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Background: Nitrogen (N) saturation theory proposes that an ecosystem might switch from N limitation to carbon (C), phosphorus (P), or other nutrient limitations if it receives continuous N input. Yet, after N limitation is removed, which nutrient is the most limited and whether topography modulates such change is rarely tested at a microbial level. Here, we conducted a two-year N addition experiment under two different topography positions (i.e. a slope and a valley) in a N-saturated subtropical forest. Soil enzyme activity was measured, and ecoenzymatic stoichiometry indexes were calculated as indicators of microbial resource limitation. Results: In the valley, two-year N addition changed the activity of all studied enzymes to various degrees. As a result, microbial C limitation was aggravated in the valley, and consequently microbial decomposition of soil labile organic C increased, but microbial P limitation was alleviated due to the stoichiometry balance. On the slope, however, N addition did not significantly change the activity of the studied enzymes, and did not alter the status of microbial resource limitation. Conclusions: These results indicate that C is a more limited element for microbial growth than P after removing N limitation, but we also highlight that topography can regulate the effect of N deposition on soil microbial resource limitation in subtropical forests. These findings provide useful supplements to the N saturation theory. Keywords: Nitrogen deposition, Topography, Nutrient limitation, N saturation, Enzyme activity, Enzymatic stoichiometry Introduction from temperate forests (Aber et al. 1989). This theory Over the past few decades, atmospheric nitrogen (N) de- proposes that continuous N input may reduce system re- position increased rapidly due to fossil fuel combustion quirements for N, and finally the system may become N- and widespread use of chemical fertilizer N (Galloway saturated. In this case, carbon (C), phosphorus (P) or et al. 2008). It is documented that N deposition is alter- water limitation is expected to occur or be aggravated ing the structure and function of terrestrial ecosystems, (Aber et al. 1989). This is a useful theory for predicting including the plant growth, carbon and nutrient cycling dynamics of terrestrial ecosystems in response to in- (Galloway et al. 2008). To describe effects of N depos- creased N deposition (Chen et al. 2018d). ition on ecosystem processes, especially the N cycling, a This proposition, however, has not specified which nu- N saturation theory was developed basing on studies trient would be the most limited after removing N limi- tation. In plants, this topic has been studied widely. * Correspondence: firstname.lastname@example.org; email@example.com Recent studies tend to suggest that N deposition may Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of switch N limitation to P limitation (Gress et al. 2007; Subtropical Agriculture, Chinese Academy of Sciences, 410125 Changsha, Hunan, Braun et al. 2010; Crowley et al. 2012; Li et al. 2016; China Huanjiang Observation and Research Station for Karst Ecosystems, Institute of Deng et al. 2017), although some studies find no effects Subtropical Agriculture, Chinese Academy of Sciences, 547100 Huanjiang, China Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Chen et al. Forest Ecosystems (2021) 8:68 Page 2 of 9 (Finzi 2009; Weand et al. 2010) or other limitations, (Zhang et al. 2021), while few experiments estimated such as calcium (Ca) limitation (McNulty et al. 1996). In whether and how topography regulates the responses of microbes, however, the discussion is relatively few, and microbial processes to N deposition (Zhang et al. 2013). the situation might differ from plants. For microbes, C is In a previous study, divergent responses of soil asymbio- often a more limited element compared to N, P or other tic N fixation to N addition has been found between nutrients (Soong et al. 2020). Thus, when N limitation is the valley and slope, implying the important effect of removed after continuous N input, C may be the most topography on ecosystem processes (Wang et al. 2019), limited factor for microbial growth, instead of P or other but further conclusions were not drawn on whether mi- nutrients. However, such an expectation is not consist- crobial resource limitation status had similar responses. ent with many recent studies, which suggest that N In general, soil N level was found lower in the upslope addition may aggravate microbial P limitation (Marklein (due to erosion) than in the downslope or valley (Wein- and Houlton 2012). Since microbes are as important as traub et al. 2015), resulting in a higher sensitivity of mi- plants in an ecosystem, more studies are undoubtedly crobes to the addition of N in the upslope than in the needed to address how microbial resource limitation downslope or valley. Nevertheless, the N leaching was changes after increased N deposition. reported higher in the upslope than in the downslope or The ecoenzymatic stoichiometry method provides a valley (Wang et al. 2019), which in turn weakened the N new tool to study this topic. Compared to traditional addition effects. The ambiguous effects of topography as methods that measure effects of substrate additions on a result of these contrary mechanisms has prevented microbial biomass or respiration as indicators of micro- predictions regarding responses of terrestrial ecosystem bial resource limitation (Traoré et al. 2016), ecoenzy- processes to atmospheric N deposition. matic stoichiometry has following advantages. First, it is Therefore, in this study we conducted a two-year N much faster, because it measures activities of only four addition experiment in a subtropical karst forest, where enzymes, including β-D-glucosidase (BG), L-leucine ami- soil microbes have been proven to be N-saturated (Chen nopeptidase (LAP), β-N-acetylglucosaminidase (NAG), et al. 2018b), to test how N deposition changes the sta- and acid/alkaline phosphatase (AP). Second, using en- tus of microbial resource limitation in such a N- zymes as proxy indicators of C, N, and P acquisition, it saturated situation. In order to investigate whether top- is much easier for us to understand which nutrient is ography regulates effects of N additions on microbial re- more limited to an ecosystem (Sinsabaugh 1994). How- source limitation status, the N-addition experiment was ever, most previous studies reported effects of N set up at two topography positions, a valley and a slope. addition on the activity of the single enzyme (Marklein To our knowledge, this is the first N-deposition simula- and Houlton 2012; Chen et al. 2016b; Jian et al. 2016), tion experiment site that considers effects of topography very few studies have assessed responses of ecoenzy- in the subtropical forest. We hypothesized that (1) nitro- matic stoichiometry to N additions (Wang et al. 2015). gen additions may aggravate microbial C limitation ac- By collecting published data regarding single enzyme ac- cording to the previous studies (Chen et al. 2018d), but tivity in response to N addition, a previous meta-analysis (2) the response of microbial limitation to nitrogen addi- reported that nitrogen deposition may aggravate micro- tions may be more sensitive in the valley than on the bial C limitation (Chen et al. 2018d). However, there slope due to the higher soil N level in the valley and were two major limitations in this study: firstly, the se- higher N leaching on the slope in the studied area. lected published studies rarely reported C, N, and P ac- quisition enzymes at the same time, which largely Materials and methods limited the data’s comparability and the conclusion’s Site description generality; secondly, most selected studies were con- The studied forest locates in Mulun National Nature ducted in N-limited systems rather than N-saturated sys- Reserve of Huanjiang County, southwest China (24°54′‒ tems, and thus could not answer the above question 24°07′ N, 107°56′‒108°00′ E; 299–686 m a.s.l) (Wang regarding changes of resource limitation after a system et al. 2019). This area has a monsoon climate. Mean an- has been N-saturated. These limitations highlight the nual temperature is about 19 °C, with the lowest importance of more field experiments conducted in N- monthly mean in January and the highest in July. The saturated systems and more experiments measuring en- mean annual precipitation is about 1,389 mm, with a zymes fully for calculating ecoenzymatic stoichiometry. distinct seasonal pattern: the wet season is from April to Topography is a modulator in many microbial pro- October and the dry season is from November to March. cesses, but is often ignored in the previous N-deposition The selected forest is about 35 years old after clear-cut, studies. Most N-deposition simulation experiments were dominated by Cryptocar-yachinensis (Hance) Hemsl., conducted in one topography position (mostly in flat Cinnamomum saxatile H. W. Li, Koelreuteria minor ground) or did not distinguish topography positions Hemsl., Pittosporum tobira (Thunb.) Ait., Bridelia Chen et al. Forest Ecosystems (2021) 8:68 Page 3 of 9 tomentosa Bl., Murraya exotica L. Mant. The soil is lep- properties, including soil organic C (SOC), total N (TN), tosols based on the FAO World Reference Base for Soil total P (TP), dissolved organic C (DOC), total dissolved Resources (IUSS-Working-Group 2006). More detailed N (TDN), and available P (AVP) (Carter and Gregorich information of soil properties can be found in a previous 2006; Lasota and Błońska 2021). Another portion were study (Wang et al. 2019). kept on ice in the field and were stored at 4 °C in the la- + − boratory for analyses of soil NH and NO concentra- 4 3 Experimental design tions, microbial biomass C (MBC), N (MBN), and P The experiment was initiated in April 2016 (Wang et al. (MBP), and extracellular enzyme activity (Chen et al. + − 2019). The experiment was conducted in the valley and 2018b). Soil NH and NO concentrations were ana- 4 3 on the slope, respectively, where the soil properties are lyzed by an autoanalyzer (FIAstar 5000, FOSS, Sweden). different (Table 1): there are higher concentrations of MBC, MBN, and MBP were determined by the chloro- − − soil organic C, total N, NO , and Ca and greater NO form fumigation extraction method (Brookes et al. 1982, 3 3 leaching rate on the slope than in the valley. In each 1985; Vance et al. 1987). In 2018, the nitrification rate position, a randomized block design was adopted with was measured using the intact tube incubation method, three N treatments and three blocks: control (CT, 0 kg and NO leaching rate was measured using ion- − 1 − 1 − 1 − 1 N·ha ·yr ), low-N addition (LN, 50 kg N·ha ·yr ) exchange resin bags method (Chen et al. 2016a). − 1 − 1 and high-N addition (HN, 100 kg N·ha ·yr ), which Enzyme activity assays were conducted within two were widely used in the N addition experiments (Mo weeks after soil sampling. β-D-glucosidase (BG), L- et al. 2007). Thus, there were 18 plots (10 m × 10 m Leucine aminopeptidase (LAP), β-N- each) in total (Fig. S1). Each plot was surrounded by a acetylglucosaminidase (NAG), and acid phosphatase 10-m wide buffer strip. NH NO was weighed and (AP) were assayed with published microplate protocols. 4 3 mixed with 10 L of water (equals to 1.2 mm annual pre- In addition, in order to model decomposition rates, we cipitation) for each N-treatment plot, and the NH NO also measured the activity of ligninolytic enzyme (i.e. 4 3 solution was sprayed every month to the forest floor polyphenol oxidase [POX]) with a spectrophotometrical with a backpack sprayer. The control plots received 10 L method. Detailed assay processes can be found in our of water without fertilizer. previous studies (Chen et al. 2017, 2018b). Enzyme activ- ity was expressed to units per g of soil organic carbon, − 1 − 1 Soil sampling and lab experiments i.e., µmol·g SOC·h . Soil sampling was conducted twice in May, 2017 and 2018, respectively. In each plot, five soil cores were col- Ecoenzymatic stoichiometry lected and mixed into a composite sample. Soils were Several methods were used to reflect microbial resource limi- passed through a 2-mm mesh sieve after picking out tation status. First, simple ratios of enzyme activities were roots and stones. The sieved soil samples were divided used. Higher BG/(LAP + NAG) and BG/AP indicate lower N into two portions for further processes. One portion was and P limitation, respectively (Waring et al. 2014). Second, air-dried at room temperature for analyzing soil vector length and angle of ecoenzymatic stoichiometry was conducted as follows (Moorhead et al. 2013): Table 1 The differences in soil properties between slope and sﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ + − 2 2 valley. BD, SOC, TN, TP, NH ,NO , Ca and Mg are bulk density, 4 3 lnBG lnBG Vector lengthðÞ unitless¼ þ ð1Þ soil organic carbon, total nitrogen, nitrate, ammonium, calcium ln½ NAG þ LAP lnAP and magnesium, respectively. Values represent means with lnBG lnBG standard errors in the parentheses. Different letters denote the Vector angleð Þ¼ Degrees ATAN2 ; lnAP ln½ NAG þ LAP significant difference between valley and slope ð2Þ Valley Slope − 3 BD (g·cm ) 0.90 (0.04) 0.95 (0.05) A longer vector length indicates greater C limitation; pH 7.19 (0.17) 7.32 (0.54) vector angles of < 45° and > 45° indicate N and P limita- − 1 SOC (g·kg ) 40.46 (3.77) b 52.24 (5.01) a tion, respectively (Moorhead et al. 2013). When vector − 1 TN (g·kg ) 3.74 (0.35) b 4.72 (0.50) a angle is < 45°, a greater vector angle indicates smaller N − 1 limitation. When vector angle is > 45°, a greater vector TP (g·kg ) 2.26 (0.16) 1.91 (0.22) + − 1 angle indicates greater P limitation. NH -N (mg·kg ) 7.60 (0.41) 5.68 (0.26) Third, C:N and C:P of the available resources (DOC/ − − 1 NO -N (mg·kg ) 19.91 (3.01) a 13.33 (1.07) b TDN (R ) and DOC/AVP (R ), respectively) were C:N C:P − 1 Ca (g·kg ) 7.04 (0.70) b 14.80 (2.1) a compared to the Threshold Elemental Ratios (TER) for NO leaching rate 3.27 (0.01) b 5.01 (0.01) a C:N and C:P (TER and TER , respectively). When C:N C:P − 1 − 1 (mg·d ·kg dry resin) R – TER or R – TER is less than zero, soil C:N C:N C:P C:P Chen et al. Forest Ecosystems (2021) 8:68 Page 4 of 9 microbes are not limited by N or P. When the R – following analyses we only focused on the results after C:N TER or R – TER is greater than zero, microbes two years of N additions. One-way analysis of variance C:N C:P C:P are N or P limited. In the latter case, higher R – (ANOVA) with least significant difference (LSD) test C:N TER or R – TER indicate higher N or P limita- was used to test the effects of the two-year N addition C:N C:P C:P tion (Sterner and Elser 2002). TER and TER were on soil properties, enzyme activities and ecoenzymatic C:N C:P calculated as follows (Sinsabaugh et al. 2009; Guo et al. stoichiometry indexes. A one-sample t-test was used to 2020; Zhao et al. 2021): test the difference between R – TER (or R – C:N C:N C:P TER ) and zero, and the difference between vector C:P BG angle and 45°. The data was tested homogeneity of vari- TER ¼ B =n ð3Þ C:N C:N 0 NAG þ LAP ance and normality of distribution prior to statistical analyses. All statistical analyses were conducted using BG TER ¼ B =p ð4Þ C:P C:P SPSS 16.0 statistical software (SPSS Inc., Chicago, IL, AP USA). All reported significant differences are at P < 0.05. where B and B are C:N and C:P ratios of micro- C:N C:P Results bial biomass, and n and p are the intercepts calculated 0 0 In the control, mean enzyme activities were 1.9 ± 0.1, from regressions of ln(BG) vs. ln(NAG + LAP) and − 1 31.7 ± 4, 0.01 ± 0.01, 0.6 ± 0.04, and 1.8 ± 0.1 µmol·g ln(BG) vs. ln(AP), respectively. − 1 SOC·h for BG, POX, LAP, NAG, and AP, respectively, We also modeled organic C decomposition (M, − 1 and there was no significant difference between slope %·day ) (Sinsabaugh and Moorhead 1994): and valley (Fig. 1). On the slope, N addition did not ½ NAG þ LAP change activities of all studied enzymes (Fig. 1). In the M ¼ðCUE ENZ Þ=ð1 þ TOT BG valley, N addition changed activities of all studied en- AP zymes after two years of N treatments (Fig. 1). The BG þÞð5Þ BG and NAG activities increased significantly in the high-N treatment compared to the control (Fig. 1a and d), and where ENZ is the sum of BG, POX, (NAG + LAP), TOT AP activity increased significantly in both high-N and and AP normalized to their maximum values. CUE is low-N addition treatments (Fig. 1e), but POX and LAP carbon use efficiency, which was calculated as follows activities decreased significantly in the high-N treatment (Sinsabaugh et al. 2013; Chen et al. 2018b): (Fig. 1c and b). As for ecoenzymatic stoichiometry, N addition did CUE ¼ CUE max 0:5 not change any indexes of ecoenzymatic stoichiometry S S C:N C:P ð6Þ on the slope position (Figs. 2 and 3). However, in the ðÞ K þ S ðÞ K þ S C:N C:N C:P C:P valley, N additions significantly increased BG/AP and vector length (Fig. 2a and b), but did not change the where BG/(LAP + NAG) in both low and high N treatments BG B C:N compared to the control (Fig. 2c). R – TER was S ¼ð1=ð Þ ð Þ C:N C:N C:N LAP þ NAG L C:N significantly lower than zero in the control, and N addition significantly decreased R – TER in C:N C:N BG B C:P S ¼ð1=ð ÞÞ C:P both low-N and high-N treatments (Fig. 2d). Vector AP L C:P angle in the control was significantly greater than 45°, CUE is the upper limit for microbial growth effi- and N addition significantly decreased vector angle in max ciency (0.6). S or S is a scalar that represents the the high-N treatment (not in the low-N treatment). C:N C:P extent to which the allocation of ecoenzymatic activities R – TER in the control was significantly greater C:P C:P offsets the disparity between the elemental composition than zero, and both low-N and high N treatments of available resources and the composition of microbial significantly increased R – TER (Fig. 2eand f). C:P C:P biomass. K and K are the half-saturation constants In addition, N addition significantly increased the C:N C:P (0.5). B and B are C/N and C/P of microbial bio- modeled decomposition rate (M) in both low-N and C:N C:P mass. L and L are available C:N (i.e. DOC/TDN) high-N treatments, and significantly decreased BG/ C:N C:P and C:P (i.e. DOC/ AVP). POX in the high-N treatment (Fig. 3aand b). Two-year N addition also changed soil properties Data analysis (Fig. 4). In the valley, N addition significantly increased In this study, soil enzyme activity had no responses to the concentrations of DOC and TDN in the high-N one-year N addition (Table S2), but had significant treatment (but not for the low-N treatment), and signifi- changes after two years of N addition. Thus, in the cantly increased nitrification rate and NO leaching rate 3 Chen et al. Forest Ecosystems (2021) 8:68 Page 5 of 9 − 1 − 1 Fig. 1 Changes of activities of studied enzymes (µmol·g SOC·h ) after two years of N addition. (a)BG(β-D-glucosidase) and (b) POX (polyphenol oxidase) are two C-acqusition enzymes; (c) LAP (L-leucine aminopeptidase) and (d) NAG (β-N-acetylglucosaminidase) are two N- acquisition enzymes; (e) AP (acid phosphatase) is a P-acqusition enzyme. CT: control; LN: low-N addition; HN: high-N addition. Error bars denote the standard error (n = 3). Different letters indicate significant difference (P < 0.05) between treatments in both low-N and high-N treatments, but did not chan- all studied enzymes after one year of N treatments in all ged other soil variables, including the concentrations of topography positions (Table S2), but altered soil enzyme SOC, TN, TP, AVP, MBC, MBN, MBP, NH and activity two years later in the valley, especially in high-N NO . On the slope, N addition significantly increased treatments (Fig. 1), indicating that the duration and rate nitrification rate and NO leaching rate in both low-N of N addition are important factors affecting soil enzyme and high-N treatments, but had no significant effects on activity. After two years of N addition, the responses of other studied variables (Fig. 4 and Table S1). the individual enzyme activity to N addition are consist- ent with the patterns reported in the recent meta- Discussion analysis studies (Jian et al. 2016;Chenetal. 2018c): in Responses of enzyme activity to N additions the valley, the BG, NAG, and AP activity increased sig- The mean enzyme activities measured in controls are nificantly, but POX and LAP activity decreased signifi- similar to those measured in nearby forests (Chen et al. cantly with the elevated N addition (Fig. 1). This is 2018a, b). Nitrogen addition did not change activities of interesting, because if such patterns reflect a general Fig. 2 Changes of enzymatic stoichiometry indexes after two years of N addition. (a) Vector length and (b) BG/AP are two indicators of microbial C limitation. High vector length or BG/AP means greater C limitation. (c) BG/(LAP + NAG) and (d) R – TER are two indicators of microbial N C:N C:N limiation. High BG/(LAP + NAG) means lower N limitation, while higher R – TER means higher N limitation. (e) Vactor angle and (f) R – TER are C:N C:N C:P C:P two indictors of microbial P limitation. Greater vector angle indicates greater P limitation when vector angles > 45°; higher R – TER means higher P C:P C:P limitation. CT: control; LN: low-N addition; HN: high-N addition. Error bars denote the standard error (n = 3). Different letters indicate significant difference (P < 0.05) between treatments. The asterisks indicate significant different between R – TER (or R – TER ) and zero C:N C:N C:P C:P Chen et al. Forest Ecosystems (2021) 8:68 Page 6 of 9 Nitrogen aggravates microbial C limitation in this for- est, which is consistent with the first hypothesis. The evidence is from the increased vector length in N- addition plots compared to the control (Fig. 2a). Simi- larly, we also found increased vector length in a previous meta-analysis by collecting enzyme activity data from 36 published N-addition studies (Chen et al. 2018d). Aggra- vated microbial C limitation has been suggested in many previous N-addition experiments (Treseder 2008). Here, we propose two likely reasons for the aggravated micro- bial C limitation after N addition in this study. First, it is a result of nutrient balance. To reach a balance of C and N, microbes will increase decomposition of organic mat- ter to get more available C as N increases. This is sup- ported by modeled decomposition rates (M) in this study, which was increased by 43 and 54 % in low-N and high-N addition, respectively, compared to the control (Fig. 3a). As a result, increase in soil DOC concentration was also found (Table 1). However, in this study, the in- creased total decomposition rate is mainly attributed to the increased decomposition of the labile C fraction, ra- ther than the non-labile C fraction. This can be reflected Fig. 3 Effects of N additon on (a) modeled decomposition rate (M) by the increased BG and the decreased POX (as a result, and (b) the ratio of BG (β-D-glucosidase) to POX (polyphenol the decreased BG/POX, Fig. 3b), since BG and POX are oxidase). BG and POX are two C-acqusition enzymes, decomposing two different enzymes to decompose labile C (such as labile and non-labile organic C, respectively. CT: control; LN: low-N cellulose) and non-labile C fraction (such as lignin), re- addition; HN: high-N addition. Error bars denote the standard error (n = 3). Different letters indicate significant difference (P < 0.05) spectively (Chen et al. 2018c). Therefore, the reduced between treatments decomposition of non-labile C fraction may be another reason for the aggravated microbial C limitation in this change of enzyme activity in response to N addition, our study, because it decreases the sources of available C. results on ecoenzymatic stoichiometry can be represen- Compared to the aggravated C limitation, however, N tative for regions of larger spatial scales. addition may alleviate microbial P limitation, because vector angle decreased but R – TER increased in C:P C:P N-treatments relative to controls (Fig. 2). This result re- Responses of microbial resource limitation to N additions jects our general belief that N addition leads to P limita- in the valley tion (Marklein and Houlton 2012). Increased AP may be We confirm that the studied forest is N-saturated, and a reason for the alleviated P limitation (Fig. 1e). How- show that N addition makes the phenomenon of N- ever, this might be a paradox, because increased AP not saturation more obvious. There are several lines of evi- only reflects the increased organic P decomposition dence to indicate that the studied forest is N-saturated: (meaning alleviated P limitation), but also reflects the in- R – TER was less than zero in controls (in both creased P demand of microbes (meaning aggravated P C:N C:N valley and slope), indicating that the forest is not limited limitation). Instead, we propose that alleviated P limita- by N (Fig. 2d); by contrast, this forest may be P-limited tion can be explained more directly by the increased because vector angle was greater than 45º and the R – BG/AP (Fig. 2b), which indicates that microbes need C:P TER was greater than zero in controls (Fig. 2e and f). more C than P under N inputs. Therefore, N addition C:P These evidence are also found in the previous studies aggravates C limitation, rather than P limitation, accord- (Chen et al. 2018b). Since this forest has been N- ing to Liebig’s law of the minimum that suggests only saturated, it is not surprising that continuous N addition one nutrient can limit plant productivity at a time make the symptom more obvious, which is evident from (Liebig 1842). that R – TER was significantly lower in N treat- C:N C:N ments than in the control (Fig. 2d). In addition, in- Topography modulates effects of N additions creased nitrification rate and NO leaching rate (Fig. 4) The aggravated C limitation or alleviated P limitation also support the aggravated N-saturation according to N were not found on the slope after two years of N saturation theory (Aber et al. 1989). addition, indicating that topography plays a role in Chen et al. Forest Ecosystems (2021) 8:68 Page 7 of 9 Fig. 4 Effects of two-year N addition on the disoveled organic carbon (DOC), total disoveled nitrogen (TDN), available phosphorus (AVP), nitrification rate, and leaching rate of nitrate (NO ). Error bars denote the standard error (n = 3). Different letters indicate significant difference (P < 0.05) between treatments modulating the effects of N addition on microbial re- Considering that soil NO is more susceptible to leach- source limitation. The second hypothesis is thus sup- ing than NH , and that more rock outcrops and the ported. Most previous studies did not consider greater inclination on the slope, more NO would be topography effects when they study the responses of leached from the slope, which weakens effects of N ecosystem processes to atmospheric N deposition. This addition on microbial activity on the slope. bias may over- or under-estimate effects of N deposition Soil N level is higher on the slope than in the valley in given that most nature ecosystems are not completely our studied forest (Table 1), which might be another flat. In a previous study with the same experimental site, mechanism making the valley position more sensitive to we have found the divergent responses of soil asymbiotic N addition relative to the slope. There is evidence that N fixation to N additions between the valley and slope N additions change ecosystem processes, such as soil C (Wang et al. 2019). Here, we also found similar pattern and N availability, more pronouncedly in N limited than for microbial activity, further highlighting the import- in N rich ecosystems (Aber et al. 1998; Chen et al. ance of topography in regulating the effects of N depos- 2015). As for microbial resource limitation, similar re- ition on ecosystem processes. sponses might exist as well. Higher soil N status on the As mentioned, there might be two contrary mecha- slope is not common in nature, but has been found in nisms (i.e., different soil N levels and N leaching poten- the karst region of south China (Liu et al. 2011). The di- tials) controlling the effects of the topography. In the vergent Ca contents between valley and slope might be current study, a reason for the weaker impacts of N responsible to this phenomenon. In karst forests, soil on addition on the slope may be due to the higher NO the slope generally has a higher Ca content due to the leaching in this topography position (Table 1). Soil gross higher rock explosion (Table 1). Because of the protec- nitrification rate in the limestone soil of the karst forest tion effect of Ca in storing soil organic matter (SOM) is very fast (Li et al. 2017), so that deposited NH would (Wen et al. 2016, 2017), soil on the slope has higher C be rapidly transformed to NO upon entering the soil. and N content relative to the valley. 3 Chen et al. Forest Ecosystems (2021) 8:68 Page 8 of 9 Conclusions Consent for publication Not applicable. Our findings that the continuous N addition to the eco- system in the valley switches N limitation to other limi- tations complements the N saturation theory. Most Competing interests The authors declare that they have no competing interests. studies suggest that P would be the critical element lim- iting plant growth after N limitation is removed, but this Author details pattern is rarely discussed at the microbial level. Carbon State Key Laboratory of Biocontrol, School of Ecology, Sun Yat-sen University, 510275 Guangzhou, China. Key Laboratory of Agro-ecological and P are two elements likely to limit microbial growth, Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese and here we show that C will be the more limited elem- Academy of Sciences, 410125 Changsha, Hunan, China. Huanjiang Observation ent after N limitation is removed, which is different from and Research Station for Karst Ecosystems, Institute of Subtropical Agriculture, Chinese Academy of Sciences, 547100 Huanjiang, China. the patterns observed in plants. This finding has import- ant implications for the global C cycling by explaining Received: 8 July 2021 Accepted: 27 August 2021 why N addition stimulates the early stages of litter de- composition but in general does not affect longer term decomposition rates (Knorr et al. 2005). 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"Forest Ecosystems" – Springer Journals
Published: Oct 12, 2021
Keywords: Nitrogen deposition; Topography; Nutrient limitation; N saturation; Enzyme activity; Enzymatic stoichiometry
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