DOI: 10.5061/dryad.69p8cz8xb
title: Data from "Removal of grazers alters the response of tundra soil carbon to warming and enhanced nitrogen availability", Ecological Monograps in October 2019

description: Here we present the data used in the manuscript "Removal of
 grazers alters the response of tundra soil carbon to warming and enhanced
 nitrogen availability", Ecological Monograps, Early view in October
 2019 by H. Ylänne, E. Kaarlejärvi, M. Väisänen, M. K. Männistö, S. H. K.
 Ahonen, J. Olofsson & S. Stark. In this paper we studied,
 how five years of experimental warming and increased soil
 nitrogen availability interact with both long- and short-term differences
 in grazing intensity in shaping ecosystem carbon stocks and the processes
 underlying the changes. We used an over 50-year-old reindeer fence that
 separates a lightly grazed shrub-dominated tundra from a heavily grazed
 graminoid-dominated tundra, where the different grazing histories on the
 two sides of the fences have created different ecosystem states. In
 addition to the long-term grazing difference, we also established
 short-term grazer exclosures on the heavily grazed side of the fence to
 account for the effect of a sudden grazing cessation. This
 file includes data of ecosystem carbon stocks, soil properties,
 and fungal and bacterial copy numbers. It also provides data on
 development of the vegetation through the course of the experiment
 (2010-2014) and presents the activities of six extracellular
 enzymes measured on three occasions in 2013.
description: <strong>Experimental set-up and site
 characteristics</strong> The data is
 from an experimental set-up, created in 2010 to a tundra heath on
 the northern slope of  a hill Raisduoddar in northern Norway (69°31’29 N,
 21°19’16 E; altitude 430–570 m a.s.l.). This is in a non-permafrost area
 with glacial tills as the dominant mineral soil type. Only weak podzolic
 profiles are developed and a continuous soil organic layer of
 approximately 4.25 cm is found above the mineral soil layer. The area is
 bisected by a reindeer pasture rotation fence, that has been in place at
 least since 1966, therby creating a legal border between the reindeer
 summer ranges and their migration range. The summer range side of the
 fence experiences heavy grazing (HG) intensity for a few weeks during
 annual reindeer migrations in August, whereas the migration-range is
 subjected to grazing for only a short period of time (referred to as light
 grazing intensity, LG). The
 set-up consists of eight blocks bisecting the fence (5 × 10 m large,
 spaced 20 m apart). On each block to both sides of the fence (HG and LG),
 treatments of control (Ctrl), warming (W), fertilization (F) and combined
 warming and fertilization (WF) were performed on plots sized betweem 0.935
 and 1 m<sup>2</sup>. Additionally, plots under HG were divided
 into grazed (HG) and ungrazed (HGexc) subplots with short-term exclosures
 (height 0.9 m, mesh 40 × 40 mm) that were built each year (2010–2014)
 before reindeer migration and removed after the grazing event to avoid
 winter-time snow accumulation. Warming was implemented with open-top
 chambers (OTCs, diam. 120 cm, height 40 cm) set to the plots after snow
 melt (early June) and kept in place until the arrival of reindeer (early
 August) to avoid OTCs affecting the feeding behaviour of reindeer. By this
 way, the warming treatment’s direct effect was limited to approximately
 two months, encompassing the early and peak growing season.
 Fertilization was applied yearly,
 early in  the growing season, by dissolving ammonium nitrate
 (NH<sub>4</sub>NO<sub>3</sub>) equivalent to 10 g
 N m<sup>-2</sup> in one L of water and applying it to the
 plots. Due to logistical reasons and the high mean annual precipitation in
 the study area, the unfertilized plots were not watered.    <strong>Ecosystem carbon
 stocks in 2014</strong> Ecosystem carbon stocks were sampled after
 five years of the experiment, in 2014, before the annual grazing period at
 the study site. Therefore, these do not account for the immediate
 grazer-induced biomass removal. Vascular aboveground biomass was collected
 from an area of 250 cm<sup>2</sup> and sorted into growth
 forms. Bryophyte, lichen and litter biomass was hand-picked from cored
 ground layer samples (diam. 119 mm). Litter included both cryptogam and
 vascular litter. Soil and root biomass was collected with 3–4 soil cores
 (diam. 29 mm) underneath the litter layer until the corer hit large stones
 and separated later into organic and mineral soil layers. The soil layers
 were later homogenized separately (2 mm sieve), and the root biomass was
 obtained from the sieving residue after washing. All vegetative, litter
 and soil biomass was dried (60 °C, 70 h), weighed, milled (RETSCH 2000)
 and analysed for carbon and nitrogen contents (C-H-N Element Analyser
 EA1110, CE Instruments, SFS-EN 15104:2011).   <strong>Soil properties in
 2014</strong> From the sieved soil samples, we determined
 soil moisture (drying at 100 °C, 12 h), organic matter content (loss on
 ignition at 475 °C, 4 h) and bulk density (dw
 volume<sup>-1</sup>). We also conducted soil extractions with
 0.5 M K<sub>2</sub>SO<sub>4</sub> for 2 h (within
 48–72 h after sampling), and analyzed the inorganic soil ammonium
 (NH<sub>4</sub><sup>+</sup>-N) and nitrate
 (NO<sub>3</sub><sup>–</sup>-N) concentrations with
 colorimetrical analyses (SFS 3032, Shimadzu UV-1700 spectrophotometer for
 NH<sub>4</sub><sup>+</sup>-N; SFS-EN ISO
 133395CFA, Seal Analytical AA3 for
 NO3<sup>–</sup>-N).   <strong>Fungal and bacterial
 copy numbers in 2013</strong> Total genomic DNA for bacterial and fungal
 quantitative PCR (qPCR) was determined from plots under LG and HG in July
 2013 from the same composite samples from which N concentrations were
 analyzed. The composite soil samples were sieved in the field and
 subsamples of  0.1–0.5 g were immediately frozen in liquid nitrogen for
 later analysis. The DNA was extracted using a modified
 phenol-chloroform-isoamylalcohol (PCI) protocol (Griffiths and Whiteley
 2000) with 600 µL of CTAB buffer in a mixture of beads (0.7 g ceramic
 beads (1.0 mm), 0.3 g glass beads (0.1 mm) and two large glass beads (3.5
 mm); BioSpec). The fungal ITS2 and bacterial 16S rRNA gene copies in the
 soil samples were quantiﬁed using a Bio-Rad CFX96 Real-time thermal cycler
 (Bio-Rad, Hercules, CA, USA). All qPCRs were run in technical triplicates
 of 20 µL containing 10 µL SSoFast EvaGreen qPCR Supermix (Bio-Rad) for
 bacterial and SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) for
 fungal reactions, 0.5 µL of forward and reverse primers (10 mM) (Eub341F
 and Eub534R)for bacteria and fITS7 and ITS4 for fungi), 7 µL
 ddH<sub>2</sub>O and 2 µl template in a 100-fold dilution. The
 conditions were 98 °C for 2 min, followed by 40 cycles of 98 °C for 5 s,
 56 °C for 20 s for bacterial qPCR and 98 °C for 3 min, followed by 40
 cycles of 98 °C for 15 s, 61 °C for 60 s for fungal qPCR with a melt curve
 analysis as the ﬁnal step. The standard curves were generated using
 genomic DNA from bacterial isolate <i>Granulicella
 mallensis</i> and fungal isolate <i>Laccaria
 laccata</i>.   <strong>Vegetation
 abundance (2010-2014)</strong> Vegetation abundance was analyzed
 throughout the course of the experiment (2010–2014), with a modified point
 intercept method, where ten vertical pins are positioned 10 cm away from
 each other on 50 cm wide rows (eight rows on LG, four rows on HGexc and HG
 in 2010–2013, and two rows in LG, HGexc and HG in 2014) and where all hits
 per species are counted. The recordings were done in the first half of
 August every year. We normalized the number of hits to hits per 100 pins
 and report species abundances pooled into growth forms (evergreen shrubs,
 deciduous shrubs, graminoids, forbs, bryophytes and lichens).
   <strong>Extracellular enzyme
 activity in June, July and August 2013 </strong>
 To determine soil  microbial
 activity, we collected composite soil samples (3–7 soil cores, diam. 25
 mm) from the organic layer three times during the growing season in 2013
 (June 19<sup>th</sup>, July 16<sup>th</sup> and
 August 6<sup>th</sup>). We recorded depth of the organic
 layer, sieved the samples (mesh 2 mm) and stored these at +4 °C for
 further analyses. We analyzed the potential activities of β-glucosidase
 (BG), β-N-acetylglucosaminidase (NAG), leucine aminopeptidase (LAP), acid
 phosphatase (AP), amidohydrolase (commonly known as urease; U) and phenol
 oxidase (POX) within 5 days after sampling. We used the following
 chromogenic substrates: 5 mM paranitrophenyl(pNP)-β-glucopyranoside for
 BG, 2 mM pNP-β-N-acetylglucosaminide for NAG, 5 mM leucine p-nitroanilide
 for LAP, 5 mM pNP-phosphate for AP, 30 mM urea for U, and 50 mM pyrogallol
 for POX and conducted the assays in sodium acetate buffer (50 mM, pH 5.0
 that corresponds to the study site’s mean soil pH, 5.13). The
 soil-substrate aliquots were incubated at room temperature, five
 microliters of 1.0 M NaOH was added to AP, BG and NAG before the
 supernatants were measured spectrophotometrically for their potential
 activities (405 nm for BG, NAG, LAP and AP, 450 for POX, Multiscan FC
 microplate reader, Thermo Scientific). U activity was verified by
 measuring the formation of
 NH<sub>4</sub><sup>+</sup> after 5 h. The 100 µL
 aliquots of supernatant were incubated with 10 µL of sodium citrate,
 phenol nitroprusside and hypochlorite (19 °C, 1 h), and absorbance was
 measured at 620 nm (Multiscan FC microplate reader, Thermo Scientific).
 The absorbances of homogenate and substrate controls were subtracted from
 the assay absorbance. Extinction coefficients for calculating potential
 activities were obtained based on standard curves for paranitrophenol (BG,
 NAG, AP), paranitroaniline (LAP) and NH<sub>4</sub>Cl (U), and
 oxidation of pyrogallol by mushroom tyrosinase (POX). All potential
 activities were counted as μmol
 h<sup>–</sup><sup>1</sup> g
 SOM<sup>–</sup><sup>1</sup>.