We collected soil samples from each treatment in the DH (Small fence, Large fence and control, all non-fertilized plots)  on 22-Jul-2017 and MAT (Small fence and control, all non-fertilized plots)   on 24-Jul-2017. For the DH, we collected three randomly located samples per plot; using a serrated bread knife we collected 10 x 10 cm columns of the organic horizon to a depth of 5 cm. In the MAT, three 10 x 10 cm columns of soils were cut from each plot to a depth of approximately 30 cm or to the depth of active layer (i.e., frozen soil was not sampled), whichever was less. For the DH only a single layer of soil was present (upper organic layer). We separated each column of MAT soil into the upper organic layer (top five cm of the organic layer), the lower organic layer (when present), and the top five cm of the mineral layer (when accessible) in the field. We sampled the mineral layer to the bottom of the active layer, or the top 5 cm, whichever was less.

For each soil column and depth, we dried a subsample of each core of known volume (approximately 5 cm3) at 50°C for 48 hours to assess bulk density (BD) and gravimetric water content (GWC). When volume could not be accurately assessed only GWC measurements were taken.  We individually homogenized the remainder of each soil sample by hand, removing all large roots (> 1mm diameter),  and partitioned samples for analysis within two days of collection, and then froze samples before shipping to the University of Texas at El Paso, where they were stored at - 20°C until analysis.

We analyzed soil samples for total C and N, inorganic nutrients (NH4+, NO3-, PO43-); organic nutrients (extractable organic C (EOC), extractable total N (ETN), extractable organic P (EOP)); microbial biomass C, N, P; and extracellular enzyme activity using the following methods.

We dried, ground, and processed soil subsamples for total %C and %N content using a dry combustion C and N analyzer (ElementarPyroCube ®). To determine soil inorganic nutrients, we thawed and extracted frozen subsamples (5 g) in 25 ml of 0.5 M K2SO4 for 2 hours, filtered through glass filter paper and analyzed extractant using colorimetric microplate assays (BioTEK Synergy HT microplate reader, Winooski, Vermont, USA). NH4+-N (NH4+) was determined using a modified Berlethot assay (Rhine et al. 1998), NO3--N (NO3-) using a modified Griess assay (Doane and Horwath 2003), and PO43--P (PO43-) using a malachite green assay (D'Angelo et al. 2001).

EOC was determined colorimetrically after an Mn (III)‐reduction assay (Bartlett and Ross 1988). ETN and EOP were determined using a modified alkaline persulfate digestion using a 1:1 ratio of oxidizing reagent to sample and autoclaved for 40 min at 121°C. (Lajtha et al. 1999) followed by analysis for NO3- and PO43- respectively as above. To determine microbial biomass C, N, and P, we conducted the above assays on samples using a direct chloroform-addition modification of the fumigation-extraction method (Brookes et al. 1985; Voroney et al. 2006), where 5 g of thawed soil was incubated for 24 hours with 2 mL of ethanol-free chloroform, followed by extraction in 25 mL of 0.5 M K2SO4. We calculated microbial biomass for C, N, and P (MBC, MBN, and MBP) by subtracting ETN, EOP or EOC respectively of non-fumigated samples from that of fumigated samples. No correction factor was applied for incomplete CHCl3-release, or sorption of P because these values are not known for K2SO4-extraction for these two ecosystems.

Extracellular enzyme (exoenzyme) activity was assessed for 10 exoenzymes involved in the microbial acquisition of C, N, and P: C-acquiring enzymes (β-glucosidase, β-cellobiosidase, β -xylosidase, α-glucosidase), N-acquiring enzymes (N-acetyl-glucosaminidase (NAG), leucine amino peptidase (LAP)) and P-acquiring enzymes (phosphatase, phosphodiesterase), as well as the oxidative enzymes phenol oxidase, and peroxidase. One g of soil was blended with a sodium acetate buffer to reflect natural soil conditions (pH = 5), and pipetted onto 96 well plates with eight replicates per soil.  Substrate tagged with fluorescing 4-methylum-belliferone (MUB) or 7-amido-4-methyl coumarin (MC) (LAP only) was added to soil slurrys. Samples were incubated at 20⁰C and enzyme activity (fluorescence) measured every 30 minutes for 3.5 hours following methods adapted from Saiya-Cork et al. (2002) and McLaren et al. (2017).   For each substrate, we measured the background fluorescence of soils and substrate and the quenching of MUB or MC by soils and used standard curves of MUB or MC to calculate the rate of substrate hydrolyzed.  Florescence was measured at 365 mm excitation and 450 nm emission using a BioTek Synergy HT microplate reader (BioTek Instruments Inc., Winooski, VT, USA). Oxidative enzyme analysis was performed using an L-3,4-dihydroxyphenylalanine (L-DOPA) substrate for phenol oxidase and peroxidase. Color absorbance was measured at 460 nm using a reader after 24 hours of incubation.

References

Bartlett RJ, Ross DS (1988) Colormetric determination of oxidizable carbon in acid soil solutions. Soil Science Society of America Journal 52:1191-1192.

Brookes PC, Landman A, Pruden G, Jenkinson DS (1985) Chloroform fumigation and the release of soil nitrogen: a rapid direct extraction method to measure microbial biomass nitrogen in soil. Soil Biology   and Biogeochemistry 17:837-842.

D'Angelo E, Crutchfield J, Vandiviere M (2001) Rapid, sensitive, microscale determination of phosphate in water and soil. Journal of Environmental Quality 30:2206-2209.

Doane TA, Horwath WR (2003) Spectrophotometric determination of nitrate with a single reagent. Analytical Letters 36:2713-2722.

Lajtha K, Driscoll CT, Jarrell WM, Elliot ET (1999) Soil phosphorus: characterization and total element analysis. In: Robertson GP, Coleman DC, Bledsoe CS, Sollins P (eds) Stand Soil Methods for Long-term   Ecological Research. Oxford University Press, New York, pp 115-142.

McLaren JR, Buckeridge KM, Van de Weg MJ, Shaver GR, Schimel JP, Gough L (2017) Shrub encroachment in Arctic tundra: Betula nana effects on above- and belowground litter decomposition. Ecology   98:1361-1376.

Rhine ED, Mulvaney RL, Pratt EJ, Sims GK (1998) Improving the Berthelot reaction for determining ammonium in soil extracts and water. Soil Science Society of American Journal 62:473-480

Saiya-Cork KR, Sinsabaugh RL, Zak DR (2002) The effects of long term nitrogen deposition on extracellular enzyme activity in an Acer saccharum forest soil. Soil Biology and Biochemistry 34:1309-1315

Voroney RP, Brooks PC, Beyaert RP (2006) Soil microbial biomass C, N, P, and S. In: Carter MR, Gregorich EG (eds) Soil sampling and methods of analysis. Lewis, Boca Raton.