DOI: 10.5061/dryad.s7h44j1h3
title: Data from: Rainfall events stimulate episodic associative nitrogen fixation in switchgrass

description: Associative N2 fixation (ANF) is widespread but poorly characterized,
 limiting our ability to estimate global inputs from N2 fixation. In some
 places, ANF rates are at or below detection most of the time, but
 occasionally and unpredictably spiking to very high rates. Here we test
 the hypothesis that plant phenology and rainfall events stimulate ANF
 episodes. We measured ANF in intact soil cores in switchgrass (Panicum
 virgatum L.) in Michigan, USA. We used rain exclusion shelters to impose
 three rainfall treatments with each receiving 60 mm of water over a 20-day
 period but at different frequencies. We concurrently established a
 treatment that received ambient rainfall, and all four treatments were
 replicated four times. To assess the effects of plant phenology, we
 measured ANF at key phenological stages in the ambient treatment. To
 assess the effects of rainfall, we measured ANF immediately before and
 immediately after each wetting event in each treatment involving rainfall
 manipulation. We found that the previous day’s rainfall could explain 29%
 of the variation in ANF rates within the ambient treatment alone, and that
 bulk soil C:N ratio was also positively correlated with ANF, explaining
 18% of the variation alone. Wetting events increased ANF and the magnitude
 of response to wetting increased with the amount of water added and
 decreased with the amount of inorganic N added in water. ANF episodes thus
 appear to be driven primarily by wetting events. Wetting events likely
 increase C availability, promote microbial growth, and make rhizosphere
 conditions conducive to ANF.
description: <em>Site description</em> This
 study was conducted at the W.K. Kellogg Biological Station (KBS) Long-Term
 Ecological Research (LTER) Interactions site in Hickory Corners, MI
 (42°24’11” N, −85°22’26” W) from May through October 2019. Mean annual
 temperature at KBS is 10.1°C and average annual precipitation includes
 1005 mm of rain and 1300 mm of snow (Robertson and Hamilton 2015). Soils
 developed on glacial outwash with intermixed loess (Sprunger and Robertson
 2018) and are classified as mixed, mesic Typic Hapludalfs of co-mingled
 Kalamazoo and Oshtemo series (Crum and Collins 1995).
 The Interactions Experiment, which was originally planted with
 continuous corn, was established in 1985 to examine interactions between
 tillage history (historically tilled or never tilled), tillage treatment
 (conventional tillage or no tillage) and fertilizer treatment (with and
 without N). In 2005, the 16 historically tilled plots (27 x 40 m) were
 switched over to a corn-soybean-wheat rotation, while maintaining their
 tillage and fertilizer treatments assigned 20 years earlier. Finally, all
 16 plots were planted with switchgrass (Cave-in-rock variety) in the
 spring of 2016. Switchgrass is harvested annually after senescence with
 all aboveground biomass greater than 10 cm in height removed from the
 plots. <em>Study design</em>
 Our study used four switchgrass plots that had not been tilled or
 fertilized for over 30 years. Each plot was subdivided into four smaller
 subplots, including an ambient subplot (5 x 2 m) for phenological
 measurements and three subplots (5 x 4 m) for manipulation of rainfall
 frequency. Subplots were assigned to their location (i.e., NW, NE, SW, or
 SE) within the four plots using a stratified random sampling approach such
 that each subplot type never occupied the same corner in multiple
 plots. To examine potential phenological effects, we
 measured N<sub>2</sub> fixation throughout the switchgrass
 growing season in the four ambient (unmanipulated rainfall) subplots.
 Because Roley et al. (2018) found that root-associated
 N<sub>2</sub> fixation rates were higher after switchgrass
 senescence than earlier in the season, we sampled more frequently during
 senescence. We measured N<sub>2</sub> fixation on almost a
 monthly basis until senescence began in mid-August, at which point
 sampling events took place every two weeks. More specifically, 2019
 sampling occurred as follows – 1) May 1: pre-emergence, 2) May 27: tiller
 stage, 3) June 18: stem elongation, 4) July 22: flowering, 5) August 5:
 seed-set, 6) August 19: senescence, 7) September 2: senescence, 8)
 September 16: senescence, 9) September 30: senescence, and 10) October 15:
 senescence (see (Sanderson 1992) for definition of stages). For analysis,
 tiller and stem elongation were considered part of the vegetative stage,
 while flowering and seed-set were considered part of the reproductive
 stage; all other time points were considered either dormancy
 (pre-emergence sampling) or senescence (all others). To
 investigate the effects of rainfall magnitude and frequency, we induced
 wetting events after various drying intervals using rainfall exclusion
 shelters coupled with irrigation systems and then measured
 N<sub>2</sub> fixation. During mid-June to mid-July 2019, we
 replicated three rainfall frequency treatments in four switchgrass
 subplots for a total of 12 subplots. All treatments received the same
 amount of total rainfall (~ 60 mm) over a 20-day period, which corresponds
 to the site’s average weekly rainfall, but they received it at different
 frequencies. The constant moisture (CM) treatment received 15 mm every
 five days for a total of four wetting events, while the moderate drying
 (MD) treatment received 30 mm every ten days for a total of two wetting
 events and the extended drying (ED) treatment received 60 mm on day 20
 (Figure 1). To capture the response of N<sub>2</sub> fixation
 to a wetting event, we sampled before and after subplots were irrigated,
 usually within eight hours of each other, but long enough after irrigation
 for the water to move into soil (Blazewicz et al. 2014). We also measured
 N<sub>2</sub> fixation in a subset of the plots after a drying
 interval of 5, 10, 15, and 20 days. Finally, we continued sampling the
 plots after the final wetting event (i.e., Day 22, 25, 30) to determine
 the effect of rainfall frequency treatment as the soils dried
 down. <em>Rainout shelters and
 irrigation</em> Twelve of the 16 subplots (three
 in each plot) were covered with a rainfall exclusion shelter, and
 rainwater was collected from gutters attached to each shelter. The shelter
 was constructed with a galvanized steel frame (4.9 x 4.3 x 1.8 m) and a
 roof of clear, corrugated polycarbonate (AmeriLux LEXAN, Wisconsin, USA),
 with a layer of UV protectant that also permits transmittance of 90%
 photosynthetically active radiation (see Figure 1 in (Hess et al. 2020)
 for a similar set-up). Each shelter gutter was connected to an irrigation
 tank for a total of two tanks per subplot. Rainwater was applied using a
 Rule 360 GPH/12 V bilge pump (Xylem Inc., Rye Brook, NY, USA), and the
 total amount was estimated visually to the nearest 20 L using the tank’s
 volumetric markings. We determined when to stop the pumps based on visual
 assessment of the tanks, but we failed to disconnect a bilge pump on one
 instance, accidentally doubling the water added to a single CM subplot on
 Day 5. Nevertheless, flow rates (87 ± 14 L h<sup>−1</sup>)
 were generally consistent across irrigation set-ups. Before each wetting
 event, water samples were collected from each irrigation tank with a 60-mL
 syringe and filtered with a 25-mm glass fiber filter (Pall Type A/E, pore
 size of 1 µm) for later measurement of dissolved inorganic
 nitrogen. <em>Soil sampling</em>
 For each sampling event, we collected soil for
 N<sub>2</sub> fixation and physicochemical measurements. For
 N<sub>2</sub> fixation, we used a hammer corer to collect two
 intact soil cores (3.6 cm in diameter, 10 cm in depth) per subplot within
 20 cm of a switchgrass crown and within 10 cm of each other to minimize
 plant damage and spatial heterogeneity. For physicochemical measurements,
 we collected multiple short soil cores (2 cm in diameter, 10 cm in depth)
 in the vicinity of the cores with a push probe for a total of about 200 g
 of soil. <em>N<sub>2</sub>
 fixation</em> To measure
 N<sub>2</sub>-fixation in intact cores, we conducted a
 three-day <sup>15</sup>N<sub>2</sub> incorporation
 assay by adapting commercial stainless steel liners used for retrieval of
 hammer core samples. We made the cores gas-tight for the assay by topping
 each end of the liner with a rubber stopper and an open Luer-Lok fitting,
 sealing it with a waterproof adhesive (Amazing Goop®, All-Purpose), and
 allowing the adhesive to dry overnight in the laboratory. To begin the
 assay, we either vacuumed the headspace of the soil cores and then added
 artificial headspace (for the first two phenology sampling events) or
 purged the cores with the appropriate artificial headspace (for the rest
 of the phenology and all rainfall sampling events) before filling the core
 headspace to 3 psi using a pressure gauge. We verified that neither
 N<sub>2</sub> fixation nor carbon mineralization rates varied
 due to vacuuming versus purging the cores. All other
 N<sub>2</sub> fixation procedures remained the same across
 sampling events. The paired cores (1 control, 1
 enriched) from each sampling period were used to determine the
 N<sub>2</sub> fixation rate for each subplot. All cores
 received an artificial headspace of 78% N<sub>2</sub>, 12%
 O<sub>2</sub>, and 10% H<sub>2</sub>. Enriched
 cores received half of their N<sub>2</sub> as 99%
 <sup>15</sup>N<sup>15</sup>N (Cambridge Isotope
 Laboratories, Tewksbury, MA, USA) resulting in a
 <sup>15</sup>N atom percent of about 50%. While contamination
 of <sup>15</sup>N<sub>2</sub> can artificially
 inflate N<sub>2</sub> fixation rates (Dabundo et al. 2014),
 the commercial source we chose exhibits very little contamination (Roley
 et al. 2018), which we independently verified for our batch. Because we
 wanted to examine the effects of wetting on N<sub>2</sub>
 fixation, we used H<sub>2</sub> instead of more typical energy
 sources such as a glucose solution (Gupta et al. 2014) or a carbon
 cocktail (Smercina et al. 2019a).  Such carbon sources are typically added
 to soil in aqueous solution, which would introduce a wetting artifact,
 rendering such additions problematic given our focus on understanding the
 effects of rainfall treatments on N<sub>2 </sub>fixation
 rates. H<sub>2</sub> is readily consumed by soil microbes,
 diazotrophs can use H<sub>2</sub> as a substrate and electron
 donor in N<sub>2</sub> fixation (Robson and Postgate 1980),
 and it is added as a gas so that it will not alter soil moisture. We
 ensured that H<sub>2</sub> was not depleted during the assay
 by adding 2 mL of pure H<sub>2</sub> after each day of
 incubation except on the final day of the assay. Our methods testing
 showed that we were able to detect N<sub>2</sub> fixation
 rates in cores receiving an artificial atmosphere with 10%
 H<sub>2</sub>, but not those with 5%
 H<sub>2</sub>. In addition, we observed that saturating cores
 with a 4% glucose solution enhanced N<sub>2</sub> fixation
 rates up to four orders of magnitude, indicating that glucose addition
 results in substantial growth of diazotrophs, dramatically inflating
 estimates of N<sub>2</sub> fixation. We found that
 H<sub>2</sub> is an energy source that raises
 N<sub>2</sub> fixation rates to a detectable level without
 altering soil moisture content or causing substantial growth during the
 period of measurement. To calculate
 N<sub>2</sub> fixation rates, we first determined the
 <sup>15</sup>N concentration of the core’s headspace and that
 of the switchgrass root tissue and surrounding soil. Core headspace was
 sampled on the second day of the assay by removing 5 mL and injecting it
 into a 3-mL exetainer, which was stored upside down in water until
 isotopic analysis. The headspace volume was immediately replaced with the
 appropriate artificial headspace after sampling to maintain positive
 pressure in the core. After three days had passed, the assay was
 terminated by opening the Luer-Lok fittings and immediately sieving the
 soil using a 4-mm mesh. Roots and soil were separated and weighed. Then we
 allowed the roots and soil to air-dry, weighed them again, and ground them
 to a fine powder using a combination of scissors, a mortar and pestle, and
 a coffee grinder. We then packed the finely powdered samples into tin
 capsules for isotopic analysis (δ<sup>15</sup>N, %C, %N).
 Solid samples were analyzed by the GLBRC Stable Isotope Facility at
 Michigan State University on a Vario ISOTOPE cube Elemental Analyzer
 (Elementar, Langenselbold, Germany) coupled to an Isoprime visION Isotope
 Ratio Mass Spectrometer (Elementar), while the gas samples were analyzed
 on a HP 5890 Gas Chromatograph (Agilent, Santa Clara, CA, USA) coupled to
 a isoprime Dual Inlet-Isotope Ratio Mass Spectrometer
 (Elementar).             Rates of
 N<sub>2</sub> fixation were calculated on a root or soil
 basis, in μg N d<sup>−1</sup>, as (<em>AE<sub>i 
 </sub>x <em>TN<sub>i</sub>) /
 (<em>AE<sub>atm </sub>x
 t),</em></em></em>  
 where <em>AE<sub>i</sub></em> is the
 <sup>15</sup>N atom % excess in the roots or soil of the
 enriched core relative to the paired control core,
 <em>TN<sub>i</sub></em> is the total amount of N
 in the roots or soil of the enriched core,
 <em>AE<sub>atm</sub></em> is the
 <sup>15</sup>N atom % excess in the headspace of the enriched
 core relative to the control core, and <em>t</em> is
 incubation time (Warembourg 1993). We then divided each rate by the dry
 mass of the roots or soil in the enriched core, resulting in root or soil
 N<sub>2</sub> fixation rates expressed as μg N
 g<sup>−1</sup> d<sup>−1</sup>. To convert to an
 areal estimate (mg N m<sup>−2</sup>
 d<sup>−1</sup>), we added the root and soil
 N<sub>2</sub> fixation rates for a core (μg N
 d<sup>−1</sup>), divided it by the area of the core’s top
 surface area (i.e., 10.2 cm<sup>2</sup>), and converted units.
 These areal rates are likely an underestimate because they assume an
 active depth of 10 cm (i.e., the depth of our core), but
 N<sub>2</sub> fixation can occur deeper in the soil profile,
 sometimes even at high rates (e.g., Casselman et al., 1981; Kreibich and
 Kern, 2003). For those samples (~23% of roots and 44% of soil) where
 N<sub>2</sub> fixation could not be detected, we assigned root
 and areal rates of 0.0001 μg N g<sup>−1</sup>
 d<sup>−1</sup> and 0.0001 mg N m<sup>−2</sup>
 d<sup>−1</sup>, respectively, which were lower than any rates
 observed (root: 0.00013 μg N g<sup>−1</sup>
 d<sup>−1</sup>; areal: 0.00023 mg N m<sup>−2</sup>
 d<sup>−1</sup>). <em>Soil
 physicochemical properties</em> Following
 collection, the small soil cores from a single subplot were composited,
 sieved (4-mm mesh), and divided for measurement of gravimetric soil
 moisture, C mineralization, and soil inorganic N concentrations, all in
 triplicate. While we quantified gravimetric soil moisture and C
 mineralization for all soil samples, we assessed inorganic N
 concentrations only for the ambient subplots. Ammonium
 (NH<sub>4</sub><sup>+</sup>-N) and nitrate
 (NO<sub>3</sub><sup>−</sup>-N) concentrations were
 evaluated using a 24-hr 1 M KCl extraction of triplicate soil subsamples
 (Robertson et al., 1999). We determined gravimetric soil moisture by
 weighing triplicate subsamples before and after they were completely
 air-dried. Then we measured short-term C mineralization following the
 re-wetting of the air-dried soils as an indicator of soil C availability
 (Robertson et al. 1999; Franzluebbers et al. 2000). To
 measure C mineralization, we placed triplicate soil subsamples (10 g of
 air-dry soil) into separate 235-mL mason jars equipped with gas-sampling
 septa and re-wet them to 50% water-filled pore space (Franzluebbers et al.
 2000). Then we sampled 5 mL of headspace from each jar at five intervals
 (0, 2, 4, 8, and 24 hrs), put the gas sample in a 3-mL exetainer, and
 replaced the headspace with laboratory air. We analyzed the
 CO<sub>2</sub> samples within three days using a LI-820
 CO<sub>2</sub> Gas Analyzer (LI-COR Biosciences, Lincoln, NE,
 USA). We accounted for headspace dilution, performed a linear regression
 of CO<sub>2</sub> concentrations through time, and converted
 the slope to a standardized C mineralization rate using the ideal gas law.
 Because wetter soil samples can lose more CO<sub>2</sub> while
 air-drying, which can affect the interpretation of this assay, we
 corrected the C mineralization rates for soil moisture as described in
 Belanger et al. (2021). Soil C:N ratio was determined
 during isotopic analysis of core samples (see <em>N<sub>2
 </sub>fixation</em>, above); total C and N content of each
 sample was determined via the elemental analyzer and the C:N subsequently
 calculated by dividing the total C content by the total N
 content. <em>Statistical
 analyses</em> To identify the factors that
 influenced N<sub>2</sub> fixation throughout the switchgrass
 growing season, we performed mixed effect model selection after Zuur et
 al. (2009) on N<sub>2</sub> fixation rates in the ambient
 subplots. We individually analyzed root fixation rates and areal fixation
 rates (i.e., soil and roots combined). We did not analyze soil
 N<sub>2</sub> fixation rates separately because soil comprised
 most of the cores’ mass, and the areal N<sub>2</sub> fixation
 rates were highly dependent on soil N<sub>2</sub> fixation
 rates (r<sup>2</sup> &gt; 0.99). We
 log<sub>10</sub>-transformed both areal and root
 N<sub>2</sub> fixation rates to better meet assumptions of
 normality and heteroscedasticity. During model selection, we assessed
 whether a spatial structure, or subplot as a random factor, should be
 added to the model, based on comparisons between models with and without
 those factors. Additionally, we included date, air temperature, the
 previous day’s precipitation, the phenological stage of switchgrass, and
 soil moisture as fixed factors in the model selection for both response
 variables. For the areal rates, we also considered soil chemical factors
 in model selection (i.e., soil moisture, C:N, inorganic N, and C
 mineralization). Results did not vary when we used ammonium and nitrate as
 separate factors in our statistical analyses, so we used total soil
 inorganic N as an indicator of soil N availability. Total soil inorganic N
 was highly correlated with ammonium concentrations (r = 0.99; P &lt;
 0.0001) because ammonium (3.6 ± 1.1 μg N
 g<sup>−</sup><sup>1</sup> dry soil) tended to be
 more than 10x higher than nitrate (0.26 ± 0.17 μg N
 g<sup>−</sup><sup>1</sup> dry soil).
 To determine whether wetting increased N<sub>2</sub>
 fixation, we compared both areal and root N<sub>2</sub>
 fixation rates in paired samples before and after we induced a wetting
 event in the rainfall exclusion shelter subplots. Because the response
 variables violated the assumptions of a paired t-test, we instead
 conducted two Wilcoxon signed-rank tests
 (<em>H<sub>a</sub></em>: difference &gt; 0) on
 N<sub>2</sub> fixation rates before and after wetting events,
 one for areal and one for root N<sub>2</sub> fixation rates.
 Then we calculated the nonparametric effect size for both areal and root
 N<sub>2</sub> fixation using Vargha and Delaney’s <em>Â
 </em>(Peng and Chen 2014). According to (Vargha and Delaney 2000),
 <em>Â</em> values of 0.56, 0.64, and 0.71 correspond to the
 small (0.2), medium (0.5), and large (0.8) values of Cohen’s
 <em>d</em>.   To identify factors that
 influenced the wetting response, we performed mixed effect model selection
 for the response of areal N<sub>2</sub> fixation to wetting.
 Specifically, we used the difference in N<sub>2</sub> fixation
 (i.e., after wetting − before wetting) as the response variable; no
 transformation was necessary. We included the amount of water and
 inorganic N added in an irrigation event and their interaction because
 they were correlated, the change in soil moisture, the number of times
 that a subplot had been wetted, and initial soil characteristics that
 might shape its response to wetting (i.e., soil moisture, C:N, and C
 mineralization) as fixed factors. Finally, we considered both day and
 subplot as potential random factors. We removed two outliers whose wetting
 responses were more than 3x the interquartile range because while their
 presence in the analysis did not change the factors in the best model,
 their inclusion made the model violate assumptions of normality.
 To determine the effect of rainfall frequency on soils as they
 dried down, we conducted mixed effect model selection on the areal
 N<sub>2</sub> fixation rates from all sampling events
 following the final wetting. We log<sub>10</sub>-transformed
 the response variable to meet assumptions of normality and we included
 rainfall frequency treatment, soil moisture, soil C:N, and C
 mineralization as potential fixed factors in model selection. We also
 considered date and subplot as potential random factors.
 For all mixed effect model selection exercises, we quantified the
 fit of the best model. Specifically, we performed a linear regression of
 the observed N<sub>2</sub> fixation rates versus those
 predicted by the best model and calculated an r<sup>2</sup>
 value. If a random factor was included in the best model, we also
 performed a linear regression of the observed N<sub>2</sub>
 fixation rates versus those predicted by the model without any fixed
 factors (i.e., only random factor and variance structure, if applicable)
 and calculated an r<sup>2</sup> value for this model. These
 two analyses allowed us to compare the influence of the fixed versus the
 random factors. We also verified that the best model did not violate
 assumptions of a generalized linear model (GLM) or generalized least
 squares (GLS) regression.
description: # Rainfall events stimulate episodic associative nitrogen fixation in
 switchgrass
 [https://doi.org/10.5061/dryad.s7h44j1h3](https://doi.org/10.5061/dryad.s7h44j1h3) ## Description of the data and file structure Data are from a 2019 study of nitrogen fixation rates in switchgrass (*Panicum virgatum*) at W.K. Kellogg Biological Station in Michigan. Nitrogen fixation rates from intact soil cores were measured across the growing season and during a rainfall frequency experiment.  ### Files and variables #### File: delta.csv **Description:** These data were from the rainfall frequency experiment. Specifically, these data were used to examine the effects of wetting before and after a rainfall event. ##### Variables * Core_ID: This is a unique core ID for identification purposes. * Plot_ID: This is the number of the plot at Kellogg Biological Station Interactions Experiment combined with the rainfall frequency experimental treatment. * Plot_No: This is the number of the plot at Kellogg Biological Station. [https://lter.kbs.msu.edu/research/long-term-experiments/lter-switchgrass-nitrogen-fixation-experiment/](https://lter.kbs.msu.edu/research/long-term-experiments/lter-switchgrass-nitrogen-fixation-experiment/) * Rain_Freq: This is the rainfall frequency treatment: C = constant moisture; M = moderate drying; E = extended drying. See figure 1 of the manuscript for more information. * Day: This is the day of the rainfall experiment. See figure 1 of the manuscript for more information. * Wet_No: This is the number of wetting events that a plot had received at this point in the experiment. * A_Soil_CN: This is the carbon to nitrogen ratio of the soil after a wetting event. * A_Root_Nfix: This is the N fixation rate of the roots after a wetting event. Units are in μg N/g dry root/d.  * A_Core_Nfix: This is the N fixation rate of the entire core (soil + roots) after a wetting event. Units are in mg N/m2/d. * A_Soil_Moisture: This is the soil moisture after a wetting event. Units are g of water/g of dry soil. * A_C_Cmin: This is the carbon mineralization rate after a wetting event. Units are μg CO2-C/g dry soil/day. Because wetter soil samples can lose more carbon dioxide while air-drying, which can affect the interpretation of this assay, we corrected the carbon mineralization rates for soil moisture as described in Belanger et al. (2021). * B_Soil_CN: This is the carbon to nitrogen ratio of the soil before a wetting event. * B_Root_Nfix: This is the N fixation rate of the roots before a wetting event. Units are in μg N/g dry root/d.  * B_Core_Nfix: This is the N fixation rate of the entire core (soil + roots) before a wetting event. Units are in mg N/m2/d. * B_Soil_Moisture: This is the soil moisture before a wetting event. Units are g of water/g of dry soil. * B_C_Cmin: This is the carbon mineralization rate before a wetting event. Units are μg CO2-C/g dry soil/day. Because wetter soil samples can lose more carbon dioxide while air-drying, which can affect the interpretation of this assay, we corrected the carbon mineralization rates for soil moisture as described in Belanger et al. (2021). * D_Root_Nfix: This is the delta N fixation rate of the roots (after minus before a wetting event) or the change due to wetting. Units are in μg N/g dry root/d.  * D_Core_Nfix: This is the delta N fixation rate of the cores (after minus before a wetting event) or the change due to wetting. Units are in mg N/m2/d. * D_Soil_Moisture: This is the delta soil moisture (after minus before a wetting event) or the change due to wetting. Units are g of water/g of dry soil. * D_C_Cmin: This is the delta carbon mineralization rate of the cores (after minus before a wetting event) or the change due to wetting. Units are μg CO2-C/g dry soil/day. Because wetter soil samples can lose more carbon dioxide while air-drying, which can affect the interpretation of this assay, we corrected the carbon mineralization rates for soil moisture as described in Belanger et al. (2021). * Irri_N: This is the total amount of inorganic nitrogen added during the wetting event. Units are kg N/ha. * Irri_H2O: This is the volume of water (L) added at each wetting event.  #### File: delta\_matrix.csv **Description:** This is data from delta.csv pared down into a matrix with the numeric variables we considered analyzing. This matrix was used to check for collinearity among numerical predictor variables. ##### Variables * Day: This is the day of the rainfall experiment. See figure 1 of the manuscript for more information. * Wet_No: This is the number of wetting events that a plot had received at this point in the experiment. * A_Soil_CN: This is the carbon to nitrogen ratio of the soil after a wetting event. * A_Soil_Moisture: This is the soil moisture after a wetting event. Units are g of water/g of dry soil. * A_C_Cmin: This is the carbon mineralization rate after a wetting event. Units are μg CO2-C/g dry soil/day. Because wetter soil samples can lose more carbon dioxide while air-drying, which can affect the interpretation of this assay, we corrected the carbon mineralization rates for soil moisture as described in Belanger et al. (2021). * B_Soil_CN: This is the carbon to nitrogen ratio of the soil before a wetting event. * B_Soil_Moisture: This is the soil moisture before a wetting event. Units are g of water/g of dry soil. * B_C_Cmin: This is the carbon mineralization rate before a wetting event. Units are μg CO2-C/g dry soil/day. Because wetter soil samples can lose more carbon dioxide while air-drying, which can affect the interpretation of this assay, we corrected the carbon mineralization rates for soil moisture as described in Belanger et al. (2021). * D_Soil_Moisture: This is the delta soil moisture (after minus before a wetting event) or the change due to wetting. Units are g of water/g of dry soil. * D_C_Cmin: This is the delta carbon mineralization rate of the cores (after minus before a wetting event) or the change due to wetting. Units are μg CO2-C/g dry soil/day. Because wetter soil samples can lose more carbon dioxide while air-drying, which can affect the interpretation of this assay, we corrected the carbon mineralization rates for soil moisture as described in Belanger et al. (2021). * Irri_N: This is the total amount of inorganic nitrogen added during the wetting event. Units are kg N/ha. * Irri_H2O: This is the volume of water (L) added at each wetting event.  * D_Root_Nfix: This is the delta N fixation rate of the roots (after minus before a wetting event) or the change due to wetting. Units are in μg N/g dry root/d.  * D_Core_Nfix: This is the delta N fixation rate of the cores (after minus before a wetting event) or the change due to wetting. Units are in mg N/m2/d. #### File: post.csv **Description:** These data were from the rainfall frequency experiment. Specifically, these data were used to look at what happened to nitrogen fixation as the soils dried down following the final wetting event on Day 20. ##### Variables * Core_ID: This is a unique core ID for identification purposes. * Plot_ID: This is the number of the plot at Kellogg Biological Station Interactions Experiment combined with the rainfall frequency experimental treatment. * Plot_No: This is the number of the plot at Kellogg Biological Station. [https://lter.kbs.msu.edu/research/long-term-experiments/lter-switchgrass-nitrogen-fixation-experiment/](https://lter.kbs.msu.edu/research/long-term-experiments/lter-switchgrass-nitrogen-fixation-experiment/) * Rain_Freq: This is the rainfall frequency treatment: C = constant moisture; M = moderate drying; E = extended drying. See figure 1 of the manuscript for more information. * Day: This is the day of the rainfall experiment. See figure 1 of the manuscript for more information. * Soil_C_N: This is the carbon to nitrogen ratio of the soil. * Root_Nfix: This is the N fixation rate of the roots. Units are in μg N/g dry root/d.  * Core_Nfix: This is the N fixation rate of the cores. Units are in mg N/m2/d. * Soil_Moisture: This is the soil moisture. Units are g of water/g of dry soil. * C_Cmin: This is the carbon mineralization rate of the cores. Units are μg CO2-C/g dry soil/day. Because wetter soil samples can lose more carbon dioxide while air-drying, which can affect the interpretation of this assay, we corrected the carbon mineralization rates for soil moisture as described in Belanger et al. (2021). * Irri_N: This is the total amount of inorganic nitrogen added during the wetting event. Units are kg N/ha. * Irri_H2O: This is the volume of water (L) added at each wetting event.  #### File: post\_matrix.csv **Description:** This is data from post.csv pared down into a matrix with the numeric variables we considered analyzing. This matrix was used to check for collinearity among numerical predictor variables. ##### Variables * Day: This is the day of the rainfall experiment. See figure 1 of the manuscript for more information. * Soil_C_N: This is the carbon to nitrogen ratio of the soil. * Soil_Moisture: This is the soil moisture. Units are g of water/g of dry soil. * C_Cmin: This is the carbon mineralization rate of the cores. Units are μg CO2-C/g dry soil/day. Because wetter soil samples can lose more carbon dioxide while air-drying, which can affect the interpretation of this assay, we corrected the carbon mineralization rates for soil moisture as described in Belanger et al. (2021). * Irri_N: This is the total amount of inorganic nitrogen added during the wetting event. Units are kg N/ha. * Irri_H2O: This is the volume of water (L) added at each wetting event.  * log_Root_Nfix: This is the log10-transformed N fixation rate of the roots. Units are in μg N/g dry root/d.  * log_Core_Nfix: This is the log10-transformed N fixation rate of the cores. Units are in mg N/m2/d. #### File: root.csv **Description:** These data were from unmanipulated, ambient plots throughout the growing season. Specifically, these data were used to examine whether seasonality affected nitrogen fixation in the roots. ##### Variables * Core_ID: This is a unique core ID for identification purposes. * Plot: This is the number of the plot at Kellogg Biological Station. [https://lter.kbs.msu.edu/research/long-term-experiments/lter-switchgrass-nitrogen-fixation-experiment/](https://lter.kbs.msu.edu/research/long-term-experiments/lter-switchgrass-nitrogen-fixation-experiment/) * Period: This is the number of sampling events that had occurred at this plot as of this date. * Date: This is the date of the sampling event. * Stage: This is the morphological stage of switchgrass on the date. For analysis, tiller and stem elongation were considered part of the vegetative stage, while flowering and seed-set were considered part of the reproductive stage; all other time points were considered senescence. * DOY: This is the Julian date of the year. * Root_C_N: This is the carbon to nitrogen ratio of the root tissue. * Soil_C_N: This is the carbon to nitrogen ratio of the soil. * Root_Nfix: This is the N fixation rate of the roots. Units are in μg N/g dry root/d.  * Core_Nfix: This is the N fixation rate of the cores. Units are in mg N/m2/d. * Soil_Moisture: This is the soil moisture. Units are g of water/g of dry soil. * C_Cmin: This is the carbon mineralization rate of the cores. Units are μg CO2-C/g dry soil/day. Because wetter soil samples can lose more carbon dioxide while air-drying, which can affect the interpretation of this assay, we corrected the carbon mineralization rates for soil moisture as described in Belanger et al. (2021). * Soil_IN: This is the amount of inorganic nitrogen (ammonium and nitrate+nitrite) in the soil. Units are μg N g−1 dry soil. * temp: This is the air temperature in degrees Celsius.  * prev_precip: This is the previous day's rainfall in mm. #### File: phen\_matrix.csv **Description:** This is data from phen.csv pared down into a matrix with the numeric variables we considered analyzing. This matrix was used to check for collinearity among numerical predictor variables. ##### Variables * DOY: This is the Julian date of the year. * Root_C_N: This is the carbon to nitrogen ratio of the root tissue. * Soil_C_N: This is the carbon to nitrogen ratio of the soil. * Soil_Moisture: This is the soil moisture. Units are g of water/g of dry soil. * C_Cmin: This is the carbon mineralization rate of the cores. Units are μg CO2-C/g dry soil/day. Because wetter soil samples can lose more carbon dioxide while air-drying, which can affect the interpretation of this assay, we corrected the carbon mineralization rates for soil moisture as described in Belanger et al. (2021). * Soil_IN: This is the amount of inorganic nitrogen (ammonium and nitrate+nitrite) in the soil. Units are μg N g−1 dry soil. * temp: This is the air temperature in degrees Celsius.  * prev_precip: This is the previous day's rainfall in mm. * log_Root_Nfix: This is the log10-transformed N fixation rate of the roots. Units are in μg N/g dry root/d.  * log_Core_Nfix: This is the log10-transformed N fixation rate of the cores. Units are in mg N/m2/d. * Ammonium: This is the amount of ammonium in the soil. Units are μg N g−1 dry soil. * Nitrate: This is the amount of nitrate+nitrite  in the soil. Units are μg N g−1 dry soil. * A_N: This is the ratio of ammonium to nitrate+nitrite in the soil. #### File: phen.csv **Description:** These data were from unmanipulated, ambient plots throughout the growing season. Specifically, these data were used to examine whether seasonality affected nitrogen fixation in the soil cores. ##### Variables * Core_ID: This is a unique core ID for identification purposes. * Plot: This is the number of the plot at Kellogg Biological Station. [https://lter.kbs.msu.edu/research/long-term-experiments/lter-switchgrass-nitrogen-fixation-experiment/](https://lter.kbs.msu.edu/research/long-term-experiments/lter-switchgrass-nitrogen-fixation-experiment/) * Period: This is the number of sampling events that had occurred at this plot as of this date. * Date: This is the date of the sampling event. * Stage: This is the morphological stage of switchgrass on the date. For analysis, tiller and stem elongation were considered part of the vegetative stage, while flowering and seed-set were considered part of the reproductive stage; all other time points were considered senescence. * DOY: This is the Julian date of the year. * Root_C_N: This is the carbon to nitrogen ratio of the root tissue. * Soil_C_N: This is the carbon to nitrogen ratio of the soil. * Root_Nfix: This is the N fixation rate of the roots. Units are in μg N/g dry root/d.  * Core_Nfix: This is the N fixation rate of the cores. Units are in mg N/m2/d. * Soil_Moisture: This is the soil moisture. Units are g of water/g of dry soil. * C_Cmin: This is the carbon mineralization rate of the cores. Units are μg CO2-C/g dry soil/day. Because wetter soil samples can lose more carbon dioxide while air-drying, which can affect the interpretation of this assay, we corrected the carbon mineralization rates for soil moisture as described in Belanger et al. (2021). * Soil_IN: This is the amount of inorganic nitrogen (ammonium and nitrate+nitrite) in the soil. Units are μg N g−1 dry soil. * temp: This is the air temperature in degrees Celsius.  * prev_precip: This is the previous day's rainfall in mm. * Ammonium: This is the amount of ammonium in the soil. Units are μg N g−1 dry soil. * Nitrate: This is the amount of nitrate+nitrite  in the soil. Units are μg N g−1 dry soil. * A_N: This is the ratio of ammonium to nitrate+nitrite in the soil. ## Code/software R software is needed to open the code files. The rain_manuscript file is the script used to analyze the data from the rainfall frequency experiment. The phen_manuscript file is the script used to analyze the data from the unmanipulated, ambient subplots throughout the growing season. ## Access information Other publicly accessible locations of the data: * NA Data was derived from the following sources: * NA