Experimental Design: The study was designed to determine the effects of decreased soil moisture availability associated with short-term (seasonal) drought on soil nutrient dynamics and trace gas fluxes. Our goals were to reduce, but not eliminate, water input to the soil, which would be an unrealistic scenario for this region. To do this, we used small shelters (1.54 m2 plots; 1.24 m × 1.24 m each) located between tree stems; plots were not trenched to allow lateral water flow into the plots. This also minimized soil disturbance and allowed root activity, which plays a key role in soil C dynamics, CO production and can influence N2O and CH4 fluxes Brienen et al., 2010; Keller et al., 2000]. Roots of mature trees were able to access water outside of the exclusions, potentially decreasing the effects of soil moisture loss on root respiration. We established ten plots in each of the three topographic zones (10 plots × 3 sites = 30 plots total). The forest sites were not located along a catena, where hydrology would potentially cause treatment interactions. Within each zone, five plots were designated as controls and five as treatment plots. Plots were established a minimum of 4 m apart and were randomly interspersed to avoid treatment interactions. To minimize the effect of repeated soil sampling, we divided each plot into four equal square quadrants. We then randomly selected one quadrant within each plot to be reserved for destructive sampling (e.g., soil nutrients, gravimetric soil moisture, pH). To evaluate the community composition of the different sites, we measured the diameter at breast height (DBH) and identified all tree species (DBH > 5 cm) within a 5 m radius of all 30 plots (See Table 1).
We excluded throughfall using clear, corrugated plastic panels (1.54 m2) that were 0.5 m above the ground. Each panel drained into a polyvinyl chlorate (PVC) gutter that was used to transfer water away from the treatment plots. The panels were flipped every 3–7 days to transfer litter back onto the forest floor beneath. The shelters were installed on June 4, 2008 and removed August 26, 2008 (12 weeks total). We chose this period as it represented the length of a typical dry season in the adjacent, moist forest life zone [Holdridge, 1967].
Throughfall. We sampled throughfall to determine the amount of water and nutrients excluded from the treatment plots. While most rainfall exclusion experiments focus only on water reduction, decreased rainfall can also potentially reduce nutrient inputs by excluding the nutrients dissolved in throughfall. We measured throughfall adjacent to each of the 30 plots using identical 24.6 mm 2 funnels attached to sterile 1-gallon plastic containers [Heartsill-Scalley et al., 2007].Throughfall was measured weekly for each plot. Sub-samples from a composite of five throughfall collectors in each topographic zone were analyzed every two weeks. The composite samples were filtered using methods described in Heartsill-Scalley et al. [2007] and frozen at 21C until analysis. Solutions were digested in persulfate and analyzed colorometrically on a spectrophotometer for N and P (Milton Roy, Ivyland, PA, USA) according to McDowell et al. [1990].
Soil Sampling and Analyses. We used time domain reflectometery (TDR, Campbell Scientific Model CS616) to estimate volumetric soil moisture in each of 6 plots per forest site (3 control, 3 treatment; 0–30 cm depth). We additionally installed one soil temperature probe (10 cm depth; Campbell Scientific, Model 108L) in one control and one exclusion plot in each topographic zone. Volumetric soil moisture and temperature were measured hourly starting one month prior to throughfall exclusion and ending three months after the shelters were removed (May through November 2008).Soils (0–10 cm depth) were collected once every two weeks concomitantly with trace gas sampling (see below) from all plots using 2.5 cm diameter corer. The soil was immediately processed for gravimetric soil moisture, inorganic N [Hart et al., 1994], sodium bicarbonate extractable P (a widely used index of exchangeable P, and thus generally considered biologically available), exchangeable Fe,and soil pH. Gravimetric soil moisture was determined on 5 g of field-wet soil dried in a 105C oven for 24 h. Soil pH was determined in a slurry of 2:1 potassium chloride (KCl) and deionized (DI) water and measured with a pH meter. We used a KCl extraction to estimate inorganic nitrogen (N) [Hart et al., 1994;Yang et al., 2012]. Samples were analyzed on a Lachat QuickChem FIA + 8000 series(Lachat Instruments, Loveland, CO, USA) for ammonium (NH4+) and nitrate plus nitrite (NO3 and NO2). Approximately 10 g field-wet soil samples were processed and analyzed the day of collection at the International Institute of Tropical Forestry (IITF). Exchangeable P and Fe were easured using an Olsen-EDTA (NH4-EDTA-NaHCO3) extraction [Anderson and Ingram, 1993] followed by analysis on an ICP-Spectro Ciros CCD (Spectro Analytical Instruments, Kleve, Germany). We measured Fe because previous research at this site identified significant relationships among redox, Fe and P availability [Chacon et al., 2006; Liptzin and Silver , 2009].
Trace Gas Measurements. We measured CO2fluxes on a weekly basis in all 30 plots using the Li-Cor LI-6400 Soil Respiration System (Li-Cor Biosciences, Lincoln, NE, USA). We consistently sampled during the morning hours to control for time of day. On days when there was rainfall, we waited 30 min before sampling CO2 emissions once the rain stopped. On days when rainfall persisted for more than 20 min, we sampled CO2 the following day. We randomly selected one of thethree remaining quadrants for initial CO2 sampling (exclud- ing the quadrant designated for destructive soil sampling). After the initial sampling, we followed a clockwise rotation for each subsequent collection to minimize any sampling bias within the shelter. We began CO2 measurements one month prior to throughfall exclusion to quantify background fluxes and continued measurements three months after the shelters were removed (April 29 through November 18) to determine if throughfall exclusion had lasting effects. Soil collars for the LI-6400 were installed a minimum of 30 min prior to CO2measurements. The collars were removed following sampling to avoid artifacts from permanent chamber bases[Varner et al., 2003]. The soil-atmosphere exchange of N2O and CH4 was estimated in each plot using a standard and well-tested static flux chamber method [Davidson, 1993; Keller et al., 1993; Livingston et al., 2005;Silver et al., 2005], rotating quadratsas above. Chambers consisted of a polyvinyl chloride (PVC) ring (25.4 cm diameter 20 cm height) and a vented PVC cover. The PVC rings were pushed into the soil to a depth of 2–3 cm and a minimum of 30 min prior to sampling. We re-deployed the chambers during each field campaign to avoid artifacts from permanent chamber bases [Varner et al.,2003]. We controlled for time of day and the timing of rainfall using the same protocol as for soil CO2 emissions (discussed above). N2O and CH4 samples were collected from all 30 plots two weeks prior to shelter installation. Subsequent gas samples were collected every two weeks. We this collection regime for the three-month duration of the drought treatment as well as for three months post-treatment for a total of 12 collection dates. For each collection date five 30 mL air samples per plot were collected with an air-tight syringe over a 40 min period (t = 0, 5, 15, 25, 40 min) and were injected into pre-evacuated 20 mL glass vials fitted with Geo-Microbial septa (GMT, Ochelata, OK, USA). Three replicates for each standard gas (CH4, N2O and CO2) were also injected into evacuated vials on each collection date to test for effects of storage and shipping on sample quality. Vials were shipped to University of California-Berkeley where they were analyzed within six months of sample collection by gas chromatography (GC) on a Shimadzu GC-14A (Shimadzu Scientific Inc., Columbia, MD, USA), equipped with a Porpak-Q column, using a flame ionization detector (FID) for CH4 detection, and an electron capture detector (ECD) for N2O detection. Methane and N2O fluxes were calculated from the concentration change overtime, and were determined using an exponential curve-fitting procedure (iterative model) described by Matthias et al. [1978]). Fluxes were considered to be zero when the relationship between time and concentration was not significant at p = 0.5.
Statistical Analyses. We analyzed soil moisture, temperature, trace gas fluxes (CO2,CH4 and N2O), soil pH, and soil nutrient concentrations for a response to soil drying using repeated measures analysis of variance (ANOVA; Proc Mixed Repeated in SAS; SAS for Windows V8.0, 2002, SAS Institute [Littell et al., 1998]). We used this analysis to and soil nutrient concentrations for a response to soil drying using repeated measures analysis of variance (ANOVA; Proc Mixed Repeated in SAS; SAS for Windows V8.0, 2002, SAS Institute [Littell et al., 1998]). We used this analysis to determine whether there were significant differences among the three topographic positions and if there was a broad treatment effect across all sites. We used a post-hoc test (Tukey-Duncan) to determine where significant differences occurred. A repeated measures ANOVA blocked by topographic zone (ridge, slope, valley) was used to determine whether there was a significant treatment effect within each of the study sites. If the homogeneity of variance assumption was not met, we log transformed the data, which successfully corrected this problem in all instances. We used regression analyses to determine relationships between mean trace gas fluxes (averaged by plot over exclusion period, n = 30) and mean soil characteristics (e.g., gravimetric soil moisture, temperature, pH, as well as soil N and P; averaged by plot over exclusion period, n = 30). All regressions were performed using SigmaPlot 10 (SigmaPlot for Windows,v. 7.101, 2001, SPSS Inc.).[16] We calculated the mean treatment effect for trace gas fluxes as the percent difference between the treatment and control throughout the entire study period (exclusion and non-exclusion). The total trace gas fluxes were calculated by interpolating data from each plot over the study period and then averaging by treatment and topographic zone. We converted the total CH4 and N2O emissions to CO2 equivalents by multiplying total CH4 and N2O emissions by their respective 100-year warming potentials (25 and 298, respectively [Forster et al., 2007]). Statistical significance was determined as P < 0.05 unless otherwise noted. Valuesreported in the text are means +/=1 standard error.
