Location of Sampling: Soil underneath ten Cornus drummondii islands in the lowlands of watershed 020c

Frequency of Sampling: One sampling event

Variables measured: C concentration, N concentration, inorganic N concentration, P concentration, organic matter content, microbial biomass C, microbial biomass N,  potential β-glucosidase activity, potential phosphatase activity, potential NAG-ase activity, and potential LAP-ase activity, shrub size, potential C mineralization rate, δ13C value of respired CO2.

Field methods: Ten distinct dogwood islands were randomly selected within watershed 020c. Five cm diameter x 15 cm deep soil cores were taken at four locations within each dogwood island along a linear transect: the center, the midpoint, the edge, and the ecotone with grassland. The center was defined as the intersection of perpendicular transects running the length and width of the dogwood island. The midpoint was defined as the halfway point between the center and the furthest edge. The edge was defined as the outer perimeter of the dogwood island canopy, and the ecotone was defined as the halfway point between the edge of the dogwood island of interest and the nearest neighboring dogwood island. Shrub island size was calculated by ellipse area equation using the lengths of the two perpendicular transects. All soil was kept on ice until it could be placed in short-term storage at 4 °C.

Laboratory methods for SMP011 dataset (soil chemistry and soil microbial processe): Laboratory methods: On the day after sampling, soils were passed through a 4 mm sieve. A small subsample of soil from each location was dried at 60 °C for 48 hours to determine gravimetric water content. Soil used for nutrient analyses was stored at 4 °C. Soil used for extracellular enzymatic activity analyses was stored at -20 °C.

Total C and N content of each soil sample was determined via dry combustion using a LECO TruSpec CN combustion analyzer. Total organic matter was determined by a slightly modified loss on ignition protocol outlined in Combs and Nathan (1998). Briefly, 1 g of soil was dried at 150 ⁰C for two hours and then combusted at 400 ⁰C for three hours. Available P was determined by the Mehlich-3 procedure (Mehlich 1984). The above analyses were performed at the Soil Testing Lab at Kansas State University.
Fifty ml of 2N KCl was added to 12 g of field moist soil and placed on an orbital shaker table at 200 rpm for 60 minutes to extract soil NH4+ and NO3- (Bremmer and Keeney 1966). Extracts were passed through a 0.45 µm polycarbonate filter and stored at -20°C until they were analyzed colorimetrically for NO3- and NH4+ in a flow analyzer at the Kansas State University Soil Testing Lab.

Microbial biomass C was determined using the chloroform fumigation-extraction method (Jenkinson and Powlson 1976). A subsample of each soil sample was placed into a chamber, fumigated by boiling chloroform under a vacuum, and kept in the fumigation chamber under a vacuum for 48 hours. A vacuum pump was used to remove all chloroform from the chamber after fumigation was completed. Fumigated and unfumigated samples were extracted by combining 15 g of field moist soil and 75 ml 0.5 M K2SO4 and placing on an orbital shaker table at 200 rpm for 60 minutes, then passing through a 0.45 µm polycarbonate filter. Extracts were stored at -20°C until they were analyzed for total organic C with a Shimadzu TOC-L. Microbial biomass C was defined as the difference in dissolved organic C between fumigated and unfumigated subsamples.
Total nitrogen content in microbial biomass was determined by taking a subsample of the K2SO4 extracts and subjecting them to a persulfate digest (D’Elia et al. 1977; Cabrera and Beare 1993). This oxidizes all forms of nitrogen to NO3-. After being reduced to NO2- by a cadmium coil, N concentrations in the extracts were determined colorimetrically using an Alpkem OI Analytical Flow Solution IV. Microbial biomass N was defined as the difference in N between fumigated and unfumigated subsamples.

We tested the potential activity of four extracellular enzymes (Sinsabaugh et al. 1999; German et al. 2011): β-glucosidase (BG; a C-acquiring enzyme), N-acetyl-glucosaminidase (NAG; a N-acquiring enzyme), phosphatase (PHOS; a P-acquiring enzyme), and leucine-aminopeptidase (LAP; a N-acquiring enzyme). We used 200 µM solutions of 4-methylumbelliferone-b-D-glucoside, 4-methylumbelliferone-N-acetyl-b-glucosaminide, 4-methylumbelliferone-phosphate, and L-leucine 7-amido-4-methylcoumarin as substrates, respectively. For each soil sample in each assay, we created a slurry of 1 g of soil in 100 ml of 50 mM acetate buffer (pH 5). In 96-well plates, we pipetted 200 µl of the soil slurry with 50 µl of the substrate solution. There were six analytical replicates for each sample in each assay as well as a blank, a negative control, a reference standard, three quench standards, and six soil blanks. For BG, NAG, and PHOS assays, we incubated the plates in the dark at room temperature for 2 hours. Assays for LAP activity were incubated for 16 hours. Once the incubations were complete, we added 10 µl of 0.5 N NaOH solution to raise the pH and stop the assays. Finally, we used a FilterMax F5 plate reader to collect fluorescence data.

References:
Bremmer JM, Keeney DR (1966) Determination and isotope-ratio analysis of different forms of nitrogen in soils: 3. Exchangeable ammonium, nitrate, and nitrite by extraction-distillation methods. Soil Sci Soc Am J 30:577–582. https://doi.org/10.2136/sssaj1966.03615995003000050015x
Cabrera ML, Beare MH (1993) Alkaline persulfate oxidation for determining total nitrogen in microbial biomass extracts. Soil Sci Soc Am J 57:1007–1012. https://doi.org/10.2136/sssaj1993.03615995005700040021x
Combs SM, Nathan M V. (1998) Soil organic matter. In: Recommended chemical soil test procedures for the north central region. Missouri Ag. Exp. Stn. SB 1001., Colombia, MO, pp 53–58
D’Elia CF, Steudler PA, Corwin N (1977) Determination of total nitrogen in aqueous samples using persulfate digestion1. Limnol Oceanogr 22:760–764. https://doi.org/10.4319/lo.1977.22.4.0760
German DP, Weintraub MN, Grandy AS, et al (2011) Optimization of hydrolytic and oxidative enzyme methods for ecosystem studies. Soil Biol Biochem 43:1387–1397. https://doi.org/10.1016/j.soilbio.2011.03.017

Laboratory methods for SMP012 dataset (potential carbon mineralization): On the day after sampling, soils were passed through a 4 mm sieve. A small subsample of soil from each location was dried at 60 °C for 48 hours to determine gravimetric water content.
Approximately 300 g of soil from each sample was placed into an 8 cm wide x 15 cm deep mason jar (Day 0). Total CO2 respired and the δ13C-CO2 value was measured at 1, 3, 5, 7, 10, 34, and 77 days after the start of the incubations. Starting at Day 0, soils were wetted to 60% water-filled pore space. Throughout the duration of the incubation, each jar was regularly weighed and rewetted to maintain constant soil moisture. Before each measurement, the time at which the lids were sealed was recorded. Total CO2 concentration and the δ13C-CO2 value was determined by taking a gas sample of the headspace of each jar through a rubber septum. Two blanks were also measured on each day to account for background CO2 of the room. The CO2 concentration and stable isotopic value of the gas sample was analyzed with a Picarro Cavity Ringdown Spectrometer (model G2101-i, Picarro Inc., Santa Clara, CA). The isotopic ratio of samples was calculated using delta notation as:
 
δ = [(Rsample/Rstandard)-1]*1000

where R is the ratio of the heavy to light isotope for the sample and standard, respectively. Delta values were calculated relative to the international standard for carbon, VDBP. For measurements of CO2 concentration, the spectrometer had a precision of ± 200 ppb for 12C-CO2 and ± 10 ppb 13C-CO2. The precision for δ13C values was ± 0.3‰.