This is the accepted version of the journal article: Kuang, Luhui; Mou, Zhijian; Li, Yue; [et al.]. «Depth-driven responses of micro- bial residual carbon to nitrogen addition approaches in a tropical forest: Canopy addition versus understory addition». Journal of Environmental Management, Vol. 340 (Aug. 2023), art.118009. DOI 10.1016/j.jenvman.2023.118009 This version is available at https://ddd.uab.cat/record/287447 under the terms of the license 1 Depth-driven responses of microbial residual carbon to nitrogen addition approaches in a tropical 2 forest: Canopy addition versus understory addition 3 Luhui Kuanga, b,c, Zhijian Moua,b,c,d, Yue Lia,b,c, Xiaofei Lue, Yuanwen Kuanga,b,c, Jun Wanga,b,c, Faming 4 Wanga,b,c, Xi’an Caia,b,c, Wei Zhanga,b,c, Shenglei Fuf, Dafeng Huig, Hans Lambersh, Jordi Sardansi,j, Josep 5 Peñuelasi,j, Hai Rena,b,c, Zhanfeng Liua,b,c* 6 a Key Laboratory of Vegetation Restoration and Management of Degraded Ecosystems & CAS 7 Engineering Laboratory for Vegetation Ecosystem Restoration on Islands and Coastal Zones, South 8 China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China 9 b South China National Botanical Garden, Guangzhou 510650, China 10 c Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, 11 Guangzhou510650, China 12 d University of Chinese Academy of Sciences, Beijing 100049, China 13 e School of Environmental Science and Engineering, Nanjing University of Information Science & 14 Technology, Nanjing 210044, China 15 f College of Environment and Planning, Henan University, Kaifeng 475004, China 16 g Department of Biological Sciences, Tennessee State University, Nashville, TN 37209, USA 17 h School of Biological Sciences, University of Western Australia, Perth, WA 6009, Australia 18 i CSIC, Global Ecology Unit CREAF-CSIC-UAB, Cerdanyola del Valles, 08193 Catalonia, Spain 19 j CREAF, Cerdanyola del Valles, 08193 Catalonia, Spain 20 * Corresponding author: Zhanfeng Liu 21 E-mail: liuzf@scbg.ac.cn; Tel: +86-20-37252592 22 1 23 Abstract 24 Canopies play an important role in nitrogen (N) redistribution in forest ecosystems, and ignoring the 25 canopy’s role might bias estimates of the ecological consequences of anthropogenic atmospheric N 26 deposition. We investigated the effects of the approach of N addition (Canopy addition vs. Understory 27 addition) and level of N addition (25 kg N ha -1yr-1 vs. 50 kg N ha-1yr-1) on microbial residual carbon 28 (MRC) accumulation in topsoil and subsoil. We found that the response of MRC to both approach and 29 level of N addition varied greatly with soil depth in a tropical forest over eight years of continuous N 30 addition. Specifically, N addition enhanced the accumulation of fungal and total MRC and their 31 contribution to soil organic C (SOC) pools in the topsoil, whereas it decreased the contribution of fungal 32 and total MRC to SOC in the subsoil. The contrasting effects of N addition on MRC contribution at 33 varying soil depths were associated with the distinct response of microbial residues production. 34 Understory N addition showed overall greater effects on MRC accumulation than canopy N addition did. 35 Our results suggest that the canopy plays an important role in buffering the impacts of anthropogenic 36 atmospheric N deposition on soil C cycling in tropical forests. The depth-dependent response of 37 microbial residues to N addition also highlights the urgent need for further studies of different response 38 mechanisms at different soil depths. 39 Keywords: nitrogen addition, canopy nitrogen addition, understory nitrogen addition, microbial residual 40 carbon, soil depth, tropical forest 41 42 43 44 2 45 1. Introduction 46 Tropical forests play an important role in mitigating global climate change via carbon (C) sequestration. 47 Although the growth of tropical forests is usually constrained by phosphorus (P), rather than nitrogen (N) 48 availability (Du et al., 2020; Hou et al., 2020), a growing body of evidence suggests that anthropogenic 49 atmospheric N deposition is having profound effects on soil C-cycling processes in tropical forests 50 (Zellweger et al., 2020). Furthermore, increasing N deposition causes an imbalance of N and P 51 availability and aggravates P limitation in tropical forests which might affect soil C stocks and 52 sequestration (Crews et al., 1995; Vitousek et al., 2010; Baccini et al., 2017). However, most studies 53 about the effects of N deposition on soil C dynamics were conducted in N-limited temperate forests, and 54 data on tropical forests are scarce which has led to a debate on the role of tropical forests in terrestrial C 55 cycling (Janssens et al., 2010; Pan et al., 2011; Baccini et al., 2017). Nitrogen deposition might not only 56 alter the production of plant litter and fine roots, but also change the decomposition rate of organic 57 material, thereby affecting the input and output of forest SOC (Lu et al., 2021b). A recent study in a 58 tropical forest showed that decades of N addition increased soil C stock by 7-21% via decreasing C losses 59 and enhancing physical protection (Lu et al., 2021d). However, the information about the origin (plant- 60 or microbial-derived) and stability of the enhanced soil C sequestration under N addition in tropical 61 forests remains unclear. 62 Microorganisms are central to the soil C-cycling process and mediate the response of soil C dynamics to 63 N deposition (Frey et al., 2014; Averill and Waring, 2018; Chen et al., 2020). As decomposers, 64 microorganisms drive soil C transformation and sequestration through microbial biomass and activity 65 (Liang et al., 2017). As contributors, while living microbial biomass contributes very little to SOC (<5%), 66 microorganisms can transform organic matter into their biomass through assimilation, and microbial 3 67 residues resulting from cell death and lysis can be preserved for a long time by stabilizing with soil 68 minerals; hence microorganisms are an important source of C input and affect the size and stability of 69 SOC pools (Joergensen, 2018; Liang et al., 2019; Ma et al., 2020a). Especially in recent decades, the role 70 of microbial residues in mediating SOC accumulation and stabilization has attracted increasing attention 71 and has been extensively studied across different ecosystems (He et al., 2011; Liang and Balser, 2012; 72 Angst et al., 2021). Microbial residual C (MRC) can even contribute up to 50% to SOC pools (Liang et 73 al., 2019) and its contribution is highly context-dependent and affected by land use, climate, soil type, 74 substrate quality, soil fauna, and sampling depth (Angst et al., 2021). Some studies have shown that the 75 response of MRC contribution to N deposition is highly context-dependent, and N deposition may lead 76 to an increase in fungal MRC contribution in temperate forests (Ma et al., 2017; Chen et al., 2020), a 77 decrease in tropical rainforests (Zhang et al., 2016; Ma et al., 2020b) or no change in tropical coastal 78 forests (Yuan et al., 2021). The divergent responses of the contribution of MRC to SOC in N-limited and 79 N-rich forests highlight the complex interactions between microbial effects on soil C sequestration and 80 ecosystem nutrient limitation. Furthermore, previous studies on the response of microbial residues to N 81 addition have exclusively focused on the 0-10 cm or 0-20 cm of topsoil, while ignoring the effect on the 82 subsoil (Ni et al., 2020). The subsoil (deeper than 20 cm) contains more than 50% of the SOC stock and 83 has a greater MRC contribution, which is critical to the global C budget (Rumpel and Kögel-Knabner, 84 2011, Ni et al., 2020). Differences in nutrient status, spatial distribution, and substrate quality along the 85 soil profile might also result in divergent responses of microbial residues to N addition (Angst et al., 86 2021). Therefore, addressing the role of microorganisms in soil C sequestration in tropical forests would 87 help to elucidate the current divergences about the effects of N deposition on forest C sequestration. 4 88 Tree canopies play an important role in maintaining forest ecological processes and functioning and 89 mediating forest responses and feedback to global climate change (Zellweger et al., 2020). Tree canopies 90 can intercept 44-52% of dissolved N and mediate the N redistribution in tropical forests (Gaige et al., 91 2007; Sievering et al., 2007; Zhang et al., 2015; Shi et al., 2016; Liu et al., 2020a; Liu et al., 2020b). Our 92 knowledge and predictions about the forest responses to atmospheric N deposition are largely based on 93 manipulative experiments involving understory N fertilization (Mo et al., 2008; Cusack et al., 2011; Frey 94 et al., 2014). However, ignoring canopy-associated processes might bias the estimation of the ecological 95 consequences of anthropogenic atmospheric N deposition in forest ecosystems (Zhang et al., 2015). 96 Therefore, canopy N addition is an ideal approach to mimic the real status of atmospheric N deposition 97 in forest ecosystems. Experiments based on canopy N addition will also help improve our mechanistic 98 understanding of how forest ecosystems respond and feedback to elevated atmospheric N deposition. 99 In this study, an eight-year N-manipulative experiment (including canopy N addition and understory N 100 addition) was conducted in a tropical forest. We aimed to investigate the effects of the N addition 101 approach (Canopy N addition versus Understory N addition) and N addition level (25 kg N ha -1yr-1 versus 102 50 kg N ha-1yr-1) on MRC and its contribution to SOC in topsoil and subsoil in a tropical forest. We 103 hypothesized that: (1) Excessive N addition would decrease the contribution of MRC to SOC via 104 inhibiting microbial biomass and activity (Lu et al., 2021d). (2) The tree canopy can buffer the effects of 105 N addition on MRC contribution to SOC with the effects of understory N addition being much greater 106 than those of canopy N addition (Lu et al., 2021a; Lu et al., 2021b; Lu et al., 2021c). (3) The contribution 107 of MRC to SOC as a result of N addition would vary between topsoil and subsoil due to the differences 108 in microclimate and nutrient availability, especially because the nutrient status of most tropical subsoils 109 is very low (Ni et al., 2020). The results in combination will improve our mechanistic understanding of 5 110 the microbial effects on soil C sequestration in response to elevated atmospheric N deposition in tropical 111 forests. 112 2. Materials and methods 113 2.1 Site description and experiment design 114 The study site was located in a tropical evergreen broadleaved forest at Shimentai (SMT) National Nature 115 Reserve (24°26´N-113°08´E), Guangdong Province, South China (Figure 1a). The climate of the study 116 site is tropical monsoon. The mean annual air temperature and precipitation are 20.9°C and 2364 mm, 117 respectively, with 71% of the precipitation occurring in the wet season (April-September). The 118 background atmospheric wet N deposition rate at the study site was 34.0 kg N ha −1 yr−1 (Zhang et al., 119 2015). The soil is classified as Acrisol developed from quaternary red earth according to the Soil 120 Taxonomic System of FAO-UNESCO. The vegetation is a tropical broadleaved evergreen forest (about 121 55 years old) with Cryptocarya concinna, Schima superba, Machilus chinensis, Castanea henryi, and 122 Engelhardtia roxburghiana dominating the tree canopy. The mean canopy height and diameter at breast 123 height of the studied forest are 13.8 m and 18.6 cm, respectively (Lu et al., 2021b). More detailed 124 information about the study site is included in Zhang et al. (2015). 125 In 2013, a field fertilization experiment with canopy N addition (CN) and understory N addition (UN) 126 was set up (Figure 1). A detailed description of this experiment can be found in Zhang et al. (2015). A 127 complete randomized block design was used, and N was added by two approaches (CN and UN) and 128 levels (25 kg N ha−1 yr−1 and 50 kg N ha−1 yr−1). Within each of the four replicated blocks, canopy 129 addition of 25 kg N ha-1yr-1 (CN25), canopy addition of 50 kg N ha-1 yr-1 (CAN50), understory addition 130 of 25 kg N ha-1 yr-1 (UN25), understory addition of 50 kg N ha -1 yr-1 (UAN50) and control (CK, without 131 N addition) were randomly assigned to five circular plots (17 m in diameter and 907 m2 in area). Within 6 132 each block, polyvinylchloride (PVC) boards were inserted between two adjacent plots, and a buffer zone 133 of more than 20 m was set up to avoid lateral N movement. Canopy N addition was achieved by a forest 134 canopy spraying system (35 m in height, 5-8 m above the canopy; Figure 1b) built in the center of each 135 plot, and understory N addition was achieved by five evenly distributed sprinklers (1.5 m above the 136 ground; Figure 1c) in each plot. A detailed description of the spraying system was included in Zhang et 137 al. (2015). Starting from 2013, NH4NO3 solutions with targeted concentrations were added monthly in 138 the growing season (from April to October). The total volume of the added water was equivalent to 21 139 mm of precipitation per year, which represents only ~1% of the total annual precipitation. Therefore, the 140 potentially confounding effect caused by water addition was negligible. 141 2.2 Soil sampling 142 Topsoil (0-10 cm) and subsoil (10-40 cm) were sampled in November 2020. In each plot, five soil cores 143 at each sampling depth were collected randomly and mixed homogeneously to form one composite 144 sample. After carefully removing visible organic materials and fine roots, all fresh soils were passed 145 through a 2-mm mesh and divided into two subsamples. The first subsample was air-dried for soil 146 physicochemical and amino sugar analyses, and the second was stored at 4°C for microbial analyses. 147 2.3 Soil physicochemical properties 148 Soil bulk density was determined by the core ring method. Soil water content (WC) was determined 149 gravimetrically by weighing it after drying in an oven at 105°C for 48 h. Soil pH was determined in a 150 1:2.5 soil: water slurry using a glass pH electrode (FiveEasyPlusTM FE28, Mettler Toledo, Greifensee, 151 Switzerland). Soil organic carbon (SOC) and total nitrogen (TN) were determined using the dichromate 152 oxidation method and the Kjeldahl method (Bremner and Mulvaney, 1982), respectively. Total 153 phosphorus (TP) was digested by H2SO4-HClO4 and Olsen-P was extracted by NH4F-HCl, and 7 154 determined by the molybdenum blue method (Olsen and Sommers, 1982). Dissolved organic C (DOC) 155 and dissolved organic N (DON) were extracted by 0.5 M K 2SO4 and determined with a TOC analyzer 156 (TOC-VCSH, Shimadzu, Japan). To determine the concentrations of NO 3--N and NH4+-N, soils were 157 extracted with 2 M KCl and measured by a flow injection analyzer (AA3, Bran Luebbe, Norderstedt, 158 Germany). 159 2.4 Soil microbial community composition 160 The soil microbial community was characterized by the phospholipid fatty acid (PLFAs) method 161 according to Bossio and Scow (1998) with minor modification. Briefly, lipids were extracted from the 162 samples using a chloroform: methanol: phosphate buffer (0.8:1:2 v/v/v). Phospholipids were separated 163 from neutral lipids and glycolipids on a silicic acid column and then subjected to alkaline methanolysis. 164 An internal standard of 19:0 methyl ester was added to the extract before the derivatization of the PLFAs. 165 Fatty acids were analyzed using gas chromatography (6890N, Agilent Technologies, Santa Clara, USA) 166 with MIDI peak identification software (MIDI Inc., Newark, USA). The biomarkers of 10Me16:0, 167 10Me17:0, and 10Me18:0 were used to indicate actinomycetes (AC). The biomarkers of i15:0, α15:0, 168 i16:0, i17:0, and α17:0 were used to indicate the biomass of Gram-positive (GP) bacteria, and the 169 biomarkers of 16:1ω9c, 16:1ω7c, 18:1ω7c, cy17:0, and cy19:0 were used to indicate the biomass of 170 Gram-negative (GN) bacteria. The biomarker 18:2ω6c was used to represent saprotrophic fungi. The 171 fungal to bacterial PLFA ratio (F:B) and GP to GN PLFA ratio (GP:GN) was also calculated. All PLFAs 172 were expressed as nmol g-1 dry soil (Tunlid et al., 1989). 173 2.5 Soil enzyme activities 174 The activities of four C-cycling-related enzymes (β-1,4-glucosidase, BG; cellobiohydrolase, CBH; 175 peroxidase, PER; phenol oxidase, POX), two N-cycling-related enzymes (β-1,4-N-acetyl- 8 176 glucosaminidase, NAG; L-leucine aminopeptidase, LAP) and one P-cycling-related enzyme (acid 177 phosphatase, AP) were measured according to German et al. (2011) and Bach et al. (2013) with minor 178 modification. Fluorometric assays were used to measure the activities of AP, BG, CBH, NAG, and LAP, 179 and fluorescence was determined by a microplate fluorometer with 365 nm excitation and 450 nm 180 emission filter. The activities of PER and POX were determined by a microplate spectrophotometer at 181 450 nm (Multiskan FC, Thermo Fisher Scientific, Germany). The unit for the seven soil extracellular 182 enzymes was nmol g-1 dry soil h-1. 183 2.6 Soil amino sugars 184 Soil amino sugars were extracted and determined according to Indorf et al. (2011) with minor 185 modifications (Mou et al., 2020). Briefly, aliquots of 0.5 g of freeze-dried soil were hydrolyzed with 10 186 ml of 6 M HCl for 6 h at 105°C, then filtered, and evaporated to dryness by N2 gas at 40-45°C. The 187 residues were dissolved in 0.5 ml deionized water and stored at -20°C until analysis. The extracted amino 188 sugars were derivatized using ortho-phthaldialdehyde (OPA) and determined by high-performance liquid 189 chromatography equipped with a Dionex RS 3400 fluorescence detector (Dionex Ultimate 3000, Thermo 190 Fisher Scientific, Waltham, USA). The emission wavelength was 445 nm, and the excitation wavelength 191 was 330 nm. The concentrations of glucosamine (GluN), galactosamine (GalN), and muramic acid 192 (MurN) and the actual biomass ratio of GP to GN bacteria measured by PLFA analysis were used to 193 calculate microbial residual C (MRC) according to Appuhn and Joergensen (2006), Engelking et al. 194 (2007), Liang et al. (2019) and Chang et al. (2021): 195 Fungal MRC (mg kg-1) = (460/49) × (GluN-2 ×MurN × 179.17/ 253.23) 196 Bacterial MRC (mg kg-1) = MurN × (460/ ((13.9 × F+3.7 × (1-F))) 9 197 Where, 460 is based on the assumption that the C concentration of bacterial and fungal biomass, mg/g 198 dry mass; 49 is an average GluN concentration in different fungal species, mg/g dry mass; 2 is based on 199 the assumption that MurN and GluN occur at a ratio of 1:2 in bacterial cells; 179.2 and 251.2 are the 200 molecular weights of GluN and MurN, respectively; 13.9 and 3.7 are average MurN concentrations in 201 Gram-positive bacteria and Gram-negative bacteria, respectively; F means the proportion of Gram- 202 positive based on the PLFAs. 203 2.7 Statistical analyswes 204 The statistical analyses were conducted using R 4.0.3. The Shapiro-Wilk test and Levene test were 205 performed to check the normality and homogeneity of variance of the data. Data were logarithmically or 206 square-root transformed when data were not normally distributed. Two-way analysis of variance 207 (ANOVA) of randomized block design was used to test the effect of the N-addition approach (canopy N 208 addition vs. understory N addition), N-addition level (25 vs. 50 kg N ha−1 yr−1), and their interactions on 209 measured soil abiotic and microbial properties in each soil depth; block served as a random effect. 210 Whenever ANOVA yielded significant effects, Tukey's HSD test was used to assess the differences 211 among treatments at P < 0.05. A Spearman correlation analysis was performed to explore the linkages 212 between MRC and other measured soil properties within the same sampling depth. Partial least squares 213 path modeling (PLS-PM) was conducted with the “plspm” package to further identify the pathways 214 contributing to the response of MRC to N addition at different sampling depths. 215 3. Results 216 3.1 Soil abiotic and microbial properties 217 Overall, the measured soil abiotic and microbial properties were more affected by sampling depth than 218 by N addition eight years after the start of the treatment (Table 1 and Figure 2). Soil bulk density (BD), 10 219 pH, and nitrate (NO3--N) concentrations were significantly lower and values for other abiotic properties 220 were greater in the topsoil than in the subsoil (Table 1). Similarly, topsoil showed greater microbial 221 biomass, F:B ratio, and enzyme activities than subsoil did, except for GP: GN ratio and L-leucine 222 aminopeptidase (Figure 2). The overall effects of N addition were quite weak, with some significant 223 effects occurring for specific properties at specific N-addition treatments or sampling depths. UN50 224 significantly increased the concentration of NO3--N in the topsoil (Table1; P=0.03) and UN25 225 significantly increased BG activity in the subsoil (Figure 2f; P=0.003). 226 3.2 Soil microbial residual carbon and its contribution 227 Soil MRC concentrations and fungal to bacterial MRC ratio decreased significantly with increasing 228 sampling depth (Figure 3). The response of soil MRC to N addition was highly dependent on the N- 229 addition approach, N-addition level, sampling depth, and their interactions (Table 2). In the topsoil, N 230 addition increased fungal and total MRC concentrations with the values in the UN50 treatment being 231 much greater than those in the control (Figure 3a, c). In the subsoil, although fungal MRC concentration 232 was unaffected by N addition, the values in the CN50 treatment were much lower than those in the CN25 233 treatment (Figure 3a). Similarly, the fungal to bacterial MRC ratio in the CN50, UN25, and UN50 234 treatments was greatly decreased by N addition in the subsoil, whereas in the CN25 treatment, it was 235 unaffected by N addition (Figure 3d). The concentrations of fungal and total MRC were also affected by 236 the N addition approach with the effects of understory addition being much greater than those of canopy 237 additions in the topsoil (Table 2; P<0.05). Understory N addition significantly decreased the fungal to 238 bacterial MRC ratio in the subsoil compared with canopy N addition (Figure 3d and Table 2; P<0.001). 239 Fungal MRC concentration and fungal to bacterial MRC ratio were significantly affected by N addition 240 level, with the effects of a high N-addition level being much greater than those of the low N-addition 11 241 level in the subsoil (Table 2; P<0.05). Bacterial MRC concentration was overall unresponsive to N 242 addition, at both sampling depths (Table 2). 243 Contrary to soil MRC concentrations, the contributions of bacterial and total MRC to SOC increased 244 significantly with increasing sampling depth, but the fungal MRC contribution was unaffected (Figure 245 4). However, similar to the soil MRC concentration, the response of the MRC contribution to N addition 246 was also affected by the N-addition approach, N-addition level, sampling depth, and their interactions 247 (Table 2). Overall, N addition increased the contributions of fungal and total MRC to SOC in the topsoil, 248 whereas it decreased their contributions in the subsoil (Figure 4a, c). The bacterial MRC contribution 249 was significantly decreased by N addition in the subsoil with the values in the UN25 treatment, but 250 unaffected in the topsoil (Figure 4b). Understory N addition showed greater effects on fungal and total 251 MRC contributions than canopy N addition did (Table 2). High-level N addition showed greater effects 252 on fungal and total MRC contributions than low-level N addition did in the topsoil (Figure 4 and Table 253 2). 254 3.3 Linkages between microbial residual carbon and environmental predictors 255 Partial least squares path modeling (PLS-PM) further demonstrated the divergent responses of the 256 contribution of MRC to SOC under N addition in the topsoil (Figure 5a) and subsoil (Figure 5b). In the 257 topsoil, N addition showed a positive effect on the contribution of MRC to SOC (Figure 5c). The N- 258 boosting effect on fungal MRC contribution was mainly caused by N addition and its consequent increase 259 in soil TN, and bacterial MRC contribution was mainly caused by soil pH and enzyme activity, with 260 fungal MRC being more enhanced than bacterial MRC in the topsoil. Nitrogen addition not only 261 significantly increased soil enzyme activity but also tended to increase soil TN, nutrient ratio, and 262 microbial biomass which may have further facilitated the accumulation and contribution of MRC to SOC 12 263 indirectly in the topsoil. In contrast to topsoil, N addition showed a negative effect on the contribution 264 of MRC to SOC in the subsoil (Figure 5d). The N-suppressing effect on fungal MRC contribution was 265 mainly caused by microbial biomass and enzyme activity, and bacterial MRC contribution was mainly 266 caused by nutrient ratio and microbial biomass in the subsoil. Although N addition had no significant 267 effect on soil abiotic and microbial properties, N addition tended to decrease microbial biomass and 268 increase enzyme activity which further indirectly inhibited the accumulation and contribution of MRC 269 to SOC in the subsoil. Furthermore, N addition had a negative effect of microbial biomass on enzyme 270 activity in the topsoil, but a positive effect in the subsoil which may have further exacerbated the 271 divergent responses of MRC concentration to SOC at different sampling depths (Figure 5a, b). 272 4. Discussion 273 4.1 Buffering effects of tree canopy on the microbial residual carbon response to nitrogen addition 274 The experimental design in our study made it possible to compare the consequences of canopy N addition 275 and understory N addition in a forest ecosystem. Consistent with our second hypothesis, our data show 276 that understory N addition had greater effects on soil MRC contribution to SOC than canopy N addition 277 did (Table 2), both in topsoil and subsoil, suggesting that the tree canopy acts as a buffer to mediate 278 microbial residues response in soil. This is also consistent with a previous study at the same site, where 279 forest canopy intercepted 44-52% of N addition and maintained soil community composition (Liu et al., 280 2020a), suggesting that the consequences of canopy N addition and understory N addition differ. 281 Furthermore, a greater contribution of fungal MRC to SOC in 50 kg N ha -1 yr-1 treatment than in 25 kg 282 N ha-1 yr-1 treatment in the topsoil also suggested that higher level N addition favored the accumulation 283 of fungal MRC (Table 2). Similarly, Ma et al. (2017) found that a high rate of understory N addition (100 284 kg N ha−1 yr−1) greatly increased the contribution of bacterial MRC to SOC compared with a low rate of 13 285 understory addition (50 kg N ha−1 yr−1) in topsoil (0-10 cm) of subtropical plantation forests. A recent 286 meta-analysis also showed that a long-term high rate of N addition enhances the accumulation of MRC 287 in croplands (Hu et al., 2022). 288 4.2 Depth-driven response of microbial residual carbon to N addition 289 Partially contrary to our first hypothesis, we found that eight years of N addition in a tropical forest 290 tended to increase microbial biomass and activity and greatly increased fungal MRC contribution in the 291 topsoil (Fig. 5a). However, partially supporting our first hypothesis and fully supporting our third 292 hypothesis, we found that N addition significantly decreased soil MRC contribution in the subsoil, in 293 contrast with that in the topsoil (Fig. 5b). Previous studies on topsoil (0-10 or 0-20 cm) of N-saturated 294 tropical and subtropical forests have also shown that fungal MRC contribution is more sensitive to N 295 addition than bacterial, but N addition tends to decrease the fungal MRC contribution (Kjoller et al., 296 2012; Zhang et al., 2016; Ma et al., 2020a; Ma et al., 2020b), which is contrary to our results. Nitrogen 297 addition might decrease MRC contribution to SOC via inhibiting microbial activity and biomass (Lu et 298 al., 2021d), or might decrease the accumulation and contribution of MRC in the SOC pool via increasing 299 bioavailable soil N, microbial biomass, and enzyme activity (Chen et al., 2020; Ma et al., 2020b). The 300 divergent response of MRC contribution to SOC under N addition among sampling depths, suggests that 301 the mechanism driving the response of MRC to N addition differs between topsoil and subsoil. 302 The accumulation of soil MRC depends on the balance between the synthesis and decomposition of 303 microbial metabolic by-products as well as their residence time (Six et al., 2006; Joergensen, 2018). The 304 differences in nutrient status, spatial distribution, and sensitivity to substrate quality of microbial residues 305 along the soil profile might also affect the divergent response of the contribution of MRC to N addition 306 (Angst et al., 2021). In the topsoil, the positive effects of N addition on MRC concentration and 14 307 contribution may be explained by the N-induced increase in soil TN, which simultaneously stimulated 308 the production and decomposition of MRC, but the production was faster than the decomposition (Fig. 309 5c). In contrast, the negative effects of N addition on the contribution of MRC in the subsoil may be 310 explained by the increase in SOC but no change in MRC concentration (Fig. 3 and Table 1). Nitrogen 311 addition might increase the movement of dissolved organic matter from plants and decomposing litter to 312 deeper soil layers which might dilute the MRC in the subsoil in regions with high precipitation (Moritz 313 et al., 2009; Mikutta et al., 2019). And N-induced decreases in microbial biomass, but increases in 314 enzyme activity in the subsoil, might inhibit MRC production, but accelerate MRC decomposition, 315 thereby further decreasing the contribution of soil MRC to SOC (Fig. 5d). Overall, the opposite effect of 316 N addition on microbial biomass between topsoil and subsoil, rather than enzyme activity is the main 317 driver of the divergent response of the contribution of MRC under N addition. Although N addition 318 tended to increase enzyme activity, it did not alter NAG, which breaks down MRC (Sinsabaugh et al., 319 2008). Furthermore, soil permeability and spatially distinct inputs of organic matter may further 320 influence the response of MRC to N addition by affecting their amount and distribution in the subsoil 321 (Chabbi et al., 2009; Heinze et al., 2018; Preusser et al., 2019). Although substrates with high quality 322 generally stimulate the accumulation of MRC in the SOC pool, the concurrently increasing soil 323 respiration rates might offset the C gain by the accumulation of MRC (Córdova et al., 2018). 324 Consequently, the response of soil MRC to N addition was highly context-dependent and varied greatly 325 with soil depth, microclimate, microbial community, and atmospheric N deposition level. The divergent 326 responses of soil MRC to N addition between topsoil and subsoil also highlights that the information on 327 deeper soil layers should be considered in future research to obtain a more complete picture. 328 5. Conclusions 15 329 Our study provided experimental evidence that the tree canopy buffered the effects of N addition on soil 330 microbial residual carbon (MRC), suggesting that tree canopies play an important role in mediating the 331 response of soil C cycling to atmospheric N deposition. We found that N addition enhanced the 332 accumulation of fungal and total MRC and their contribution to SOC in the topsoil, but decreased the 333 contribution of fungal and total MRC to SOC in the subsoil, demonstrating that the response of microbial 334 residues to N addition strongly depended on soil depth. The distinct responses of microbial residues 335 production at different soil depths resulted in contrasting effects of N addition on soil MRC. These 336 findings are critical for reconciling discrepancies in existing studies and highlight the urgent need to 337 consider canopy and soil depth in future forest N-deposition studies. 338 Acknowledgments 339 This work was supported by the Key-Area Research and Development Program of Guangdong Province 340 (No. 2022B1111230001), the National Natural Science Foundation of China (No.42177289 and No. 341 42007045), the Youth Innovation Promotion Association CAS (2019340), the Spanish Government 342 projects PID2019-110521GB-I00 and PID2020115770RB-I, Fundación Ramón Areces project 343 CIVP20A6621 and Catalan government project (SGR2017-1005). 344 Author Contributions 345 ZFL and LHK conceived and designed the study and led the writing of the manuscript. LHK, ZJM, YL, 346 XFL, and XAC performed the experiment and analyzed the data. YWK, JW, FMW, WZ, SLF, DFH, HL, 347 JS, JP and HR contributed substantially to manuscript revisions. All authors reviewed and edited the 348 manuscript. 349 Data availability 350 All data that support the findings of this study are available on request. 16 351 Conflict of Interests 352 The authors declare that they have no conflicts of interest. 353 References 354 Angst, G., Mueller, K.E., Nierop, K.G.J., Simpson, M.J., 2021. Plant- or microbial-derived? A review on 355 the molecular composition of stabilized soil organic matter. Soil Biology and Biochemistry 156, 356 108189. 357 Appuhn, A., Joergensen, R.G., 2006. Microbial colonisation of roots as a function of plant species. Soil 358 Biology and Biochemistry 38, 1040-1051. 359 Averill, C., Waring, B., 2018. Nitrogen limitation of decomposition and decay: How can it occur? Global 360 Change Biology 24, 1417-1427. 361 Baccini, A., Walker, W., Carvalho, L., Farina, M., Sulla-Menashe, D. Houghton, R.A., 2017. Tropical 362 forests are a net carbon source based on aboveground measurements of gain and loss. Science 363 358, 230-234. 364 Bach, C.E., Warnock, D.D., Van Horn, D.J., Weintraub, M.N., Sinsabaugh, R.L., Allison, S.D., German, 365 D.P., 2013. Measuring phenol oxidase and peroxidase activities with pyrogallol, L-DOPA, and 366 ABTS: Effect of assay conditions and soil type. Soil Biology and Biochemistry 67, 183-191. 367 Bossio, D.A., Scow, K.M., 1998. Impacts of carbon and flooding on soil microbial communities: 368 phospholipid fatty acid profiles and substrate utilization patterns. Microbial Ecology 35, 265- 369 278. 370 Bremner, J.M., Mulvaney, C.S., 1982. Nitrogen: Total. In: Page, A.L. (Eds.), Methods of Soil Analysis. 371 Part 2: Chemical and Microbial Properties. Agronomy Society of America, Madison, WI, pp. 372 595-624. 17 373 Chabbi, A., Kögel-Knabner, I., Rumpel, C., 2009. Stabilised carbon in subsoil horizons is located in 374 spatially distinct parts of the soil profile. Soil Biology and Biochemistry 41, 256-261. 375 Chang, R.Y., Liu, S.G., Chen, L.Y., Li, N., Bing, H.J., Wang, T., Chen, X.P., Li, Y., Wang, G.X., 2021. 376 Soil organic carbon becomes newer under warming at a permafrost site on the Tibetan Plateau. 377 Soil Biology and Biochemistry 152, 108074. 378 Chen, J.G., Ji, C.J., Fang, J.Y., He, H.B., Zhu, B., 2020. Dynamics of microbial residues control the 379 responses of mineral-associated soil organic carbon to N addition in two temperate forests. 380 Science of The Total Environment 748, 141318. 381 Córdova, S.C., Olk, D.C., Dietzel, R.N., Mueller, K.E., Archontouilis, S.V., Castellano, M.J., 2018. Plant 382 litter quality affects the accumulation rate, composition, and stability of mineral-associated soil 383 organic matter. Soil Biology and Biochemistry 125, 115-124. 384 Cusack, D.F., Silver, W.L., Torn, M.S., Burton, S.D., Firestone, M.K., 2011. Changes in microbial 385 community characteristics and soil organic matter with nitrogen additions in two tropical forests. 386 Ecology 92, 621-632. 387 Du, E.Z., Terrer, C., Pellegrini, A., Ahlström, A., van Lissa, C.J., Zhao, X., Xia, N., Wu, X.H., Jackson, 388 R.B., 2020. Global patterns of terrestrial nitrogen and phosphorus limitation. Nature Geoscience 389 13, 221-226. 390 Engelking, B., Flessa, H., Joergensen, R.G., 2007. Shifts in amino sugar and ergosterol contents after 391 addition of sucrose and cellulose to soil. Soil Biology and Biochemistry 39, 2111-2118. 392 Frey, S.D., Ollinger, S., Nadelhoffer, K., Bowden, R., Brzostek, E., Burton, A., Caldwell, B.A., Crow, S., 393 Goodale, C.L., Grandy, A.S., Finzi, A., Kramer, M.G., Lajtha, K., LeMoine, J., Martin, M., 394 McDowell, W.H., Minocha, R., Sadowsky, J.J., Templer, P.H., Wickings, K., 2014. Chronic 18 395 nitrogen additions suppress decomposition and sequester soil carbon in temperate forests. 396 Biogeochemistry 121, 305-316. 397 Gaige, E., Dail, D.B., Hollinger, D.Y., Davidson, E.A., Fernandez, I.J., Sievering, H., White, A., 398 Halteman, W., 2007. Changes in canopy processes following whole-forest canopy nitrogen 399 fertilization of a mature spruce-hemlock forest. Ecosystems 10, 1133-1147. 400 German, D.P., Weintraub, M.N., Grandy, A.S., Lauber, C.L., Rinkes, Z.L., Allison, S.D., 2011. 401 Optimization of hydrolytic and oxidative enzyme methods for ecosystem studies. Soil Biology 402 and Biochemistry 43, 1387-1397. 403 He, H.B., Zhang, W., Zhang, X.D., Xie, H.T., Zhuang, J., 2011. Temporal responses of soil 404 microorganisms to substrate addition as indicated by amino sugar differentiation. Soil Biology 405 and Biochemistry 43, 1155-1161. 406 Heinze, S., Ludwig, B., Piepho, H., Mikutta, R., Don, A., Wordell-dietrich, P., Helfrich, M., Hertel, D., 407 Leuschner, C., Kirfel, K., Kandeler, E., Preusser, S., Guggenberger, G., Leinemann, T., 408 Marschner, B., 2018. Factors controlling the variability of organic matter in the top- and subsoil 409 of a sandy Dystric Cambisol under beech forest. Geoderma 311, 37-44. 410 Hou, E.Q., Luo, Y.Q., Kuang, Y.W., Chen, C.R., Lu, X.K., Jiang, L.F., Luo, X.Z., Wen, D.Z., 2020. 411 Global meta‐ analysis shows pervasive phosphorus limitation of aboveground plant production 412 in natural terrestrial ecosystems. Nature Communications 11, 637. 413 Hu, J.X., Huang, C.D., Zhou, S.X., Liu, X., Dijkstra, F.A., 2022. Nitrogen addition increases microbial 414 necromass in croplands and bacterial necromass in forests: A global meta-analysis. Soil Biology 415 and Biochemistry 165, 108500. 416 Indorf, C., Dyckmans, J., Khan, K.S., Joergensen, R.G., 2011. Optimisation of amino sugar quantification 19 417 by HPLC in soil and plant hydrolysates. Biology and Fertility of Soils 47, 387-396. 418 Janssens, I.A., Dieleman, W., Luyssaert, S., Subke, J.A., Reichstein, M., Ceulemans, R., Ciais, P., 419 Dolman, A.J., Grace, J., Matteucci, G., Papale, D., Piao, S.L., Schulze, E.D., Tang, J., Law, B.E., 420 2010. Reduction of forest soil respiration in response to nitrogen deposition. Nature Geoscience 421 3, 315-322. 422 Joergensen, R.G., 2018. Amino sugars as specific indices for fungal and bacterial residues in soil. 423 Biology and Fertility of Soils 54, 559-568. 424 Kjoller, R., Nilsson, L.O., Hansen, K., Schmidt, I.K., Vesterdal, L., Gundersen, P., 2012. Dramatic 425 changes in ectomycorrhizal community composition, root tip abundance and mycelial 426 production along a stand-scale nitrogen deposition gradient. New Phytologist 194, 278-286. 427 Liang, C., Balser, T.C., 2012. Warming and nitrogen deposition lessen microbial residue contribution to 428 soil carbon pool. Nature Communications 3, 1222. 429 Liang, C., Schimel, J.P., Jastrow, J.D., 2017. The importance of anabolism in microbial control over soil 430 carbon storage. Nature Microbiology 2, 17105. 431 Liang, C., Amelung, W., Lehmann, J., Kastner, M., 2019. Quantitative assessment of microbial 432 necromass contribution to soil organic matter. Global Change Biology 25, 3578-3590. 433 Liu, T., Mao, P., Shi, L.L., Eisenhauer, N., Liu, S.J., Wang, X.L., He, X.X., Wang, Z.Y., Zhang, W., Liu, 434 Z.F., Zhou, L.X., Shao, Y.H., Fu, S.L. 2020a. Forest canopy maintains the soil community 435 composition under elevated nitrogen deposition. Soil Biology and Biochemistry 143, 107733. 436 Liu, Y., Tan, X.P., Wang, Y.Y., Guo, Z.M., He, D., Fu, S.L., Wan, S.Q., Ye, Q., Zhang, W., Liu, W., Shen, 437 W.J., 2020b. Responses of litter, organic and mineral soil enzyme kinetics to 6 years of canopy 438 and understory nitrogen additions in a temperate forest. Science of The Total Environment 712, 20 439 136383. 440 Lu, M., Zhou, X.H., Luo, Y.Q., Yang, Y.H., Fang, C.M., Chen, J.K., Li, B., 2011. Minor stimulation of 441 soil carbon storage by nitrogen addition: A meta-analysis. Agriculture, Ecosystems and 442 Environment 140, 234-244. 443 Lu, X.F., Hou, E.Q., Guo, J.Y., Gilliam, F.S., Li, J.L., Tang, S.B., Kuang, Y.W., 2021a. Nitrogen addition 444 stimulates soil aggregation and enhances carbon storage in terrestrial ecosystems of China: A 445 meta-analysis. Global Change Biology 21, 2780-2792. 446 Lu, X.F., Kuang, Y.W., Mou, L.Y., Hou, E.Q., Fu, S.L., Li, J.L., 2021b. Canopy mitigates the effects of 447 nitrogen deposition on soil carbon-related processes in a subtropical forest. Science of the Total 448 Environment 757, 143847. 449 Lu, X.F., Ren, W.D., Hou, E.Q., Tang, S.B., Zhang, L.L., Liu, Z.F., Lin, Y.B., Fu, S.L., Wen, D.Z., Kuang, 450 Y.W., 2021c. Different effects of canopy and understory nitrogen addition on soil organic carbon 451 and its related processes in a subtropical forest. Journal of Soils and Sediments 21, 235-244. 452 Lu, X.K., Vitousek, P.M., Mao, Q.G., Gilliam F.S., Luo, Y.Q., Turner, B.L., Zhou, G.Y., Mo, J.M., 2021c. 453 Nitrogen deposition accelerates soil carbon sequestration in tropical forests. Proceedings of the 454 National Academy of Sciences of the United States of America 118, e2020790118. 455 Ma, S.H., Chen, G.P., Du, E.Z., Tian, D., Xing, A.J., Shen, H.H., Ji, C.J., Zheng, C.Y., Zhu, J.X., Zhu, 456 J.L., Huang, H.Y., He, H.B., Zhu, B., Fang, J.Y., 2020a. Effects of nitrogen addition on microbial 457 residues and their contribution to soil organic carbon in China's forests from tropical to boreal 458 zone. Environmental Pollution 268, 115941. 459 Ma, S.H., Chen, G.P., Tian, D., Du, E.Z., Xiao, W., Jiang, L., Zhou, Z., Zhu, J.L., He, H.B., Zhu, B., 460 Fang, J.Y., 2020b. Effects of seven-year nitrogen and phosphorus additions on soil microbial 21 461 community structures and residues in a tropical forest in Hainan Island, China. Geoderma 361, 462 114034. 463 Ma, Z.Q., Zhang, X.Y., Zhang, C., Wang, H.M., Chen, F.S., Fu, X.L., Fang, X.M., Sun, X.M., Lei, Q.L., 464 2017. Accumulation of residual soil microbial carbon in Chinese fir plantation soils after 465 nitrogen and phosphorus additions. Journal of Forestry Research 29, 953-962. 466 Mikutta, R., Turner, S., Schippers, A., Gentsch, N., Meyer-Stüve, S., Condron, L.M., Peltzer, D.A., 467 Richardson, S.J., Eger, A., Hempel, G., Kaiser, K., Klotzbücher, T., Guggenberger, G., 2019. 468 Microbial and abiotic controls on mineral-associated organic matter in soil profiles along an 469 ecosystem gradient. Scientific Reports 9, 10294. 470 Mo, J.M., Zhang, W., Zhu, W.X., Gundersen, P., Fang, Y.T., Li, D.J., Wang, H., 2008. Nitrogen addition 471 reduces soil respiration in a mature tropical forest in southern China. Global Change Biology 472 14, 403-412. 473 Moritz, L.K., Liang, C., Wagai, R., Kitayama, K., Balser, T.C., 2009. Vertical distribution and pools of 474 microbial residues in tropical forest soils formed from distinct parent materials. 475 Biogeochemistry 92, 83-94. 476 Mou, Z.J., Kuang, L.H., Yan, B.Y., Zhang, X.Y., Wang, Y.Q., Liu, Z.F., 2020. Influences of sample 477 storage and grinding on the extraction of soil amino sugars. Soil Ecology Letters 2, 157-163. 478 Ni, X.Y., Liao, S., Tan, S.Y., Peng, Y., Wang, D.Y., Yue, K., Wu, F.Z., Yang, Y.S., 2020. The vertical 479 distribution and control of microbial necromass carbon in forest soils. Global Ecology and 480 Biogeography 29, 1829-1839. 481 Olsen, S.R., Sommers, L.E., 1982. Phosphorous. In: Page A.L. (Eds.), Methods of Soil Analysis. Part 2: 482 Chemical and Microbial Properties. Agronomy Society of America, Madison, WI, pp. 403-430. 22 483 Pan, Y.D., Birdsey, R.A., Fang, J.Y., Houghton, R., Kauppi, P. E., Kurz, W.A., Phillips, O.L., Shvidenko, 484 A., Lewis, S.L., Canadell, J.G., Ciais, P., Jackson, R.B., Pacala, S.W., McGuire, A.D., Piao, S.L., 485 Rautiainen, A., Sitch, S., Hayes, D., 2011. A large and persistent carbon sink in the world’s 486 forests. Science 333, 988-993. 487 Preusser, S., Poll, C., Marhan, S., Angst, G., Mueller, C.W., Bachmann, J., Kandeler, E., 2019. Fungi and 488 bacteria respond differently to changing environmental conditions within a soil profile. Soil 489 Biology and Biochemistry 137, 107543. 490 Rumpel, C., Kögel-Knabner, I., 2011. Deep soil organic matter-a key but poorly understood component 491 of terrestrial C cycle. Plant and Soil 338, 143-158. 492 Shi, L.L., Zhang, H.Z., Liu, T., Zhang, W., Shao, Y.H., Ha, D.L., Li, Y.Q., Zhang, C.M., Cai, X.A., Rao, 493 X.Q., Lin, Y.B., Zhou, L.X., Zhao, P., Ye, Q., Zou, X.M., Fu, S.L., 2016. Consistent effects of 494 canopy vs. understory nitrogen addition on the soil exchangeable cations and microbial 495 community in two contrasting forests. Science of The Total Environment 553, 349-357. 496 Sievering, H., Tomaszewski, T., Torizzo, J., 2007. Canopy uptake of atmospheric N deposition at a 497 conifer forest. Part I - Canopy N budget, photosynthetic efficiency and net ecosystem exchange. 498 Tellus Series B 59, 483-492. 499 Sinsabaugh, R.L., Lauber, C.L., Weintraub, M.N., Ahmed, B., Allison, S.D., Crenshaw, C., Contosta, 500 A.R., Cusack, D., Frey, S., Gallo, M.E., Gartner, T.B., Hobbie, S.E., Holland, K., Keeler, B.L., 501 Powers, J.S., Stursova, M., Takacs-Vesbach, C., Waldrop, M.P., Wallenstein, M.D., Zak, D.R., 502 Zeglin, L.H., 2008. Stoichiometry of soil enzyme activity at global scale. Ecology Letters 11, 503 1252-1264. 504 Six, J., Frey, S.D., Thiet, R.K., Batten, K.M., 2006. Bacterial and fungal contributions to carbon 23 505 sequestration in agroecosystems. Soil Science Society of America Journal 70, 555-569. 506 Tunlid, A., Hoitink, H. A. J., Low, C., White, D.C., 1989. Characterization of bacteria that suppress 507 rhizoctonia damping-off in bark compost media by analysis of Fatty Acid biomarkers. Applied 508 and Environmental Microbiology 55, 1368-1374. 509 Vitousek, P.M., Porder, S., Houlton, B.Z., Chadwick, O.A., 2010. Terrestrial phosphorus limitation: 510 mechanisms, implications, and nitrogen-phosphorus interactions. Ecological Applications 20, 511 5-15. 512 Yuan, Y., Li, Y., Mou, Z.J., Kuang, L.H., Wu, W.J., Zhang, J., Wang, F.M., Hui, D.F., Peñuelas, J., Sardans, 513 J., Lambers, H., Wang, J., Kuang, Y.W., Li, Z.A., Liu, Z.F., 2021. Phosphorus addition decreases 514 microbial residual contribution to soil organic carbon pool in a tropical coastal forest. Global 515 Change Biology 27, 454-466. 516 Zellweger, F., De Frenne, P., Lenoir, J., Vangansbeke, P., Verheyen, K., Bernhardt-Römermann, M., 517 Baeten, L., Hédl, R., Berki, I., Brunet, J., Van Calster, H., Chudomelová, M., Decocq, G., 518 Dirnböck, T., Durak, T., Heinken, T., Jaroszewicz, B., Kopecký, M., Máliš, F., Macek, M., 519 Malicki, M., Naaf, T., Nagel, T.A., Ortmann-Ajkai, A., Petrík, P., Pielech, R., Reczynska, K., 520 Schmidt, W., Standovár, T., Swierkosz, K., Teleki, B., Vild, O., Wulf, M., Coomes, D., 2020. 521 Forest microclimate dynamics drive plant responses to warming. Science 368, 772-775. 522 Zhang, W., Cui, Y.H., Lu, X.K., Bai, E., He, H.B., Xie, H.T., Liang, C., Zhang, X.D., 2016. High nitrogen 523 deposition decreases the contribution of fungal residues to soil carbon pools in a tropical forest 524 ecosystem. Soil Biology and Biochemistry 97, 211-214. 525 Zhang, W., Shen, W.J., Zhu, S.D., Wan, S.Q, Luo, Y.Q., Yan, J.H., Wang, K.Y., Liu, L., Dai, H.T., Li, 526 P.X., Dai, K.Y., Zhang, W.X., Liu, Z.F., Wang, F.M., Kuang, Y.W., Li, Z.A., Lin, Y.B., Rao, 24 527 X.Q., Li, J., Zou, B., Cai, X.A., Mo, J.M., Zhao, P., Ye, Q., Fu, S.L., 2015. Can canopy addition 528 of nitrogen better illustrate the effect of atmospheric nitrogen deposition on forest ecosystem? 529 Scientific Reports 5, 11245. 530 25 531 Table 1 Responses of soil physicochemical properties (means ± standard error, n=4) to nitrogen (N) addition in the topsoil (0-10 cm) and subsoil (10-40 cm). Different letters 532 represent significant differences among N-addition treatments within the same column (Tukey's HSD test, P<0.05). BD, bulk density; SOC, soil organic carbon (C); TN, total 533 N; TP, total phosphorus; C/P, SOC to TP ratio; N/P, TN to TP ratio; DOC, dissolved organic C; DON, dissolved organic N. CK, control without N addition; CN25, canopy N 534 addition with 25 kg N ha−1 yr−1; CN50, canopy N addition with 50 kg N ha−1 yr−1; UN25, understory N addition with 25 kg N ha −1 yr−1; UN50, understory N addition with 50 535 kg N ha−1 yr−1. BD pH SOC TN TP C/P N/P DOC DON NH4+-N NO3--N Olsen-P -3 -1 -1 -1 -1 -1 -1 -1 (g cm ) (g kg ) (g kg ) (g kg ) (mg kg ) (mg kg ) (mg kg ) (mg kg ) (mg kg-1) Topsoil CK 1.1±0.07 3.6±0.11 31.0±2.90 2.8±0.38 0.36±0.05 89.0±7.57 8.0±0.88 243.0±34.00 23.0±2.00 6.1±1.40 1.3±0.41b 6.5±0.79 CN25 1.0±0.05 3.6±0.03 36.7±2.13 3.2±0.06 0.4±0.01 99.0±3.19 8.6±0.07 274.1±22.53 26.8±1.92 5.9±1.11 1.5±0.26b 5.6±1.01 CN50 1.0±0.05 3.6±0.03 30.3±1.30 2.5±0.22 0.3±0.04 91.3±9.58 7.5±0.56 271.4±26.37 28.4±2.17 8.3±1.91 2.8±0.35ab 5.1±0.81 UN25 1.1±0.03 3.6±0.05 34.6±0.42 3.2±0.15 0.3±0.02 101.4±4.59 9.3±0.44 266.2±15.24 24.8±1.20 9.8±0.34 2.0±0.67ab 5.9±0.71 UN50 1.0±0.07 3.6±0.03 34.3±2.82 2.7±0.20 0.4±0.03 96.7±10.78 7.6±0.71 265.5±6.39 26.3±1.68 4.6±0.59 6.1±2.06a 6.5±1.25 Subsoil CK 1.4±0.06 4.0±0.09 10.7±1.64 1.1±0.15 0.3±0.04 37.9±5.71 4.0±0.43 217.3±17.91 18.7±1.13 4.5±1.00 6.3±0.93 1.7±0.28 CN25 1.3±0.06 4.0±0.01 12.7±0.46 1.4±0.04 0.3±0.02 42.0±3.46 4.5±0.36 233.7±2.93 21.2±0.63 5.1±0.12 5.4±0.92 2.8±0.40 CN50 1.4±0.10 4.0±0.04 11.9±1.36 1.1±0.13 0.3±0.03 45.9±2.48 4.2±0.27 178.1±10.69 17.6±1.48 4.0±0.63 6.9±0.85 2.2±0.35 UN25 1.4±0.05 4.0±0.04 15.1±1.95 1.4±0.17 0.3±0.01 55.6±6.43 5.2±0.55 215.1±24.41 19.2±1.82 4.6±0.57 4.3±0.46 2.3±0.26 UN50 1.3±0.10 4.0±0.06 12.9±0.89 1.3±0.10 0.3±0.02 47.3±4.40 4.9±0.37 214.4±10.09 18.8±0.37 4.8±1.21 6.1±1.60 2.0±0.25 536 26 537 Table 2 Effects of addition approach (A) and addition level (L) of nitrogen (N) addition, and their 538 interactions on microbial residual carbon (MRC) concentration and their contribution to soil organic 539 carbon (SOC) by two-way ANOVAs. F: B MRC, fungal to bacterial MRC ratio; Fungal MRC/SOC, the 540 contribution of fungal MRC to SOC; Bacterial MRC/SOC, the contribution of bacterial MRC to SOC; 541 MRC/SOC, the contribution of total MRC to SOC. Depth Variable A L A× L F (1,12) P F (1,12) P F (1,12) P Topsoil Fungal MRC 7.8 0.016 0.1 0.765 4.0 0.069 Bacterial MRC 0.0 0.968 0.2 0.628 3.0 0.110 Total MRC 6.1 0.029 0.0 0.905 5.1 0.043 F: B MRC 3.6 0.081 0.2 0.634 0.0 0.864 FMRC/SOC 6.6 0.025 5.9 0.032 0.4 0.542 BMRC/SOC 0.2 0.674 1.4 0.253 0.2 0.667 MRC/SOC 5.2 0.042 7.3 0.020 0.5 0.473 Subsoil Fungal MRC 0.4 0.540 5.0 0.045 7.2 0.020 Bacterial MRC 1.2 0.292 0.5 0.475 0.0 0.874 Total MRC 0.0 0.970 3.0 0.108 3.4 0.091 F: B MRC 29.7 <0.001 25.6 <0.001 74.2 <0.001 FMRC/SOC 28.1 <0.001 13.4 0.003 90.8 <0.001 BMRC/SOC 0.2 0.628 2.6 0.132 7.9 0.020 MRC/SOC 20.6 <0.001 6.6 0.025 75.9 <0.001 542 543 544 545 546 547 548 549 550 27 551 Figure legends 552 Figure 1 Layout of experiment design (a, credit by Jiong Li). Canopy nitrogen (N) addition (b, credit by 553 Xi’an Cai) and understory N addition (c, credit by Xi’an Cai) were used to simulate atmospheric N 554 deposition. Nitrogen was added at two-levels: 25 kg N ha-1 yr-1 and 50 kg N ha-1 yr-1. 555 Figure 2 Responses of soil microbial community composition (a, b, d, e) and enzyme activities (c and f) 556 to nitrogen (N) additions in the topsoil and subsoil. Values are means ± standard error (n=4). Different 557 letters on top of the bars represent significant differences among the N-addition treatments (Tukey's HSD 558 test, P<0.05). The links above letters indicate significant differences between canopy N addition and 559 understory N addition. CK, control without N addition; CN25, canopy N addition with 25 kg N ha −1 yr−1; 560 CN50, canopy N addition with 50 kg N ha−1 yr−1; UN25, understory N addition with 25 kg N ha −1 yr−1; 561 UN50, understory N addition with 50 kg N ha−1 yr−1. GP, Gram-positive bacteria; GN, Gram-negative 562 bacteria; Actino, actinomycetes; AMF, arbuscular mycorrhizal fungi; F/B, fungi to bacteria ratio. AP, 563 acid phosphatase; NAG, β-1,4-N-acetyl-glucosaminidase; LAP, L-leucine aminopeptidase; BG, β-1,4- 564 glucosidase; CBH, cellobiohydrolase; PER, peroxidase; POX, phenol oxidase. 565 Figure 3 Effects of nitrogen (N) addition on the concentrations of microbial residual carbon (MRC). 566 Values are means ± standard error (n=4). Different lowercase letters indicate significant differences 567 among N-addition treatments within the same sampling depth (Tukey's HSD test, P<0.05). Different 568 uppercase letters indicate significant differences between topsoil and subsoil (Tukey's HSD test, P<0.05). 569 The links above letters indicate significant differences between canopy N addition and understory N 570 addition. F: B MRC, fungal to bacterial MRC ratio. CN25, canopy N addition with 25 kg N ha −1 yr−1; 571 CN50, canopy N addition with 50 kg N ha−1 yr−1; UN25, understory N addition with 25 kg N ha −1 yr−1; 572 UN50, understory N addition with 50 kg N ha −1 yr−1. 28 573 Figure 4 Effects of nitrogen (N) addition on the contributions of microbial residual carbon (MRC) to the 574 soil organic carbon (SOC) pool. Values are means ± standard error (n=4). Different letters indicate 575 significant differences among N addition treatments within the same sampling soil depth (Tukey's HSD 576 test, P<0.05). Different uppercase letters indicate significant differences between topsoil and subsoil 577 (Tukey's HSD test, P<0.05). The links above letters indicate significant differences between canopy N 578 addition and understory N addition. CN25, canopy N addition with 25 kg N ha −1 yr−1; CN50, canopy N 579 addition with 50 kg N ha−1 yr−1; UN25, understory N addition with 25 kg N ha −1 yr−1; UN50, understory 580 N addition with 50 kg N ha−1 yr−1. 581 Figure 5 Partial least squares path modeling (PLS-PM) for the effects of nitrogen (N) addition on the 582 contribution of microbial residual carbon (MRC) to soil organic carbon (SOC) in the topsoil and subsoil. 583 Orange lines represent negative paths, blue lines represent positive paths, and the width of the lines 584 indicates the magnitude of the effect. The thickness of the line indicates the effect size and the broken 585 line represent non-significant effects (P>0.05). R2 indicates the variance of the dependent variable 586 explained by the model. The nutrient ratio includes SOC to total phosphorus (TP) ratio and total N to TP 587 ratio. Microbial biomass is indicated by fungal PLFAs and bacterial PLFAs. Microbial activity is 588 indicated by the activities of peroxidase (PER) and phenol oxidase (POX) in the topsoil, but β-1,4- 589 glucosidase (BG) in the subsoil. 590 591 29 592 593 Figure 1 594 595 596 597 598 599 600 601 602 603 604 605 30 606 607 Figure 2 608 609 610 611 612 613 614 615 616 617 618 619 620 31 621 622 Figure 3 623 624 625 626 627 628 629 630 631 632 633 32 634 635 Figure 4 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 33 651 652 Figure 5 653 34