabolism. All microbiomes remained dissimilar through 51 time, but consistent presence of similar taxa suggested dispersal and/or common selective 52 pressures among zones. Further, we demonstrated a pronounced impact of deterministic 53 assembly in all microbiomes as well as seasonal shifts from heterotrophic to autotrophic 54 microorganisms associated with increases in groundwater discharge. The abundance of one 55 statistical cluster of organisms increased with active biomass and respiration, revealing 56 o...