Biogeochemical hotspots are pervasive at terrestrial-aquatic interfaces, particularly within groundwater-surface water mixing zones (hyporheic zones), and they are critical to understanding spatiotemporal variation in biogeochemical cycling. Here, we use multi 'omic comparisons of hotspots to low-activity sediments to gain mechanistic insight into hyporheic zone organic matter processing. We hypothesized that microbiome structure and function, as described by metagenomics and metaproteomics, would distinguish hotspots from low-activity sediments by shifting metabolism towards carbohydrate-utilizing pathways and elucidate discrete mechanisms governing organic matter processing in each location. We also expected these differences to be reflected in the metabolome, whereby hotspot carbon (C) pools and metabolite transformations therein would be enriched in sugar-associated compounds. In contrast to expectations, we found pronounced phenotypic plasticity in the hyporheic zone microbiome that was denoted by similar microbiome structure, functional potential, and expression across sediments with dissimilar metabolic rates. Instead, diverse nitrogenous metabolites and biochemical transformations characterized hotspots. Metabolomes also corresponded more strongly to aerobic metabolism than bulk C or N content only (explaining 67% vs. 42% and 37% of variation respectively), and bulk C and N did not improve statistical models based on metabolome composition alone. These results point to organic nitrogen as a significant regulatory factor influencing hyporheic zone organic matter processing. Based on our findings, we propose incorporating knowledge of metabolic pathways associated with different chemical fractions of C pools into ecosystem models will enhance prediction accuracy.

In light of increasing terrestrial carbon (C) transport across aquatic boundaries, the mechanisms governing organic carbon (OC) oxidation along terrestrial‐aquatic interfaces are crucial to future climate predictions. Here we investigate the biochemistry, metabolic pathways, and thermodynamics corresponding to OC oxidation in the Columbia River corridor using ultrahigh‐resolution C characterization. We leverage natural vegetative differences to encompass variation in terrestrial C inputs. Our results suggest that decreases in terrestrial C deposition associated with diminished riparian vegetation induce oxidation of physically bound OC. We also find that contrasting metabolic pathways oxidize OC in the presence and absence of vegetation and—in direct conflict with the “priming” concept—that inputs of water‐soluble and thermodynamically favorable terrestrial OC protect bound‐OC from oxidation. In both environments, the most thermodynamically favorable compounds appear to be preferentially oxidized regardless of which OC pool microbiomes metabolize. In turn, we suggest that the extent of riparian vegetation causes sediment microbiomes to locally adapt to oxidize a particular pool of OC but that common thermodynamic principles govern the oxidation of each pool (i.e., water‐soluble or physically bound). Finally, we propose a mechanistic conceptualization of OC oxidation along terrestrial‐aquatic interfaces that can be used to model heterogeneous patterns of OC loss under changing land cover distributions.

Subsurface groundwater-surface water mixing zones (hyporheic zones) have enhanced biogeochemical activity, but assembly processes governing subsurface microbiomes remain a critical uncertainty in understanding hyporheic biogeochemistry. To address this obstacle, we investigated (a) biogeographical patterns in attached and waterborne microbiomes across three hydrologically-connected, physicochemically-distinct zones (inland hyporheic, nearshore hyporheic and river); (b) assembly processes that generated these patterns; (c) groups of organisms that corresponded to deterministic changes in the environment; and (d) correlations between these groups and hyporheic metabolism. All microbiomes remained dissimilar through time, but consistent presence of similar taxa suggested dispersal and/or common selective pressures among zones. Further, we demonstrated a pronounced impact of deterministic assembly in all microbiomes as well as seasonal shifts from heterotrophic to autotrophic microorganisms associated with increases in groundwater discharge. The abundance of one statistical cluster of organisms increased with active biomass and respiration, revealing organisms that may strongly influence hyporheic biogeochemistry. Based on our results, we propose a conceptualization of hyporheic zone metabolism in which increased organic carbon concentrations during surface water intrusion support heterotrophy, which succumbs to autotrophy under groundwater discharge. These results provide new opportunities to enhance microbially-explicit ecosystem models describing hyporheic zone biogeochemistry and its influence over riverine ecosystem function.

Community assembly processes generate shifts in species abundances that influence ecosystem cycling of carbon and nutrients, yet our understanding of assembly remains largely separate from ecosystem-level functioning. Here, we investigate relationships between assembly and changes in microbial metabolism across space and time in hyporheic microbial communities. We pair sampling of two habitat types (i.e., attached and planktonic) through seasonal and sub-hourly hydrologic fluctuation with null modeling and temporally explicit multivariate statistics. We demonstrate that multiple selective pressures—imposed by sediment and porewater physicochemistry—integrate to generate changes in microbial community composition at distinct timescales among habitat types. These changes in composition are reflective of contrasting associations of Betaproteobacteria and Thaumarchaeota with ecological selection and with seasonal changes in microbial metabolism. We present a conceptual model based on our results in which metabolism increases when oscillating selective pressures oppose temporally stable selective pressures. Our conceptual model is pertinent to both macrobial and microbial systems experiencing multiple selective pressures and presents an avenue for assimilating community assembly processes into predictions of ecosystem-level functioning.

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...