DOI: 10.5061/dryad.kh18932hd
title: Data from: Peatland fungal community responses to nutrient enrichment: a story beyond nitrogen

description: Anthropogenically elevated inputs of nitrogen (N), phosphorus (P) and
 potassium (K) can affect the carbon (C) budget of nutrient-poor peatlands.
 Fungi are intimately tied to peatland C budgets due to their roles in
 organic matter decomposition and symbioses with primary producers; however
 the influence of fertilization on peatland fungal composition and
 diversity remains unclear. Here we examined the effect of fertilization
 over 10-yrs on fungal diversity, composition, and functional guilds along
 an acrotelm (10-20 cm), mesotelm (30-40 cm), and catotelm (60-70 cm) depth
 gradient at the Mer Bleue bog, Canada. Simultaneous N and PK addition
 decreased the relative abundance of ericoid mycorrhizal fungi (ErMF) and
 increased ectomycorrhizal fungi (EcMF) and lignocellulose-degrading fungi.
 Fertilization effects were not more pronounced in the acrotelm relative to
 the catotelm, nor was there a shift towards nitrophilic taxa after N
 addition. The direct effect of fertilization significantly decreased the
 abundance of Sphagnum-associated fungi, primarily owing to the overarching
 role of limiting nutrients rather than a decline in Sphagnum cover.
 Increased nutrient loading may threaten peatland C stocks if
 lignocellulose-degrading fungi become abundant and accelerate
 decomposition of recalcitrant organic matter. Additionally, future changes
 in plant communities, strong water table fluctuations, and peat subsidence
 after long-term nutrient loading may also influence fungal functional
 guilds and depth-dependencies of fungal community structure.
description: <strong>Fertilization experiments</strong>
 Two sets of fertilization experiments were established in
 2000-2001 and 2005, respectively. Experiment 1 included six treatments
 with different rates of N additions with or without P and K. To better
 understand the effect of different rates of N addition alone, Experiment 2
 was established in 2005. In both experiments, triplicate plots (3 m x 3 m)
 were fertilized every three weeks from early May to late August (7 times
 per year) by applying ammonium nitrate (for N treatments) and/or mono
 potassium phosphate (for PK treatments). The two experiments combined
 represented a total of 27 plots (9 treatments x 3 replicates) and were
 established on large hummocks which covered 70% of the terrain. There have
 been no changes to the distribution of hummocks versus hollows with the
 fertilization treatments. <strong>Vegetation
 survey</strong> Plant communities were
 characterized by the point intercept method in July 2014. The number of
 times (‘hits’) a specific plant species and organ (leaf/woody
 stem/flower/moss shoot) contacted a metal rod (4 mm in radius) over 61
 grid points in a 60 cm x 60 cm frame were recorded.
 <em>Sphagnum</em> cover (%) was estimated by the number of
 hits divided by 61 and multiplying by 100. Vascular species abundance (not
 identical to cover) was estimated by the total number of hits of all
 organs per m<sup>2</sup> for each species.
 <strong>Peat sampling</strong> In
 mid-July 2014, a single peat core was taken from a location selected at
 random within each plot, with 3 depth increments saved per core (27 cores
 x 3 depth increments = 81 samples total). The upper two depth increments
 of peat (10-20 and 30-40 cm below the peat surface, 10 cm length x 10 cm
 width x 10 cm height) were collected using a clean bread knife, and an
 Eijkelkamp auger was used for deeper peat (60-70 cm, 5.2 cm diameter x 10
 cm height). At Mer Bleue, the uppermost layer (10-20 cm) represents the
 typical acrotelm peat which is rarely saturated (i.e., oxic) while the
 lowermost layer (60-70 cm) is from the catotelm where peat is rarely above
 the water table (i.e., anoxic). The middle layer (30-40 cm) is from the
 mesotelm through which the water table has seasonal oscillations and thus
 it is a biogeochemical ‘hotspot’ where roots never dry out and are rarely
 exposed to anoxic conditions in the growing season. At the time of
 collection, water table depth and the location of the water table relative
 to the mid-point of the depth of the peat sample were determined in each
 core hole, and peat temperature was measured from each depth increment.
 Samples were subdivided into two subsamples (for DNA analyses and
 physicochemical analyses), transported to the laboratory on dry ice,
 stored at -20°C, and shipped to the USDA Forest Service, Northern Research
 station (Houghton, MI, USA) where they were stored at -20°C until further
 processing. <strong>Physicochemical properties of
 peat</strong> A variety of elemental
 concentrations, pH, humification index, and organic chemical composition
 of peat were measured. Using moist peat from each sample, humification
 level was characterized using the von Post humification index, and peat pH
 was measured using a 2:1 ratio (volume:volume) of distilled water to peat.
 A subsample of peat was oven-dried at 60 °C to a constant weight and
 ground using a Wiley Mini Mill with size 60 mesh. Total C, N, and S
 concentrations were determined by dry combustion on a VarioMacro CNS
 Analyzer in the Watmough lab at Trent University. P and K were analyzed
 using ICP-MS (Varian 810, now part of Agilent Technologies, Santa Clara,
 CA, USA) in the Spiers lab at Laurentian University after combustion with
 a muffle furnace and a modified EPA3050A block digestion.
 Fourier-transform infrared spectroscopy (FTIR) analysis of dried peat and
 assignment of peaks to carbohydrate and aromatic classes was carried out
 in the Chanton lab at Florida State University.
 <strong>DNA extraction, amplicon library preparation, and
 sequencing</strong> In a 50 mL centrifuge tube,
 10 mL of wet peat was placed with twenty 3.2 mm chrome-steel beads. The
 sample was pulverized for two minutes on a modified mini-beadbeater-96. A
 subsample of 0.5 g pulverized peat was used for DNA extraction with a
 PowerSoil® DNA Isolation Kit. The extraction procedure followed the
 manufacturer’s instructions, with the addition of a 10-min vortex followed
 by incubation at 65 °C for 30-min, all in the C1 lysis buffer. DNA was
 cleaned using a MoBio PowerClean® Pro DNA Clean-Up Kit and quantified with
 a Qubit Fluorometer. Cleaned DNA extracts were pooled
 and sequenced at the U.S. Department of Energy Joint Genome Institute. The
 amplicon library preparation and sequencing were performed following Joint
 Genome Institute Protocols for Illumina MiSeq community amplicon
 sequencing. Briefly, the fungal ITS2 region was targeted with the forward
 primer fITS9 (GAACGCAGCRAANNGYGA) and the reverse primer ITS4
 (TCCTCCGCTTATTGATATGC). The full-length primer was composed of an Illumina
 adapter, an 11-bp barcode unique to each sample on the reverse primer, a
 10-bp primer pad, a 3-bp spacer pad and the primer sequence. DNA samples
 were combined in equimolar aliquots and sequenced on an Illumina Miseq
 platform with 2×300 bp chemistry. Eight samples were lost due to poor
 sequencing, which occurred independently and were not treatment- or
 depth-specific.
description: # Peatland fungal community responses to nutrient enrichment: a story
 beyond nitrogen
 [https://doi.org/10.5061/dryad.kh18932hd](https://doi.org/10.5061/dryad.kh18932hd) ## Description of the data and file structure ### **Description:**  This dataset contains two tabs. The first (“Ancillary_data”) contains the Sequence Read Archive (SRA) experiment identifier for each sample and the ancillary data for the hierarchical partitioning analysis. The second (“OTU_table_w_annotations”) contains the rarefied OTU table and the putative functional annotations based the taxonomic levels of each OTU. Fungal OTUs were finally assigned to putative functional guilds using FUNGuild and FungalTraits, and the assignments were refined, if necessary, based on literature searches and our expertise in fungal ecology in peatlands. #### **“Ancillary\_data” tab** ##### Variables * sample_ID: a unique ID for each sample. * SRX_IDs: Sequence Read Archive (SRA) experiment identifier. * treatment: nine fertilization treatments; C1 and C2 represent the two sets of control groups for Experiment 1 and Experiment 2, respectively; PK represents the treatment with the addition of 5 g of phosphorus and 6.3 g of potassium per m2 per year; 1.6N, 3.2N and 6.4N represent treatments with the additions of 1.6, 3.2 and 6.4 g of nitrogen per m2 per year; 1.6N+PK, 3.2N+PK and 6.4N+PK represent treatments with the additions of 1.6, 3.2 and 6.4 g of nitrogen plus 5 g of phosphorus and 6.3 g of K per m2 per year, respectively. * depth: sampling depths, i.e., 10-20, 30-40 and 60-70 cm below the peat surface. * core: one core was sampled from each fertilized plot, named as a, b and c; there are three replicated plots for each fertilization treatment. * exp_set, two sets of fertilization experiment. * exp1 = Experiment 1, starting at 2000/2001 * exp2 = Experiment 2, starting at 2005 * C: mg of carbon per g of dry peat. * N: mg of nitrogen per g of dry peat. * P: mg of phosphorus per g of dry peat. * K: mg of potassium per g of dry peat. * Carbohydrates: estimated content (%) of carbohydrates determined by fourier-transform infrared spectroscopy, which represents the combined relative abundance of putative carbohydrates as a percentage of the total abundance of all components. * Aromatics: estimated content (%) of aromatic compounds determined by fourier-transform infrared spectroscopy, which represents the combined relative abundance of putative aromatic compounds as a percentage of the total abundance of all components. * pH: soil pH measured with a 2:1 volume ratio of water to peat. * Sphag: number of hits of *Sphagnum* mosses determined by point-intercept method; as a non-vascular plant, the number of hits divided by the total number of grids (i.e., 61) provide an estimate of *Sphagnum* moss cover. * Polyt: number of hits of *Polytrichum* mosses determined by point-intercept method; as a non-vascular plant, the number of hits divided by the total number of grids (i.e., 61) provide an estimate of *Polytrichum* moss cover. * Tot_Erica: total number of hits of all ericaceous shrubs determined by point-intercept method. * Relative.WTD: the location of the water table relative to the mid point of the depth of the peat sample. Values that are negative are therefore samples where the water table was below the sample (or at least below the midpoint), and values that are positive are for samples that are below the water table. * Von.Post.Score: Von Post scale of peat humification, from 1-10. #### **“OTU\_table\_w\_annotations” tab** ##### Variables * OTU_ID: the ID of each OTU from the OTU table. * Columns B to BV, sample_ID with numbers of OTUs for each sample. * Phylum: the taxonomic level of fungal phylum. * Class: the taxonomic level of fungal class. * Order: the taxonomic level of fungal order. * Family: the taxonomic level of fungal family. * Genus: the taxonomic level of fungal genus. * Species: the taxonomic level of fungal species. * Putative functional annotations: fungal OTUs were assigned to putative functional guilds using FUNGuild and FungalTraits, and the assignments were refined, if necessary, based on literature searches and our expertise in fungal ecology in peatlands. ## Access information Other publicly accessible locations of the data: * Sequence data are accessible via the National Center for Biotechnology Information (PRJNA1059234).