The LTER moist acidic tussock (MAT) site was set up in 1989.  The experimental design is four blocks of 5 x 20 meter plots with randomly assigned treatments within each block. Treatments include control (CT), nitrogen (N), phosphorus (P) and nitrogen plus phosphorus (NP). Fertilizer is added annually following snowmelt in June as 10 g/m2 nitrogen (as NH4NO3) and 5 g/m2 phosphorous (as triple superphosphate). Exclosure plots were set up at the MAT site in July 1996 on extra 5 x 20 meter plots within the four-block design of the 1989 LTER acidic tussock experimental plots.  On each plot a 5 x 10 meter section was fenced with large mesh (4-inch square mesh) and within this fence a 5x5-meter plot was fenced with a small mesh (1/2-inch square mesh). In each block two fenced plots were setup: a plot with no fertilizer and a plot with annual fertilization treatments as described above. Thus the treatments created are no fence, no fertilizer (NFCT); small mesh fence, no fertilizer (SFCT); large mesh fence, no fertilizer (LFCT); no fence, N plus P (NFNP); small mesh fence, N plus P (SFNP); and large mesh fence, N plus P (LFNP). Only CT, N, P,  NP censused in 2013.

 In 1997 experimental plots were established in three block at the LTER moist non-acidic tussock (MNT) site with the same methods as those used at MAT. CT, N, P and NP treatments were censused in 2013.

Sampling Description

Organic and mineral soil horizons were sampled from MAT and MNT in early July 2013. When present, tussocks of Eriophorum vaginatum were avoided when sampling and sampling occurred only in intertussock areas (moss dominated areas between tussocks) because of distinct micro-topographic differences between tussock and intertussock areas. A single ca. 10cm x 10cm column of soil was collected from each plot to the depth of the permafrost. All organic horizons were < 20 cm deep, and were separated into the upper organic (0 – 5cm depth) and lower organic (5cm-15cm depth) layers, to allow comparison with previous studies that separated by depth and because ecosystem nutrient pools and microbial biomass can vary strongly by depth in the organic horizon. The mineral layer was sampled either to permafrost, or the upper 10 cm only, whichever was less. Mineral soils were sampled for MAT only, as the permafrost extended into the organic horizon for all MNT plots, preventing mineral soil sampling.

Soils were separated into layers in the field, and then returned to the field lab where they were homogenised and all large roots (>1mm diameter) removed. Soil was then partitioned for the analyses below. Soil was immediately dried for total CNP analysis, with the remainder frozen at -20C and shipped to University of California Santa Barbara for the remaining analyses.

Lab Analysis

Microbial Biomass, ETN and EOC, Extractable Nutrients:

Fresh soil samples (10 g) were extracted with 40 mls of deionized water by shaking for 3 hours. Duplicate samples for estimates of the microbial biomass flush (‘fumigated’) were extracted in the same manner but with the addition of 1 ml CHCl3 (Fierer et al. 2003). All extracts were vacuum filtered through 1-mm pore size glass fiber filter paper and sparged for 30 min with compressed air (to remove residual C from the CHCl3), then frozen at -20oC until analysis.Extractable organic C (EOC) and total N (ETN) contents in the fumigated and non-fumigated extracts were determined by oxidative combustion and infrared (EOC; Nelson and Sommers, 1982) or chemiluminesence (ETN) analysis (TOC-TN autoanalyzer, Shimadzu, Kyoto, Japan). Extractable NH4+-N, NO3--N and PO4-P in non-fumigated extracts and PO4-P in the fumigated extracts were determined colorimetrically, using automated flow analysis (Lachat autoanalyser) and the salicylate (NH4+-N), sulphanilamide (NO3--N) and molybdate blue (PO4-P ) methods (Mulvaney 1996).

Extracellular Enzyme Analysis:

We assayed for the activity of three hydrolytic enzymes that acquire carbon, nitrogen and phosphorous at the terminal stages of organic matter decomposition: cellulose-degrading b-glucosidase, chitin-degrading N-acetyl-glucosaminidase (NAG) and phosphatase. Soil was thawed and blended with 0.05M acetate buffer (MAT: pH 5; MNT: pH 6). Soil slurries were pipeted onto 96-well plates to which fluorescing 4-methylum-belliferone (MUB) tagged substrate (b-D-glucoside, N-acetyl-b-D-glucosaminide and phosphate) was added, with 8 analytical replicates per soil. The assays were incubated at 22 oC for ~2-4.5h (previously determined, by substrate, for these soils, to be during the phase of linear increase in activity) and then the reaction was stopped by adding 20 mL of 0.5M NaOH. Sample fluorescence was read with a TECAN Infinite Pro 200 plate reader (Tecan Group Ltd., Männedorf, Switzerland) at 365 nm excitation, 450 nm emission.

Total soil CNP analysis

Soil CN was analysed on dried, ground soil for each soil layer using a dry combustion total CN analyser (Perkin Elmer 2400 at NC State University Environmental Testing Service). Soil total P was analysed using a strong-acid soluble digest (EPA method 3050B digested at NC State University Environmental and Agricultural Testing Service Laboratory).  The digestates were analysed using an Inductively-coupled plasma-optical emission spectrometer (ICP-OES; Perkin Elmer Model 8000) with a cross- flow nebulizer.

Protocol:

Fierer, N., Allen, A.S., Schimel, J.P. & Holden, P.A. (2003) Controls on microbial CO2 production: a comparison of surface and subsurface soil horizons. Global Change Biology, 9, 1322–1332.

Nelson, D.W. & Sommers, L.E. (1982) Total carbon, organic carbon, and organic matter., 2nd ed (ed D.L. Sparks), pp. 539–579. Soil Science Society of America and American Society of Agronomy, Madison, WI.

Mulvaney, R.L. (1996) Nitrogen - Inorganic Forms. (ed D.L. Sparks), p. pp 1123-1184. Soil Science Society of America and American Society of Agronomy, Madison, WI.