Study location: The study will be conducted at the University of Michigan Biological Station from May—November 2014. A site dominated by speckled alder will be selected from populations growing along Little Carp Creek, in the Gorge Watershed, Cheboygan County, MI. The headwaters of Carp Creek emerge at the base of a 30-45m ravine eroded by subsurface flow seeping from the hillside. Sampling Method: I propose measuring (1) alder above ground N-uptake, (2) in situ Frankia nodule N-fixation rates on alder roots, (3) soil net N-mineralization rates, and (4) groundwater NO3- and NH4+ concentrations and the depth of the saturated zone. Sampling will be conducted across the study location in five randomly located plots, to capture spatial variability in measured variables. Biomass: Great Lakes specific allometry (Parker and Schneider 1974, Parker and Schneider 1975) based on diameter at breast height (DBH) will be used to estimate alder above ground biomass (stem, bark, branches, leaves, and total). Fine root (< 2cm dia.) biomass per m2 will be estimated using soil cores sampling to a depth of 25cm. Frankia nodules excavated with root core samples will be used to estimate nodule mass per m2. Uptake and Demand: Alder N-uptake from soils is equal to the sum of annual storage in alder perennial tissues (stem, bark, and branches pools) + N losses from leaf senescence (Schlesinger and Bernhardt 2012) and determined at the plot level. A sub sample of trees in each plot will be felled, separated into component parts, and dried. The dry matter N will be determined using an auto-analyzer, and extrapolated to the plot level by multiplying mean biomass by mean nitrogen concentrations (mg g-1). Alder N demand is defined as the sum of N-uptake + resorption + Frankia N-fixation + N-mineralization (Uri et al. 2004). Resorption will be determined by sampling fresh leaves, and comparing differences in leaf and litter N concentration using an auto-analyzer (Uri et al. 2002). Leaf litter will be collected using 25 litter traps as described by Muller-Landau and Wright (2010). Fixation: The vast majority of N2 fixing organisms utilize the enzyme nitrogenase to reduce atmospheric N2 to NH3-. Dilworth (1966), also demonstrated that nitrogenase has the capacity to reduce C2H2 (acetylene) gas to C2H4 (ethylene) gas. Therefore, acetylene reduction assays are useful in estimating nitrogenase activity in Frankia. Because the assay indirectly measures N2 fixation, a conversion factor for acetylene reduction to N2 fixation is required. This study assumes the theoretical conversion factor of 4:1 estimated by Schwintzer and Tjepkema (1997). Acetylene reduction assays (ARA) are performed using a modified flow-through method described by Hurd et al. (2001). Three to five nodules including subtending roots and adhering soil will be excavated at random and placed in a 60ml cuvette. A Cole/Parmer Masterflex L/S peristaltic pump is used to create a 10% acetylene environment in the cuvette at a rate of ~300 ml min-1. Gas samples are extracted from the cuvette’s outlet every minute for four minutes following the introduction of acetylene. Ethylene production peaks are expected at the second and third minute following exposure to acetylene, and decline after four minutes (Schwintzer and Tjepkema 1997). Samples will be analyzed using a gas chromatograph with a flame ionization detector. Minus-nodule controls will be used to detect ethylene contamination. Ethylene detected in the controls will be subtracted from peak rates. Four ARAs per plot will be performed on a weekly basis between the hours of 11am and 4pm, and a subset of measurements will be made at 8 p.m., midnight, and 8 a.m. to capture seasonal and diurnal N-fixation. Mineralization: Net N-mineralization will be estimated in situ in the upper 15cm soil where most speckled alder fine roots are located (Hosie 1969), using the buried polyethylene bag method described by Nadelhoffer et al. (1985). Two soil cores per sample, replicated six times per plot will be extracted from the field. One core is placed into a polyethylene bag, sealed, and incubated in the soil. NO3- -N and NH4+ -N concentrations of the harvested core will be extracted with KCL and analyzed on an auto-analyzer. The N-content of incubated cores will be measured after six weeks. N-mineralization will be determined a total of three times (18 weeks) from May to September. Net N-mineralization is equal to the difference in inorganic N between incubated and measured samples. Groundwater: Piezometers will be used to sample saturated zone water, and to determine the depth and thickness of the soil saturated zone under base and rain event conditions. The protocol for piezometer construction and use is based on methods described by (Saunders et al. 2006). The depth and thickness of the saturated zone qualitatively assesses root access to ground water N, and the availability of N contained therein. Three transects of 5 equally spaced piezometers will be installed to a depth of 50cm, parallel to the stream bank at distances of 1, 5, and 15m from the stream. Groundwater samples will be collected weekly, acidified, and frozen for analysis using a Shimadzu Total N analyzer.