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Nitrogen addition reduces dissolved organic carbon leaching in a montane forest
Accepted Manuscript Nitrogen addition reduces dissolved organic carbon leaching in a montane forest Ruiying Chang, Na Li, Xiangyang Sun, Zhaoyong Hu, Xuesong Bai, Genxu Wang PII:
S0038-0717(18)30301-8
DOI:
10.1016/j.soilbio.2018.09.006
Reference:
SBB 7273
To appear in:
Soil Biology and Biochemistry
Received Date: 21 April 2018 Revised Date:
24 August 2018
Accepted Date: 5 September 2018
Please cite this article as: Chang, R., Li, N., Sun, X., Hu, Z., Bai, X., Wang, G., Nitrogen addition reduces dissolved organic carbon leaching in a montane forest, Soil Biology and Biochemistry (2018), doi: 10.1016/j.soilbio.2018.09.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title: Nitrogen addition reduces dissolved organic carbon leaching in a montane forest
2 Authors: Ruiying Chang 1, Na Li 2, Xiangyang Sun 1, Zhaoyong Hu 1, Xuesong Bai 1,
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Genxu Wang 1
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Hazards and Environment, Chinese Academy of Sciences, Chengdu, China
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Ecological Restoration and Biodiversity Conservation Key Laboratory of SichuanProvince,
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CAS Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization &
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Key Laboratory of Mountain Environment Evolvement and Regulation, Institute of Mountain
Chengdu Instituteof Biology, Chinese Academy of Sciences, Chengdu, China
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Corresponding author, Genxu Wang, E-mail: [email protected];
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ACCEPTED MANUSCRIPT Abstract: Dissolved organic carbon (DOC) plays a significant role in the forest soil carbon cycle and
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can be regulated by nitrogen (N) addition. However, the regulatory direction, mechanism and seasonal
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pattern of DOC under N addition are less clear. Here, in a montane evergreen forest located at the
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eastern edge of the Tibetan Plateau, 2 levels of N were applied over 2 years to determine the effects of
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N addition on DOC release from organic (O layer) and mineral soil. Frequent sampling revealed that
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high levels of N addition could decrease the concentration of DOC and the flux from the O layer but
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not from mineral soil and that moderate N addition had no effect on DOC leaching from either the O or
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mineral layer. The effect of N addition on DOC leaching from the O layer was seasonally dependent,
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showing a significant reduction in DOC leaching during autumn/winter but no changes during summer
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and spring. This seasonally different response of DOC to N addition affected the seasonal pattern of
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DOC leaching. Soil and leachate pH were not influenced by N addition in the short term, indicating
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that there was not enough difference in DOC retention by mineral soil to significantly affect DOC
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leaching under N addition. In contrast, N addition-derived reduction in DOC leaching was likely to be
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due to suppressed fresh litterfall–derived DOC production during autumn/winter; this speculation was
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supported by lower values of O layer water-extractable organic carbon and microbial biomass carbon
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as well as lower saccharase and cellulose activities found with high N addition. These results suggested
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that the processes in control of DOC leaching and their responses to N addition were different for O
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and mineral soil and that short-term N addition could decrease O-layer DOC leaching, which is likely
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associated with decreased DOC production rather than greater DOC retention.
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Key words: Anthropogenic nitrogen deposition; Zero-tension lysimeters; Seasonal pattern; DOC
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production; DOC retention; Extracellular enzyme activity 2
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1.
Introduction Elevated active nitrogen (N) deposition is occurring in many areas around world, especially in
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China (Liu et al., 2013). The effects of elevated N deposition on forest soil carbon (C) pools and cycles
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have been widely documented; soil C pools are generally found to be enhanced under N addition
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(Janssens et al., 2010; Greaver et al., 2016; Entwistle Elizabeth et al., 2017), which is associated with
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higher C input derived from above-and/or below-ground litter and lower C-CO2 loss (Janssens et al.,
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2010; Huang et al., 2011). However, leaching of dissolved organic carbon (DOC), an important
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component in the C cycle that plays a significant role in soil C transport, transformation and formation
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in the mineral soil (Kalbitz and Kaiser, 2008; Kramer Marc et al., 2012), is less documented, and the
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responses of DOC to N addition are debatable and unclear (Evans et al., 2008).
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Some authors have reported positive effects of N addition on DOC leaching (Pregitzer et al., 2004;
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Camino-Serrano et al., 2016), while others have reported negative effects (Hagedorn et al., 2012; Lu et
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al., 2013) and yet others report neutral effects (McDowell et al., 2004; Lovett et al., 2013). These
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varying responses of DOC leaching to N addition have been attributed to the effects of various forms of
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N on soil pH and DOC sorption in the mineral soil (e.g., Evans et al., 2008). Ammonium salt addition
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could generally reduce DOC leaching by enhancing DOC retention in the mineral soil through a lower
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soil and/or leachate pH with N addition, while the increase in DOC leaching with sodium nitrate
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addition is associated with an increase in pH (Pregitzer et al. 2004; Evans et al., 2008; Lu et al. 2013).
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The responses of DOC to N addition are also soil depth-dependent. Hagedorn et al. (2012) reported
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reduced DOC release from mineral soil layers after NH4NO3addition, but leaching of DOC from the
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litter layer did not change. The processes that might affect DOC leaching from the organic (O) and
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mineral layers are likely to be different; however, previous research has not yet fully defined the
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differences. Patterns of DOC leaching also display seasonal trends, showing generally high levels of DOC
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during summer and/or fall (Kaiser et al., 2002; Dittman et al., 2007), high DOC concentrations in the
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spring (Pregitzer et al., 2004; Smemo et al., 2007), or no clear trend (Hagedorn et al., 2012). Seasonal
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changes in DOC are often in response to biotic and abiotic factors, such as the decomposition of
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litterfall, which is responsible for the high DOC release in spring (Pregitzer et al., 2004), and are likely
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to be influenced by N addition; consequently, the changes in DOC leachate due to N addition may also
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be seasonally dependent, and the seasonal pattern of DOC is also altered by N addition. Nevertheless,
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there is limited understanding of the seasonal differences in DOC levels with N addition: more in-depth
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study of this subject will improve the understanding of mechanisms of DOC production, retention and
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leaching.
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In this study, an NH4NO3-addition experiment was conducted in a subalpine evergreen conifer
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forest with moderate N deposition, and the DOC leachate was collected at frequent intervals of 5-7
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days from the forest O layer and mineral soil for 2 years. The objectives of this study are (1) to detect
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the differences in the responses of DOC leaching from the O and mineral layers and to distinguish the
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processes affecting DOC leaching in different horizons; and (2) to examine the seasonality of the
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responses of DOC to N addition.
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2.
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2.1 Study area
Material and methods
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We carried out our work at the long-term observation plot of the Alpine Ecosystem Observation
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and Experiment Station (29°34′N, 101°0′E) located on the eastern slope of Mt. Gongga, at the eastern 4
ACCEPTED MANUSCRIPT edge of the Tibetan Plateau. The study plot is at an altitude of 3000 m asl, with a gentle slope of less
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than 6 degrees. The area is dominated by the south eastern (Pacific) monsoon, with a long-term mean
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annual temperature (MAT) of 3.8 °C and a mean annual precipitation (MAP) of 1940 mm, 60% of
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which falls during June to September. The temperature and precipitation data used in this study were
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obtained from a nearby weather station (Fig. 1). The snow depth in the winter is approximately 50 cm,
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and all the snow melt occurs from March to April. The plant community is dominated by Abies fabri
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(Mast.) Craib. The soil type is classified as a Cambisol, according to the World Reference Base for Soil
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Resources (WRB), with low clay content (Table 1). The average depth of the organic (O) layer is
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approximately 8 cm. The total atmospheric N-deposition (wet and dry deposition) rate is approximate
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8.0 kg N/ha/year for the area (Liu et al., 2008).
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Figure 1
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Table 1
2.2 Leaching experiment and sampling
In this plot, five 6×6m subplots were selected randomly, and in each subplot, three 2×2m quadrats
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were established. In April 2013 (approximately 1 year prior to the experiment), in each quadrat, two
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zero-tension tray lysimeters (755 cm2 per lysimeter, made of silicon) were installed: one beneath the O
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layer and the other beneath the mineral layer (O layer + 20 cm of mineral soil), in adjacent areas. Each
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lysimeter was connected with Tygon tubing to a 4L brown bottle (installed at approximately 40 cm
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underground) to collect the leachate. In 2014 and 2015, the leachate was collected and sampled at an
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interval of 5-7 days from late March to late December of each year and at an interval of every 15 days
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during the remaining time (no water was collected during this period due to limited precipitation, as
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shown in Fig. 1). However, water samples were also taken the day after heavy rains. The leachate
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station laboratory immediately for pH, DOC and total dissolved nitrogen (TDN) analyses. All
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collectors were washed with deionized water following each collection. The concentrations of DOC
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and TDN for all water samples were analysed using a liquid CN analyser (Vario TOC, Elementar
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Instruments Inc, Germany) after filtering with a 0.45-µm PTFE filter.
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2.3 Nitrogen addition experiment
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The nitrogen addition experiment was established in 2014, with 3 treatments based on the
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atmospheric N-deposition rate: ambient (0 N addition), moderate N (8.0 kg of N/ha/year N addition)
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and high N (40 kg of N/ha/year N addition). In each subplot, three quadrats were randomly assigned to
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different N-addition treatments. In total, there were five replications for each of the three treatments.
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During the summer (from late June to September) in 2014 and 2015, an NH4NO3 solution was added by
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hand three times to the floor of the quadrats in 3 equal amounts. The fertilizer was dried at 60 °C,
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weighed, mixed with 500 ml of deionized water (approximately 0.1 mm of rainfall) and added by
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spraying evenly on the quadrats of the moderate and high N-addition treatments; ambient quadrats
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received an equivalent volume of deionized water.
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2.4 Soil sampling and analysis
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In September 2015, the O layer (30 × 30 cm area) and mineral soil samples (0-5, 5-10 and 10-20
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cm increments, using a 251cm3 hollow steel tube) were collected in each quadrat. All samples were
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stored in a plastic bag in a refrigerator at 4 °C and transported quickly to the laboratory for further
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analysis. Fresh organic and mineral soil samples were passed through a 2mm sieve for
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water-extractable organic carbon and nitrogen (WEOC and WETN), microbial biomass carbon and
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nitrogen (MBC and MBN) and extracellular enzyme activity analyses. WEOC and WETN were 6
ACCEPTED MANUSCRIPT analysed in some studies as a potential source of DOC and TDN (Fröberg et al., 2003; Zsolnay, 2003)
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and were measured in 10-g subsamples by extracting with 2 M KCl and analysing with a liquid CN
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analyser (Elementar vario TOC, Germany) after filtering through a 0.45-µm PTFE filter (Perakis and
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Hedin, 2001). MBC and MBN were determined using the chloroform fumigation extraction method
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(Vance et al., 1987), with a 1:4 ratio of soil: solution (w/v) for extraction with 0.5 M K2SO4; an
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extraction efficiency coefficient of 0.45 and 0.54 was taken into account when calculating MBC and
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MBN, respectively (Beck et al., 1997; Riggs and Hobbie, 2016). Saccharase (EC 3.2.1.26; SAC) and
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cellulase (EC 3.2.1.4; CEL) activities were measured according to the methods of Guan et al., (1986).
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Briefly, saccharase activity (using 5 g of fresh sample) was determined using3,5-dinitrosalicylic acid
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colorimetry with sucrose as the substrate. The amount of 3-amino-5-nitrosalicylicacid released over 24
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h was detected colourimetrically at 508 nm. Cellulase (using 5 g of fresh sample) was determined by
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nitrosalicylic acid colorimetry, and the amount of glucose released over 72 h was assayed
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colourimetrically at 540 nm. Soil pH was determined in a 2.5:1 deionized water-to-soil mixture using a
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pH electrode (HASH, HQ30D, American), and water pH values of the leachate were also measured by
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a pH electrode.
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2.5 Data analysis and statistics
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DOC and TDN fluxes (mg C/m2 and mg N/m2, respectively) of the leachate in each sample were
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calculated by multiplying the water volume by the DOC and TDN concentration, respectively. Annual
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DOC and TDN fluxes (mg C/m2/year and mg N/m2/year) were calculated, respectively, by summing
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the DOC and TDN fluxes throughout the year.
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A mixed linear model with repeated measures was used to analyse the effect of N addition on soil
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water DOC and TDN concentrations, efflux, and leachate pH. Treatment (3 levels, including ambient, 7
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spring from late March to late June, summer from late June to late September and autumn/winter from
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late September to late December; there was no water leachate during late December to late March) and
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their interactions were treated as fixed factors; sampling date (nested within season) and quadrats
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(treated as subject and nested within treatment) were treated as random factors. Sampling date was
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entered as a repeated effect of the first-order autoregressive covariance type. Treatment, soil depth,
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season, and the season × treatment × soil depth interaction term as the main effects were used to
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compare the DOC and TDN concentrations, efflux and leachate pH among treatments at different
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depths and different seasons and to compare the DOC and TDN concentrations, efflux, DOC:TDN
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ratio and leachate pH among seasons within the same depth and treatment, using a Bonferroni test
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(significant at 0.05). Two-way ANOVA was used to compare soil pH, MBC and MBN, WEOC and
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WETN and extracellular enzyme activities among treatments using a general linear model (GLM).
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Treatment, soil depth and the interaction between treatment and soil depth were treated as fixed factors,
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and the main effects were compared using the Bonferroni test. The data were transformed, when
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necessary, to meet the assumptions of ANOVA for a mixed linear model and two-way ANOVA. To
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detect the effect of precipitation on soil water DOC concentration, the amount of precipitation between
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the two soil water sampling times was calculated and used in a simple ordinary least squares (OLS)
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linear regression associated with the DOC concentration. All analyses were performed using SPSS
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19.0.
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3.1 DOC and TDN leaching and seasonal pattern for the ambient plot
Results
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ACCEPTED MANUSCRIPT The concentration and flux of DOC and TDN decreased significantly from the forest O layer to
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the mineral layer (p< 0.05 for all comparisons) in the ambient plot, but the C:N (DOC:TDN) ratio of
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the leachate was similar between depths (p> 0.10 for all comparisons). For example, the average
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concentrations of DOC and TDN decreased from 38.2 mg/L (with a range of 7.2 to 175.1 mg/L) and
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1.1 mg/L (with a range of 0.3 to 6.6 mg/L) in the O layer to 26.2 mg/L (with a range of 10.2 to 77.4
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mg/L) and 0.8 mg/L (with a range of 0.3 to 2.8 mg/L) in the mineral soil, respectively, and the average
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C:N ratios were 35.5 and 34.6 in the O and mineral soil, respectively. The annual average DOC release
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from the O layer and mineral layer (17.3 g C/m2/year and 8.9 g C/m2/year, respectively) accounted for
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approximately 0.6% and 0.2% of the soil organic carbon storage in the O layer (with C storage being
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2900 g C/m2) and the O layer plus the top 20 cm of the mineral soil (with C storage being 4150g C/m2),
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respectively, indicating a limited effect of DOC leaching on soil C status in the top soils of this forest.
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DOC and TDN concentrations were low during the summer, gradually increased during autumn
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and winter and quickly increased in the spring (Fig. 2A). The low DOC and TDN concentrations in the
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summer may partly be due to dilution from higher precipitation during this season, as a negative
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relationship between DOC concentration and precipitation was observed (Fig. 3). DOC flux had a
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similar seasonal pattern to that of DOC concentration, but the flux decreased in the later stage of
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autumn/winter (Fig. 2B), which was due to reduced precipitation during this stage (Fig. 1). The
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seasonal average O layer DOC flux increased from spring to summer to autumn/winter, but there was
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no clear trend in DOC flux in the mineral soil (Fig. S1). The C:N ratio of the leachate in the O layer
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increased significantly from spring (with a ratio of 33) to summer (with a ratio of 36) to autumn/winter
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(with a ratio of 39) (p< 0.05 for the comparisons).
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Figure 2 and 3
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3.2 The effect of N addition on DOC and TDN leaching High nitrogen addition reduced the concentration of DOC in the O layer with marginal
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significance (p = 0.062) but decreased the O layer DOC flux significantly (p = 0.017; Table 2) due,
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likely, to the changes in the treatment variations resulting partly from high spatial and temporal
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variations in water efflux, even though there was no difference in annual water efflux among the
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treatments in either the O or mineral layers (p> 0.1 for comparisons among treatments; Table 2). By
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contrast, high nitrogen addition had no effect on the concentration and flux of DOC released from
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mineral soil (p> 0.1 for both concentration and flux), nor did moderate nitrogen addition significantly
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affect the concentration and flux of DOC from the O layer or from the mineral soil (p> 0.1 for both
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concentrations and flux at both depths; Table 2).
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Nevertheless, the effects of N addition on DOC leaching showed seasonal dependency. N addition
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did not affect the DOC flux from the O layer during spring or the DOC flux from mineral soil during
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any season, but in autumn/winter, moderate and high N addition significantly decreased the O layer
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DOC flux (p = 0.04 and p = 0.003 for moderate and high N addition, respectively). High N addition
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also had a marginally negative effect on O layer DOC flux in the summer (p = 0.07). There was a no
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clear seasonal pattern of DOC flux under N addition different from that observed in the ambient
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treatment (Fig. S1).
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In contrast to DOC, N addition increased the TDN concentration and flux in the summer when
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NH4NO3 was added (p < 0.05). This phenomenon was correlated with a quick increase in TDN release
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after each N addition (Fig. 2). In the other two seasons, TDN release was not significantly affected by 10
ACCEPTED MANUSCRIPT N addition (p > 0.10). High N addition significantly increased the annual TDN release from the O layer
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(p < 0.05; Table 2). The C:N ratio of the leachate decreased significantly under N addition only in the O
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layer during summer (p = 0.024 and p = 0.001 for the moderate and high N addition, respectively; data
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not shown); this result was not observed in the other seasons or in the mineral soil (p> 0.1 for all
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comparisons).
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Table 2
3.3 The effect of N addition on MBC and extracellular enzyme activities
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In the O horizon, high N addition significantly reduced the MBC and MBN compared to the
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ambient treatment (Two-way ANOVA, p <0.01 for both MBC and MBN; Fig. 4), but there was no
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difference in the MBC: MBN ratio among the three treatments (p =0.79). High N addition also
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decreased saccharase and cellulase activities (p =0.048 and p = 0.012 for saccharase and cellulase,
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respectively; insert figures in Fig. 4). High N addition significantly decreased both WEOC and WETN
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concentrations in the O layer (two-way ANOVA test, p = 0.04 for WEOC and p = 0.002 for WETN;
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data not shown), and moderate N addition had no significant effect on either WEOC or WETN (p >
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0.10 for both comparisons). There was no difference in soil pH among treatments after two years of N
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addition (p > 0.10; Table 1). Additionally, there was no difference in leachate pH among treatments by
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season or year (p > 0.10; Fig. 5).
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Figure 4 and 5
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4.
Discussion Annual fluxes of DOC and TDN in the ambient plots of this montane forest were 17.3 g 11
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mineral layer, respectively, which were within the range for temperate forests (Michalzik et al., 2001;
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Neff and Asner, 2001). The annual TDN efflux from the O and mineral layers accounted for
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approximately 63% and 37%, respectively, of the annual N deposition of 0.8 g N/m2/year, which
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indicated net retention and possibly N limitation. Additionally, annual TDN efflux from the O layer
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accounted for approximately 37% (in the moderate N-addition plot) and 33% (in the high N-addition
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plot) of the total amount of N input from experimental addition and atmospheric deposition, indicating
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that the temporarily elevated N did not change the N-limited status of this moderate-N-deposition
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forest.
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4.1 N addition decreases DOC efflux
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The biotic mechanism responsible for DOC production and the abiotic factors responsible for
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DOC retention in the soil are both generally used to explain the changes in DOC leaching from N
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addition. For example, in an NH4NO3-addition experiment in the Harvard Forest, elevated DOC
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concentration in the hardwood forest floor was suggested to be due to increased biodegradable DOC
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production derived from an increased number of fine roots (Yano et al., 2000). In another hardwood
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forest in Michigan, USA, nitrate (NaNO3) addition was found to significantly increase DOC leachate
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from the mineral layer (Pregitzer et al., 2004), and this increased DOC leaching was suggested to be
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responsible for an increase in soluble phenolics in the leachate associated with N-induced suppression
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of phenol oxidase (DeForest et al., 2004; Sinsabaugh et al., 2004; Waldrop and Zak, 2006). However,
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in a later study by Smemo et al. (2007) in the same Michigan hardwood forest, an increase in soluble
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phenolics (increased by 1 mg C/L) contributed to less than 10% of the increments in DOC (an increase
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of 6.6 mg C/L in the ambient to 18.5 mg C/L from N addition); thus, these authors concluded that the
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decreased retention of DOC in mineral soil associated with higher pH from NaNO3 addition could
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explain the increase in DOC efflux. With NH4NO3addition, by contrast, a greater retention of DOC, due to reduced pH, has usually
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been suggested to result in a reduction in DOC release from mineral soil (Evans et al., 2008; Hagedorn
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et al., 2012; Lu et al., 2013). In this study, however, NH4NO3addition did not affect soil pH, leachate
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pH and DOC concentration or flux from mineral soil, indicating that DOC retention during transport
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along the soil profile may play a limited role in reducing DOC by N addition, at least in the short term.
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The low soil clay content in this montane forest may constrain DOC retention in the mineral soil, as
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soil clay is suggested to be an important factor in absorbing DOC (Kalbitz et al., 2000; Neff and Asner,
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2001). In addition, the effect of N addition on DOC retention was difficult to detect using zero-tension
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lysimeters; rapidly flowing water, which was captured by zero-tension lysimeters, could decrease the
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absorption/precipitation of DOC by mineral soil (Kaiser and Kalbitz, 2012) and thus diminish the effect
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of N addition on DOC retention.
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In contrast, short-term high N addition decreased the DOC concentration and flux from the O
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layer, and this decrease was thought to be likely associated with a reduction in DOC production in
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addition to greater retention with the N-addition treatment. First, WEOC, being a product associated
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with soil C decomposition (Kalbitz et al., 2000), was found to decrease under high N addition in the O
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layer. Second, N addition decreased the microbial biomass in the O layer, which is consistent with the
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results of many other studies and several meta-analyses (DeForest et al., 2004; Treseder, 2008; Greaver
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et al., 2016; Yue et al., 2016). On one hand, low microbial biomass could suppress litter decomposition
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(Riggs and Hobbie, 2016) and consequently reduce the DOC production in the O layer. On the other
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hand, lower MBC could lead to lower DOC, as MBC is a source of DOC (Kalbitz et al., 2000). Third,
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of cellulase, thus reducing cellulase-derived DOC, especially during the early stage of litter
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decomposition. For example, lower DOC export from the O layer with N addition from litterfall during
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the autumn/winter season may partially support this speculation (litterfall is higher in autumn in this
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forest, Fig. S2).
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4.2 Origin of DOM for the ambient plot and factors affecting seasonal patterns of DOC
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The highest DOC concentration is usually observed in the summer (Kalbitz et al., 2000; Fröberg
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et al., 2009; Wu et al., 2014), as many processes, including soil organic carbon decomposition and fine
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root and microbe activities associated with DOC production increase with summer temperatures
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(Kalbitz et al., 2000)(Gielen et al., 2011).In this and other forests (Pregitzer et al., 2004; Lu et al.,
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2013), however, DOC production might follow litterfall seasonality (higher litterfall in autumn than in
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spring and summer in this forest, as shown in Fig. S2, data from Luo et al., (2003)), showing a higher
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DOC or TDN concentration in autumn/winter other than in summer. This speculation was supported to
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a certain extent by a significantly higher DOC:TDN ratio in leachate in autumn/winter (with a ratio of
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39) than in other seasons (with a ratio of 33 and 36 in spring and summer, respectively) and by the
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observation that the leachate DOC:TDN ratio was similar to the C:N ratio in fresh litterfall (with a ratio
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of 43), except for that of the O layer (with a ratio of 14). However, here, the TDN was not separated
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into dissolved inorganic N (including NH
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DOC from atmospheric deposition was also not separated: therefore, the DOC:TDN ratio used here
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may introduce some error to understanding the dynamic of DOC production, and the DOC:DON ratio
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of the soil leachate, without atmospheric deposition, should be calculated and used. Additionally, if the
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and NO3-) and dissolved organic N (DON), and TDN and
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litterfall has a significant effect on DOC production, there would be a high DOC concentration in the
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early spring, as was observed in this study, which corresponds to litter senescence in the previous fall
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and soil flushing during snow melt. Nevertheless, some studies using 13C-or 14C-labelled litter reported that DOC derived from fresh
305
litter contributed to a small proportion of the quantity of DOC released from the O layer because of
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DOC sorption and decomposition during transport (Fröberg et al., 2007; Müller et al., 2009). However,
307
in their later study with higher amounts of precipitation (approximately 1300 mm) compared to their
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earlier studies with lower amounts of precipitation (688 mm in Fröberg et al., (2007) and 712 mm
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Fröberg et al.,(2003)), Fröberg et al. (2009) found that approximately 50% of the DOC that leached
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from the litter and the organic humus layer originated from
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litter-derived DOC retention was limited in the O horizon. Thus, the contribution of fresh litter to DOC
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efflux in the deeper layer was dependent on the amount of precipitation, and under conditions of high
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precipitation, such as in this montane forest with an MAP of 1940 mm, fast water infiltration could
314
decrease sorption and microbial decomposition, resulting in a greater amount of litter-derived DOM
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transported and to deeper soil (Cleveland et al., 2006; Kaiser and Kalbitz, 2012). Furthermore,
316
zero-tension lysimeters, as used in both layers in this study, were reported to capture greater amounts
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of more recent C than were suction cups at the same depth (Müller et al., 2009) because zero-tension
318
lysimeters can more successfully capture the rapidly flowing soil water from rainfall and from
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macropores.
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C-enriched litter, which suggested that
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Dilution by precipitation had a significant effect on the seasonal patterns of DOC concentration,
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which is consistent with other findings (Fang et al., 2009; Sleutel et al., 2009). Nevertheless, the
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seasonal patterns of DOC flux still showed a gradual increase from spring to summer to autumn/winter 15
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in the O layer in the ambient plot, indicating a limited magnitude of the effect of precipitation dilution.
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Additionally, N addition also could change the DOC seasonal pattern due to a reduction in DOC in
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autumn/winter from N addition, as discussed above.
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5.
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In this montane forest, high (5 times more than the atmospheric N deposition) and moderate
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(equal to the atmospheric N deposition) N deposition decreased the DOC concentration and flux from
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the O layer during the autumn/winter season in the short term, but N addition had no effect on the DOC
331
in mineral soil, indicating a different response of DOC to N addition between the O and mineral layers.
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Moreover, the observed decrease in microbial biomass C and saccharase and cellulose activities
333
suggested that the decrease in DOC under N addition could most likely be attributed to reduced
334
production of DOC from fresh litter. There was a non-significant change in DOC retention in the short
335
term, but long-term observation was needed to examine a likely change in DOC sorption in a forest soil
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composed of such a high proportion of sand. The observed decrease in DOC leaching was in
337
accordance with a common phenomenon-a decrease in CO2 emission under N addition - indicating that
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N deposition may likely result in a decrease in both gaseous and liquid C loss. Finally, the
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results emphasized that fresh litter-derived DOC plays a significant role in the seasonal patterns of
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DOC flux and showed that N addition can alter seasonal patterns as a result of a decrease in DOC
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leaching, which is likely associated with the inhibition of DOC production during autumn/winter.
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Acknowledgements
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We thank Dr. Zhang Shuang for his help and suggestions in the statistical analysis. This work was 16
ACCEPTED MANUSCRIPT supported by the National Key Research and Development Program of China (2016YFC0502105);
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Key Research Program of Frontier Sciences, CAS (QYZDJ-SSW-DQC006), the 135 Strategic Program
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of the Institute of Mountain Hazards and Environment, CAS (No. SDS-135-1702), the Youth
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Innovation Promotion Association CAS (2018406), and the Natural Science Foundation of China
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(41301219).
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Waldrop, M.P., Zak, D.R., 2006. Response of Oxidative Enzyme Activities to Nitrogen Deposition Affects Soil Concentrations of Dissolved Organic Carbon. Ecosystems 9, 921-933. Wu, H., Peng, C., Moore, T., Hua, D., Li, C., Zhu, Q., Peichl, M., Arain, M., Guo, Z., 2014. Modeling dissolved organic carbon in temperate forest soils: TRIPLEX-DOC model development and validation. Geoscientific Model Development 7, 867-881. Yano, Y., McDowell, W.H., Aber, J.D., 2000. Biodegradable dissolved organic carbon in forest soil solution and effects of chronic nitrogen deposition. Soil Biology and Biochemistry 32, 1743-1751. Yue, K., Peng, Y., Peng, C., Yang, W., Peng, X., Wu, F., 2016. Stimulation of terrestrial ecosystem carbon storage by nitrogen addition: a meta-analysis. Scientific Reports 6, 19895. Zsolnay, Á., 2003. Dissolved organic matter: artefacts, definitions, and functions. Geoderma 113, 187-209.
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Figure 1. Seasonal temperature and precipitation patterns during the study period. The red line
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and histogram indicate temperature and precipitation, respectively, and the blue histogram indicates
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that, during this period with limited precipitation, there was no water leachate collected under the
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O-layer or mineral layer.
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Figure 2. Seasonal patterns of dissolved organic carbon (DOC) and total dissolved nitrogen (TDN)
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concentrations (A) and fluxes (B) from the organic layer and mineral layer (O layer + the top 20
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cm of mineral soil) under different treatments. Values are the mean ± 1SE. The black dashed line
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indicates the date of N addition.
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Figure 3. Association of precipitation and dissolved organic carbon (DOC) in the organic layer
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and mineral layer under ambient conditions.
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Figure 4.Comparison of microbial biomass carbon and nitrogen and saccharase and cellulase
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activities (inset figures) among different treatments. Values are the mean ± 1SE (n = 5 for each
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treatment at each depth). Different letters mean that there are significant differences among different
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treatments at the same depth.
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491 492
Figure 5.Seasonal leachate pH patterns from the organic layer and mineral layer under different
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treatments. Values are the mean ± 1SE. 21
ACCEPTED MANUSCRIPT 494 Figure S1. Seasonal average of dissolved organic carbon (DOC) and total dissolved nitrogen
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(TDN) concentrations (A) and fluxes (B) from the organic layer and mineral layer under different
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treatments. Values are the mean ± 1SE. Different lowercase letters mean that there are significant
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differences among different seasons for the same treatment, and different uppercase letters mean that
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there are significant differences among different treatments in the same season in a mixed linear model.
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Figure S2.Seasonal litterfall pattern in this forest. Data are from Luo et al.(2003).
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ACCEPTED MANUSCRIPT Table 1 Soil properties under different N-addition treatments Treatment Organic layer
Mineral soil (top 5 cm) STN
SOC
STN
(g/kg)
(g/kg)
pH
(g/kg)
(g/kg)
Ambient
343.4 ± 33.3
12.7 ± 1.5 6.06 ± 0.01 18.9 ± 1.3
1.2 ± 0.1 5.56 ± 0.04 <0.1
14.1 ± 0.9 85.9 ± 0.9
Moderate N
393. 3 ± 34.4 15.0 ± 0.9 6.07 ± 0.01 25.2 ± 4.3
1.7 ± 0.3 5.49 ± 0.08 <0.1
13.0 ± 1.5 86.9 ± 1.5
High N
386.6 ± 8.9
17.6 ± 0.9 6.05 ± 0.01 19.6 ± 3.2
1.4 ± 0.2 5.45 ± 0.07 <0.1
14.9 ± 1.4 85.0 ± 1.3
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pH
Clay
Silt
Sand
(%)
(%)
(%)
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SOC
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Note: Ambient, Moderate N and High N indicate ambient treatment with 0 N addition, 8.0 kg N/ha/year addition and 40 kg N/ha/year addition, respectively; SOC and STN are soil organic carbon and soil
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ACCEPTED MANUSCRIPT Table 2 Annual dissolved organic carbon (DOC) and total dissolved nitrogen (TDN) released from the organic (O) and mineral (O + 20 cm of mineral soil) layers TDN flux
DOC flux 2
Water flux
2
(g C/m /year, mean ± SE)
(g N/m /year, mean ± SE)
a
Mineral layer
a
8.9± 1.1
0.5 ± 0.1 a
0.3± 0.1
Moderate N 16.1± 1.9 ab
9.8± 2.0
0.6 ± 0.1 a
0.4± 0.1
High N
6.4± 1.8
1.6 ± 0.4 b
0.5± 0.1
Ambient
O layer
17.3± 2.6 a 13.4± 2.0 b
O layer
(mm/year, mean ± SE)
Mineral layer O layer
Mineral layer
543± 37 344± 44
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Treatment
517± 24 371± 65
503± 20 344± 81
Note: a, Different letters indicate significant differences among the treatments at the 0.05 significance
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addition and 40 kg N/ha/year addition, respectively.
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level; Ambient, Moderate N and High N indicate ambient treatment with 0 N addition, 8.0 kg N/ha/year
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ACCEPTED MANUSCRIPT Highlights: 1.
Short-term N addition decreased DOC leaching from the organic layer but not from mineral soil
2.
N addition reduced microbial biomass, saccharase and cellulose activities, likely inducing lower
3.
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DOC production N-induced reduction in DOC leaching was most likely due to lower DOC production and not to DOC retention
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Seasonally different changes in DOC under N addition affected the seasonal pattern of DOC
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leaching
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S0038-0717(18)30301-8
DOI:
10.1016/j.soilbio.2018.09.006
Reference:
SBB 7273
To appear in:
Soil Biology and Biochemistry
Received Date: 21 April 2018 Revised Date:
24 August 2018
Accepted Date: 5 September 2018
Please cite this article as: Chang, R., Li, N., Sun, X., Hu, Z., Bai, X., Wang, G., Nitrogen addition reduces dissolved organic carbon leaching in a montane forest, Soil Biology and Biochemistry (2018), doi: 10.1016/j.soilbio.2018.09.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Title: Nitrogen addition reduces dissolved organic carbon leaching in a montane forest
2 Authors: Ruiying Chang 1, Na Li 2, Xiangyang Sun 1, Zhaoyong Hu 1, Xuesong Bai 1,
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Genxu Wang 1
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1
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Hazards and Environment, Chinese Academy of Sciences, Chengdu, China
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2
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Ecological Restoration and Biodiversity Conservation Key Laboratory of SichuanProvince,
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CAS Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization &
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Key Laboratory of Mountain Environment Evolvement and Regulation, Institute of Mountain
Chengdu Instituteof Biology, Chinese Academy of Sciences, Chengdu, China
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Corresponding author, Genxu Wang, E-mail: [email protected];
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ACCEPTED MANUSCRIPT Abstract: Dissolved organic carbon (DOC) plays a significant role in the forest soil carbon cycle and
16
can be regulated by nitrogen (N) addition. However, the regulatory direction, mechanism and seasonal
17
pattern of DOC under N addition are less clear. Here, in a montane evergreen forest located at the
18
eastern edge of the Tibetan Plateau, 2 levels of N were applied over 2 years to determine the effects of
19
N addition on DOC release from organic (O layer) and mineral soil. Frequent sampling revealed that
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high levels of N addition could decrease the concentration of DOC and the flux from the O layer but
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not from mineral soil and that moderate N addition had no effect on DOC leaching from either the O or
22
mineral layer. The effect of N addition on DOC leaching from the O layer was seasonally dependent,
23
showing a significant reduction in DOC leaching during autumn/winter but no changes during summer
24
and spring. This seasonally different response of DOC to N addition affected the seasonal pattern of
25
DOC leaching. Soil and leachate pH were not influenced by N addition in the short term, indicating
26
that there was not enough difference in DOC retention by mineral soil to significantly affect DOC
27
leaching under N addition. In contrast, N addition-derived reduction in DOC leaching was likely to be
28
due to suppressed fresh litterfall–derived DOC production during autumn/winter; this speculation was
29
supported by lower values of O layer water-extractable organic carbon and microbial biomass carbon
30
as well as lower saccharase and cellulose activities found with high N addition. These results suggested
31
that the processes in control of DOC leaching and their responses to N addition were different for O
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and mineral soil and that short-term N addition could decrease O-layer DOC leaching, which is likely
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associated with decreased DOC production rather than greater DOC retention.
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Key words: Anthropogenic nitrogen deposition; Zero-tension lysimeters; Seasonal pattern; DOC
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production; DOC retention; Extracellular enzyme activity 2
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1.
Introduction Elevated active nitrogen (N) deposition is occurring in many areas around world, especially in
39
China (Liu et al., 2013). The effects of elevated N deposition on forest soil carbon (C) pools and cycles
40
have been widely documented; soil C pools are generally found to be enhanced under N addition
41
(Janssens et al., 2010; Greaver et al., 2016; Entwistle Elizabeth et al., 2017), which is associated with
42
higher C input derived from above-and/or below-ground litter and lower C-CO2 loss (Janssens et al.,
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2010; Huang et al., 2011). However, leaching of dissolved organic carbon (DOC), an important
44
component in the C cycle that plays a significant role in soil C transport, transformation and formation
45
in the mineral soil (Kalbitz and Kaiser, 2008; Kramer Marc et al., 2012), is less documented, and the
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responses of DOC to N addition are debatable and unclear (Evans et al., 2008).
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Some authors have reported positive effects of N addition on DOC leaching (Pregitzer et al., 2004;
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Camino-Serrano et al., 2016), while others have reported negative effects (Hagedorn et al., 2012; Lu et
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al., 2013) and yet others report neutral effects (McDowell et al., 2004; Lovett et al., 2013). These
50
varying responses of DOC leaching to N addition have been attributed to the effects of various forms of
51
N on soil pH and DOC sorption in the mineral soil (e.g., Evans et al., 2008). Ammonium salt addition
52
could generally reduce DOC leaching by enhancing DOC retention in the mineral soil through a lower
53
soil and/or leachate pH with N addition, while the increase in DOC leaching with sodium nitrate
54
addition is associated with an increase in pH (Pregitzer et al. 2004; Evans et al., 2008; Lu et al. 2013).
55
The responses of DOC to N addition are also soil depth-dependent. Hagedorn et al. (2012) reported
56
reduced DOC release from mineral soil layers after NH4NO3addition, but leaching of DOC from the
57
litter layer did not change. The processes that might affect DOC leaching from the organic (O) and
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mineral layers are likely to be different; however, previous research has not yet fully defined the
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differences. Patterns of DOC leaching also display seasonal trends, showing generally high levels of DOC
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during summer and/or fall (Kaiser et al., 2002; Dittman et al., 2007), high DOC concentrations in the
62
spring (Pregitzer et al., 2004; Smemo et al., 2007), or no clear trend (Hagedorn et al., 2012). Seasonal
63
changes in DOC are often in response to biotic and abiotic factors, such as the decomposition of
64
litterfall, which is responsible for the high DOC release in spring (Pregitzer et al., 2004), and are likely
65
to be influenced by N addition; consequently, the changes in DOC leachate due to N addition may also
66
be seasonally dependent, and the seasonal pattern of DOC is also altered by N addition. Nevertheless,
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there is limited understanding of the seasonal differences in DOC levels with N addition: more in-depth
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study of this subject will improve the understanding of mechanisms of DOC production, retention and
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leaching.
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In this study, an NH4NO3-addition experiment was conducted in a subalpine evergreen conifer
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forest with moderate N deposition, and the DOC leachate was collected at frequent intervals of 5-7
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days from the forest O layer and mineral soil for 2 years. The objectives of this study are (1) to detect
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the differences in the responses of DOC leaching from the O and mineral layers and to distinguish the
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processes affecting DOC leaching in different horizons; and (2) to examine the seasonality of the
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responses of DOC to N addition.
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2.
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2.1 Study area
Material and methods
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We carried out our work at the long-term observation plot of the Alpine Ecosystem Observation
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and Experiment Station (29°34′N, 101°0′E) located on the eastern slope of Mt. Gongga, at the eastern 4
ACCEPTED MANUSCRIPT edge of the Tibetan Plateau. The study plot is at an altitude of 3000 m asl, with a gentle slope of less
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than 6 degrees. The area is dominated by the south eastern (Pacific) monsoon, with a long-term mean
83
annual temperature (MAT) of 3.8 °C and a mean annual precipitation (MAP) of 1940 mm, 60% of
84
which falls during June to September. The temperature and precipitation data used in this study were
85
obtained from a nearby weather station (Fig. 1). The snow depth in the winter is approximately 50 cm,
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and all the snow melt occurs from March to April. The plant community is dominated by Abies fabri
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(Mast.) Craib. The soil type is classified as a Cambisol, according to the World Reference Base for Soil
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Resources (WRB), with low clay content (Table 1). The average depth of the organic (O) layer is
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approximately 8 cm. The total atmospheric N-deposition (wet and dry deposition) rate is approximate
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8.0 kg N/ha/year for the area (Liu et al., 2008).
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Figure 1
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2.2 Leaching experiment and sampling
In this plot, five 6×6m subplots were selected randomly, and in each subplot, three 2×2m quadrats
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were established. In April 2013 (approximately 1 year prior to the experiment), in each quadrat, two
96
zero-tension tray lysimeters (755 cm2 per lysimeter, made of silicon) were installed: one beneath the O
97
layer and the other beneath the mineral layer (O layer + 20 cm of mineral soil), in adjacent areas. Each
98
lysimeter was connected with Tygon tubing to a 4L brown bottle (installed at approximately 40 cm
99
underground) to collect the leachate. In 2014 and 2015, the leachate was collected and sampled at an
100
interval of 5-7 days from late March to late December of each year and at an interval of every 15 days
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during the remaining time (no water was collected during this period due to limited precipitation, as
102
shown in Fig. 1). However, water samples were also taken the day after heavy rains. The leachate
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station laboratory immediately for pH, DOC and total dissolved nitrogen (TDN) analyses. All
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collectors were washed with deionized water following each collection. The concentrations of DOC
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and TDN for all water samples were analysed using a liquid CN analyser (Vario TOC, Elementar
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Instruments Inc, Germany) after filtering with a 0.45-µm PTFE filter.
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2.3 Nitrogen addition experiment
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The nitrogen addition experiment was established in 2014, with 3 treatments based on the
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atmospheric N-deposition rate: ambient (0 N addition), moderate N (8.0 kg of N/ha/year N addition)
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and high N (40 kg of N/ha/year N addition). In each subplot, three quadrats were randomly assigned to
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different N-addition treatments. In total, there were five replications for each of the three treatments.
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During the summer (from late June to September) in 2014 and 2015, an NH4NO3 solution was added by
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hand three times to the floor of the quadrats in 3 equal amounts. The fertilizer was dried at 60 °C,
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weighed, mixed with 500 ml of deionized water (approximately 0.1 mm of rainfall) and added by
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spraying evenly on the quadrats of the moderate and high N-addition treatments; ambient quadrats
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received an equivalent volume of deionized water.
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2.4 Soil sampling and analysis
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In September 2015, the O layer (30 × 30 cm area) and mineral soil samples (0-5, 5-10 and 10-20
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cm increments, using a 251cm3 hollow steel tube) were collected in each quadrat. All samples were
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stored in a plastic bag in a refrigerator at 4 °C and transported quickly to the laboratory for further
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analysis. Fresh organic and mineral soil samples were passed through a 2mm sieve for
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water-extractable organic carbon and nitrogen (WEOC and WETN), microbial biomass carbon and
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nitrogen (MBC and MBN) and extracellular enzyme activity analyses. WEOC and WETN were 6
ACCEPTED MANUSCRIPT analysed in some studies as a potential source of DOC and TDN (Fröberg et al., 2003; Zsolnay, 2003)
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and were measured in 10-g subsamples by extracting with 2 M KCl and analysing with a liquid CN
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analyser (Elementar vario TOC, Germany) after filtering through a 0.45-µm PTFE filter (Perakis and
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Hedin, 2001). MBC and MBN were determined using the chloroform fumigation extraction method
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(Vance et al., 1987), with a 1:4 ratio of soil: solution (w/v) for extraction with 0.5 M K2SO4; an
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extraction efficiency coefficient of 0.45 and 0.54 was taken into account when calculating MBC and
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MBN, respectively (Beck et al., 1997; Riggs and Hobbie, 2016). Saccharase (EC 3.2.1.26; SAC) and
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cellulase (EC 3.2.1.4; CEL) activities were measured according to the methods of Guan et al., (1986).
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Briefly, saccharase activity (using 5 g of fresh sample) was determined using3,5-dinitrosalicylic acid
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colorimetry with sucrose as the substrate. The amount of 3-amino-5-nitrosalicylicacid released over 24
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h was detected colourimetrically at 508 nm. Cellulase (using 5 g of fresh sample) was determined by
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nitrosalicylic acid colorimetry, and the amount of glucose released over 72 h was assayed
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colourimetrically at 540 nm. Soil pH was determined in a 2.5:1 deionized water-to-soil mixture using a
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pH electrode (HASH, HQ30D, American), and water pH values of the leachate were also measured by
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a pH electrode.
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2.5 Data analysis and statistics
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DOC and TDN fluxes (mg C/m2 and mg N/m2, respectively) of the leachate in each sample were
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calculated by multiplying the water volume by the DOC and TDN concentration, respectively. Annual
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DOC and TDN fluxes (mg C/m2/year and mg N/m2/year) were calculated, respectively, by summing
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the DOC and TDN fluxes throughout the year.
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A mixed linear model with repeated measures was used to analyse the effect of N addition on soil
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water DOC and TDN concentrations, efflux, and leachate pH. Treatment (3 levels, including ambient, 7
ACCEPTED MANUSCRIPT moderate and high N addition), soil depth (2 levels, O layer and mineral layer) and season (3 levels,
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spring from late March to late June, summer from late June to late September and autumn/winter from
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late September to late December; there was no water leachate during late December to late March) and
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their interactions were treated as fixed factors; sampling date (nested within season) and quadrats
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(treated as subject and nested within treatment) were treated as random factors. Sampling date was
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entered as a repeated effect of the first-order autoregressive covariance type. Treatment, soil depth,
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season, and the season × treatment × soil depth interaction term as the main effects were used to
154
compare the DOC and TDN concentrations, efflux and leachate pH among treatments at different
155
depths and different seasons and to compare the DOC and TDN concentrations, efflux, DOC:TDN
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ratio and leachate pH among seasons within the same depth and treatment, using a Bonferroni test
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(significant at 0.05). Two-way ANOVA was used to compare soil pH, MBC and MBN, WEOC and
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WETN and extracellular enzyme activities among treatments using a general linear model (GLM).
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Treatment, soil depth and the interaction between treatment and soil depth were treated as fixed factors,
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and the main effects were compared using the Bonferroni test. The data were transformed, when
161
necessary, to meet the assumptions of ANOVA for a mixed linear model and two-way ANOVA. To
162
detect the effect of precipitation on soil water DOC concentration, the amount of precipitation between
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the two soil water sampling times was calculated and used in a simple ordinary least squares (OLS)
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linear regression associated with the DOC concentration. All analyses were performed using SPSS
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19.0.
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3.
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3.1 DOC and TDN leaching and seasonal pattern for the ambient plot
Results
8
ACCEPTED MANUSCRIPT The concentration and flux of DOC and TDN decreased significantly from the forest O layer to
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the mineral layer (p< 0.05 for all comparisons) in the ambient plot, but the C:N (DOC:TDN) ratio of
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the leachate was similar between depths (p> 0.10 for all comparisons). For example, the average
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concentrations of DOC and TDN decreased from 38.2 mg/L (with a range of 7.2 to 175.1 mg/L) and
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1.1 mg/L (with a range of 0.3 to 6.6 mg/L) in the O layer to 26.2 mg/L (with a range of 10.2 to 77.4
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mg/L) and 0.8 mg/L (with a range of 0.3 to 2.8 mg/L) in the mineral soil, respectively, and the average
175
C:N ratios were 35.5 and 34.6 in the O and mineral soil, respectively. The annual average DOC release
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from the O layer and mineral layer (17.3 g C/m2/year and 8.9 g C/m2/year, respectively) accounted for
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approximately 0.6% and 0.2% of the soil organic carbon storage in the O layer (with C storage being
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2900 g C/m2) and the O layer plus the top 20 cm of the mineral soil (with C storage being 4150g C/m2),
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respectively, indicating a limited effect of DOC leaching on soil C status in the top soils of this forest.
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DOC and TDN concentrations were low during the summer, gradually increased during autumn
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and winter and quickly increased in the spring (Fig. 2A). The low DOC and TDN concentrations in the
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summer may partly be due to dilution from higher precipitation during this season, as a negative
183
relationship between DOC concentration and precipitation was observed (Fig. 3). DOC flux had a
184
similar seasonal pattern to that of DOC concentration, but the flux decreased in the later stage of
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autumn/winter (Fig. 2B), which was due to reduced precipitation during this stage (Fig. 1). The
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seasonal average O layer DOC flux increased from spring to summer to autumn/winter, but there was
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no clear trend in DOC flux in the mineral soil (Fig. S1). The C:N ratio of the leachate in the O layer
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increased significantly from spring (with a ratio of 33) to summer (with a ratio of 36) to autumn/winter
189
(with a ratio of 39) (p< 0.05 for the comparisons).
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Figure 2 and 3
192 193
3.2 The effect of N addition on DOC and TDN leaching High nitrogen addition reduced the concentration of DOC in the O layer with marginal
195
significance (p = 0.062) but decreased the O layer DOC flux significantly (p = 0.017; Table 2) due,
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likely, to the changes in the treatment variations resulting partly from high spatial and temporal
197
variations in water efflux, even though there was no difference in annual water efflux among the
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treatments in either the O or mineral layers (p> 0.1 for comparisons among treatments; Table 2). By
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contrast, high nitrogen addition had no effect on the concentration and flux of DOC released from
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mineral soil (p> 0.1 for both concentration and flux), nor did moderate nitrogen addition significantly
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affect the concentration and flux of DOC from the O layer or from the mineral soil (p> 0.1 for both
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concentrations and flux at both depths; Table 2).
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Nevertheless, the effects of N addition on DOC leaching showed seasonal dependency. N addition
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did not affect the DOC flux from the O layer during spring or the DOC flux from mineral soil during
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any season, but in autumn/winter, moderate and high N addition significantly decreased the O layer
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DOC flux (p = 0.04 and p = 0.003 for moderate and high N addition, respectively). High N addition
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also had a marginally negative effect on O layer DOC flux in the summer (p = 0.07). There was a no
208
clear seasonal pattern of DOC flux under N addition different from that observed in the ambient
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treatment (Fig. S1).
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In contrast to DOC, N addition increased the TDN concentration and flux in the summer when
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NH4NO3 was added (p < 0.05). This phenomenon was correlated with a quick increase in TDN release
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after each N addition (Fig. 2). In the other two seasons, TDN release was not significantly affected by 10
ACCEPTED MANUSCRIPT N addition (p > 0.10). High N addition significantly increased the annual TDN release from the O layer
214
(p < 0.05; Table 2). The C:N ratio of the leachate decreased significantly under N addition only in the O
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layer during summer (p = 0.024 and p = 0.001 for the moderate and high N addition, respectively; data
216
not shown); this result was not observed in the other seasons or in the mineral soil (p> 0.1 for all
217
comparisons).
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Table 2
3.3 The effect of N addition on MBC and extracellular enzyme activities
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In the O horizon, high N addition significantly reduced the MBC and MBN compared to the
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ambient treatment (Two-way ANOVA, p <0.01 for both MBC and MBN; Fig. 4), but there was no
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difference in the MBC: MBN ratio among the three treatments (p =0.79). High N addition also
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decreased saccharase and cellulase activities (p =0.048 and p = 0.012 for saccharase and cellulase,
225
respectively; insert figures in Fig. 4). High N addition significantly decreased both WEOC and WETN
226
concentrations in the O layer (two-way ANOVA test, p = 0.04 for WEOC and p = 0.002 for WETN;
227
data not shown), and moderate N addition had no significant effect on either WEOC or WETN (p >
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0.10 for both comparisons). There was no difference in soil pH among treatments after two years of N
229
addition (p > 0.10; Table 1). Additionally, there was no difference in leachate pH among treatments by
230
season or year (p > 0.10; Fig. 5).
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Figure 4 and 5
232 233 234
4.
Discussion Annual fluxes of DOC and TDN in the ambient plots of this montane forest were 17.3 g 11
ACCEPTED MANUSCRIPT C/m2/year and 0.5 g N/m2/year from the O layer and 8.9 g C/m2/year and 0.3 g N/m2/year in the
236
mineral layer, respectively, which were within the range for temperate forests (Michalzik et al., 2001;
237
Neff and Asner, 2001). The annual TDN efflux from the O and mineral layers accounted for
238
approximately 63% and 37%, respectively, of the annual N deposition of 0.8 g N/m2/year, which
239
indicated net retention and possibly N limitation. Additionally, annual TDN efflux from the O layer
240
accounted for approximately 37% (in the moderate N-addition plot) and 33% (in the high N-addition
241
plot) of the total amount of N input from experimental addition and atmospheric deposition, indicating
242
that the temporarily elevated N did not change the N-limited status of this moderate-N-deposition
243
forest.
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4.1 N addition decreases DOC efflux
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The biotic mechanism responsible for DOC production and the abiotic factors responsible for
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DOC retention in the soil are both generally used to explain the changes in DOC leaching from N
247
addition. For example, in an NH4NO3-addition experiment in the Harvard Forest, elevated DOC
248
concentration in the hardwood forest floor was suggested to be due to increased biodegradable DOC
249
production derived from an increased number of fine roots (Yano et al., 2000). In another hardwood
250
forest in Michigan, USA, nitrate (NaNO3) addition was found to significantly increase DOC leachate
251
from the mineral layer (Pregitzer et al., 2004), and this increased DOC leaching was suggested to be
252
responsible for an increase in soluble phenolics in the leachate associated with N-induced suppression
253
of phenol oxidase (DeForest et al., 2004; Sinsabaugh et al., 2004; Waldrop and Zak, 2006). However,
254
in a later study by Smemo et al. (2007) in the same Michigan hardwood forest, an increase in soluble
255
phenolics (increased by 1 mg C/L) contributed to less than 10% of the increments in DOC (an increase
256
of 6.6 mg C/L in the ambient to 18.5 mg C/L from N addition); thus, these authors concluded that the
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decreased retention of DOC in mineral soil associated with higher pH from NaNO3 addition could
258
explain the increase in DOC efflux. With NH4NO3addition, by contrast, a greater retention of DOC, due to reduced pH, has usually
260
been suggested to result in a reduction in DOC release from mineral soil (Evans et al., 2008; Hagedorn
261
et al., 2012; Lu et al., 2013). In this study, however, NH4NO3addition did not affect soil pH, leachate
262
pH and DOC concentration or flux from mineral soil, indicating that DOC retention during transport
263
along the soil profile may play a limited role in reducing DOC by N addition, at least in the short term.
264
The low soil clay content in this montane forest may constrain DOC retention in the mineral soil, as
265
soil clay is suggested to be an important factor in absorbing DOC (Kalbitz et al., 2000; Neff and Asner,
266
2001). In addition, the effect of N addition on DOC retention was difficult to detect using zero-tension
267
lysimeters; rapidly flowing water, which was captured by zero-tension lysimeters, could decrease the
268
absorption/precipitation of DOC by mineral soil (Kaiser and Kalbitz, 2012) and thus diminish the effect
269
of N addition on DOC retention.
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In contrast, short-term high N addition decreased the DOC concentration and flux from the O
271
layer, and this decrease was thought to be likely associated with a reduction in DOC production in
272
addition to greater retention with the N-addition treatment. First, WEOC, being a product associated
273
with soil C decomposition (Kalbitz et al., 2000), was found to decrease under high N addition in the O
274
layer. Second, N addition decreased the microbial biomass in the O layer, which is consistent with the
275
results of many other studies and several meta-analyses (DeForest et al., 2004; Treseder, 2008; Greaver
276
et al., 2016; Yue et al., 2016). On one hand, low microbial biomass could suppress litter decomposition
277
(Riggs and Hobbie, 2016) and consequently reduce the DOC production in the O layer. On the other
278
hand, lower MBC could lead to lower DOC, as MBC is a source of DOC (Kalbitz et al., 2000). Third,
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280
of cellulase, thus reducing cellulase-derived DOC, especially during the early stage of litter
281
decomposition. For example, lower DOC export from the O layer with N addition from litterfall during
282
the autumn/winter season may partially support this speculation (litterfall is higher in autumn in this
283
forest, Fig. S2).
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4.2 Origin of DOM for the ambient plot and factors affecting seasonal patterns of DOC
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The highest DOC concentration is usually observed in the summer (Kalbitz et al., 2000; Fröberg
287
et al., 2009; Wu et al., 2014), as many processes, including soil organic carbon decomposition and fine
288
root and microbe activities associated with DOC production increase with summer temperatures
289
(Kalbitz et al., 2000)(Gielen et al., 2011).In this and other forests (Pregitzer et al., 2004; Lu et al.,
290
2013), however, DOC production might follow litterfall seasonality (higher litterfall in autumn than in
291
spring and summer in this forest, as shown in Fig. S2, data from Luo et al., (2003)), showing a higher
292
DOC or TDN concentration in autumn/winter other than in summer. This speculation was supported to
293
a certain extent by a significantly higher DOC:TDN ratio in leachate in autumn/winter (with a ratio of
294
39) than in other seasons (with a ratio of 33 and 36 in spring and summer, respectively) and by the
295
observation that the leachate DOC:TDN ratio was similar to the C:N ratio in fresh litterfall (with a ratio
296
of 43), except for that of the O layer (with a ratio of 14). However, here, the TDN was not separated
297
into dissolved inorganic N (including NH
298
DOC from atmospheric deposition was also not separated: therefore, the DOC:TDN ratio used here
299
may introduce some error to understanding the dynamic of DOC production, and the DOC:DON ratio
300
of the soil leachate, without atmospheric deposition, should be calculated and used. Additionally, if the
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+ 4
and NO3-) and dissolved organic N (DON), and TDN and
14
ACCEPTED MANUSCRIPT 301
litterfall has a significant effect on DOC production, there would be a high DOC concentration in the
302
early spring, as was observed in this study, which corresponds to litter senescence in the previous fall
303
and soil flushing during snow melt. Nevertheless, some studies using 13C-or 14C-labelled litter reported that DOC derived from fresh
305
litter contributed to a small proportion of the quantity of DOC released from the O layer because of
306
DOC sorption and decomposition during transport (Fröberg et al., 2007; Müller et al., 2009). However,
307
in their later study with higher amounts of precipitation (approximately 1300 mm) compared to their
308
earlier studies with lower amounts of precipitation (688 mm in Fröberg et al., (2007) and 712 mm
309
Fröberg et al.,(2003)), Fröberg et al. (2009) found that approximately 50% of the DOC that leached
310
from the litter and the organic humus layer originated from
311
litter-derived DOC retention was limited in the O horizon. Thus, the contribution of fresh litter to DOC
312
efflux in the deeper layer was dependent on the amount of precipitation, and under conditions of high
313
precipitation, such as in this montane forest with an MAP of 1940 mm, fast water infiltration could
314
decrease sorption and microbial decomposition, resulting in a greater amount of litter-derived DOM
315
transported and to deeper soil (Cleveland et al., 2006; Kaiser and Kalbitz, 2012). Furthermore,
316
zero-tension lysimeters, as used in both layers in this study, were reported to capture greater amounts
317
of more recent C than were suction cups at the same depth (Müller et al., 2009) because zero-tension
318
lysimeters can more successfully capture the rapidly flowing soil water from rainfall and from
319
macropores.
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C-enriched litter, which suggested that
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Dilution by precipitation had a significant effect on the seasonal patterns of DOC concentration,
321
which is consistent with other findings (Fang et al., 2009; Sleutel et al., 2009). Nevertheless, the
322
seasonal patterns of DOC flux still showed a gradual increase from spring to summer to autumn/winter 15
ACCEPTED MANUSCRIPT 323
in the O layer in the ambient plot, indicating a limited magnitude of the effect of precipitation dilution.
324
Additionally, N addition also could change the DOC seasonal pattern due to a reduction in DOC in
325
autumn/winter from N addition, as discussed above.
327
5.
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In this montane forest, high (5 times more than the atmospheric N deposition) and moderate
329
(equal to the atmospheric N deposition) N deposition decreased the DOC concentration and flux from
330
the O layer during the autumn/winter season in the short term, but N addition had no effect on the DOC
331
in mineral soil, indicating a different response of DOC to N addition between the O and mineral layers.
332
Moreover, the observed decrease in microbial biomass C and saccharase and cellulose activities
333
suggested that the decrease in DOC under N addition could most likely be attributed to reduced
334
production of DOC from fresh litter. There was a non-significant change in DOC retention in the short
335
term, but long-term observation was needed to examine a likely change in DOC sorption in a forest soil
336
composed of such a high proportion of sand. The observed decrease in DOC leaching was in
337
accordance with a common phenomenon-a decrease in CO2 emission under N addition - indicating that
338
N deposition may likely result in a decrease in both gaseous and liquid C loss. Finally, the
339
results emphasized that fresh litter-derived DOC plays a significant role in the seasonal patterns of
340
DOC flux and showed that N addition can alter seasonal patterns as a result of a decrease in DOC
341
leaching, which is likely associated with the inhibition of DOC production during autumn/winter.
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342 343
Acknowledgements
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We thank Dr. Zhang Shuang for his help and suggestions in the statistical analysis. This work was 16
ACCEPTED MANUSCRIPT supported by the National Key Research and Development Program of China (2016YFC0502105);
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Key Research Program of Frontier Sciences, CAS (QYZDJ-SSW-DQC006), the 135 Strategic Program
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of the Institute of Mountain Hazards and Environment, CAS (No. SDS-135-1702), the Youth
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Innovation Promotion Association CAS (2018406), and the Natural Science Foundation of China
349
(41301219).
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Cleveland, C.C., Reed, S.C., Townsend, A.R., 2006. Nutrient regulation of organic matter decomposition in a tropical rain forest. Ecology 87, 492-503.
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Dittman, J.A., Driscoll, C.T., Groffman, P.M., Fahey, T.J., 2007. Dynamics of nitrogen and dissolved organic carbon at the Hubbard Brook Experimental Forest. Ecology 88, 1153-1166. Entwistle Elizabeth, M., Zak Donald, R., Argiroff William, A., 2017. Anthropogenic N deposition
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ACCEPTED MANUSCRIPT Figure captions
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Figure 1. Seasonal temperature and precipitation patterns during the study period. The red line
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and histogram indicate temperature and precipitation, respectively, and the blue histogram indicates
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that, during this period with limited precipitation, there was no water leachate collected under the
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O-layer or mineral layer.
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Figure 2. Seasonal patterns of dissolved organic carbon (DOC) and total dissolved nitrogen (TDN)
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concentrations (A) and fluxes (B) from the organic layer and mineral layer (O layer + the top 20
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cm of mineral soil) under different treatments. Values are the mean ± 1SE. The black dashed line
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Figure 3. Association of precipitation and dissolved organic carbon (DOC) in the organic layer
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Figure 4.Comparison of microbial biomass carbon and nitrogen and saccharase and cellulase
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activities (inset figures) among different treatments. Values are the mean ± 1SE (n = 5 for each
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treatment at each depth). Different letters mean that there are significant differences among different
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treatments at the same depth.
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Figure 5.Seasonal leachate pH patterns from the organic layer and mineral layer under different
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ACCEPTED MANUSCRIPT 494 Figure S1. Seasonal average of dissolved organic carbon (DOC) and total dissolved nitrogen
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(TDN) concentrations (A) and fluxes (B) from the organic layer and mineral layer under different
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treatments. Values are the mean ± 1SE. Different lowercase letters mean that there are significant
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differences among different seasons for the same treatment, and different uppercase letters mean that
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there are significant differences among different treatments in the same season in a mixed linear model.
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Figure S2.Seasonal litterfall pattern in this forest. Data are from Luo et al.(2003).
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ACCEPTED MANUSCRIPT Table 1 Soil properties under different N-addition treatments Treatment Organic layer
Mineral soil (top 5 cm) STN
SOC
STN
(g/kg)
(g/kg)
pH
(g/kg)
(g/kg)
Ambient
343.4 ± 33.3
12.7 ± 1.5 6.06 ± 0.01 18.9 ± 1.3
1.2 ± 0.1 5.56 ± 0.04 <0.1
14.1 ± 0.9 85.9 ± 0.9
Moderate N
393. 3 ± 34.4 15.0 ± 0.9 6.07 ± 0.01 25.2 ± 4.3
1.7 ± 0.3 5.49 ± 0.08 <0.1
13.0 ± 1.5 86.9 ± 1.5
High N
386.6 ± 8.9
17.6 ± 0.9 6.05 ± 0.01 19.6 ± 3.2
1.4 ± 0.2 5.45 ± 0.07 <0.1
14.9 ± 1.4 85.0 ± 1.3
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pH
Clay
Silt
Sand
(%)
(%)
(%)
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SOC
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ACCEPTED MANUSCRIPT Table 2 Annual dissolved organic carbon (DOC) and total dissolved nitrogen (TDN) released from the organic (O) and mineral (O + 20 cm of mineral soil) layers TDN flux
DOC flux 2
Water flux
2
(g C/m /year, mean ± SE)
(g N/m /year, mean ± SE)
a
Mineral layer
a
8.9± 1.1
0.5 ± 0.1 a
0.3± 0.1
Moderate N 16.1± 1.9 ab
9.8± 2.0
0.6 ± 0.1 a
0.4± 0.1
High N
6.4± 1.8
1.6 ± 0.4 b
0.5± 0.1
Ambient
O layer
17.3± 2.6 a 13.4± 2.0 b
O layer
(mm/year, mean ± SE)
Mineral layer O layer
Mineral layer
543± 37 344± 44
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Treatment
517± 24 371± 65
503± 20 344± 81
Note: a, Different letters indicate significant differences among the treatments at the 0.05 significance
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addition and 40 kg N/ha/year addition, respectively.
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level; Ambient, Moderate N and High N indicate ambient treatment with 0 N addition, 8.0 kg N/ha/year
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ACCEPTED MANUSCRIPT Highlights: 1.
Short-term N addition decreased DOC leaching from the organic layer but not from mineral soil
2.
N addition reduced microbial biomass, saccharase and cellulose activities, likely inducing lower
3.
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DOC production N-induced reduction in DOC leaching was most likely due to lower DOC production and not to DOC retention
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Seasonally different changes in DOC under N addition affected the seasonal pattern of DOC
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leaching
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4.