DOI: 10.5061/dryad.xksn02vqh
title: Canopy and understory nitrogen additions differently affect soil microbial residual carbon in a temperate forest

description: Atmospheric nitrogen (N) deposition in forests can affect soil microbial
 growth and turnover directly through increasing N availability and
 indirectly through altering plant-derived carbon (C) availability for
 microbes. This impacts microbial residues (i.e., amino sugars), a major
 component of soil organic C (SOC). Previous studies in forests have so far
 focused on the impact of understory N addition on microbes and microbial
 residues, but the effect of N deposition through the plant canopy, the
 major pathway of N deposition in nature, has not been explicitly explored.
 In this study, we investigated whether and how the quantities (25 and 50
 kg N ha-1 yr-1) and modes (canopy and understory) of N addition affect
 soil microbial residues in a temperate broadleaf forest under 10-yr N
 additions. Our results showed that N addition enhanced the concentrations
 of soil amino sugars and microbial residual C (MRC) but not their relative
 contributions to SOC, and this effect on amino sugars and MRC was closely
 related to the quantities and modes of N addition. In the topsoil, high-N
 addition significantly increased the concentrations of amino sugars and
 MRC, regardless of the N addition mode. In the subsoil, only canopy N
 addition had a positive effect on amino sugars and MRC, implying that the
 indirect pathway via plants plays a more important role. Neither canopy
 nor understory N addition significantly affected soil microbial biomass
 (as represented by phospholipid fatty acids), community composition, and
 activity, suggesting that enhanced microbial residues under N deposition
 likely stem from increased microbial turnover. These findings indicate
 that understory N addition may underestimate the impact of N deposition on
 microbial residues and SOC, highlighting that the processes of canopy N
 uptake and plant-derived C availability to microbes should be taken into
 consideration when predicting the impact of N deposition on the C
 sequestration in temperate forests.
description: <strong>Soil physicochemical properties </strong>Soil
 pH was measured in a slurry (soil: water = 1: 2.5, w/v) with a pH meter
 (FiveEasy Plus<sup>TM</sup> FE28, Mettler Toledo). SOC, soil
 total nitrogen (TN), and total phosphorus (TP) concentrations were
 determined by the concentrated sulfuric acid-potassium dichromate external
 heating method, the concentrated sulfuric acid digestion-phenol blue
 colorimetric method, and the concentrated sulfuric acid
 digestion-molybdenum antimony anti-colorimetric method, respectively (Lu,
 1999). <strong>Soil microbial PLFAs The
 </strong>Soil microbial community was characterized by the
 phospholipid fatty acids (PLFAs) method (Bossio & Scow 1998). The
 concentration of each PLFA was calculated based on the 19:0 internal
 standard concentration. The PLFAs i14:0, i15:0, a15:0, i16:0, a16:0,
 i17:0, a17:0, a18:0, i18:0, a19:0, 16:1ω7c, 16:1ω9c, 17:1ω8c, 18:1ω7,
 cy17:0, and cy19:0 were used to indicate bacterial biomarkers. The PLFAs
 18:1ω9c, 18:2ω6,9c, and 18:3ω6,9,12c were applied to denote fungal
 biomarkers. The PLFA 16:1ω5c was considered an arbuscular mycorrhizal
 fungal (AMF) biomarker. The PLFAs 10Me 16:0, 10Me 17:0, and 10Me 18:0 were
 used as actinomycetes biomarkers. Total microbial biomass was represented
 by the sum of bacterial, fungal, AMF, and actinomycetes biomarkers. Soil
 microbial community structure was represented by the ratio of fungal to
 bacterial PLFAs (F:B ratio) (Frostegård & Bååth 1996).
 <strong>Soil microbial activity </strong>Soil
 microbial activity was assessed through determining microbial respiration
 under incubation (Hu & van Bruggen, 1997). A 50 ml beaker
 containing 30.00 g (fresh weight) soil sample was placed in a 1000 ml jar.
 Inside this jar, a 25 ml beaker containing 5.00 mL 1 M NaOH solution was
 placed to trap CO<sub>2</sub> produced by incubated soil
 sample. Four replicates of each treatments and four control jars with no
 soil samples were prepared. Each jar was sealed and then kept at 25℃ for
 10 days in one incubation period, 44 jars in total. Subsequently, the
 beaker containing NaOH solution was taken out and the NaOH solution was
 titrated by 0.20 M HCl solution. After each measurement, each sample was
 returned to ambient CO<sub>2</sub> level by leaving the
 incubation jars open for 1 h and NaOH was refreshed. Meanwhile, the soil
 moisture content was maintained by weighing the 50 ml beaker and adding
 the distilled water. These soil samples were subjected to six incubation
 periods (60 days of incubation period in total). The released
 CO<sub>2</sub> in each incubation period was calculated as the
 follows: R<sub>CO2</sub> = (V<sub>0 </sub>-
 V<sub>k</sub>) × <em>C</em><sub>HCl
 </sub>× 44 / (2 × w) / 10, where R<sub>CO2</sub> is the
 rate of CO<sub>2</sub> release (mg g<sup>-1</sup>
 d<sup>-1</sup>). V<sub>0</sub> and
 V<sub>k</sub> are the volumes of consumed HCl in the blank
 controller (without soil) and the incubated soil treatments (ml),
 respectively. <em>C</em><sub>HCl</sub> is the
 calibrated concentration of HCl (mol L<sup>-1</sup>). 44 is
 the molar mass of CO<sub>2</sub>; w is the dry weight of the
 fresh soil (g); 10 is the days of each incubation period (days).
 <strong>Soil amino sugars </strong>The concentrations
 of soil amino sugars, including muramic acid (MurN), galactosamine (GalN),
 and glucosamine (GluN), were determined as described by Indorf et al.
 (2011). In brief, amino sugars were hydrolyzed, extracted, and derivatized
 with ortho-phthaldialdehyde, determined by high-performance liquid
 chromatography (Dionex Ultimate 3000, Thermo Fisher Scientific). The
 detailed relevant information was described by Yuan et al.
 (2021). <strong>Data calculation and statistical
 analysis</strong> Microbial residual C (MRC) was
 calculated by the following formulas (1)-(4): (1) F-GluN (μg
 g<sup>−1</sup>) = total GluN (μg g<sup>−1</sup>) –
 2 × MurN (μg g<sup>−1</sup>) × (179.2/251.2), where F-GluN is
 fungi-derived GluN. It was assumed that MurN and GluN occurred at a molar
 ratio of 1 to 2 in bacterial cell walls (Engelking et al., 2007). Where
 179.2 and 251.2 are the molecular weights of GluN and MurN, respectively
 (Shao et al., 2017). (2) Fungal MRC (μg g<sup>−1</sup>) =
 F-GluN × 9, (3) Bacterial MRC (μg g<sup>−1</sup>) = MurN × 45,
 (4) Total MRC (μg g<sup>−1</sup>) = Fungal MRC + Bacterial
 MRC, where 9 and 45 are conversion factors (Appuhn & Joergensen,
 2006). Fungal MRC, bacterial MRC, and total MRC are fungi-derived,
 bacteria-derived, and total microbial residual C, respectively.
 A univariate general linear model (GLM) with a random factor
 (blocks) was used to examine the effects of N deposition treatments on
 concentrations of soil total amino sugars, MurN, GalN, GluN, fungal MRC,
 bacterial MRC, total MRC, and the ratio of fungal to bacterial MRC, while
 the effect of blocks was not significant. The effects of N deposition on
 the mean of SOC mineralization rate, relative contributions of total amino
 sugars, fungal MRC, bacterial MRC, and total MRC to SOC were tested by a
 general linear model as well. The impacts of N deposition on soil
 physicochemical properties (pH, SOC, TN, and TP) and soil microbial
 parameters (the PLFAs of bacteria, fungi, AMF, actinomycetes, and total
 microbes, and the F:B ratio) were examined by one-way ANOVA. Multiple
 comparison analyses (LSD) were used after general linear model and one-way
 ANOVA. Pearson correlation analysis was performed to assess the
 relationships of measured soil microbial residues (amino sugars and
 residual C) with soil physicochemical properties and soil microbial
 parameters (microbial PLFAs and community structure). All statistical
 analyses were carried out with SPSS 18.0 (SPSS, Chicago, Illinois, USA),
 and results were considered statistically significant
 at <em>p</em> &lt; 0.05.
description: # Canopy and understory nitrogen additions differently affect soil
 microbial residual carbon in a temperate forest
 [https://doi.org/10.5061/dryad.xksn02vqh](https://doi.org/10.5061/dryad.xksn02vqh) ## Description of the data and file structure The data from 20 experimental plots including five treatments of control (without N addition, CT), canopy low-N addition ((25 kg N ha-1 yr-1, CN25), canopy high-N addition (50 kg N ha-1 yr-1, CN50), understory low-N addition (25 kg N ha-1 yr-1, UN25), and understory high-N addition (50 kg N ha-1 yr-1, UN50), four replicated plots for each treatment, were collected. The data include soil physiochemical properties (soil pH, soil organic carbon (SOC), soil total nitrogen (TN), soil total phosphorus (TP) in 0-10 and 10-20 cm layers), soil microbial biomass ( represented by phospholipid fatty acids, PLFAs), microbial activities (determined by microbial respiration under incubation), soil amino sugar concentration, the relative contribution of total amino sugars to SOC, microbial residual carbon (MRC) concentration, and the relative contribution of MRC to SOC. Please see the note in the dataset file for abbreviations. Units are included with the variables in the file.