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Experimental Design

This study was designed to assess Siberian alder N-fixation input variability across post-fire age, fire severity levels, and stand types. Study plots were randomly located to capture the variability of environmental factors across the landscape. Detailed site information for each research location in this study is available within the BNZ-LTER Data Set entitled Post-fire Variability in Siberian Alder in Interior Alaska: Distribution Patterns, Nitrogen Fixation Rates, and Ecosystem Consequences X - Research Project Site Information 2014.

Methods

FIELD: A subsample of the 2014 alder density plots was chosen for N-fixation sampling between June 29th and July 29th of 2015. The 2014 plots were stratified by burn scar and stand type. We alternated sampling between burn scars daily so that sampling dates for each burn scar were evenly spread throughout the 30-day sampling period. On each day we randomly selected a stand type and then randomly selected a plot within the chosen stand type for measurement. This method of selecting plots controlled for variation in N-fixation activity throughout the day and throughout the 30-day sampling period. We subsampled as many of the 2014 plots as possible during the 30-day sampling period. Within the BF, nodules from 48 alders across 19 plots and four stand types were sampled for N-fixation, and in the WDF, nodules from 48 alders across 21 plots and three stand types were sampled. At each plot, experimental or control nodules were collected from alders of representative height and vigor. Experimental nodules were then incubated in 15N2 gas following previously established methods for alder nodule 15N2 uptake. Experimental and control nodules from individual alders were separately stored in 5 ml cryovials which were preserved in liquid nitrogen during transfer from the field to the lab. LAB: The N-fixation rate was measured using a 15N2 uptake method (Anderson et al. 2004, Ruess et al. 2009, and Ruess et al. 2013). A nodule sample was excavated within 10 minutes immediately adjacent to an individual alder, at a random vector from the centroid, and taken from the organic horizon. Approximately 1 gram of live nodule was cut away from the root and placed in a vacuum-sealed polyethylene syringe containing 50ml of ambient air. Exactly 10ml of 99 atom % 15N2 gas was injected into the incubation syringe, and the syringe was placed beneath the litter layer at the same depth the nodules were located. Following ten minutes of incubation, the enriched nodules were cleaned thoroughly of root and dirt using very delicate brush, and deionized water. Immediately after rinsing, nodules were placed into cryovials, frozen in liquid N2. A plot control sample was collected in the same manner, however, not incubated in the 99 atom % 15N2 atmosphere. In the laboratory, nodule samples were oven-dried at 65 deg C and ground and pulverized before mass spectrometry analysis on a Sercon stable isotope mass spectrometer (Sercon Limited, Crewe, Cheshire, UK) for both total nitrogen (atom % N) and 15N (atom % 15N) content. The APE equation follows as: APEnodule = 15Nenriched nodules - 15Ncontrol nodules where both 15N content measures are in atom %. Determination of atom percent enrichment (APE) for each nodule sample was determined using the same methods outlined in Ruess et al. 2006 and Ruess et al. 2009. By combining APE with total nodule N content, dividing by incubation time, and correcting for the composition of the initial incubation atmosphere, we calculated the specific N fixation activity of the nodule samples (SNF = micromole N assimilated x gDWT nodule-1 x h-1) as follows: SNF = (APEnodule x % Nnodule) (incubation time (h) x % 15Natomsphere) where % Nnodule is the mass percent N content of the enriched nodule sample and %15Natmosphere is the atom percent 15N content of the incubation atmosphere at the beginning of the incubation.