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Effects of long-term nitrogen addition on the δ15N and δ13C of Larix gmelinii and soil in a boreal forest
Ecological Processes volume 11, Article number: 37 (2022)
Natural abundance of carbon (C) and nitrogen (N) stable isotope ratios (δ13C and δ15N) has been used to indicate the state and cycle of ecosystem C and N. However, it is still unclear how C and N cycle of boreal forests respond to the N deposition.
We conducted an 8-year continuous N addition field experiment in a Larix gmelinii forest in Greater Khingan Mountains, Northeast China. Four N treatments (0, 25, 50, 75 kg N ha−1 year−1) were built. The effects of N addition on the δ13C and δ15N of needle, branch, bark, and fine root of Larix gmelinii and soil were studied. The result of the balance between the N input and output flux showed that N addition significantly increased the δ15N in each organ of Larix gmelinii, but did not change the δ15N of soil. We also found that the N absorption by needles of Larix gmelinii could increase the needle photosynthesis rate and δ13C by increasing carboxylation, but N addition had no significant effect on the δ13C of soil and other organs. In addition, both the soil δ15N and δ13C increased with the soil depth.
Long-term N addition may lead to more open C and N cycles and further affect plant nutrient acquisition strategies in boreal forest ecosystems.
Due to fossil fuel combustion and agricultural emissions, anthropogenic nitrogen (N) deposition has been increased dramatically in the past century and is expected to intensify further in the coming decades (Galloway et al. 2008; Liu et al. 2013). Excessive N input can lead to many negative effects, including increased N leaching to water, soil acidification, increased N gas losses from soil and changes in biodiversity (Gao et al. 2014; Chen et al. 2016; Lucander et al. 2021). Therefore, it is necessary to understand the impact direction, intensity and future trend of N deposition on ecosystem C and N cycle, and formulate effective C and N management measures under enhanced global N deposition.
The natural abundance of stable C and N isotope ratios (δ13C and δ15N) is an effective method to explore the C and N dynamics of different ecosystems (Liu et al. 2017; Rao et al. 2017). 15N natural abundance (δ15N) is the comprehensive result of the dynamic process of N cycle (Högberg 1997; Zhang et al. 2018). However, the fractionation of N isotopes in the process of N transformation, absorption, assimilation, distribution, and loss, leads to plants δ15N being different from the δ15N of N source (Bai et al. 2012). Therefore, δ15N of plants can reflect the δ15N of source N and the isotopic fractionation during N cycling processes (Robinson 2001; Kahmen et al. 2008). For example, during nitrification, when the chemical bond of the substrate breaks, the heavier 15N will be transformed into the product more slowly than the lighter 14N, so that the product will have a lower 15N/14N ratio than that of the remaining residual nitrate (Niu et al. 2016; Reis et al. 2016). Therefore, when N saturation causes increased nitrification, soil and plant will become more and more enriched in 15N (Wang et al. 2014). For ecosystem N pool, the N transformation rate is determined by the input and output rates (Högberg et al. 2014). If the increased N input leads to increased N transformation, stronger fractionation and loss of 15N-depleted N, it would affect enrichment of soil and subsequently plant tissue. However, if both the rates of N input and output changes with the increase of N deposition, the responses of plant and soil δ15N values would be complex. Results showed that N additions increased N losses, and the change in plant δ15N after N addition might be related to the balance between higher soil δ15N and 15N depleted N from atmospheric deposition (Kriszan et al. 2009; Gurmesa et al. 2017; Hobbie et al. 2017). Therefore, evaluating the changes in ecosystem δ15N under N addition treatments can provide valuable information on how N flux, assimilation and distribution respond to the increase in N input in forest ecosystems.
Atmospheric air represents a constant C pool with relatively stable isotopic composition and concentration (Galewsky et al. 2016). Therefore, the change in 13C reflects plant physiology: the balance between CO2 supply (conduction) and CO2 demand (assimilation) (Ehleringer 1993). For example, the decrease in stomatal conductance and the restriction of photosynthetic enzyme activity will eventually lead to an increase in plant δ13C value (Jia et al. 2016; Lavergne et al. 2020). Several studies have suggested that foliar uptake of atmospheric reactive N could enhance the rates of photosynthesis and altered foliar δ13C through increased carboxylation (Siegwolf et al. 2001; Guerrieri et al. 2011), although some studies reported no changes in plant δ13C as a result of N enrichment (Wang et al. 2021). Carbon isotope exchange reaction enriches 13C in inorganic salts (such as carbonate), while the dynamic fractionation effect of photosynthesis in the organic cycle of C leads to the 13C-depletion in biogenic C (organic matter), leading to a change in C isotope composition during the exchange of CO2 between organisms and terrestrial ecosystems (Cao et al. 2012; Busch et al. 2020). In the process of microbial decomposition and mineralization of soil organic carbon (SOC) into CO2, the isotope effect is small (only 1–3‰), while strong fraction (10–70‰) occurs during methane production from SOC under anaerobic condition (Conrad et al. 2010). Compared with the fractionation of CO2 fixation during plant photosynthesis, the fractionation during SOC decomposition is almost negligible (Crow et al. 2006). Therefore, by measuring the change in C isotope ratio in each C pool, we can quantify the source, distribution and turnover of C in the “atmosphere–plant–soil” continuum.
Boreal forests account for about 15% of the earth’s total land area and 30% of the forest area, making them the second largest forest biota on land (Vitousek et al. 2002). The boreal forest plays an important role in regulating the global C and N balance and slowing down the rise of greenhouse gas concentration in the atmosphere, and it is also considered to be one of the ecosystems seriously limiting N (Randerson et al. 2006; Houghton 2007). The Greater Khingan Mountains is the only boreal forest zone of China. The widely distributed Larix gmelinii forest is one of the most sensitive vegetation types to global climate change. However, the impacts of N enrichment on the C and N cycle in this ecosystem need to be evaluated. Therefore, we carried out N addition field experiment in Larix gmelinii forest in Greater Khingan Mountains. In 2011, According to the current N deposition rate in northern China (25 kg N ha−1 year−1) (Liu et al. 2013), we established four N addition treatments (0, 25, 50, 75 kg N ha−1 year−1) to simulate the increase of atmospheric N deposition by 0, 1, 2, 3 times in the future. In this experiment, we measured the total C (TC), total N (TN), δ13C and δ15N of needle, branch, bark, and fine root of Larix gmelinii and soil under different N addition treatments. We also measured the photosynthetic rate and photosynthetic enzyme activity. The purpose of this study is to answer the following questions: (1) Whether N addition will change the composition of δ13C and δ15N in plants and soil? (2) What are the effects of different N addition levels on the changes of δ13C and δ15N composition in plants and soil? We hypothesized that (1) N addition will increase the loss of N through gaseous pathway and increase the soil δ15N value. (2) N addition will increase the absorption of N by Larix gmelinii and increase δ15N value in each organ. (3) N addition can increase the δ13C value of Larix gmelinii needles by increasing related processes of photosynthesis.
This study was conducted in a natural larch forest of Nanwenghe National Nature Reserve in the Greater Khingan Mountains, Northeast China (51° 05′–51° 39′ N, 125° 07′–125° 50′ E) (Fig. 1). The region has a typical temperate continental climate, with an average temperature of − 2.7 ℃, an annual precipitation of about 500 mm, a plant growth period of about 110 days and an annual sunshine time of 2500 h. The soil is dark brown sandy gravel soil with an average depth of 20 cm. The dominant tree species is Larix gmelinii, whose growth density is 2852 (± 99) trees/ha, and the average diameter at breast height (DBH) was 8.98 (± 0.32) cm, a stand age of 40 years and a canopy density of 0.76 ± 0.30. The vegetation under the forest includes Rhododendron dauricum and Rosa davurica.
In 2011, we randomly set up three sample blocks (Fig. 1), and each sample block includes four 20 m × 20 m plots. The buffer between any two plots is greater than 10 m to avoid interference with nearby plots. We randomly set up four levels of N addition treatment, control (CK, 0 kg N ha−1 year−1), low N (LN, 25 kg N ha−1 year−1), medium N (MN, 50 kg N ha−1 year−1), and high N (HN, 75 kg N ha−1 year−1). The N deposition levels were chosen to simulate a future N deposition of 1–3 folds of the current N deposition level in the study region (25 kg N ha−1 year−1) (Liu et al. 2013). For each N addition plot, NH4NO3 was dissolved in 32 L water in the growing season (from May to September, a total of 5 times each year), and sprayed evenly on the forest floor of each plot using a prayer. To compensate water supply, the control plot was sprayed with the same amount of pure water. The natural abundance of the N fertilizer (NH4NO3) in this study was 0.21% ± 0.43‰.
The sample collection was carried out in mid July 2019. Three trees with similar height and DBH (diameter at breast height) in each plot were randomly selected. Current-year needle, branch, and bark of Larix gmelinii from the top most were cut with a pole pruner. The sampled tree species were marked with numbers. Under the marked trees in each plot, we randomly drilled three soil cores with a soil auger (5 cm in diameter and 20 cm in length), then picked out the root. To obtain soil samples, we randomly selected three points under each labeled tree species, and used a soil auger to get the soil (divided into Oa+e layer and mineral layer, in which the “O” layer is the surface layer of the soil, composed of forest litter, the “a” layer represents the humus layer, and the “e” layer represents the leaching layer). The samples of the same soil layer from each plot were evenly combined to obtain a representative soil sample in each plot. All samples were dried to constant weight at 65 °C and then ground into a fine powder using a Tecator sample mill (Subang, Shanghai, China) for analysis.
Three Larix gmelinii trees with similar DBH and height were randomly selected in each plot, with a total of 36 trees (trees for sampling). The healthy needles were selected at different positions (from upper, middle, and lower canopy layers) of the tree crown, and the photosynthetic rate was measured in situ by a hand-held photosynthesis measurement system (CI-340, CID, USA). For each tree, the photosynthesis measurement was done three times in each layer to produce average photosynthetic rate of the whole plant. The photosynthetic rate of all leaves was measured from 9:00 to 11:00 every morning. Because the needles of Larix gmelinii are irregular, it is difficult to accurately determine its leaf area. It is inappropriate to use the photosynthesis per unit area of leaves to express the photosynthetic rate. Therefore, after each measurement of photosynthesis, the measured needles were removed with scissors and weighed to calculate the photosynthetic rate per unit mass. The following equation was used (Qiu 2011):
where Pn (FW) is the photosynthetic rate per unit fresh weight; Pn is the measured value displayed by photosynthetic instrument; S is the leaf chamber area; FW is the fresh weight of leaves in leaf chamber.
The C, 13C, N, and 15N contents of all samples were measured on an isotope ratio mass spectrometer (Isoprime 100, Isoprime Ltd.) coupled to an automatic, online elemental analyzer (Vario ISOTOPE cube). The reproducibility of the isotope determination was analyzed in multiple runs at several laboratories using an internal standard soil and plant sample from boreal forests. The accuracy of element content and isotope test is 0.01‰, and the repeated test error (± 2σ) is < 0.02% and < 0.05‰, respectively. Natural abundance δ15N and δ13C in samples were reported in the conventional delta (δ) notation, with units of per mil (‰). When measuring the 13C abundance of the soil, we acidified the soil with 1 N HCl at room temperature for 24 h to remove any inorganic C. The stable isotope ratio (δ15N and δ13C) is expressed as parts per thousand differences from a standard: δ15N/δ13C = (Rsample/Rstandard − 1) × 1000 (‰). Rsample represents the isotope ratio of 15N/14N or 13C/12C in the plant or soil sample, and Rstandard represents the standard ratio of 15N/14N or 13C/12C, usually expressed as 15N/14N of N2 in the atmosphere, Rstandard = 0.011237 is the 13C/12C ratio of international standard (PeeDee Belemnite—PDB).
To determine the content of NH4+–N and NO3−–N in the soil, 5 g fresh soil samples were extracted with 100 mL 0.2 M KCl, oscillated 60 min in the oscillator, and then collected the supernatant. All analyses were performed on a Smartchem instrument 200 (Westco Scientific Instruments, Inc., Italy). Stomatal conductance was measured by hand-held photosynthesis measurement system (CI-340, CID, USA), chlorophyll content and Rubisco activity were measured by ultraviolet–visible spectrophotometer (SHIMADZU UV-1780, Japan).
The Kolmogorov–Smirnov test was used to detect whether the data conformed to the normal distribution, and data homogeneity was detected with Levene’s test. One-way analysis of variance (ANOVA) was used to analyze the effects of N addition on different components C %, δ13C, N %, and δ15N of the soil and Larix gmelinii, and the effects of N addition on needle photosynthesis related parameters. Pearson correlation analysis was used to analyze the correlation between different components C %, δ13C, N %, and δ15N of the soil and Larix gmelinii, and the correlation between photosynthetic related parameters and needle δ13C. All analyses were performed with SPSS 22.0 software package (SPSS, Inc., Chicago, Illinois, USA). Diagrams were drawn using Sigmaplot 13.0 software (Systat Software Inc., Chicago, IL, USA). Tukey’s post hoc test was used to test differences between treatments. Statistically significant differences were set at p < 0.05.
Effects of N addition on the N content and δ15N of plant and soil
Compared with the control, the δ15N and N content of Oa+e layer showed a significant increase under MH and HN treatments (p < 0.05, Table 1). There was no significant difference in the δ15N between 0–10 cm and 10–20 cm soil, but MN and HN treatments significantly increased TN content in 0–10 cm soil (p < 0.05). HN treatment significantly increased NH4+–N and NO3−–N in 0–10 cm soil, while all the three levels of N addition had no significant effect on TN, NH4+–N, and NO3−–N in 10–20 cm soil (Table 1). Under different N addition treatments, the concentration of NH4+–N in Oa+e layer showed significant differences, and the concentration of NH4+–N under HN treatment was significantly higher than that of the control (p < 0.05, Table 1). Besides, MN treatment significantly increased NO3−–N in Oa+e layer (p < 0.05, Table 1).
The δ15N of each organ of Larix gmelinii increased with N addition. MN and HN treatments significantly increased δ15N of needle, bark, and fine root (p < 0.05), while HN treatment significantly increased the δ15N of branch and litter (p < 0.05, Fig. 2A). HN treatment significantly increased TN of needle, bark, and litter (p < 0.05), MN and HN treatments significantly increased TN of fine root (p < 0.05), while N addition had no significant effect on TN of branch (Fig. 2B). The soil δ15N and litter δ15N were significantly negatively correlated with the needle δ15N, while the needle δ15N was significantly positively correlated with the δ15N of Oa+e, bark, branch, litter, and fine root. There was a significant positive correlation between the δ15N of fine root and litter, branch, and bark (Fig. 5).
Effects of N addition on the C content and δ13C of plant and soil
Nitrogen addition did not significantly change TC and δ13C in Oa+e and 0–20 cm soil layers (Table 1). For Larix gmelinii, N addition significantly increased the δ13C of the needle (p < 0.05). The δ13C of branch and bark decreased with N addition, while the δ13C of litter and fine root increased with N addition (Fig. 3A). The N addition had no significant effect on the TC of organs of Larix gmelinii (Fig. 3B). The TC and TN of Oa+e were significantly positively correlated, and the TC of litter was significantly positively correlated with the δ13C of litter (Fig. 5).
Effects of N addition on the photosynthetic rate of Larix gmelinii
MN treatment significantly increased the photosynthetic rate of Larix gmelinii (Fig. 4A). HN treatment significantly increased the chlorophyll content (Fig. 4C). LN and MN treatments significantly increased the activity of Rubisco enzyme (Fig. 4D). Nitrogen addition had no significant effect on the stomatal conductance of the needle (Fig. 4B). The photosynthetic rate was significantly positively correlated with Rubisco enzyme, chlorophyll, and needle δ13C (p < 0.05), and chlorophyll and stomatal conductance were significantly positively correlated with the needle δ13C (p < 0.05, Table 2).
Effects of N addition on the N content and δ15N of plant and soil
The input process (e.g., N deposition and biological N fixation) and output process (e.g., denitrification, leaching and plant absorption) of N in soil determine the size of N pool (Dawson et al. 2002; Fernandez-Alonso et al. 2018; Rivero-Villar et al. 2021). In this study, addition of N with δ15N close to that of atmospheric N2 (≈ 0), significantly increased the δ15N of Larix gmelinii (Fig. 2). Our results showed that the N addition significantly increased the soil TN (Table 1), but had no significant effect on the δ15N of soil mineral horizon, which is inconsistent with previous studies (Gurmesa et al. 2017; Wang et al. 2021) and our first hypothesis. The δ15N of soil is regulated by N input and N output fluxes (Liu et al. 2017). Based on the mass balance model of Brenner et al. (2001), under the condition of constant N output flux, N addition reduces the δ15N of soil. Therefore, the lack of significant changes in soil δ15N after long-term N addition is likely to be the result of the balance between input and output flux. This is probably due to the loss of 15N-depleted N through denitrification and leaching. Under such condition, the residual soil (and plant) would be 15N-enriched. MN and HN treatments significantly increased NH4+–N and NO3––N, and increased soil mineralization rate (Table 1), which might lead to increased denitrification and leaching. Gaseous losses of 15N-depleted N by denitrification would enrich 15N in soil N (Houlton et al. 2006). It has been proved that NH3 volatilization process will cause 40–60‰ isotope fractionation effect (Robinson 2001; Liu et al. 2017). MN and HN treatments significantly increased the δ15N of Oa+e, which might be because we added a relatively 15 N-enriched N to the Oa+e that was relatively 15N-depleted. In addition, soil organic matter rich in 14N was decomposed during soil microbial decomposition, resulting in soil 15N enrichment (Bostrm et al. 2007). 15N enriched microorganisms and microbial residues are produced in the process of microbial assimilation for organic matter, which will also lead to the gradual enrichment of 15N in soil organic matter over time (Dijkstra et al. 2010).
Nitrogen addition significantly increased the δ15N in plant compartments (Fig. 2), and the δ15N in needles was positively correlated with branch, bark, fine root, litter, and humus (Fig. 5), which was consistent with our second hypothesis. The increased plant δ15N as a result of N addition could be the imprint of the added fertilizer N which was 15N-enriched compared to the plant tissues (Gurmesa et al. 2017). Our results also showed that the increase of δ15N of Larix gmelinii was directly related to the soil δ15N (Fig. 5). In the process of N cycle, N addition leads to an increase in plant N utilization rate, and then increases the δ15N of each organ of Larix gmelinii due to isotope fractionation (Sheng et al. 2019). In addition, when the soil N availability is low, plants may be more dependent on mycorrhizal fungi (Vallano and Sparks 2013). Mycorrhizal fungi usually transfer 15N depleted N to plants, resulting in high enrichment of 15N in mycorrhizal mycelium, resulting in 15N depletion in plant tissues (Schweiger, 2016). The δ15N of fine roots of Larix gmelinii is significantly lower than that of needle, branch, and bark (Fig. 2), which may be caused by the fractionation effect of mycorrhizal. The increase of δ15N in needles can also be used to indicate the increase of N cycle rate. Previous studies have also shown that there is a strong correlation between the enrichment level of 15N in leaves and the increase in N cycle rate (Garten 1993; Emmett et al. 1998). The greater the difference in δ15N between the shoot and the root, the greater the proportion of inorganic N absorbed by the leaves (Kalcsits et al. 2014).
We also found that the soil δ15N increased with an increase in soil depth, which is consistent with a large number of studies (Kramer et al. 2017; Högberg et al. 2019; Potapov et al. 2019). This may be because 15N depleted litter and plant residues enter the surface soil, and the accumulation of 15N enriched mycorrhizal fungal residues in the deep soil can lead to a large enrichment of 15N in the deep soil relative to the surface litter.
Effects of N addition on the C content and δ13C of plant and soil and photosynthetic rate
Our results showed that N addition significantly increased the δ13C of needle, but had no significant effect on the δ13C of other plant organs (Fig. 3). The C isotope fractionation caused by photosynthesis mainly takes place in the following two steps: first, there is a certain resistance when 13CO2 enters mesophyll cells. Due to the influence of kinetic fractionation effect, 12CO2 diffuses faster and is easily absorbed by plants; Second, under the action of carboxyl polypeptidase, 12CO2 dissolved in cells and Rubisco carboxylase are preferentially combined into phosphoglyceric acid, while the remaining CO2 is enriched in 13C (Park and Epstein 1960; Flexas et al. 2006; Ulli et al. 2008). Previous studies have shown that the decrease in plant stomatal conductance leads to a decrease of CO2 concentration in plant leaves and the increase of δ13C in leaves (Morecroft and Woodward 1990; Rao et al. 2017). In our study, N addition had no significant effect on stomatal conductance (Fig. 4B). Therefore, we speculate that the increase in needle δ13C may be caused by the assimilation of 12CO2. The increase of 13C in needles could be explained by the addition of N increased Rubisco activity (Fig. 4D) and chlorophyll (Fig. 4C), increased photosynthetic rate (Fig. 4A), and gradually increased the consumption of 12CO2, which is consistent with our third hypothesis. Tomaszewski and Sieving (2007) also showed that N addition increased photosynthetic rate, which could enhance CO2 assimilation but also reduce the thermally dissipated light.
Our previous results found that N addition significantly increased CH4 and CO2 emissions from the Larix gmelinii forest (Yan et al. 2018), and the increase of C output flux usually led to the enrichment of soil 13C. The lack of significant change in soil 13C in response to N addition (Fig. 3A) could be a trade-off between increased levels of plant residue (more 13C depleted than soil C) inputs and 13C enrichment from enhanced soil C decomposition. Litter, fine root, and root exudates are the main sources of C input to soil (Waldrop et al. 2004; Cusack et al. 2010). The decrease of litter δ13C (Fig. 3A) may be related to the fractionation effect of C release during litter decomposition. Easily decomposable organic compounds (such as sugar, starch and cellulose) in litter usually show high δ13C value. In the process of microbial decomposition, this leads to the loss of 12C faster than 13C, resulting in the enrichment of 13C in litter (Cotrufo et al. 2005; Bowling et al. 2008). The retention efficiency of root C in soil is much higher than that of litter C (Clemmensen et al. 2013). Therefore, the change in equilibrium δ13C output may mainly come from the input of roots and root exudates. Gougherty et al. (2018) found that the greater allocation of relatively 13C depleted (i.e., lower δ13C values) non-structure carbohydrate (NSC) from leaves to roots when the photosynthesis was high. Our previous studies also showed that N addition increased the biomass of underground roots of Larix gmelinii and strengthened the transport function of fine roots to overcome the P limitation (Liu et al. 2021). A meta-analysis by Finzi et al. (2015) showed that the contribution of root C input to the C–N mineralization process of forest soil was as high as 33%. The study by Bais et al. (2006) also found that up to 40% of photosynthetic fixed C was secreted into the rhizosphere. And high nutrient availability can promote the release of fine root exudates (Neumann and Romheld 2000). Although we have not measured root exudates, the fractionation effect of root exudates on soil C input cannot be ignored.
Gessler et al. (2012) showed that the difference of δ13C in leaves and δ13C in phloem of branch reached 30%, due to the fractionation effect of photosynthesis. In our results, although there is no significant change in δ13C of branch and bark, it shows a decreasing trend with the N addition, and the δ13C of branch and bark is also 8.4–13% lower than that of needle (Fig. 3A), which may be due to the increased transport of organic matter rich in 12C to the aboveground.
Our study shows that long-term N addition changes the C and N cycle of boreal forest ecosystem. The N addition increased the δ15N value and TN of each organ of Larix gmelinii and also increased the N mineralization of soil, but had no significant effect on the soil δ15N. Compared with N, the C concentration and 13C of Larix gmelinii were less affected. The N addition only increased the δ13C of needle, which was the result of the increase of photosynthetic efficiency of needle. By exploring the stable C and N isotopic signatures in plant–soil compartments in boreal forest ecosystems, this study confirms that the C and N state and cycle of ecosystem may be fluctuated under the projected N enrichment.
Availability of data and materials
Data are available from the corresponding author on reasonable request.
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We gratefully acknowledge Professor Yunting Fang and Dr. Feifei Zhu from the Institute of Applied Ecology, Chinese Academy of Sciences for their comments and suggestions on an earlier draft of this manuscript.
This research was supported by grants from the National Natural Science Foundation of China (42230106, 41773075, 41575137) and Open Grant for Key Laboratory of Sustainable Forest Ecosystem Management (Northeast Forestry University), Ministry of Education (KFJJ2021ZD01).
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Liu, G., Yin, Z., Yan, G. et al. Effects of long-term nitrogen addition on the δ15N and δ13C of Larix gmelinii and soil in a boreal forest. Ecol Process 11, 37 (2022). https://doi.org/10.1186/s13717-022-00382-0
- Nitrogen deposition
- Stable isotope technology
- Natural abundance of carbon
- Natural abundance of nitrogen
- Boreal forest