Article The Additions of Nitrogen and Sulfur Synergistically Decrease the Release of Carbon and Nitrogen from Litter in a Subtropical Forest Liehua Tie 1,2,3,4 , Rao Fu 1,2 , Josep Peñuelas 3,4 , Jordi Sardans 3,4 , Shibin Zhang 1,2,5 , Shixing Zhou 1,2 , Junxi Hu 1,2 and Congde Huang 1,2, * 1 National Forestry and Grassland Administration Key Laboratory of Forest Resources Conservation and Ecological Safety on the Upper Reaches of the Yangtze River, College of Forestry, Sichuan Agricultural University, Chengdu 611130, China; tiefromchina@163.com (L.T.); furaosc@163.com (R.F.); b20162501@stu.sicau.edu.cn (S.Z.); szhou@sicau.edu.cn (S.Z.); 18681628120@163.com (J.H.) 2 Sichuan Province Key Laboratory of Ecological Forestry Engineering on the Upper Reaches of the Yangtze River, College of Forestry, Sichuan Agricultural University, Chengdu 611130, China 3 CSIC, Global Ecology Unit CREAF-CSIC-UAB, Edifici C, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain; josep.penuelas@uab.cat (J.P.); j.sardans@creaf.uab.cat (J.S.) 4 CREAF, Cerdanyola del Vallès, 08193 Barcelona, Spain 5 Sichuan Forestry and Grassland Inventory and Planning Institute, Chengdu 611130, China * Correspondence: lyyxq100@aliyun.com; Tel.: +86-0288-629-1456  Received: 22 September 2020; Accepted: 27 November 2020; Published: 29 November 2020  Abstract: Atmospheric nitrogen (N) and sulfur (S) deposition in subtropical forests has increased rapidly and the current level is very high, thus seriously affecting nutrient (e.g., N and phosphorus (P)) release from litter. However, the specific effects of S addition and its interaction with N on the release of carbon (C), N, and P from litter in subtropical evergreen broadleaved forests are unclear. Therefore, a two-year field experiment was performed using a litterbag method in a subtropical evergreen broadleaved forest in western China to examine the responses of litter decomposition and nutrient release to the control (CK), added N (+N), added S (+S), and added N and S (+NS) treatments. The results showed that the remaining litter mass, lignin, cellulose, C, N, P, and litter N/P ratio were higher, whereas the litter C/N ratio and soil pH were lower in the fertilization treatments than in CK. The annual decomposition coefficients (k-values) in the +N, +S, and +NS treatments were 0.384 ± 0.002, 0.378 ± 0.002, and 0.374 ± 0.001 year−1 , respectively, which were significantly lower than the k-values in CK (0.452 ± 0.005 year−1 , p < 0.05). The remaining mass, lignin, cellulose, C, and litter N/P ratio were higher, whereas the soil pH was lower in the +NS treatment than in the +N and +S. The interactive effects of N addition and S addition on the remaining litter lignin, cellulose, C, N, and P; the litter C/N, C/P, and N/P ratios; and the soil pH were significant (p < 0.05). In conclusion, the addition of N and S synergistically decreased the degradation of lignin and cellulose and the release of C and N and increased the litter N/P ratio, suggesting that external N and S inputs synergistically slowed the release of C and N from litter and exacerbated litter P limitation during decomposition in this forest. Keywords: acid deposition; litter decomposition; C, N and P cycles; subtropical forest; nutrient limitation 1. Introduction Nitrogen (N) and sulfur (S) deposition has increased rapidly because of the burning of fossil fuels and the application of fertilizers since the Industrial Revolution [1,2] and has become an important Forests 2020, 11, 1280; doi:10.3390/f11121280 www.mdpi.com/journal/forests Forests 2020, 11, 1280 2 of 16 contributor to global climate change [3–5]. The estimated global N and S depositions in 2010 were 123 Tg N y−1 and 84 Tg S y−1 , respectively [1]. China accounts for >15% of the global atmospheric N and S deposition and has become one of the world’s highest N deposition countries [1,6]. Although N and mainly S depositions are declining in some parts of the world [1], N and S deposition is increasing rapidly in the subtropical forests in southern China and the mean level of atmospheric depositions are two- to three-fold higher than the national average [6]. The previous studies reported that N and S depositions in southern China increased approximately 300% and 50%, respectively, from the 1990s to 2010 [6,7]. Therefore, high-level and rapidly increasing atmospheric N and S deposition may seriously affect the C, N, and P cycles in these subtropical forests in southern China. The release of nutrients from litter is key to the C, N, and P cycles of forests because >50% of plant primary net production returns to the soil through the decomposition of litter [8–10]. Inputs of external N can alter the structures of microbial communities [11,12], the activities of enzymes [12,13], and the nutrient balance of forest ecosystems [14–16], affecting the release of nutrients during litter decomposition [17,18]. Globally, different results have been reported in different studies. For example, Mo et al. [19] reported that N addition accelerated litter decomposition in a subtropical forest due to the increase in soil N availability, whereas a study by Bejarano et al. [20] showed that N addition did not significantly change litter decomposition in a tropical forest. In addition, Lv et al. [21] found that N addition inhibited litter decomposition and the release of nutrients in a subtropical forest because of the reduced microbial activity. Compared with N addition, studies focusing on how S addition affects litter nutrient release are relatively limited, but S addition has been found to increase [22], decrease [21,23], or not affect [4] litter mass loss. Moreover, N and S usually interact [22,24], increasing the uncertainty of the responses of litter decomposition and nutrient release to N and S addition. However, the specific effects of S addition and its interaction with N on the release of C, N, and P from litter in subtropical evergreen broadleaved forests are unclear. The rainy zone of Western China has received considerable N and S depositions, and it represents one of the major regions of acid deposition in China [25]. Additionally, the levels of S input (193 kg S ha−1 y−1 ) from atmospheric deposition are twice as high as the levels of N input (95 kg N ha−1 y−1 ) in this rainy area [25,26], which most likely changes the litter decomposition and nutrient release processes in forests in this area. However, the effects of external S input or a combination of N and S input on litter decomposition and nutrient release have never been investigated in evergreen broadleaved forests in this area. In this study, we carried out a two-year field experiment in a subtropical evergreen broadleaved forest in this rainy area to examine the responses of the litter decomposition and the release of C, N, and P to the addition of N and S. The aims of this study are to improve the understanding of the effects of N and S deposition on litter nutrient release in subtropical evergreen broadleaved forests in the studied area and to provide a reference for the management of these forests under the scenario of a sustained rise in N and S depositions. Previous studies in broadleaved forests in this rainy area have shown that the addition of N decreased soil microbial biomass [27–29] and the activities of soil C-cycle enzymes [30], suggesting that N input may negatively affect litter decomposition and nutrient release [21,31,32]. We thus hypothesized that (1) N addition would inhibit litter decomposition and decrease the release of C, N, and P in the study forest. Most previous studies have shown that S addition negatively affects litter mass loss in tropical and subtropical forests [21,23,33]. Knowledge on the responses of litter nutrient release to S addition is nevertheless still limited. We also hypothesized that (2) S addition would suppress litter decomposition and reduce the release of C, N, and P in the study forest. Typically, N and S interactively decrease decomposition activity in terrestrial ecosystems [21,22,30] and may synergistically affect the release of nutrients from litter. Therefore, we further hypothesized that (3) N and S additions would synergistically inhibit litter decomposition and nutrient release in this forest. Forests 2020, 11, 1280 3 of 16 Forests 2020, 11, x FOR PEER REVIEW 3 of 16 2. 2. Materials Materialsand and Methods Methods 2.1. 2.1. Study StudySite SiteDescription Description The The rainy rainy zone zone of of Western Western China China is an ecotone ecotone between between the the Sichuan Sichuan Plateau Plateau and and the the Sichuan Sichuan Basin Basin (Figure 1)1) [28,34]. [28,34]. TheThemean meanannual annualwet wetNNand and S depositions S depositions fromfrom 2008 2008 to 2010 to 2010 in this in this rainyrainy area area werewere 95 kg95Nkg haN−1 ha y−1 y−1 and −1 and kg SkghaS−1hay−1−1y,−1respectively 193193 , respectively [25,26]. [25,26]. AnAn evergreen evergreen broadleaved broadleaved forest forest on on Jinfengsi Jinfengsi Mountain Mountain ◦ 0 (30 (30°02′ 02 N, 103 ◦ 0 N, 103°03′ 03 E; 800E; 800 m a.s.l.), m a.s.l.), Yucheng Yucheng County,County, was used was used as theasobject the object of of study. study. The meanTheannual mean annual temperaturetemperature (MAT) and (MAT) mean and meanprecipitation annual annual precipitation (MAP) of (MAP) the last of 30 the yearslast 30 were 16.1 ◦ Cwere years and 16.1 1700 °C mm, and 1700 mm,The respectively. respectively. dominant tree The species dominant tree in the species study forestinare theLindera study megaphylla, forest are Lindera Quercusmegaphylla, serrata, andQuercus serrata, and Choerospondias Choerospondias axillaris, with standard axillaris, ageswith standard ages 40 of approximately of approximately years. The soil 40 is ayears. LithicThe soil is a Lithic Dystrudept (USDADystrudept Soil Taxonomy).(USDAThe Soilsoil Taxonomy). layer in our The soil forest study layer inwasour study forest approximately was 80 cm.approximately 80 cm. The topsoil (0–20 cmThesoiltopsoil horizon)(0–20 pHcm wassoil horizon) 4.50 ± 0.09 (HpH 2 Owas 4.50 ± 0.09 extraction) (H2beginning at the O extraction) at of the the beginningSee experiment. of the experiment. Tie et al. [30] for See Tie et a more al. [30]description detailed for a more detailed of the studydescription site. of the study site. Figure 1. Figure Location of 1. Location of the the study study site site on on the the Google Google Satellite Satellite Map. Map. The Thearea area within within the the solid solid red red line line is is the rainy the rainy zone zone of of Western Western China [34]. [34]. The area area within within the the solid solid blue blue line line is is Yucheng YuchengCounty. County. 2.2. Experimental Design 2.2. Experimental Design 2.2.1. Leaf Collecting and Bagging 2.2.1. Leaf Collecting and Bagging Freshly fallen litter leaves were collected from the ground from October 2012 to December 2012. Freshly fallen litter leaves were collected from the ground from October 2012 to December 2012. The collected fallen leaves were transported to the laboratory and air-dried. In this study, the mixed The collected fallen leaves were transported to the laboratory and air-dried. In this study, the mixed litter was used to simulate the decomposition process of litter in a natural scenario. The ratio of litter was used to simulate the decomposition process of litter in a natural scenario. The ratio of mixed mixed air-dried litter leaves was 5:3:2 for L. megaphylla, Q. serrate, and C. axillaris. Then, we randomly air-dried litter leaves was 5:3:2 for L. megaphylla, Q. serrate, and C. axillaris. Then, we randomly weighed 15 bags of the mixed air-dried litter and placed the litter into a 65 ◦ C oven for 96 h prior to weighed 15 bags of the mixed air-dried litter and placed the litter into a 65 °C oven for 96 h prior to determining the initial litter quality. Then, 10.0 g of the mixed air-dried litter were then placed into determining the initial litter quality. Then, 10.0 g of the mixed air-dried litter were then placed into litterbags (20 × 20 cm, 0.05 mm pore size). A total of 432 litterbags were filled in February 2013. The Forests 2020, 11, x; doi: FOR PEER REVIEW www.mdpi.com/journal/forests Forests 2020, 11, 1280 4 of 16 litterbags (20 × 20 cm, 0.05 mm pore size). A total of 432 litterbags were filled in February 2013. The initial concentrations of C, N, P, lignin, and cellulose were (averages ± standard deviations) 370 ± 6.91, 7.71 ± 0.75, 0.330 ± 0.06, 94.6 ± 3.12, and 185 ± 4.82 mg g−1 , respectively. 2.2.2. Plot Design and Fertilization Considering the high levels of atmospheric deposition and the observations of previous N and S addition studies in the study area [25,27–30], 150 kg N ha−1 y−1 and 300 kg S ha−1 y−1 were added in the present experiment, consisting of the control (CK, no added N or S), added N (+N, added 150 kg N ha−1 y−1 ), added S (+S, added 300 kg S ha−1 y−1 ), and added N and S (+NS, added 150 kg N ha−1 y−1 and 300 kg S ha−1 y−1 ) treatments. The N and S were from urea and sodium sulfate, respectively. At the beginning of March 2013, twelve plots (5 × 5 m) were established in the study forest and then were divided into three blocks perpendicular to the maximum slope. Each plot was separated by >5 m. The four treatments were randomly distributed in plots inside each block, with three plot repeats for each treatment. In early April 2013, 36 (12 samplings × 3 bags sampling−1 plot−1 ) litterbags were evenly placed on the ground of each plot. Then, the fertilizers were dissolved in 2 L of water and sprayed evenly onto the ground of each plot using a hand-held sprayer (3 L of maximum capacity) once every half-month. The CK plots were sprayed with 2 L of water. We estimated that approximately 0.192 mm per year of simulated precipitation, a negligible amount, was added to the ground of each plot during the experiment period. See Tie et al. [30] for a more detailed description. 2.3. Litter and Soil Sampling and Analysis 2.3.1. Litter and Soil Sampling Litterbags were sampled every two months, i.e., at the beginning of June, August, October, and December 2013; February, April, June, August, October, and December 2014; and February and April 2015. At each sampling time, three litterbags were randomly sampled from each plot and then immediately translated from the field to the laboratory, where the litter was dried at 65 ◦ C for 96 h. The litter sample was sieved (<0.1 mm) prior to measuring the contents of lignin, cellulose, nutrient elements, and ash. Soil samples were collected every six months, i.e., at the beginning of October 2013, April and October 2014, and April 2015. Moss, leaf litter, and the organic layer were removed from the soil surface. Then, five topsoil (0–20 cm soil horizon) samples were collected in each plot, and were fully mixed to form a composite sample. In the laboratory, the soil samples were air-dried for approximately one week and sieved (<2 mm) prior to measuring the pH. 2.3.2. Sample Analysis The cellulose and lignin (acid unhydrolyzable residue, AUR) contents were determined using an acid detergent digestion method [35]. The dichromate oxidation-external heating method was used to measure the litter’s organic C content [36]. Subsamples of the litter were digested in 10 mL of mixed sulfuric acid and perchloric acid (5:1), and then the total N and total P contents of the litter were determined using the Kjeldahl digestion method and molybdenum–antimony colorimetry method, respectively [37]. The litter ash was determined using the dry-ashing method [5]. Soil pH was determined using a glass electrode in the H2 O extracts (boiling to expel CO2 ) [37]. All determinations were repeated three times. Forests 2020, 11, 1280 5 of 16 2.4. Calculation of the Litter’s Stoichiometry, Remaining Mass, and Remaining Nutrients The stoichiometry ratios of the litter were mass-based. The remaining mass (Rm , % of the initial amount) was defined according to Equation (1), and the remaining (Ry , % of the initial amount) lignin, cellulose, C, N, and P were defined by Equation (2) [38]: Wt Rm (%) = 100 × (1) W0 Wt Ct R y (%) = 100 × (2) W0 C0 where W 0 is the initial litter dry weight, Wt is the litter dry weight at tth sampling time, C0 is the initial nutrient concentration, and Ct is the nutrient concentration at tth sampling time. The litter’s dry weight was corrected according to the ash content. A widely applied single exponential model was applied to define the decomposition coefficient, following Equation (3) [39]. Meanwhile, the time for 50% litter decomposition (T50% ) was determined following Equation (4) [40]: 1 Rm   k = − ln (3) t a − ln(1 − 0.5) T50% = (4) k where Rm is the remaining mass (% of the initial amount), a is a constant, k is the annual decomposition coefficient (year−1 ), and t is the time (year) for decomposition. 2.5. Statistical Analyses The one-sample Kolmogorov–Smirnov test and Levene’s test were applied to test the normal distribution and homogeneity of each studied variable. Then, the Box-Cox method was used to transform the unequal or non-normal variances (e.g., litter N/P ratio). One-way ANOVA using Fisher’s least significant difference (LSD), adjusted using the Bonferroni correction, was applied to examine the differences among different treatments in terms of the remaining litter mass, lignin, cellulose, C, N, and P; the C/N, C/P, and N/P ratios at the end of the experiment; the k-value; and the T50% . Two-way ANOVA using Fisher’s LSD, adjusted using the Bonferroni correction, was applied to examine the differences among different treatments on soil pH at each sampling time. In order to understand the interaction of the addition of N and S, linear mixed-effects models with the restricted maximum-likelihood estimation were applied to determine the main effects of N addition, S addition, sampling time, and their interactions on the remaining litter mass, lignin, cellulose, C, N, and P, as well as on the litter’s stoichiometry ratios during the study period. We included blocks and plots as the random effects and N addition, S addition, and sampling time as the fixed effects. The pairwise relationships among the remaining nutrients and the remaining lignin and cellulose were determined by multiple regression models, including the unary linear regression model, quadratic regression model, and exponential regression model. In addition, the optimal model was confirmed based on the coefficient of determination (R2 ). The statistical analysis was performed in SPSS 25.0 (SPSS Inc., Chicago, IL, USA). 3. Results 3.1. Remaining Litter Mass and Decomposition Coefficient The litter mass loss gradually decreased with the decomposition time (Figure 2). At the end of this experiment (24th month of decomposition), the remaining mass was 11.1%, 15.6%, and 19.5% significantly higher in the +N, +S, and +NS treatments than in CK (39.8% ± 0.65% of the initial amount; p < 0.05; histogram in Figure 2), respectively. Additionally, the main effects of N addition, S addition, Forests 2020, 11, 1280 6 of 16 sampling time, and the interactive effects of N addition × sampling time, S addition × sampling time, and N addition × S addition × sampling time on the remaining litter mass during the study period were significant (Table S1). The +N, +S, and +NS treatments decreased the k-values (p < 0.05; Table 1). The T50% values in the +N (1.804 ± 0.007 year), +S (1.832 ± 0.010 year), and +NS (1.854 ± 0.006 year) treatments were higher than those in the CK (1.535 ± 0.015 year; p < 0.05) treatment. Moreover, the k-value was lower (p < 0.05) whereas the T50% was higher (p < 0.05) in the +NS treatment than in the +N and +S treatments. These results highlight that the addition of N and S decreased the rate of litter Forestsdecomposition. 2020, 11, x FOR PEER REVIEW 6 of 16 100 60 c b a d 40 90 20 0 Remaining mass (%) 80 CK +N +S +NS 70 60 50 CK +N 40 +S +NS 30 2 4 6 8 10 12 14 16 18 20 22 24 Decomposition time (month) Figure 2. Figure 2. Remaining Remaining litter littermass mass(% (%ofofthe theinitial initial amount) amount) during during thethe decomposition decomposition process. process. Values Values are are averages the the averages of plot of three threereplicates plot replicates ± standard± standard deviations. deviations. DifferentDifferent lowercaselowercase letters letters in the in the histogram histogram denote denote significant significant differencesdifferences between between distinct distinct treatments treatments at the endat ofthe thisend of this study study based on based one-wayon ANOVA (p < 0.05; Fisher’s LSD test adjusted using the Bonferroni correction). CK, no added N or S; one-way ANOVA (p < 0.05; Fisher’s LSD test adjusted using the Bonferroni correction). CK, no added +N, N oradded S; +N,N added Nadded but no +S, added but noS;added S; +S, Sadded but noS added but no N; and +NS, added N; and +NS, N added added N and S. and S. Table 1. Decomposition coefficient (k-value) and times required for 50% (T50% ) decomposition under Table 1. Decomposition coefficient (k-value) and times required for 50% (T50%) decomposition under different treatments based on one-way ANOVA. different treatments based on one-way ANOVA. Treatments Coefficient of Determination (R ) 2 2 −1 T 50% (Year) Treatments Coefficient k-Value(Year of Determination (R ) k-Value (Year−1) ) T50% (Year) a d CK CK 0.975 0.975** ** 0.452 ±± 0.452 0.005a 1.535 ± 1.535 0.005 0.015±d0.015 c +N 0.947 ** 0.384 ± 0.002bb 1.804 ± 0.007 +S +N 0.947 0.974 ** ** 0.384 ±± 0.378 0.002 0.002 c 1.804 ±1.832 0.007±c 0.010 b +NS +S 0.974 0.955 ** ** 0.378 0.374± ± 0.002 0.010±b 0.006 a 0.001c d 1.832 ± 1.854 +NSsignificant (p < 0.01). Different ** Denotes highly 0.955 **lowercase letters in 0.374 ± 0.001 denote the superscript d 1.854 ± 0.006 a significant differences between different treatments (p < 0.05; Fisher’s least significant difference (LSD) test adjusted using the Bonferroni ** denotes highly significant (p < 0.01). Different lowercase letters in the superscript denote significant correction). Values are the averages of three plot replicates ± standard deviations. Treatment abbreviations are differences provided between in Figure 2. different treatments (p < 0.05; Fisher’s least significant difference (LSD) test adjusted using the Bonferroni correction). Values are the averages of three plot replicates ± standard 3.2. Remaining deviations.Litter Lignin Treatment and Cellulose abbreviations are provided in Figure 2. Across all treatments, the lignin was accumulated in the early stage (<16 months) of decomposition 3.2. Remaining Litter Lignin and Cellulose and then released (>16 months) (Figure 3a), and the cellulose was gradually degraded with Across all time decomposition treatments, the lignin (Figure 3b). At thewas end accumulated in the(24th of this experiment early stageof(<16 month months) of decomposition), decomposition the and then remaining lignin wasreleased 13.5%, (>16 7.9%,months) (Figure and 26.2% 3a), and the significantly cellulose higher and was gradually degraded the remaining cellulose with34.4%, was decomposition 30.1%, andtime (Figure 70.9% 3b). At thehigher significantly end of in this theexperiment +N, +S, and(24th +NSmonth of decomposition), treatments than in CK the remaining (remaining lignin lignin andwas 13.5%, cellulose 7.9%, were and±26.2% 61.3% 1.20%significantly and 14.4% ± higher 1.82% ofand thethe remaining initial amount,cellulose was respectively; 34.4%, 30.1%, and 70.9% significantly higher in the +N, +S, and +NS treatments than in CK (remaining lignin and cellulose were 61.3% ± 1.20% and 14.4% ± 1.82% of the initial amount, respectively; p < 0.05; histograms in Figure 3a,b), respectively. The remaining lignin and cellulose were significantly higher in the +NS treatment than in the treatments with the addition of N and S alone (p < 0.05). Furthermore, the main effects of N addition, S addition, and their interaction on the remaining lignin and cellulose Forests 2020, 11, 1280 7 of 16 p < 0.05; histograms in Figure 3a,b), respectively. The remaining lignin and cellulose were significantly higher in the +NS treatment than in the treatments with the addition of N and S alone (p < 0.05). Furthermore, the main effects of N addition, S addition, and their interaction on the remaining lignin and cellulose during the study period were significant (p < 0.05; Table S1). These results indicate that the addition of N and S synergistically inhibited the degradation of lignin and cellulose. Forests 2020, 11, x FOR PEER REVIEW 7 of 16 160 100 100 40 a d b c CK 140 (a) 90 (b) +N c b b a Remaining cellulose (%) 50 20 80 Remaining lignin (%) 120 +S 0 70 +NS 0 CK +N +S +NS CK +N +S +NS 100 60 80 50 60 40 CK 30 40 +N 20 20 +S +NS 10 0 0 2 4 6 8 10 12 14 16 18 20 22 24 2 4 6 8 10 12 14 16 18 20 22 24 Decomposition time (month) Decomposition time (month) Figure Figure 3.3. Remaining Remaininglitter litter (% (% ofinitial of the the initial amount)amount) lignin (a)lignin (a) and(b)cellulose and cellulose (b) during the during the decomposition decomposition process. Valuesprocess. Values are are the averages the averages of three of three plot replicates plot replicates ± standard ± standard deviations. Differentdeviations. lowercase Different letters inlowercase letters the histogram in the significant denote histogram denote significant differences between differences between distinct distinct treatments at thetreatments end of this at the end of experiment this on based experiment one-way ANOVAbased on (p < one-way ANOVA 0.05). The (p < 0.05). abbreviations The for the abbreviations treatments for the are defined in treatments Figure 2. are defined in Figure 2. 3.3.Remaining 3.3. RemainingNutrients NutrientsininLitter Litter TheC, The C,N,N,and andPPtended tendedto tobe begradually graduallyreleased releasedfrom fromlitter litterthroughout throughoutthe thedecomposition decompositiontime time (Figure 4a–c). The amounts of C and N remaining in the litter were significantly (Figure 4a–c). The amounts of C and N remaining in the litter were significantly higher in the +N, +S,higher in the +N, +S, and +NS treatments than in CK at the end of this experiment (p < 0.05; histograms and +NS treatments than in CK at the end of this experiment (p < 0.05; histograms in Figure 4a,b). in Figure 4a,b). Moreover, the Moreover, the remaining remaining litter litter CC was was significantly significantly greater greater inin the +NStreatment the +NS treatmentthan thanininthe thetreatments treatments with the addition of N and S alone (p < 0.05). The remaining litter N was slightly with the addition of N and S alone (p < 0.05). The remaining litter N was slightly higher in the higher in +NS the +NS treatmentthan treatment thanininthe thetreatments treatmentswith withthetheaddition additionofofNNand andS S alone alone (p(p > 0.05). > 0.05). TheThe main main effects effects of Nof N addition, S addition, sampling time, and their interaction on the C, N, and addition, S addition, sampling time, and their interaction on the C, N, and P remaining in the litter P remaining in the litter during during the study the study periodperiod werewere significant significant < 0.05; (p <(p0.05; Table Table S1). S1). Therefore,the Therefore, theaddition additiontreatments treatments suppressedthe suppressed therelease releaseofofC, C,N,N,and andPPfrom fromlitter, litter,and andthe theaddition additionof ofNNand andSSsynergistically synergisticallyreduced reduced the release of C and the release of C and N. N. 3.4. Litter Stoichiometry Ratios and Soil pH 3.4. Litter Stoichiometry Ratios and Soil pH The litter stoichiometry ratios fluctuated greatly with the decomposition time (Figure 4d–f). The litter stoichiometry ratios fluctuated greatly with the decomposition time (Figure 4d–f). The The litter C/N and C/P ratios were lower, whereas the litter N/P ratio was higher in the addition litter C/N and C/P ratios were lower, whereas the litter N/P ratio was higher in the addition treatments than in CK at the end of this experiment (p < 0.05; histograms in Figure 4d–f). The litter treatments than in CK at the end of this experiment (p < 0.05; histograms in Figure 4d–f). The litter N/P ratio was significantly greater in the +NS treatment than in the treatments with the addition of N N/P ratio was significantly greater in the +NS treatment than in the treatments with the addition of and S alone (p < 0.05). The main effects of N addition, S addition, sampling time, and their interactive N and S alone (p < 0.05). The main effects of N addition, S addition, sampling time, and their effects on the litter N/P ratio during the study period were significant (p < 0.01; Table S2). Therefore, interactive effects on the litter N/P ratio during the study period were significant (p < 0.01; Table S2). the addition of N and S synergistically increased the litter N/P ratio. Therefore, the addition of N and S synergistically increased the litter N/P ratio. At the end of this experiment, the main effects of N addition, S addition, and their interactive At the end of this experiment, the main effects of N addition, S addition, and their interactive effects on the soil pH were significant (p < 0.05; Table S3). The addition of N and S treatments decreased effects on the soil pH were significant (p < 0.05; Table S3). The addition of N and S treatments soil pH at the end of the study (p < 0.05), and the decrease in the +NS treatment was significantly decreased soil pH at the end of the study (p < 0.05), and the decrease in the +NS treatment was stronger than that in the treatments with the addition of N and S alone (p < 0.05). Consequently, significantly stronger than that in the treatments with the addition of N and S alone (p < 0.05). the addition of N and S synergistically decreased the soil pH. Consequently, the addition of N and S synergistically decreased the soil pH. 3.5. Release of Nutrient Elements and Degradation of Lignin and Cellulose The remaining lignin correlated quadratically with the remaining C (R2 = 0.564–0.830; p < 0.01; Figure 5a) and N (R2 = 0.350–0.549; p < 0.01; Figure 5c), and the remaining lignin was positively and linearly related with the remaining P (R2 = 0.415–0.709; p < 0.01; Figure 5e). Furthermore, the remaining cellulose was positively and linearly correlated with the remaining C (R2 = 0.609–0.817; p < Forests 2020, 11, 1280 8 of 16 Forests 2020, 11, x FOR PEER REVIEW 8 of 16 100 40 a 100 40 a a (a) d c b (b) b 90 90 c 20 20 80 80 Remaining N (%) 0 0 Remaining C (%) 70 CK +N +S +NS 70 CK +N +S +NS 60 60 50 50 40 40 CK CK 30 30 +N +N 20 +S 20 +S 10 +NS 10 +NS 0 0 2 4 6 8 10 12 14 16 18 20 22 24 2 4 6 8 10 12 14 16 18 20 22 24 Decomposition time (month) Decomposition time (month) 100 120 80 a ab a 100 c b CK 60 b c bc 110 (d) 50 +N 40 90 100 +S 20 0 0 Remaining P (%) 90 +NS Litter C/N ratio CK +N +S +NS CK +N +S +NS 80 80 70 70 60 CK 60 50 +N +S 40 50 (c) +NS 30 40 20 2 4 6 8 10 12 14 16 18 20 22 24 2 4 6 8 10 12 14 16 18 20 22 24 Decomposition time (month) Decomposition time (month) 3000 1600 60 30 b a c CK a b CK 20 d c +N +N 1200 d 2600 50 +S 10 +S 800 0 +NS +NS Litter C/P ratio Litter N/P ratio CK +N +S +NS CK +N +S +NS 2200 40 (f) (e) 1800 30 1400 20 1000 10 2 4 6 8 10 12 14 16 18 20 22 24 2 4 6 8 10 12 14 16 18 20 22 24 Decomposition time (month) Decomposition time (month) Figure Figure 4.4. Remaining Remaining litter (% of litter (% of the theinitial initialamount) amount)CC(a), (a),NN(b),(b), and and P (c) P (c) andand C/NC/N (d),(d), C/PC/P (e), (e), andand N/P N/P (f) ratios (f) ratios duringduring the decomposition the decomposition process. process. Thestoichiometry The litter litter stoichiometry ratios ratios are basedareonbased mass.on mass. Different Different lowercaselowercase letters in letters in the histogram the histogram denote significant denote significant differences differences betweentreatments between distinct distinct treatments at the end at the end of this experiment based on one-way ANOVA (p < 0.05). Values of this experiment based on one-way ANOVA (p < 0.05). Values are the averages of three are the averages of three plot replicates plot replicates ± standard deviations. The abbreviations for the treatments ± standard deviations. The abbreviations for the treatments are defined in Figure 2. are defined in Figure 2. 3.5. Release of Nutrient Elements and Degradation of Lignin and Cellulose The remaining lignin correlated quadratically with the remaining C (R2 = 0.564–0.830; p < 0.01; Figure 5a) and N (R2 = 0.350–0.549; p < 0.01; Figure 5c), and the remaining lignin was positively and linearly related with the remaining P (R2 = 0.415–0.709; p < 0.01; Figure 5e). Furthermore, the remaining Forests 2020, 11, x; doi: FOR PEER REVIEW www.mdpi.com/journal/forests Forests 2020, 11, 1280 9 of 16 cellulose was positively and linearly correlated with the remaining C (R2 = 0.609–0.817; p < 0.01; Figure 5b), N (R2 = 0.675–0.783; p < 0.01; Figure 5d), and P (R2 = 0.260–0.622; p < 0.01; Figure 5f). Forests 2020, 11, x FOR PEER REVIEW 9 of 16 CK +N +S +NS CK +N +S +NS CK +N +S +NS CK +N +S +NS 100 100 (a) (b) Remaining C (%) Remaining C (%) 80 80 60 60 CK: R2 = 0.830** CK: R2 = 0.732** 40 +N: R2 =0.564** 40 +N: R2 =0.817** +S: R2 = 0.644** +S: R2 = 0.609** 20 20 +NS: R2 =0.728** +NS: R2 =0.782** 0 0 50 75 100 125 150 175 0 20 40 60 80 100 Remaining lignin (%) Remaining cellulose (%) CK +N +S +NS CK +N +S +NS CK +N +S +NS CK +N +S +NS 100 CK: R2 = 0.549** 100 (c) Remaining N (%) (d) Remaining N (%) +N: R2 =0.350** 80 +S: R2 = 0.475** 80 60 +NS: R2 =0.549** 60 CK: R2 = 0.742** 40 40 +N: R2 =0.773** +S: R2 = 0.675** 20 20 +NS: R2 =0.783** 0 0 50 75 100 125 150 175 0 20 40 60 80 100 Remaining lignin (%) Remaining cellulose (%) CK +N +S +NS CK +N +S +NS CK +N +S +NS CK +N +S +NS 100 100 (e) (f) Remaining P (%) Remaining P (%) 80 80 CK: R2 = 0.477** 60 60 +N: R2 =0.260** 40 CK: R2 = 0.709** 40 +S: R2 = 0.344** +N: R2 =0.591** +NS: R2 =0.622** 20 +S: R2 = 0.415** 20 +NS: R2 =0.439** 0 0 50 75 100 125 150 175 0 20 40 60 80 100 Remaining lignin (%) Remaining cellulose (%) Figure 5. Correlations between between remaining remaininglitter litternutrients nutrients(%(%ofofthe theinitial initialamount) amount) and and remaining remaining (%(% of of thethe initial initial amount) amount) lignin lignin and and cellulose. cellulose. (a),a, correlation correlation between between remaining remaining litter C and remaining lignin; (b), b, correlation correlationbetween between remaining remaining litter litter C and and remaining remainingcellulose; cellulose;(c), c, correlation between remaining litter N N and and remaining remaining lignin; lignin; (d), d, correlation correlationbetween between remaining remaining litter litter N N and remaining cellulose; (e), e, correlation correlationbetween between remaining remaining litter and remaining litter P and remaining lignin; lignin;(f), f, correlation correlation between remaining litter P and remaining cellulose. ** denotes denotes that the coefficient of determination determination is highly significant (p << 0.01). Data from each sampling Data from each sampling time are aggregated. Treatment Treatment abbreviations abbreviations are defined in Figure 2. n == 144. 144. 4. Discussion 4. Discussion 4.1. Rate of Litter Decomposition 4.1. Rate of Litter Decomposition The +N treatment decreased the rate of litter decomposition in our study, supporting our first The +N treatment decreased the rate of litter decomposition in our study, supporting our first hypothesis that N addition would decrease litter decomposition in the study forest. This result was in hypothesis that N addition would decrease litter decomposition in the study forest. This result was agreement with most of the previous studies in subtropical forests [21,28]. This phenomenon can be in agreement with most of the previous studies in subtropical forests [21,28]. This phenomenon can be explained by two mechanisms. First, exogenous N input can decrease the activity of decomposers, thereby slowing litter decomposition [13,41]. The previous studies in evergreen broadleaved forests in the same study area reported that N addition decreased the C and N contents of the soil microbial biomass [27,29,42]; thus, the decrease in soil microbial biomass may contribute to the lower rate of litter decomposition under the +N treatment in the current study. Second, high levels of N input Forests 2020, 11, 1280 10 of 16 explained by two mechanisms. First, exogenous N input can decrease the activity of decomposers, thereby slowing litter decomposition [13,41]. The previous studies in evergreen broadleaved forests in the same study area reported that N addition decreased the C and N contents of the soil microbial biomass [27,29,42]; thus, the decrease in soil microbial biomass may contribute to the lower rate of litter decomposition under the +N treatment in the current study. Second, high levels of N input usually slow the decomposition of litter due to soil N enrichment, or even aggravate soil nutrient imbalances [43]. The results of our previous study in the same study forest also showed that added N exacerbated the limitations of soil C and P [30], which may inhibit the decomposition of litter. This inference was further confirmed because our follow-up study in this study area showed that P addition alleviated the limitation of soil P caused by N addition [27]. However, a previous field experiment in the same study area reported that external N input promoted the litter decomposition in a subtropical bamboo forest dominated by Pleioblastus amarus [44], contrary to our result. The addition of N generally accelerates litter decomposition when the initial litter C/N ratio is >55; otherwise (<55), the addition of N inhibits the decomposition [45]. The initial litter C/N ratio in the bamboo ecosystem studied by Tu et al. [44] was >90, whereas in our study forest it was <50; this difference may have caused the differing results between the two studies. Bamboo ecosystems need N fertilizer for the growth of shoots and are commonly N-limited [25,44]. In contrast, N was not limiting and was even saturated in our evergreen broadleaved forest [30]. The difference in nutrient dependence in the two ecosystems may be one reason accounting for the differing results between our study forest and the forest studied by Tu et al. [25]. Taken together, the response of litter mass loss to the input of N may be related not only to the activity of decomposers but also to the nutrient dependence of ecosystems and the initial litter quality. As expected from the second hypothesis, the +S treatment reduced the rate of litter decomposition, which was consistent with previous studies in subtropical forests [21,46]. The results of our previous study confirmed that the addition of sodium sulfate decreased the activities of soil invertase, cellulase, and polyphenol oxidase in the same study forest [30]. The reduction in the activities of soil C-cycle enzymes can explain the decrease in the mass loss [46–48]. However, Wang et al. [22] reported that ten years of sodium sulfate addition increased the mass loss in a temperate broadleaved forest (Populus tremuloides and Picea glauca). Moreover, Jung et al. [4] reported that seven years of sodium sulfate addition did not significantly affect the mass loss in a boreal mixedwood forest (P. tremuloides and P. glauca). First, the large differences in fertilization time may cause the different results obtained in our study (two years), the study (ten years) of Wang et al. [22] and the study (seven years) of Jung et al. [4]. Second, the forest types in the sites studied by Wang et al. [22] (a temperate broadleaved forest) and Jung et al. [4] (a boreal mixedwood forest) differed greatly from those in our study site (a subtropical evergreen broadleaved forest), which may explain the distinct responses. Thus, the effect of S addition on the litter mass loss may be correlated with the fertilization time, as well as the ecosystem type. 4.2. Degradation of Cellulose and Lignin Litter stoichiometry generally controls litter decomposition [32,49], and the stoichiometry is constantly changing during decomposition [17,43]. Nutrient element stoichiometric ratios during decomposition can thus also control subsequent decomposition and degradation of cellulose and lignin [43,50]. In our study, the addition of N and S slowed the degradation of cellulose and lignin, which was in agreement with a previous study of a subtropical forest [33]. This response may be linked to the decreased C/N ratio, as well as the increased N/P ratio, under the addition treatments. On the one hand, the decreased C/N ratio can reduce the fungi-to-bacteria ratio due to the increased proportion of opportunistic bacteria in the microbial communities [11]. Moreover, fungi are more efficient than bacteria at degrading organic C in litter [51,52]. The decreased litter C/N ratio under the +N, +S, and +NS treatments in the present experiment strongly demonstrated that N and S fertilization treatments were likely to decrease the fungi-to-bacteria ratio, thereby inhibiting the degradation of cellulose and lignin [33]. On the other hand, P is usually a major limiting element for microbial activity Forests 2020, 11, 1280 11 of 16 in subtropical forests due to the combination of P with aluminum and iron ions to form complexes in strongly weathered soils [53,54]. Increased N/P ratios are generally considered to exacerbate P limitation in microorganisms in N-rich forests [54]. Our study showed that the +N, +S, and +NS treatments enhanced the litter N/P ratio during litter decomposition, strongly suggesting that these treatments may have exacerbated microbial P limitation, reducing the degradation of cellulose and lignin. Additionally, the reformation of aromatic polymers may be another factor accounting for the slowed lignin degradation due to the addition treatments. Laccases secreted by fungi can promote the reformation of aromatic polymers (an AUR) by the generation of phenoxyl radicals [35,55,56]. The activity of fungi is susceptible to the inputs of external nutrients [57,58]. Therefore, the additions of N and S can alter the activity and structure of fungi to regulate the content of lignin. For instance, Xu et al. [33] reported that S and N addition enhanced the amount of aromatic polymers by altering the structure of fungi. In this way, the increased content of aromatic polymers by the addition of N and S may be one of the mechanisms inhibiting the degradation of lignin under the +N, +S, and +NS treatments in our study. Across all treatments, the lignin was accumulated in the early stage (<16 months). In fact, the results of a study by Berg and McClaugherty [59] showed that the lignin content was directly decreased in litters with >300 mg g−1 initial lignin concentration; otherwise, the content of lignin would increase before an absolute decrease. Subsequent studies have found that in litters with a low initial lignin concentration (<300 mg g−1 ) the lignin is usually accumulated in the early stage of decomposition because of the increased lignin-like compounds produced by microorganisms [51,60]. In our study, the remaining litter C and N decreased with reduced remaining lignin when the remaining lignin was <100% of the initial amount, but decreased with increasing remaining lignin when the lignin was accumulated (especially when the remaining lignin was >125% of the initial amount) (Figure 5a,c), suggesting that the C and N contents of lignin (or lignin-like compounds) between the non-accumulative and accumulative scenarios may be different. Thus, lignin-like compounds may be produced. Furthermore, the remaining P was positively and linearly correlated with the remaining lignin (Figure 5e), implying that the P content of lignin (or lignin-like compounds) between the non-accumulative and accumulative scenarios is probably not significantly different. Therefore, the P content of the produced lignin-like compounds is likely similar to the P content of the initial lignin. Previous studies reported that the C and N content of initial lignin and produced lignin-like compounds are different, but comparison of the P content between them has not been reported [51,56]. The P content of initial lignin and produced lignin-like compounds, however, needs further study. 4.3. Release of C, N, and P from Litter The +N treatment reduced the release of C, N, and P. This result supports our first hypothesis that N addition would decrease the release of C, N, and P from litter in this study forest. Two potential mechanisms may explain this decrease. The addition of urea generally slows the release of these elements due to the direct increase in external C and N nutrients [17,61]. Furthermore, the microbial nutrient mining theory posits that the release of nutrients is stimulated by microorganisms to meet their demands for growth and reproduction when the nutrient contents in the environment are low, but the microorganisms do not need to decompose litter to meet their nutrient demands when nutrient levels are adequate in the background [11]. Urea addition can increase nutrient availability in the ecosystem [62], which may have reduced microbial nutrient mining, resulting in a lower release of nutrients. However, a study reported that the addition of N accelerated the C and N release in N-poor leaf litters [63]. Therefore, the addition of N may work together with the initial litter N content to affect the release of nutrient elements from litter. The addition of S slowed the release of C, N, and P from litter in this study forest, similar to a previous study in a subtropical forest [46]. Soil acidification can negatively affect nutrient element releases by reducing decomposer activity [33,46]. The S addition treatments exacerbated soil Forests 2020, 11, 1280 12 of 16 acidification (decreased soil pH), which may have resulted in the slower release of nutrients in the present study. In this study, the remaining nutrient elements positively and linearly correlated with the remaining cellulose, highlighting that the remaining cellulose was a predictor for the release of C, N, and P. Furthermore, the difference in the remaining nutrient elements between fertilization treatments and the control increased with time (Figure 4a–c), suggesting that the addition of N and S may have cumulative effects on nutrient release [43]. Consequently, medium- and long-term N and S addition is likely to further inhibit the C, N, and P releases of litter in this forest. 4.4. Interactive Effects of the Addition of N and S In our study, the addition of N and S synergistically slowed the degradation of lignin and cellulose and the release of C and N, supporting our third hypothesis. This phenomenon may correspond with the lower soil pH and higher litter N/P ratio in the N and S combined-addition treatment than in the separate-addition treatments. As discussed above, soil acidification usually decreases decomposer activity [33,46]. The N and S combined-addition treatment thus can more strongly decrease the decomposer activity because the pH was lower than it was in the N and S separate-addition treatments. Moreover, the additions of N and S synergistically increased the litter N/P ratio, suggesting that the input of these two elements may have synergistically aggravated P limitation during litter decomposition, thereby lowering the degradation of cellulose and the release of C and N. A study in the same study forest also found that the addition of N and S synergistically increased the soil N/P ratio [30], further highlighting that the added N and S synergistically aggravated P limitation in this forest. Interestingly, the decrease in the litter mass loss and the release of C and N were stronger in the +S treatment than those in the +N treatment, indicating that the addition of S may be more critical than the addition of N in influencing litter decomposition and nutrient release in this forest. Therefore, we should focus not only on the responses of litter decomposition to the input of external N but also the input of external S in subtropical evergreen broadleaved forests in southern China. 5. Conclusions The addition of N and S suppressed litter decomposition and the release of C, N, and P from litter in this study forest, which was related to a decreased soil pH and litter C/N ratio and an increased litter N/P ratio. The addition of N and S synergistically decreased the release of C and N, suggesting that S deposition may have exacerbated the negative effect of N deposition on the release of C and N in this forest area. Moreover, the addition of N and S synergistically increased the litter N/P ratio and thus may have synergistically exacerbated litter P limitation during decomposition. These results highlight that if N and S deposition rates continue rising in southern China, the rate of litter decomposition and the release of C, N, and P in subtropical evergreen broadleaved forests in this area will decrease. Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4907/11/12/1280/s1, Table S1: F- and p-values of linear mixed-effects model analysis focusing on the effects of nitrogen (N) and sulfur (S) addition on remaining mass, remaining lignin, and remaining cellulose, Table S2: F- and p-values of linear mixed-effects model analysis focusing on the effects of nitrogen (N) and sulfur (S) addition on remaining carbon (C), remaining N, and remaining phosphorus (P), litter C/N ratio, litter C/P ratio, and litter N/P ratio, Table S3: The soil pH under different treatments. Values are the averages of three plot replicates ± standard deviations. Different lowercase letters denote significant differences between different treatments at each sampling time based on ANOVA (p < 0.05; Fisher’s LSD test adjusted using the Bonferroni correction). CK, no added N or S; +N, added N but no added S; +S, added S but no added N; and +NS, added N and S. Author Contributions: Data curation, R.F., S.Z. (Shibin Zhang), and C.H.; formal analysis, R.F. and S.Z. (Shibin Zhang); funding acquisition, C.H.; project administration, C.H.; software, L.T.; writing—original draft, L.T., J.P., J.S., and C.H.; writing—review and editing, L.T., J.P., J.S., S.Z. (Shixing Zhou), J.H., and C.H. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Crop Breeding Research Project of the ‘12th Five-year Plan’ of Sichuan Province [2011NZ0098-10], the Sci-tech Project of the ‘12th Five-year Plan’ of China [2010BACO1A11], Forests 2020, 11, 1280 13 of 16 the IMBALANCE-P Grant of European Research Council Synergy [ERC-SyG-2013-610028], and the China Scholarship Council [201906910006]. Acknowledgments: The authors thank Wenyu Bai for providing data analysis assistance. Conflicts of Interest: The authors declare no conflict of interest. References 1. Tan, J.; Fu, J.S.; Dentener, F.; Jian, S.; Keating, T. Multi-model study of HTAP II on sulfur and nitrogen deposition. Atmos. Chem. Phys. 2018, 18, 6847–6866. [CrossRef] 2. Duan, L.; Yu, Q.; Zhang, Q.; Wang, Z.; Pan, Y.; Larssen, T.; Tang, J.; Mulder, J. Acid deposition in Asia: Emissions, deposition, and ecosystem effects. Atmos. Environ. 2016, 146, 55–69. [CrossRef] 3. Gao, Y.; Ma, M.; Yang, T.; Chen, W.; Yang, T. Global atmospheric sulfur deposition and associated impaction on nitrogen cycling in ecosystems. J. Clean. Prod. 2018, 195, 1–9. [CrossRef] 4. Jung, K.; Kwak, J.-H.; Gilliam, F.S.; Chang, S.X. Simulated N and S deposition affected soil chemistry and understory plant communities in a boreal forest in western Canada. J. Plant Ecol. 2017, 11, 511–523. [CrossRef] 5. Adrian, W.J. A comparison of a wet pressure digestion method with other commonly used wet and dry-ashing methods. Analyst 1973, 98, 213–216. [CrossRef] 6. Du, E.; de Vries, W.; Liu, X.; Fang, J.; Galloway, J.N.; Jiang, Y. Spatial boundary of urban ‘acid islands’ in southern China. Sci. Rep. 2015, 5, 12625. [CrossRef] 7. Lu, Z.; Zhang, Q.; Streets, D.G. Sulfur dioxide and primary carbonaceous aerosol emissions in China and India, 1996–2010. Atmos. Chem. Phys. 2011, 11, 9839–9864. [CrossRef] 8. Frouz, J. Effects of soil macro- and mesofauna on litter decomposition and soil organic matter stabilization. Geoderma 2018, 332, 161–172. [CrossRef] 9. Yarwood, S.A. The role of wetland microorganisms in plant-litter decomposition and soil organic matter formation: A critical review. FEMS Microbiol. Ecol. 2018, 94. [CrossRef] 10. Sardans, J.; Peñuelas, J. The role of plants in the effects of global change on nutrient availability and stoichiometry in the plant-soil system. Plant Physiol. 2012, 160, 1741–1761. [CrossRef] 11. Craine, J.M.; Morrow, C.; Fierer, N. Microbial nitrogen limitation increases decomposition. Ecology 2007, 88, 2105–2113. [CrossRef] [PubMed] 12. Corrales, A.; Turner, B.L.; Tedersoo, L.; Anslan, S.; Dalling, J.W. Nitrogen addition alters ectomycorrhizal fungal communities and soil enzyme activities in a tropical montane forest. Fungal Ecol. 2017, 27, 14–23. [CrossRef] 13. Xiao, S.; Wang, G.G.; Tang, C.; Fang, H.; Duan, J.; Yu, X. Effects of one-year simulated nitrogen and acid deposition on soil respiration in a subtropical plantation in China. Forests 2020, 11, 235. [CrossRef] 14. Janssens, I.A.; Dieleman, W.; Luyssaert, S.; Subke, J.A.; Reichstein, M.; Ceulemans, R.; Ciais, P.; Dolman, A.J.; Grace, J.; Matteucci, G.; et al. Reduction of forest soil respiration in response to nitrogen deposition. Nat. Geosci. 2010, 3, 315–322. [CrossRef] 15. Peñuelas, J.; Sardans, J.; Rivas-Ubach, A.; Janssens, I.A. The human-induced imbalance between C, N and P in earth’s life system. Global Change Biol. 2012, 18, 3–6. [CrossRef] 16. Sardans, J.; Rivas-Ubach, A.; Peñuelas, J. The C:N:P stoichiometry of organisms and ecosystems in a changing world: A review and perspectives. Perspect. Plant Ecol. Evol. Syst. 2012, 14, 33–47. [CrossRef] 17. Manning, P.; Saunders, M.; Bardgett, R.D.; Bonkowski, M.; Bradford, M.A.; Ellis, R.J.; Kandeler, E.; Marhan, S.; Tscherko, D. Direct and indirect effects of nitrogen deposition on litter decomposition. Soil Biol. Biochem. 2008, 40, 688–698. [CrossRef] 18. Zhuang, L.; Liu, Q.; Liang, Z.; You, C.; Tan, B.; Zhang, L.; Yin, R.; Yang, K.; Bol, R.; Xu, Z. Nitrogen additions retard nutrient release from two contrasting foliar litters in a subtropical forest, southwest China. Forests 2020, 11, 377. [CrossRef] 19. Mo, J.; Brown, S.; Xue, J.; Fang, Y.; Li, Z. Response of litter decomposition to simulated N deposition in disturbed, rehabilitated and mature forests in subtropical China. Plant Soil 2006, 282, 135–151. [CrossRef] 20. Bejarano, M.; Crosby, M.M.; Parra, V.; Etchevers, J.D.; Campo, J. Precipitation regime and nitrogen addition effects on leaf litter decomposition in tropical dry forests. Biotropica 2014, 46, 415–424. [CrossRef] Forests 2020, 11, 1280 14 of 16 21. Lv, Y.; Wang, C.Y.; Jia, Y.Y.; Wang, W.W.; Ma, X.; Du, J.J.; Pu, G.Z.; Tian, X.J. Effects of sulfuric, nitric, and mixed acid rain on litter decomposition, soil microbial biomass, and enzyme activities in subtropical forests of China. Appl. Soil Ecol. 2014, 79, 1–9. [CrossRef] 22. Wang, Q.; Kwak, J.-H.; Choi, W.-J.; Chang, S.X. Decomposition of trembling aspen leaf litter under long-term nitrogen and sulfur deposition: Effects of litter chemistry and forest floor microbial properties. For. Ecol. Manag. 2018, 412, 53–61. [CrossRef] 23. Singh, R.K.; Dutta, R.K.; Agrawal, M. Litter decomposition and nutrient release in relation to atmospheric deposition of S and N in a dry tropical region. Pedobiologia 2004, 48, 305–311. [CrossRef] 24. Liu, X.; Zhang, B.; Zhao, W.R.; Wang, L.; Xie, D.J.; Huo, W.T.; Wu, Y.W.; Zhang, J.C. Comparative effects of sulfuric and nitric acid rain on litter decomposition and soil microbial community in subtropical plantation of Yangtze River Delta region. Sci. Total Environ. 2017, 601, 669–678. [CrossRef] 25. Tu, L.H.; Hu, T.X.; Zhang, J.; Li, X.W.; Hu, H.L.; Liu, L.; Xiao, Y.L. Nitrogen addition stimulates different components of soil respiration in a subtropical bamboo ecosystem. Soil Biol. Biochem. 2013, 58, 255–264. [CrossRef] 26. Lin, H.J.; Hu, T.X. Preliminary study on the Chemistry Features of Atmospheric Precipitation of three Typical Regions in Sichuan (in Chinese with English Abstract); Sichuan Agricultural University: Ya’an, China, 2011. 27. Wei, S.Z.; Tie, L.H.; Liao, J.; Liu, X.; Du, M.L.; Lan, S.X.; Li, X.R.; Li, C.S.; Zhan, H.C.; Huang, C.D. Nitrogen and phosphorus co-addition stimulates soil respiration in a subtropical evergreen broad-leaved forest. Plant Soil 2020, 450, 171–182. [CrossRef] 28. Zhou, S.X.; Xiang, Y.B.; Tie, L.H.; Han, B.H.; Huang, C.D. Simulated nitrogen deposition significantly reduces soil respiration in an evergreen broadleaf forest in western China. PLoS ONE 2018, 13, e0204661. [CrossRef] 29. Peng, Y.; Song, S.Y.; Li, Z.Y.; Li, S.; Chen, G.T.; Hu, H.L.; Xie, J.L.; Chen, G.; Xiao, Y.L.; Liu, L.; et al. Influences of of nitrogen addition and aboveground litter-input manipulations on soil respiration and biochemical properties in a subtropical forest. Soil Biol. Biochem. 2020, 142, 107694. [CrossRef] 30. Tie, L.; Zhang, S.; Peñuelas, J.; Sardans, J.; Zhou, S.; Hu, J.; Huang, C. Responses of soil C, N, and P stoichiometric ratios to N and S additions in a subtropical evergreen broad-leaved forest. Geoderma 2020, 379. [CrossRef] 31. Berg, B.; Davey, M.P.; De Marco, A.; Emmett, B.; Faituri, M.; Hobbie, S.E.; Johansson, M.B.; Liu, C.; McClaugherty, C.; Norell, L.; et al. Factors influencing limit values for pine needle litter decomposition: A synthesis for boreal and temperate pine forest systems. Biogeochemistry 2010, 100, 57–73. [CrossRef] 32. Cleveland, C.C.; Reed, S.C.; Keller, A.B.; Nemergut, D.R.; O’Neill, S.P.; Ostertag, R.; Vitousek, P.M. Litter quality versus soil microbial community controls over decomposition: A quantitative analysis. Oecologia 2014, 174, 283–294. [CrossRef] [PubMed] 33. Xu, Y.H.; Fan, J.L.; Ding, W.X.; Gunina, A.; Chen, Z.M.; Bol, R.; Luo, J.F.; Bolan, N. Characterization of organic carbon in decomposing litter exposed to nitrogen and sulfur additions: Links to microbial community composition and activity. Geoderma 2017, 286, 116–124. [CrossRef] 34. Zhang, P.; Gao, X.M. The concept of the Rainy Zone of West China and its significance to the biodiversity conservation in China (Abstract in English). Biodiv. Sci. 2002, 10, 339–344. [CrossRef] 35. Berg, B.; Matzner, E. Effect of N deposition on decomposition of plant litter and soil organic matter in forest systems. Environ. Rev. 1997, 5, 1–25. [CrossRef] 36. Schinner, F.; Ohlinger, R.; Kandeler, E.; Margesin, R. Methods in Soil Biology; Springer: Berlin, Germany, 1996. 37. Allen, S.E.; Grimshaw, H.M.; Parkinson, J.A.; Quarmby, C. Chemical Analysis of Ecological Materials; Blackwell Scientific Publications: Oxford/London, UK, 1974. 38. Bragazza, L.; Siffi, C.; Iacumin, P.; Gerdol, R. Mass loss and nutrient release during litter decay in peatland: The role of microbial adaptability to litter chemistry. Soil Biol. Biochem. 2007, 39, 257–267. [CrossRef] 39. Olson, J.S. Energy storage and the balance of producers and decomposers in ecological systems. Ecology 1963, 44, 322–331. [CrossRef] 40. Bockheim, J.G.; Jepsen, E.A.; Heisey, D.M. Nutrient dynamics in decomposing leaf litter of four tree species on a sandy soil in northwestern Wisconsin. Can. J. For. Res. 1991, 21, 803–812. [CrossRef] 41. Entwistle, E.M.; Zak, D.R.; Edwards, I.P. Long-term experimental nitrogen deposition alters the composition of the active fungal community in the forest floor. Soil Sci. Soc. Am. J. 2013, 77, 1648–1658. [CrossRef] Forests 2020, 11, 1280 15 of 16 42. Zhou, S.X.; Huang, C.D.; Han, B.H.; Xiao, Y.X.; Tang, J.D.; Xiang, Y.B.; Luo, C. Simulated nitrogen deposition significantly suppresses the decomposition of forest litter in a natural evergreen broad-leaved forest in the Rainy Area of Western China. Plant Soil 2017, 420, 135–145. [CrossRef] 43. Zhang, T.A.; Luo, Y.Q.; Chen, H.Y.H.; Ruan, H.H. Responses of litter decomposition and nutrient release to N addition: A meta-analysis of terrestrial ecosystems. Appl. Soil Ecol. 2018, 128, 35–42. [CrossRef] 44. Tu, L.H.; Hu, H.L.; Chen, G.; Peng, Y.; Xiao, Y.L.; Hu, T.X.; Zhang, J.; Li, X.W.; Liu, L.; Tang, Y. Nitrogen addition significantly affects forest litter decomposition under high levels of ambient nitrogen deposition. PLoS ONE 2014, 9. [CrossRef] [PubMed] 45. Moore, T.R.; Trofymow, J.A.; Prescott, C.E.; Fyles, J.; Titus, B.D. Patterns of carbon, nitrogen and phosphorus dynamics in decomposing foliar litter in Canadian forests. Ecosystems 2006, 9, 46–62. [CrossRef] 46. Wang, C.Y.; Guo, P.; Han, G.M.; Feng, X.G.; Zhang, P.; Tian, X.J. Effect of simulated acid rain on the litter decomposition of Quercus acutissima and Pinus massoniana in forest soil microcosms and the relationship with soil enzyme activities. Sci. Total Environ. 2010, 408, 2706–2713. [CrossRef] [PubMed] 47. Jian, S.Y.; Li, J.W.; Chen, J.; Wang, G.S.; Mayes, M.A.; Dzantor, K.E.; Hui, D.F.; Luo, Y.Q. Soil extracellular enzyme activities, soil carbon and nitrogen storage under nitrogen fertilization: A meta-analysis. Soil Biol. Biochem. 2016, 101, 32–43. [CrossRef] 48. Keuskamp, J.A.; Feller, I.C.; Laanbroek, H.J.; Verhoeven, J.T.A.; Hefting, M.M. Short- and long-term effects of nutrient enrichment on microbial exoenzyme activity in mangrove peat. Soil Biol. Biochem. 2015, 81, 38–47. [CrossRef] 49. Aerts, R.; van Bodegom, P.M.; Cornelissen, J.H.C. Litter stoichiometric traits of plant species of high-latitude ecosystems show high responsiveness to global change without causing strong variation in litter decomposition. New Phytol. 2012, 196, 181–188. [CrossRef] 50. Fioretto, A.; Di Nardo, C.; Papa, S.; Fuggi, A. Lignin and cellulose degradation and nitrogen dynamics during decomposition of three leaf litter species in a Mediterranean ecosystem. Soil Biol. Biochem. 2005, 37, 1083–1091. [CrossRef] 51. Ahmad, M.; Taylor, C.R.; Pink, D.; Burton, K.; Eastwood, D.; Bending, G.D.; Bugg, T.D.H. Development of novel assays for lignin degradation: Comparative analysis of bacterial and fungal lignin degraders. Mol. Biosyst. 2010, 6, 815–821. [CrossRef] 52. Brown, M.E.; Chang, M.C.Y. Exploring bacterial lignin degradation. Curr. Opin. Chem. Biol. 2014, 19, 1–7. [CrossRef] 53. Du, E.; Vries, W.D.; Han, W.; Liu, X.; Yuan, J. Imbalanced phosphorus and nitrogen deposition in China’s forests. Atmos. Chem. Phys. 2016, 16, 1–17. [CrossRef] 54. Mieczan, T.; Adamczuk, M.; Tarkowska-Kukuryk, M.; Wojciech, P.; Pawlik-Skowronska, B. Effects of experimental addition of nitrogen and phosphorus on microbial and metazoan communities in a peatbog. Eur. J. Protistol. 2017, 59, 50–64. [CrossRef] [PubMed] 55. Floudas, D.; Binder, M.; Riley, R.; Barry, K.; Blanchette, R.A.; Henrissat, B.; Martinez, A.T.; Otillar, R.; Spatafora, J.W.; Yadav, J.S.; et al. The paleozoic origin of enzymatic lignin decomposition reconstructed from 31 fungal genomes. Science 2012, 336, 1715–1719. [CrossRef] [PubMed] 56. Talbot, J.M.; Treseder, K.K. Interactions among lignin, cellulose, and nitrogen drive litter chemistry-decay relationships. Ecology 2012, 93, 345–354. [CrossRef] [PubMed] 57. Allison, S.D.; Vitousek, P.M. Responses of extracellular enzymes to simple and complex nutrient inputs. Soil Biol. Biochem. 2005, 37, 937–944. [CrossRef] 58. Hu, Y.L.; Jung, K.; Zeng, D.H.; Chang, S.X. Nitrogen- and sulfur-deposition-altered soil microbial community functions and enzyme activities in a boreal mixedwood forest in western Canada. Can. J. For. Res. 2013, 43, 777–784. [CrossRef] 59. Berg, B.; McClaugherty, C. Plant litter. In Decomposition, Humus Formation, Carbon Sequestration, 2nd ed.; Springer: Heidelberg, Germany, 2008. 60. Tan, B.; Yin, R.; Yang, W.; Zhang, J.; Xu, Z.; Liu, Y.; He, S.; Zhou, W.; Zhang, L.; Li, H.; et al. Soil fauna show different degradation patterns of lignin and cellulose along an elevational gradient. Appl. Soil Ecol. 2020, 155. [CrossRef] 61. Zhang, X.; Liu, Z. Responses of litter decomposition and nutrient release of Bothriochloa ischaemum to soil petroleum contamination and nitrogen fertilization. Int. J. Environ. Sci. Technol. 2019, 16, 719–728. [CrossRef] Forests 2020, 11, 1280 16 of 16 62. Chen, H.; Li, D.J.; Zhao, J.; Xiao, K.C.; Wang, K.L. Effects of nitrogen addition on activities of soil nitrogen acquisition enzymes: A meta-analysis. Agric. Ecosyst. Environ. 2018, 252, 126–131. [CrossRef] 63. Pei, G.; Liu, J.; Peng, B.; Wang, C.; Jiang, P.; Bai, E. Non-linear coupling of carbon and nitrogen release during litter decomposition and its responses to nitrogen addition. J. Geophys. Res. Biogeosciences 2020, 125. [CrossRef] Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).