inorganic nutrients

Experimental design: Randomized complete block design was established at 2 different study sites, girdled piñon-juniper (PJ) woodland and non-girdled (control) PJ woodland.  In late May, 2011, we set-up each study site to contain six complete blocks (plots), each with multiple tree-to-tree gradients.  At the girdled PJ site, each plot included five different tree-to-tree gradients: Live pine to live pine, live pine to dead pine, live pine to live juniper, dead pine to live juniper, and live juniper to live juniper.  At the control PJ site we also established 6 blocks (plots); however, at this site there were only three gradients: Live pine to live juniper, dead pine to live juniper, and live juniper to live juniper.

Setting up plots:  Plots and gradients were established by marking sampling locations with orange flagging tape and pin-flags by Daniel Warnock and Kimberly Elsenbroek on May 19 and 23, 2011. 

Sample collection, allocation and storage: Soil samples were collected monthly from the girdled PJ woodland to establish two pre-monsoonal (dry) season time points, with samples collected on June 6, 2011 and June 15, 2011 considered as being from single time point.  Soil samples collected on July 20, 2011 represented our second dry season time point.  Soil samples for our two post-monsoon moisture time points were collected on August 15, 2011 and September 28, 2011.  As with the girdled site, soils sample from the control PJ woodland site  were collected both before and after the onset of the monsoon season.  However, unlike the girdled PJ woodland site, we only have one pre-monsoon time point June 29, 2011 and one post monsoon time point, September 15, 2011. 

All soil samples were collected by combining three 0-10cm sub-samples into the same zipper-locking plastic storage bag.  Samples were collected from three different locations within each tree-to tree gradient.  Two of the three samples were collected from locations within 30cm of the trunk of each of the two focal trees within a gradient.  The other sample for each gradient was collected from a point at the center of a zone formed by the edges of the canopies from the two competing focal trees.  All samples were then transported to the lab for refrigeration.  

Within 24-72 hours of sample collection, 5mL sub-samples were taken from each bulked soil sample and placed into individual Corning 15mL screw-cap centrifuge tubes.  Each tube was then filled to the 10mL mark with an 0.8% KOH in Methanol solution.  These tubes were placed in the fridge for storage until analyzed for ergosterol content. After preparation of the samples for ergosterol analyses, 1g samples were placed into 125mL round Nalgene bottles for analyses of fungal exoenzyme actitity (EEA) rates from each sample.  All enzyme activity assays were performed within 1 to 5 days after collection. Further, for all but the final post-monsoonal time points, assays were performed within 2 to 3 days of sample collection. 

After all of the fresh, refrigerated samples were alloquated for ergosterol and EEA analyses we placed the remaining quantities of soil for each sample into labeled paper bags for air-drying on a lab bench.  After 1-2 weeks, 30g of each sample was placed into a labeled plastic bag for shipping to Ithaca, New York, USA for analyses of soil-physicochemical properties.  While taking the 30g sub-samples, a separate 5g sub-sample from the air-dried sample was placed into a labeled, no. 1 coin-envelope for storage until analysis of soil hyphal abundance.   After all sub-sampling was completed any remaining soil was kept in its sample bag and stored in the lab.

Hydrolytic exoenzyme activity (EEA) assays: All hydrolytic EEA assays were performed as follows: Each 125mL sample bottle was partially filled with 50mM sodium bicarbonate buffer solution and homogenized using a Kinematica Polytron CH 6010 (Lucerne, Switzerland).  Upon homogenization, sample bottles were filled to 125mL with buffer solution.  Sample bottles were then set aside until placement in black, 96-well, micro-plates.  At the time of placement, each sample suspension was poured into a glass crystalizing dish where it was stirred at high speed into the appropriate columns within each micro-plate.  These columns included a quench control (200 uL sample suspension + 50uL MUB or methylcoumrin substrate control), a sample control (200uL sample suspension + 50uL 50mM bicarbonate buffer) and an assay column (200uL suspension + 50ul 200mM substrate).  Samples were pipetted into four sets of plates with each set analyzing the activity rates of a single hydrolytic enzyme.  These enzymes included alanine amino peptidase, alkaline phosphatase, β-d-glucosidase, and N-acetyl-β-d-glucosiminidase.  Further, all three samples from a single gradient within a single plot were added to the same plate (e.g., all samples from the live-pine-to-live-pine gradient from plot one were pipetted into a single plate for analyzing the activity of the enzyme alkaline-phospotase.

Ultimately our plate layout was completed as follows usingt two other columns for substrate controls:  In column one, we added 200uL buffer and 50uL of a substrate standard, which accounts for the fluorescence emitted by either the MUB, or the methylcoumarin group that is a component of the substrate solution added to the assay wells.  In column six of each plate was a substrate control, which is a solution of 200uL buffer and 50uL of one the four different substrates used in our hydrolytic EEA assays.   Columns 3-5 were our quench controls, which accounts for the quantity of fluorescence emitted by the MUB or methylcoumarin molecule absorbed by the particles in the soil suspension itself.  Columns 7-9 were the sample controls and  account for the amount of fluorescence emitted by the soil suspension + buffer solution added to each well.  Finally, columns 10-12 were our assay wells.  From these wells we could determine enzyme activity by measuring the fluorescence emitted by the MUB or methylcoumarin molecules cleaved off of the substrates initially added to each well.  The substrates included in these assays included: 7-amino-4-methylcoumarin (Sigma-Aldrich), 4-MUB-phosphate (Sigma-Aldrich), 4-MUB-β-d-glucoside (Sigma-Aldrich), and 4-MUB-N-acetyl-β-d-glucosiminide (Sigma-Aldrich). 

Because the intrinsic EEA rates varied across our targeted exoenzymes, assay plates were scanned for flourscence in sets of two.  Alanine aminopeptidase plates and alkaline phosphatase plates were scanned twice, first at 30-40 minutes after substrate addion and again at 50-80 minutes after substrate addition.  β-d-glucosidase, and N-acetyl-β-d-glucosiminidase plates were all scanned at 3-4 hours after substrate addition.  The timing of the second enzyme activity time point depended on expected soil moisture conditions.  Here, the post monsoon soils were allowed to incubate for a total of 5-6 hours prior to the second scan and the pre-monsoon plates were incubated for a total of 7-9 hours. 

Fungal biomass measurements: Fungal biomass was quantified by measuring the concentration of ergosterol in a sub-sample taken from each soil sample collected from June to September.   Within 24-72 hours of sample collection, 5mL sub-samples were taken from each bulked soil sample and placed into individual Corning 15mL screw-cap centrifuge tubes.  Each tube was filled to the 10mL mark with an 0.8% KOH in methanol solution.  Tubes were refrigerated for storage until analyzed for fungal biomass by measuring the ergosterol content within each sample.  Ergosterol concentration for each sample was determined using HPLC with 100% methanol as the solvent at a flow rate of 1.5mL/ minute and a c-18 column.  Ergosterol was quantified by measuring the peak height that passed through a detector set to measure absorbance at 282nm, at 3.7min after the sample was injected into the column.  The height of each peak was then converted into μg ergosterol/g soil and finally converted to mg fungal biomass/ g soil by applying a conversion factor.  

Experimental design: 

Each study area consisted of a 10m wide, 30m long, downslope runoff plot, bound at the top and sides with aluminum flashing and fitted with water collecting guttering at the bottom so inputs and outputs could be quantified. The guttering fed water into a flume fixed into the ground at 4⁰ allowing water leaving the plot as runoff to be quantified.

The flumes were instrumented with a pump sampler to collect runoff samples leaving the plot and a bubbler module to measure discharge. In addition all runoff and associated sediment was collected in a covered stock tank (560 gallons for study area 1&2, 1000 gallons for study are 3). Rainfall onto the plot was measured using tipping-bucket rain gauges connected to the pump sampler. Following rainfall events all data was downloaded from the sampler using Flowlink v3.2 software.

Eroded sediment was collected from stock tank following rainfall events, coarse organic matter was removed via flotation, samples were oven dried at 60⁰C, weighed and sieved for particle size analysis using US. standard sieves at the Sevilleta LTER field station.

Setting up plots: 

Plots were selected on comparable planar slopes in areas believed to be representative of endmember vegetation habitats.