Global nitrogen enrichment impacts plant diversity more than soil bacterial and fungal diversity: a meta-analysis

Domain: earth_science
Published: 2026-01-10
Editorial outcome: major_revision

Abstract
Anthropogenic nitrogen enrichment has significant effects on plant and soil biodiversity across local to global scales, yet how the nitrogen-induced changes in plant diversity differ from those in soil organisms across multiple ecosystems and climatic conditions remains virtually unknown. By synthesizing plant and soil studies globally (3,816 paired observations from 458 articles), we demonstrate that nitrogen enrichment has stronger negative effects on plant diversity but surprisingly modest to negligible effects on soil bacterial and fungal diversity. On average, nitrogen enrichment results in 8.3% and 10.3% reductions in species richness and Shannon index for plants. In contrast, it leads to 1.9% and 0.2% reductions for soil bacteria and <0.4% changes for soil fungi, respectively. The nitrogen-induced soil acidification modulates soil biodiversity change and, in conjunction with aboveground biomass, regulates plant biodiversity change. Our findings provide a perspective on the differential impacts of global nitrogen enrichment on above- and below-ground biodiversity, highlighting the need for integrated biodiversity conservation strategies.

Introduction
Biodiversity is fundamental for the functioning, resilience, and productivity of ecosystems and for the health and stability of the biosphere1–5. Diverse plant communities provide various ecosystem services, from carbon sequestration, climate change mitigation and cultural services to support of a wider array of species, including soil biota, when compared with low-plant diversity ecosystems6–8. Similarly, diverse soil communities plays a critical role in key ecological processes such as decomposition, nutrient cycling, carbon storage, and disease regulation, which in turn enhance plant diversity and productivity2,4,9,10. The intricate interplay between plant and soil communities is essential for maintaining ecosystem functions and services, as well as for responding to climate change and other anthropogenic pressures11–14. Losses in either plant or soil biodiversity can trigger cascading ecosystem dysfunction, with detrimental consequences for both natural systems and human well-being15–17. While changes in plant diversity often influence soil biodiversity and composition11,13,18, plant and soil communities can exhibit divergent responses to anthropogenic stressors5, mainly due to their fundamental differences in life-history traits, environmental sensitivity, and resource dependence19,20. Therefore, we need to better understand the combined and interactive nature of plant and soil biodiversity responses to global change across multiple ecosystems and climatic conditions.

Anthropogenic nitrogen (N) enrichment (including atmospheric N deposition, N fertilization or N addition), a pervasive component of global change, is projected to continue21, which may increasingly harm plant and soil biodiversity22–27. Plant diversity is often negatively affected by N enrichment with non-linear patterns28, mainly due to the increased competition for light and other resources26,29,30, acidification25,31, and reduced niche dimensionality32,33. The magnitude of these effects is further modulated by environmental conditions and the responsiveness of primary production34,35. Nitrogen affects soil biodiversity primarily through changing soil pH and nutrients availability, which directly alter competition advantages among taxa and their niche convergence36–38. Given larger variations in taxonomic or functional groups of soil organisms compared to plants20,39, soil microbial communities have exhibited variable responses to N enrichment, ranging from positive to neutral to negative, depending on environmental context27,40,41. Moreover, the effects of N enrichment and the feedbacks between plants and soils are influenced by climatic and edaphic conditions25,34,35,42–45. The lack of holistic integrated understanding of these context-dependent responses (our goal herein) strongly limits our capacity to predict how terrestrial ecosystems might respond to ongoing global N enrichment.

Previous studies have examined the effects of global change factors (including N enrichment) on plant richness and soil biodiversity separately (Supplementary Table 1). However, they have not addressed two critical questions: (i) Do plant and soil biodiversity respond divergently to N enrichment? and (ii) How do these responses vary across different climates and biomes? These knowledge gaps hinder our ability to contrast the effects of N on these interconnected communities and to understand how plant communities mediate N effects on soil communities, and vice versa.

Despite the close association between plant and soil biodiversity, plant diversity is often more directly influenced by aboveground resource competition (e.g., light, space) and soil acidification25,26,29–31, whereas soil biodiversity is shaped by belowground conditions such as pH, nutrient stoichiometry, and organic matter inputs9,46,47, and usually depends on the taxonomic or functional groups and environmental context27,40,41. We therefore hypothesize that plant diversity may respond more consistently and strongly to N enrichment, while soil biodiversity show weaker or more context-dependent responses.

Herein, we perform a global synthesis using 3816 paired observations from 458 N enrichment studies across 366 sites in 31 countries. Our global dataset contain control and N enrichment treatments covering a wide range of N enrichment rates at sites with contrasting mean annual temperature and precipitation (i.e., binned into three major climate zones for certain purposes: tropical, temperate and boreal), soil pH and ecosystem types (i.e., croplands, grasslands and forests) (Supplementary Fig. 1 and Table 2). We first conduct a meta-analysis to evaluate the effects of N on plant and soil biodiversity, testing the climatic and biome context dependency. We report richness (e.g., the number of species) and Shannon index as metrics of plant and soil biodiversity, which jointly capture the number of taxa and their relative abundance distribution. However, due to limited data availability, we include only the Shannon index for soil nematodes. We focus on standardized effect size expressed at a common rate (25 kg N ha−1 year−1) and duration (10 years) of N treatment to minimize the bias induced by variations in these two factors (see “Methods”). We use model selection and regression analyses, followed by structural equation modeling, to identify the key factors modulating N effects. We find that N enrichment impacts plant diversity more than soil bacterial and fungal diversity, underscoring the need for integrated approaches to mitigate the impacts of N enrichment on both above- and below-ground biodiversity.

Results
Effects of nitrogen enrichment on soil properties
Nitrogen enrichment increased the concentrations of soil total and available N, as well as organic carbon (Supplementary Fig. 2), but decreased soil pH (Supplementary Fig. 3), indicating N-induced soil acidification. Soil N and organic carbon increases were greater in grasslands and forests than in croplands. Among the three climates, soil organic carbon and ammonium N increases were greater in boreal climates. In contrast, soil nitrate N increase was greater in tropical climates compared with other climates. Overall, the increase in total N was comparable among the three climates. The N enrichment-induced soil acidification was significantly greater in grasslands and croplands than in forests, and it was also notably higher in temperate climates compared to boreal and tropical climates. This pattern primarily stems from the fact that these ecosystem and climate regions generally have higher background soil pH levels (Supplementary Fig. 3), while the reduction in soil pH due to N enrichment is significantly negatively correlated with background soil pH (Supplementary Fig. 3). Furthermore, ammonium N has a more pronounced effect on soil pH than other forms of N (Supplementary Fig. 3).

Effects of nitrogen enrichment on plant diversity
Nitrogen enrichment had relatively strong negative effects on plant biodiversity (Fig. 1), in agreement with previous studies22,23,25,26,45. The negative effect of N (at the common rate of 25 kg ha−1 year−1 for 10 years) for plant species richness averaged -8.3% over forests and grasslands, being similar for these two ecosystems (mean effect size (EComN): −10.3% and −8.1%, 95% confidence interval (CI): −13.9 to −6.6% and −8.8 to −7.4%, all P < 0.05). Shannon index showed similar trends between forests and grasslands (EComN: −3.6% and −2.5%, CI: −12.4 to 6.2 and −4.2 to −0.8%).

Among the three main climate zones, the negative effect of N on plant species richness was significantly greater in boreal (including arctic) climates (EComN: −19.5%, CI: −25.4 to −13.2%, P < 0.05), followed by temperate (EComN: −8.3%, CI: −9.0 to −7.5%, P < 0.05) and tropical (including subtropical) climates (EComN: −6.3%, CI: −8.4 to −4.3%, P < 0.05). Similarly, plant Shannon index also showed a significant negative response to N enrichment in boreal climates (EComN: −16.3%, CI: −23.0 to −9.0%, P < 0.05) but not in other climates (Fig. 1). This more negative effect in boreal climates may arise from intensified interspecific competition induced by N enrichment, as boreal regions receive lower N inputs (Supplementary Fig. 4) and N limitation is common in cold high-latitude ecosystems48,49. Therefore, exogenous N inputs may stimulate the growth of certain fast-growing species, enabling them to outcompete others for limited resources, thereby reducing overall diversity. This explanation is supported by the significantly greater N enrichment-induced increase in plant aboveground biomass in boreal compared to other climates (Supplementary Fig. 5a). This increase was negatively correlated with declines in plant diversity (Supplementary Fig. 5b,c), as an increase in aboveground biomass typically enhances competition for light30. Our findings underscore the complex and context-dependent effects of N enrichment on plant diversity across ecosystems and climate zones.

Effects of nitrogen enrichment on soil biodiversity
When averaged across ecosystems and climates, N enrichment significantly but very modestly reduced the diversity of soil bacteria (EComN: −1.9% and −0.2%, CI: −3.3 to −0.5% and −0.2 to −0.1%, for richness and Shannon index, respectively, all P < 0.05) and soil nematodes (EComN: −2.2%, CI: −3.0 to −1.5% for Shannon index, P < 0.05) but not of total soil fungi (EComN: +0.2% and +0.4%, CI: −0.4 to 0.8% and 0–0.7%) (Fig. 1). The negative effects on soil bacteria and nematodes are associated with increased soil acidification and elevated soil N concentration50,51 (Fig. 2 and Supplementary Figs. 2, 3, 6), which disrupt bacterial energy metabolism by impairing proton gradients36,37 and reduced the ability of nematodes to regulate osmotic pressure52. These processes modestly enhance the competitive advantage of well-adapted taxa in acidified soils, resulting in ecological niche convergence and thereby decreased diversity36–38. Conversely, fungal cell membranes exhibit greater tolerance to low pH conditions, allowing fungi to thrive45 and occupy niches that are less suitable for bacteria37,52. Furthermore, the symbiotic relationships between fungi and plants may also provide a buffering effect against soil acidity53. The lack of N enrichment effects on total fungi may also be related to contrasting N effects on different fungal guilds54–56.

Among the three ecosystem types, N enrichment reduced bacterial and nematode diversity in grasslands and/or croplands (Fig. 1). This trend likely stems from greater reductions in soil pH in these two ecosystems (Supplementary Fig. 3), in as much as the biodiversity of both bacteria and nematodes is strongly affected by soil pH (Fig. 2). In forests, N enrichment significantly increased the overall fungal Shannon index (EComN: +1.6%, CI: +0.6 to +2.7%, P < 0.05). We ascribe this increase to elevated soil phosphorous availability, which positively correlates with fungal diversity57–59. Nitrogen enrichment usually increases phosphorus availability by increasing phosphatase activities60,61 and promoting the dissolution of immobile inorganic phosphorous to more available forms62. This explanation is supported by meta-analytical evidence showing that N enrichment results in consistently increase of soil phosphatase activities in forests63.

Nitrogen enrichment had divergent effects on soil biodiversity in different climates. In temperate regions, N enrichment reduced bacterial richness (EComN: −1.7%, CI: −3.2 to −0.2%, P < 0.05) and Shannon index (EComN: −0.2%, CI: −0.5 to 0%, P > 0.05) and nematode Shannon index (EComN: −2.5%, CI: −3.3 to −1.8%, P < 0.05) (Fig. 1). This is likely related to the stronger acidification in this climate zone compared to the other climates (Supplementary Fig. 3), as the biodiversity of both bacteria and nematodes is strongly influenced by soil pH reduction (Fig. 2). In contrast, N enrichment significantly increased fungal richness (EComN: +5.9%, CI: +2.0 to +9.9%, P < 0.05) and Shannon index (EComN: +4.7%, CI: +2.5 to +6.9%, P < 0.05) in boreal region (Fig. 1), likely due to the increased plant productivity (Supplementary Fig. 5) and soil organic carbon (Supplementary Fig. 2) in this resource-limited region40,64. Fungi, which are highly sensitive to resource availability50, may benefit from these changes. This process, however, may not apply to temperate and tropical regions, where fungi have adapted to a relatively high N environment.

More substantial nitrogen effects on plant diversity than on soil bacterial and fungal diversity
To compare the effects of N enrichment on plants versus soil biota, we conducted Z-tests across the entire global dataset and within specific ecosystem types or climate zones (Table 1). Across the global dataset, N enrichment-induced changes in the Shannon index and species richness were significantly greater in plants than in soil bacteria (Z = 2.9 and 8.1, P < 0.01) and fungi (Z = 3.5 and 17.6, P < 0.001). However, the change in Shannon index for plants (−2.6%) was comparable to that for soil nematodes (−2.2%) (Z = 0.4, P = 0.69). Within ecosystem types or climate zones, 15 out of 20 comparisons between plants and soil bacteria/fungi were significant (P < 0.05) or marginally significant (P < 0.10), and these all showed greater declines for plants.

To enable a more direct comparison, we extracted independent studies from our global dataset that included both plant and soil biota (bacteria, fungi, or nematodes) and performed an additional meta-analysis (Table 2). Our Z-test on this meta-analysis showed that N enrichment-induced changes in plant diversity were significantly greater than those in soil bacteria (Z = 6.8 and 5.4 for Shannon index and richness, P < 0.001) and fungi (Z = 6.5 and 6.9, P < 0.001), but were similar to those in soil nematodes (Z = 0.4, P = 0.71).

We further examined how changes in biodiversity with N enrichment rates vary between plants and soil biota by using meta-regression. We found that plant and soil biodiversity exhibited more pronounced negative effects at higher N rates, but the decline in plant diversity was significantly steeper than that in soil biodiversity (P < 0.01 for the comparison between plant and any group of soil biota, Fig. 3). This indicates that plant diversity is more sensitive to elevated N enrichment rates than soil biodiversity.

These comparisons collectively demonstrate that N enrichment has more pronounced negative effect on plant diversity than soil bacterial or fungal diversity. The increase in plant aboveground biomass under N enrichment (Supplementary Fig. 5) creates shading effects that disproportionately impact plant communities, while soil biota remain relatively unaffected (Fig. 2). Soil organisms, embedded in complex food webs with diverse trophic interactions and niche differentiation65–67, exhibit greater ability to withstand N enrichment68,69. In contrast, plants, as autotrophs, have less complex interactions and niche differentiation, making them more vulnerable to N-induced changes67,70,71. Additionally, for plant communities, N enrichment-induced shading effects result in asymmetric light competition where larger plants dominate over smaller ones72. Whereas, soil biota communities are less affected by light competition as their dynamics are independent on light availability73.

Factors regulating nitrogen enrichment effects on plant and soil biodiversity
Our meta-regression analysis revealed that N enrichment-induced changes in plant diversity were negatively correlated with increases in soil N and plant biomass, but positively correlated with reductions in soil pH (Fig. 2). Similarly, changes in soil bacterial and nematode diversity were also negatively correlated with increases in soil N and positively correlated with soil pH reduction.

The results from structural equation modeling and model selection analyses identified soil mineral N (ammonium or nitrate) concentration, plant aboveground biomass, and soil pH reduction as the primary factors regulating changes in plant diversity (Figs. 4, 5). Soil ammonium N concentration and pH also served as key moderators for N enrichment-induced changes in bacterial diversity, with the effects of pH being greater than those of ammonium N. For nematode diversity, soil pH was the sole moderating factor. Conversely, N enrichment-induced changes in fungal diversity were unaffected by these factors. Climate indirectly influenced biodiversity changes by modulating soil pH (Fig. 4).

Methods
Data collection
We compiled a global dataset to examine N enrichment’s effects on plant and soil (bacteria, fungi and nematode) biodiversity (Supplementary Data 1). We followed the guidelines of PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) (Supplementary Fig. 7)82 to systematically search for peer-reviewed journal articles and theses that report the effects of N enrichment on soil properties, soil microbes, nematodes and plants in various terrestrial ecosystems, using the Web of Science (Core Collection; https://www.webofknowledge.com), Google Scholar (https://scholar.google.com), and the China National Knowledge Infrastructure (CNKI; https://www.cnki.net) to August 1st, 2023. The search strings were composed of “OR” and “AND” statements combining terms related to N enrichment experiments and diversity of plants species and soil biota, i.e., (“nitrogen fertilization”, “nitrogen deposition”, “nitrogen addition” OR “nitrogen enrichment”) AND (“richness” OR “diversity”) AND (“plant” OR “soil microbial” OR “soil bacteria” OR “soil fungi” OR “soil nematode”).

We evaluated each study to determine whether it met the following criteria to ensure the quality of the data. (1) N was directly added to the ecosystems and at least one of the considered variables was measured (including plant/bacterial/fungal richness and Shannon index, nematode Shannon index). (2) N-enrichment and control plots were established under the same abiotic and biotic conditions in the field. Furthermore, laboratory incubation studies were excluded. (3) In studies involving additional global change drivers (e.g., warming, altered precipitation, phosphorus fertilization), only N-enrichment and control plots under matched field conditions were included. Plots with non-nitrogen treatments were excluded to isolate the effects of N enrichment. (4) Experiments applying herbicide, fungicide, or pesticide were retained only if treatments were consistent across both N-enrichment and control plots; otherwise they were excluded. (5) Plant diversity in croplands was not included because plant communities are highly modified and managed for agricultural purposes. (6) N treatment was applied continuously from the first application to the measurement, with duration calculated in years based on the interval between the first and final application, including repeated treatments within a year. (7) The means, standard deviation (SD) and sample sizes (n) of the variables selected were reported or could be calculated from the related results, and the standard error (SE) was converted to SD (SD = SE × √n). (8) If multiple independent experiments conducted under different environmental conditions or ecosystem types were reported in the same publication, each experiment was considered an independent data record. (9) The type of N added, rate and duration of N enrichment were directly reported. The N enrichment rate and duration units were converted to common units of kg N ha−1 year−1 and year, respectively. (10) We included only observations where soil samples were collected from the mineral soil layer, typically within the top 0–20 cm.

Metrics describing plant and soil biodiversity, climates, and soil properties
The metrics of plant diversity included plant species richness and the Shannon index. The plant species richness was investigated by the total number of species recorded in its quadrat, and the Shannon index was calculated based on this measurement. The diversity metrics of soil bacteria and fungi included richness (Chao1 and/or OTU number) and the Shannon index, and that of nematodes included the Shannon index. Soil nematodes were identified to genera based on morphological characteristics per 100 g of dry soil from each sample using a modified Baermann wet funnel technique, and the Shannon index was calculated based on this measurement. In our dataset, the microbial diversity assessments varied in sequencing depth, taxonomic resolution, and methodological approaches among independent studies. However, since all comparisons were performed between treatment and control conditions within the same study, the difference in technical variations did not affect our comparison and could be neglected in this meta-analysis.

We also collected ancillary data, including climatic variables, soil properties, ecosystem types, type of N used in the experiment, and rate and duration of N enrichment. The climatic variables used in this study included mean annual temperature (MAT, °C), mean annual precipitation (MAP, mm), and the aridity index (AI, unitless). The MAT and MAP were directly obtained from the publications, or in case they were not reported, from the database at http://www.worldclim.org/ using the location information (i.e., latitude and longitude) covering the years over which each experiment was conducted. The aridity index was extracted from the Global Aridity and Potential Evapotranspiration database at https://csidotinfo.wordpress.com/data/global-aridity-and-pet-database/. The soil properties were directly extracted from the publications. The soil properties included soil pH (1:2.5 or 1:5 soil:water (w/v), or KCl or CaCl2 mixture), soil moisture, and the concentrations of soil organic carbon (dichromate oxidation or elemental analyzer), total N (Kjeldahl or elemental analyzer), nitrate and ammonium N (flow injection analysis or spectrophotometric). The indices from different analytical methods were standardized to common units to ensure comparability. The experimental protocols (i.e., rate of N enrichment, duration of the experiment, type of N used in the experiment) were also directly extracted from the publications.

Our final dataset contained 3816 paired observations from 458 N-enrichment studies at 366 sites in 31 countries. The dataset covered a wider range of N enrichment rates, latitude, longitude, MAP and MAT, and soil background pH (Supplementary Fig. 1 and Table 2). All the original data were extracted from the publications’ text, tables, figures, and appendices. The Engauge software 4.1 obtained numeric data (http://getdata-graph-digitizer.com) when the data were graphically presented.

We grouped the data in the set by ecosystem type and climatic region to determine how the effects of N enrichment varied with ecosystem types and climatic regions. The ecosystem types were grouped into croplands, forests and grasslands. The diversity in forests was referred to understory vegetation. The climatic regions were grouped into tropical (tropical and subtropical regions), temperate, and boreal (boreal and polar regions) regions based on the 2010 map of Global Ecological Zones (https://data.apps.fao.org/).

Publication bias
We performed a series of analyses to evaluate the potential for publication bias. First, we conducted a funnel plot analysis to assess the bias against publishing negative results, and we tested the asymmetry of the funnel plot using Egger’s regression test83. If asymmetry was detected, we further evaluated the sensitivity of our results to publication bias with the Rosenberg fail-safe number84. Additionally, to evaluate potential publication time-lag bias, where studies with larger or significant effects are published earlier, we included publication year as a continuous moderator in the meta-regression. This allowed us to test whether effect sizes declined over time, which could indicate inflated early estimates and assess the robustness of the overall results85. Finally, we assessed the robustness of our results through a leave-one-out analysis.

Our analyses revealed no evidence of publication bias regarding the effects of N enrichment on the richness and Shannon index of plants and soil bacteria, as well as on the Shannon index of soil nematodes (Supplementary Table 3 and Fig. 8; Egger’s test: P > 0.05). However, we detected asymmetry in the funnel plot for the effects of N enrichment on the richness and Shannon index of soil fungi (Supplementary Table 3 and Fig. 8; Egger’s test: P < 0.05). Despite this asymmetry, the Rosenberg fail-safe numbers for these two metrics were significantly larger than the threshold of 5k+10 (Supplementary Table 3), suggesting that publication bias is unlikely to impact our findings regarding the effects of N enrichment on plant and soil biodiversity.

Our examination on publication time-lag bias did not show larger positive effect sizes in earlier studies compared to more recent studies for nearly all biodiversity indices, except for the Shannon index of soil bacteria (P = 0.027) (Supplementary Fig. 9). Given that we strengthen our conclusions by incorporating two metrics of diversity (i.e., richness and the Shannon index), the publication year may have had limited impact on our results. Moreover, the results from the leave-one-out analysis indicated that the effect size was not significantly altered by the removal of any single independent study (Supplementary Fig. 10), further confirming that the effects of N enrichment in our study are not driven by any single influential study. We further examined the publication bias for each climate-zone and ecosystem-type subgroup for all metrics. We found that, except for soil bacterial richness in forests and bacterial Shannon index in grasslands, 43 out of 45 examinations showed symmetric funnel plots and exceeded the 5k+10 fail-safe threshold (Supplementary Table 4), indicating no publication bias for the vast majority of subgroups. Collectively, the outcomes of these three analyses suggest that our findings are robust against publication bias.

Meta-analysis
We quantified the response of each variable to N enrichment across the global dataset by calculating the effect size as the natural logarithm of the response ratio (lnRR), that is, the natural logarithm of the ratio of the mean of the treatment groups to the mean of the control groups86. The lnRR was calculated as follows:

lnRR = ln(XN / XCK) = ln XN − ln XCK (1)

where XN and XCK are the mean values of a given variable in the N-enrichment and control treatment, respectively. Soil pH uses a logarithmic scale, so N enrichment’s effects on pH were calculated as the difference between the treatment and control groups (pHN − pHCK).

We also quantified such response within various groups of ecosystems and climates by weighting the lnRR with the inverse of the variance and a random-effect model. The weighted mean response ratio (lnRR+) was calculated as follows:

lnRR+ = (∑i=1m ∑j=1n ij w ij lnRR ij) / (∑i=1m ∑j=1n ij w ij) (2)

where m is the number of observations in every corresponding compared group, and n represents the number of groups.

The weighting factor (wij) of the individual observation was calculated as:

wij = 1 / (vij + τ2) (3)

where τ2 is the between observations variance.

The variance (vij) of individual observation was calculated as:

vij = (SN2 / (nN XN2)) + (SCK2 / (nCK XCK2)) (4)

where nN and nCK are the sample sizes for the N-enrichment and control treatments, respectively, and SN and SCK are the standard deviations for the N-enrichment and control treatments, respectively, of the ith observation.

The standard error of lnRR+ was calculated as:

s(lnRR+) = 1 / √(∑i=1m ∑j=1n ij wij) (5)

The 95% confidence interval (CI) for lnRR+ was calculated as:

95% CI = lnRR+ ± 1.96 s(lnRR+) (6)

The weighted percent effect size for plant and soil variables induced by N enrichment in a group of ecosystems or climates was calculated as:

Effect size (%) = (exp(lnRR+) − 1) × 100% (7)

We first conducted a meta-analysis using our global dataset to assess the overall effects of N enrichment on plant and soil variables and to assess how these effects vary with ecosystem types and climate regions. We conducted between-group heterogeneity tests (QB) to compare the effects of N enrichment on each metrics among subgroups of ecosystem types or climate regions87, and conducted Z-test to compare the effects of N enrichment on diversity between plant and soil biota88. We then extracted the independent studies that include both plant and any group of soil biota (bacteria, fungi, or nematodes) from the whole global dataset to compose a sub-dataset. In this sub-dataset, any pair of plant and each group of soil biota has the same sampling size. We conduct an additional meta-analysis with this sub-dataset to assess the effects of N enrichment, and conducted a Z-test to compare the effects of N between plant and any group of soil biota.

Both meta-analyses and QB test were conducted using the software MetaWin 2.189 with a categorical random effect model89. The effects of N enrichment were considered significantly positive (lnRR+ > 0, P < 0.05) or negative (lnRR+ < 0, P < 0.05) if the 95% CIs did not overlap to zero. A significant QB indicates that the metrics are significantly different among subgroups74. The Z-test was conducted using the equation Z = ΔlnRR / SEΔ, where ΔlnRR represents the difference in the log response ratio (lnRR) between plant and soil biodiversity, and SEΔ is the pooled standard error calculated as √(SEPlant2 + SEsoil2). The test assumes that the compared effect sizes are independent. Statistical significance was determined based on the resulting Z value and corresponding P value, with P < 0.05 considered significant88.

Given that the rate and duration of N treatment varied largely among independent studies within the dataset (Supplementary Table 2), this will lead to significant bias when comparing directly calculated effect size among subgroups (e.g., ecosystem types or climate regions) because this effect size is largely depended on N treatment rate and duration (Supplementary Figs. 11, 12). We thus calculated a standardized effect size (EComN) of N enrichment at a common rate and duration of N enrichment to eliminate such bias and ensure the effect of N enrichment is comparable across subgroups, and thus to provide a meaningful advance regarding how the effects of N enrichment vary with ecosystem types and climate regions. Prior to calculation, we evaluated the linear and non-linear (logarithmic, quadratic) functions for modeling the effects of N enrichment rate and duration. Our comparison revealed comparable explanatory power across these functions (Supplementary Tables 5–6). To facilitate the standardization of the effect size to a common N enrichment rate and duration, we selected the linear function, but not intend this as an indication that linear function is preferable to non-linear functions. We calculated EComN as follows:

EComN (%) = (exp(lnRR+) − 1) / (R+ × T+) × 100% (8)

R+ and T+ are the weighted mean N enrichment rate and duration of a group of ecosystems or climates.

We also calculated the standardized effect size of N enrichment of each paired observation in our dataset with Eqs. 9 and 10, and finally calculated the standardized effect size of each subgroup by conducting a meta-analysis using the software MetaWin 2.189.

lnRRComN = ln(XN / XCK) / (R × T) (9)

Effect sizeComN (%) = ((XN − XCK) / (XCK × R × T)) × 100% (10)

Where R and T are the N enrichment rate and duration of the paired observation.

Our comparison of the results from Eqs. 8, 9 and 10 showed that the standardized effect size for each subgroup from Eq. 8 was significantly positively correlated to the results from Eq. 9 and Eq. 10 (Supplementary Fig. 13). We therefore only reported the results from Eq. 8 because the rate and/or duration of N enrichment is included as influential factors when conducting meta-regression, model selection and structural equation model (SEM) analyses and global projection for the effect of N enrichment. If we would use results from Eqs. 9 and 10, the rate and duration of N enrichment would have to be recalculated in these analyses. Given that the calculation on the EComN was based on the effect size and N enrichment rate and duration, scaling the effect size to a common rate and duration of N treatment does not affect the statistical significance of the effect size.

In our global dataset, the mean, mode, and median N enrichment rates are ~100 kg N ha−1 year−1 (Supplementary Fig. 14). However, the N deposition rate in most parts of the world is currently less than 50 kg N ha−1 year−179. In order to link our results to global N deposition, we calculated a standardized effect size of N enrichment at a common rate of 25 kg N ha−1 year−1 and duration of 10 years.

Meta-regression
We performed mixed-effect meta-regression modeling to examine the relationships of the response ratio (lnRR) of N enrichment on plant and soil biodiversity with the rate and duration of N enrichment, lnRR of soil mineral N (ammonium and nitrate N) and plant aboveground biomass, and change in pH. Our preliminary analysis showed that N-induced changes in biodiversity metrics were significantly affected by change in soil pH but not by initial soil pH (soil pH in control treatment) (Supplementary Fig. 6). We therefore included pH change rather than initial pH as an influencing factor. We conducted this meta-regression analysis using the lmer function in the lme4 R package90 by including “studyID” as a random factor in the model. Given that most variables (9 out of 12) showed no significant relationship with experiment duration (Supplementary Figs. 11, 12), we did not include duration as a random effect in our mixed-effect meta-regression models. We then bootstrapped the fitted coefficients by 1000 iterations because many of our models violated the assumption of normality based on the Shapiro-Wilk test on model residuals.

Structural equation modeling
We constructed a SEM using “piecewiseSEM”91 to examine the direct effects of N enrichment and climate on plant and soil biodiversity and indirect effects through N enrichment-induced changes in soil properties. In doing this, we selected MAT and aridity index as variables for climate, the changes in soil pH, nitrate and ammonium N concentrations as variables for soil properties. We also selected change in aboveground biomass as variable when examining the effects on plant diversity.

The pathways of the piecewise SEM were fitted as linear mixed-effects models with the study and observation IDs considered as a random factor. We first hypothesized a conceptual model (Supplementary Fig. 15) that included all reasonable pathways, as well as the effect of N and its interaction with each pathway. Tested pathways that were statistically non-significant (P > 0.05) were excluded from the model and the model was further optimized to account for more variation. The final optimized model was selected based on the lowest AIC score, and chi-square statistics were run to evaluate the model goodness-of-fit92. If the chi-square was statistically non-significant (P > 0.05) the model was a good fit to the data. The standardized coefficient for each path from each component model and the Fisher’s C and P value for the final optimized SEM were displayed in the Fig. 4. All statistical analyses were conducted in the R environment (v4.1.2, http://www.r-project.org/).

Model-selection analysis
We used a model selection and multimodel inference to examine the relative importance of multiple factors influencing the effects of N enrichment on plant and soil biodiversity93,94. We conducted this analysis using the glmulti R package95. The relative importance value of each predictor was calculated as the sum of the Akaike weights (probability that a model is the most plausible model) of the model in which that predictor appears. Hence, predictors with large Akaike weights in the models received a high importance value. These importance values can be considered as the overall support for each variable in the model41. We selected 0.8 as the cut-off for importance to differentiate between important and nonessential predictors, according to Terrer et al.94. A factor with an importance value > 0.8 is generally significant, and a factor with a value < 0.8 is generally not significant. We examined the relative importance of the influencing factors, including N enrichment rate, N enrichment duration, MAT, AI, change in soil pH and response ratios of soil nutrient concentrations, and plant biomass (only for plant diversity).

We extracted subset databases from the whole dataset based on the indicators of plant and soil biodiversity with the least amount of data and then interpolated the missing data rows for the remaining predictors using the missRanger R package93,96 before conducting SEM and model selection analyses. This approach for missing value imputation has often been used in biological studies97–100. We conducted missing value imputation for each diversity metric of each group of biodiversity separately.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this MS (NCOMMS-25-30290: Global nitrogen enrichment impacts plant diversity more than soil bacterial and fungal diversity), Song et al. reported a meta-analysis regarding how nitrogen enrichment affects plant and soil biodiversity by using an impressive global dataset with 3816 paired observations from 458 publications. By conducting various comparisons between the effects on plant and soil biodiversity, they found that N enrichment has stronger negative effects on plant diversity but surprisingly modest to negligible effects on soil bacterial and fungal diversity. They also found that the N-induced soil acidification modulates soil biodiversity change and, in conjunction with aboveground biomass, regulates plant biodiversity change. By predicting global patterns of N effects on biodiversity, they identified Europe and Northeastern Asia as critical regions where plant and soil biodiversity are most negatively affected by current and/or historical N deposition. Biodiversity is critical for the functioning, resilience, and productivity of ecosystems and for the health and stability of the biosphere, and is significantly negatively affected by global N enrichment. Additionally, there is close association between plant and soil biodiversity. However, currently, how the effects of N enrichment on plant diversity is contrast to that on soil biodiverse is not compared. This study provides the first comprehensive global comparison of such effects across various ecosystems and climates, which is fundamental for us to understand how N and biodiversity interacts in a changing planet, and is urgently needed for developing integrated biodiversity conservation strategies. Moreover, the authors reported standardized effect size expressed at a common rate and duration of N treatment, which minimize the bias induced by variations in these two factors, and thus is significantly advanced from current meta-analysis regarding N treatment. Taken together, this study is the great extension of the current focus on the changes in above- and belowground biodiversity. This work will have great interest for broad readership and is closely fall within the scope of nature communications. The manuscript is well written, structured, concise, and impactful. The methodology is clearly described and is repeatable. The results are well present.\nI have some minor comments need to be addressed prior to publication.\n1. It would be better to add a subtitle of “Results and Discussion” after Introduction section.\n2. Line 46: Impressive dataset.\n3. Lines 70-71: Please add a sentence to explain “plant and soil communities can exhibit divergent responses to anthropogenic stressors”.\n4. Line 89: “Several meta-analyses” to “Previous studies”.\n5. Line 93: Remove (Supplementary Table S1).\n6. Line 94: “This knowledge gap hinders” to “These knowledge gaps hinder”.\n7. Line 103: “microbial diversity” to “soil biodiversity”\n8. Lines 146-147: could you add a citation to support this statement?\n9. Line 148: “pollution” to “enrichment”\n10. Lines 152-156: Please clarify that these are in soils, i.e., soil bacteria, soil fungi, soil nematodes.\n11. Lines 164-165: Please add a citation to support this statement.\n12. Line 179: remove “microbial”, just say “soil biodiversity”.\n13. Line 182: remove “diversity”.\n14. Line 193: The authors conducted three comparisons to address that N had more substantial effects on plant diversity than soil bacterial and fungal diversity, great!\n15. Lines 212-214: Remove this sentence. It says the same thing to the following sentence.\n16. Lines 212-213: “biodiversity” to “diversity”.\n17. Line 298: “nitrogen (N)” to “N”.\n18. Line 325: remove one period.\n19. Lines 339-340: I guess it should be “Chao1 and/or OUT number”.\n20. Line 381: I like this subsection.\n21. Lines 465-474: Good idea to minimize the influence of N rate and time on the overall effects of N when comparing among sub-groups.\n22. Line 495: “(79)” to “79”.\n23. Lines 498-501: I think you need to address this issue just after the introduction of your dataset at the end of the Introduction section (Lines 111-113). Maybe one sentence is enough.\n24. Line 606: I like this subsection to address the potential limitation of this study.", "reviewer_2": "This manuscript presents a global synthesis of nitrogen (N) enrichment studies, aiming to understand the differential impacts of N on plant diversity versus soil bacterial and fungal diversity across various ecosystems and climatic conditions. The study leverages a large dataset of 3,816 paired observations from 458 articles to demonstrate that N enrichment has stronger negative effects on plant diversity, while showing surprisingly modest to negligible effects on soil bacterial and fungal diversity. A significant merit of this work is its comprehensive approach, utilizing meta-analysis, structural equation modeling, and machine-learning models to identify key modulating factors like N-induced soil acidification and aboveground biomass. The identification of Europe and Northeastern Asia as critical regions experiencing the most negative impacts is a valuable finding for conservation strategies. The study provides novel insights into the contrasting responses of above- and below-ground biodiversity to N enrichment, emphasizing the need for integrated conservation efforts.\nThe primary concern with the manuscript lies in the representation and interpretation of results, particularly concerning the climate-specific findings. While robust analyses for publication bias are performed for the overall dataset (e.g., all observations of plant richness), the manuscript subsequently subsets these groups (e.g., boreal plant richness) without explicitly examining publication biases within these specific subsets. The effect sizes derived from these potentially biased subsets are then utilized to train machine learning models and to inform Z-tests, which rely on the accuracy of these subgroup-calculated effect sizes.\nFurthermore, the manuscript at times appears to claim significant differences for certain climatic subgroups based on visual interpretation or other means, even when the reported Qb test for heterogeneity does not statistically support such a distinction among climate zones (e.g., for soil bacterial Shannon index). This inconsistency between claims and the results of heterogeneity tests undermines the robustness of the conclusions regarding climate-specific impacts.\nIf a meta-analysis draws distinct conclusions about effects within subgroups, the validity of those subgroup-specific conclusions must be independently assessed for common biases, including publication bias. Failing to do so presents a less robust and potentially misleading picture of the evidence for those specific conditions. It appears that the confidence in claims about differences among climate zones, especially for areas like boreal studies, may be overstated if the underlying subgroups are not adequately tested for publication bias. This is particularly relevant when heterogeneity tests show significant differences among climate zones, but these differences might be driven by regions with very few case studies, which are more susceptible to publication bias. Detecting publication bias in these subgroups would significantly caution against direct comparisons, as a biased subgroup will distort the between-group heterogeneity.\nPlease find my specific critiques below:\n• Line 134 – The statement \"significantly greater in boreal\" regarding plant richness appears to be contradicted if your between-group heterogeneity test (e.g., for Figure 1a) found plant richness to not significantly differ among climate zones. Please clarify this apparent discrepancy.\n• Line 180 – I have serious misgivings about the comparisons of soil microbial biodiversity in temperate regions. While N enrichment reduced bacterial Shannon index in temperate regions, your own heterogeneity test (Qb = 0.41, P = 0.816, as stated in your notes) found no significant difference among climate zones. While it's fair to report a significant effect within temperate regions, the lack of publication bias testing on these subgroups means that publication bias could have as much, if not more, of an effect on this observed difference than the acidification you suggest. This issue is further illustrated by the climate comparisons for soil fungi, where heterogeneity tests did find significant differences among climate zones, but these seem to be driven by boreal forests which are relying on far fewer case studies from very few studies. If publication biases are detected in these subgroups, it would significantly weaken claims about observed differences among climate zones, as a biased subgroup would bias the between-group heterogeneity.\n• Line 372 – The provided link \"http://digitizer.sourceforge.net\" for the Engauge software leads to a page with a broken GitHub link. Authors should attempt to find a current webpage or repository link for this software to ensure others are able to evaluate and reproduce your methods.\n• Line 403 – You document a significant effect of publication year on the Shannon index of soil bacteria in your analysis. However, you immediately declare that “publication timing did not influence our results” without explanation. Your own analysis suggests this is not the case for the Shannon index of soil bacteria. Some explanation or a nuanced interpretation of this finding is appropriate here.\n• Line 501 – The statement \"Our analyses indicated that the effects of N on richness and the Shannon index were consistent across the entire dataset as well as within specific ecosystem types and climate regions (Fig. 1)\" is unclear. The effect of N is, in fact, significantly different within certain ecosystem types and climate regions according to your heterogeneity tests. If this is a comment about publication biases, Figure 1 does not explain any of that. This statement needs to be rephrased for clarity and accuracy.\n• Line 606 – While the inclusion of a discussion on data limitations is appreciated, it is unclear why it is relegated to the end of the methods and cites no other publications. This section, which highlights important gaps in the literature and cautions the limitations in the predictive power of these models, frankly, belongs somewhere prominent in the Discussion section to effectively couch the results and their implications.\n• Line 925 – This should refer to Figure S6, not S7.\n• Figure S2 – It seems somewhat irresponsible to compare datasets with 1627 and 271 points respectively to a subset with only 3 data points. The authors should be very sure, based on their publication bias tests, that this very small subset is robust enough to justify making such a comparison. Otherwise, there should be significantly more caveats regarding this response in the discussion section than is currently present.\nThank you for the opportunity to review this manuscript. I hope these comments are helpful in improving the quality and clarity of your important work.", "reviewer_3": "General comments\nI like the meta-analysis part of the study comparing effects of N-enrichment induced diversity changes among plants, soil bacteria and soil fungi. The size of that part of the study is impressive. I think this makes a nice and relevant contribution to the literature, and I would like to see the paper published. However, I also have two major concerns:\nFirst the results of the study are largely based on regressions between diversity and N enrichment levels. The N application levels range between 2-800 kg N ha/year. This range is no problem for studying effects of N enrichment in general but becomes tricky when the results are placed in the context of anthropogenic N enrichment. The authors do make predictions for a standardized level of 25 kg N/ ha.year, which is good, but the regressions are to a very large extent based on the outcome of studies that were carried out with far higher N enrichment levels. I guess if effects of N enrichment in only the this ‘anthropogenic N enrichment range’ (e.g. 2-50 kg N/ ha.year) would have been quantified it might not have yielded significantly differences compared to the unfertilized control (considering the large variation of effects in this N-range in Figs 3 and S11). This is at least something to include in a revision and needs to be discussed in the main text.\nSecond, I have some doubts about the global model. As the authors state the model is largely determined on observations from the highest N deposition regions, and again is mainly based on results of studies that were carried out with far higher N application levels than the ‘anthropogenic N enrichment range’. The prediction of the model that positive effects of N enrichment are to be expected in some regions of the Southern hemisphere to my opinion illustrates that these predictions should be handled with care. The authors do mention this latter aspect but don’t really discuss the implications of it. I wonder whether the paper wouldn’t be better off without this relatively weak part of the study, and just fully concentrate on the outcome of the N-enrichment in general. In that case my first concern would also be less problematic, and its implications could just be discussed in the text. By adding this global model emphasis is put on effects of N-enrichment in the ‘anthropogenic N enrichment range and that is tricky.\nAdditional comments\nLn 45: “how the nitrogen-induced changes in plant diversity differ from those in soil organisms across multiple ecosystems and climatic conditions remains virtually unknown” – Isn’t this a bit exaggerating, if there are 458 articles with paired observations about this (see next sentence)?\nLn 42-54: To my opinion the abstract could provide more detailed statements about the outcome of the study. Now it is very general and a bit vague.\nLn 75-85: I like the way the effects of anthrophenic N enrichment on plants is formulated in this section. Also the cited papers for the plants are all about effects of N enrichment by atmospheric N deposition. However, for the soil communities, the formulations are about effects of N enrichment in general, and also the cited papers don’t focus on N enrichment from N depotion (based on the titles). Are you comparing effects of the same levels of N enrichment for plants and soil communities, or do the soil studies include also higher levels of N -enrichment?\nLn 92-99: Are you still focusing on the effects of N-enrichment from enhanced atmospheric N deposition (hence the level corresponding to ‘global N enrichment’), or are you slightly changing to effects of N enrichment in general? That should be clear, because the rate of N enrichment probably matters, and levels of N enrichment in experimental studies often go far beyond that of enhanced atmospheric N deposition.\nLn 100-104: The hypothesis is poorly supported with arguments. The fact that factors influencing plant or soil biodiversity are ‘different’ doesn’t by definition imply that effects of N enrichment on soil communities should be weaker than effects on plants.\nLn 106 and Table S2: the ranges of N enrichment are extremely wide (from 1-2 to >500 kg N ha / year). That means ranging from hardly any ecological meaningful enrichment to probably strongly toxic levels. I expected to read some discussion about this aspect of the study, but didn’t really see it. (see main comments).\nLn 114-116. It is good to standardise for N enrichment level (25 kg ha/year) and time (10 years), but it was not directly clear to me how this was done (at least not here). Also, I would like to know how many of the studies were indeed carried out with an N enrichment level of about 25 kg N per ha/year. I noted that this information is provided in Fig. 3 and Fig. S11. It would be good to refer to this additional figures here (and please add the scale information at the x-axes Figs S11 A, C and E.)\nAlso, are these relationships in Figs. 3 and S11 linear? That doesn’t need to be the case with potentially growth-stimulation effects on low to mediate N application levels and potentially toxic effects at medium to high N-application levels.\nHence, looking at Figs 3 and S11 I note that the fast majority of the studies were carried out with N-enrichment levels clearly above the rates of atmospheric N; i.e, 25 kg N per ha and year is completely at the left site of the x-axes. Are effects in the range of enhanced atmospheric N deposition (c. 0-40 kg N kg N / ha and year) different from zero? This is worth to be mentioned somewhere in the text.\nLn 133-149: How do these climate zones correspond to regional variation in atmospheric N deposition? Also, the effect of N enrichment on plants is acribed to effects of productivity and competition for light, but what about effects of acidification?\nLn 164-165: add a reference for this statement.\nLn 172-178: The effect of N enrichment on P availability might be valid, but it is a bit speculative. Moreover, I doubt that N enrichment only increases phosphatase activity in forests and not in grasslands. Does forests vs croplands and graslsands in ref 56 perhaps (partly) coincide with unfertilized vs fertilized sites?.\nLn 179-191: see comment to ln 133-149.\nLn 204-210: It is good that you added this direct comparison.\nLn 211-219: This meta-regression approach is important in this paper. See my comments above.\nLn 222-228: Perhaps you could refer to competition for light causing a-symmetric competive effects for plants (see Weiner, TREE 5: 360-364)? The one-directional resource light doesn’t play a role for the soil biota communities.\nLn 236-244 and Fig 4. There are large differences in the effect sizes of the paths that are included in all 7 SEMs (paths between N enrichment, climate, NH4, NO3 and pH). For instance in some SEMs (panels C and E) there is a strong effect of N enrichment on NO3 and on others (panel G) there is no effect at all, etc. How can you explain these large differences, and what do they imply?\nLn 247-256. I think one should be careful with making predictions about the effects of N deposition rates on the base of a linear regression which is very strongly determined by effect of N enrichment that is far higher than the current N deposition levels. Also, the confidence interfal of such a prediction for the N enrichment range 0-50 kg N ha/year is probably not significantly different from no effect (see e.g. the large variation in Fig 3 in this N enrichment range). In that sense, I don’t understand that the 95% confidence interfal is always positive in the Northern hemisphere in Fig. S6. Furthermore, I don’t understand how the model can predict an increase in species richness in some regions of the Southern hemisphere, since the regression lines in Figs 3 and S11 are never >0. How to explain this? (see main concerns)\nLn 259-260: I am not sure if you can state that boreal biomes are in general naturally N-limited. The two references also mainly focus on N, and ref 41 doesn’t exclude other nutrients to be potentially limiting as well.\nLn 268-270: Isn’t the prediction that Europe en Northern Asia are the most critical regions for effects of N deposition just a reflection of the high levels of Atmospheric N deposition in these regions? What does the model basically add to global maps of variation in atmospheric N deposition?\nLn 321: What about about herbicide, fungicide, pesticide etc treatments in croplands. Were these sites or studies excluded?\nLn 550-553: Is this value of 0.8 robust? This sounds a bit arbitrary. At least some extra information from Ref 84 seems appropriate here.\nLn 612-619: It would be appropriate to place this important remark about the limitations of the study in the main text.", "reviewer_author_response": "Point-by-point response to the reviewers’ comments\nDear Reviewers:\nWe sincerely thank the three Reviewers for your constructive and insightful\ncomments on our manuscript of Global nitrogen enrichment impacts plant diversity\nmore than soil bacterial and fungal diversity (NCOMMS-25-30290). We carefully\nconsidered all of the comments in our revision. Please find the detailed revisions in the\nrevised manuscript. The point-to-point responses to your comments are shown in blue\nin the following, corresponding changes in the manuscript are highlighted in yellow.\nReviewer #1\nGeneral comments\nIn this MS (NCOMMS-25-30290: Global nitrogen enrichment impacts plant diversity\nmore than soil bacterial and fungal diversity), Song et al. reported a meta-analysis\nregarding how nitrogen enrichment affects plant and soil biodiversity by using an\nimpressive global dataset with 3816 paired observations from 458 publications. By\nconducting various comparisons between the effects on plant and soil biodiversity, they\nfound that N enrichment has stronger negative effects on plant diversity but surprisingly\nmodest to negligible effects on soil bacterial and fungal diversity. They also found that\nthe N-induced soil acidification modulates soil biodiversity change and, in conjunction\nwith aboveground biomass, regulates plant biodiversity change. By predicting global\npatterns of N effects on biodiversity, they identified Europe and Northeastern Asia as\ncritical regions where plant and soil biodiversity are most negatively affected by current\nand/or historical N deposition.\nBiodiversity is critical for the functioning, resilience, and productivity of ecosystems\nand for the health and stability of the biosphere, and is significantly negatively affected\nby global N enrichment. Additionally, there is close association between plant and soil\nbiodiversity. However, currently, how the effects of N enrichment on plant diversity is\ncontrast to that on soil biodiverse is not compared. This study provides the first\ncomprehensive global comparison of such effects across various ecosystems and\nclimates, which is fundamental for us to understand how N and biodiversity interacts\nin a changing planet, and is urgently needed for developing integrated biodiversity\nconservation strategies. Moreover, the authors reported standardized effect size\nexpressed at a common rate and duration of N treatment, which minimize the bias\ninduced by variations in these two factors, and thus is significantly advanced from\ncurrent meta-analysis regarding N treatment. Taken together, this study is the great\nextension of the current focus on the changes in above- and belowground biodiversity.\nThis work will have great interest for broad readership and is closely fall within the\nscope of nature communications. The manuscript is well written, structured, concise,\nand impactful. The methodology is clearly described and is repeatable. The results are\nwell present.\nReviewer #2\nGeneral comments\nCOMMENT #1: This manuscript presents a global synthesis of nitrogen (N)\nenrichment studies, aiming to understand the differential impacts of N on plant diversity\nversus soil bacterial and fungal diversity across various ecosystems and climatic\nconditions. The study leverages a large dataset of 3,816 paired observations from 458\narticles to demonstrate that N enrichment has stronger negative effects on plant diversity,\nwhile showing surprisingly modest to negligible effects on soil bacterial and fungal\ndiversity. A significant merit of this work is its comprehensive approach, utilizing meta-\nanalysis, structural equation modeling, and machine-learning models to identify key\nmodulating factors like N-induced soil acidification and aboveground biomass. The\nidentification of Europe and Northeastern Asia as critical regions experiencing the most\nnegative impacts is a valuable finding for conservation strategies. The study provides\nnovel insights into the contrasting responses of above- and below-ground biodiversity\nto N enrichment, emphasizing the need for integrated conservation efforts.\nReviewer #2 (Remarks on code availability):\nCOMMENT #13: I downloaded and opened the code attached as well as the data for\nthe code, but did not attempt to run any of it nor checked for accuracy of the\nimplementation. There seemed to be adequate documentation for someone to compile\nthe code, though I did not attempt this myself.\nReviewer #3\nGeneral comments\nCOMMENT #1: I like the meta-analysis part of the study comparing effects of N-\nenrichment induced diversity changes among plants, soil bacteria and soil fungi. The\nsize of that part of the study is impressive. I think this makes a nice and relevant\ncontribution to the literature, and I would like to see the paper published. However, I\nalso have two major concerns:"}, "author_response": ""}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have made good revisions to this manuscript. All the questions and concerns I raised in the previous review have been fully addressed. In terms of scientific quality, the manuscript now meets the high standards required by Nature Communications. I strongly the acceptance of this manuscript for publication.", "reviewer_2": "I have reviewed the revised version of the manuscript and the authors' response to the reviewers' comments. I find that all of my previous comments have been sufficiently addressed, and the manuscript has been significantly improved as a result.\nI have also reviewed the responses to the other reviewers' comments and am satisfied with the changes made in the manuscript to address their concerns. The authors have done an excellent job of incorporating the feedback and strengthening the paper's arguments and presentation.\nBased on this comprehensive revision, I believe the manuscript is now ready for publication. I commend the authors on their thorough and professional response to the feedback.", "reviewer_3": "The authors addressed my comments mostly very well, including the suggestion to skip the global modal component. I feel sorry for the work that was related with it, but I think you made a good decision to focus on the meta-analysis part. The paper now reads very well, and I only have a few many points left.\n1. Lines 123-124 and previous comments #11 and #20. As previously stated in my previous comment #20. I doubt that it can be concluded from the two cited papers (which mostly focus on N and not much on other elements) that boreal systems are generally N-limited. Also Supplementary Fig 2 does not provide evidence for that. Hence, either provide better references for this or tune down this point and it’s implications for the text.\n2. Lines 197-182, and previous comment #13. I agree that N enrichment can stimulate phosphatase activity. I have experimentally demonstrated that myself for grassland plants (Plant and Soil 347: 137-146). However, I think that generally it can not be concluded that this only counts for forests and not for grasslands. See my mentioned paper for the grassland plants, and also your own response to this point in which you mention that the meta-analysis of Gou et al (2024 ) showed that the effect of N enrichment on acid phosphatase was clearly not different between forests and grassland, and that only the alkaline phosphatase response differed. But in the latter case it was even reduced upon N-enrichment in grasslands soils. Was that due to an N effect or to something else (e.g. pH). Anyway, I think the nuance made by the authors in their response is fine, but the single sentence in the manuscript (Line 179-182) should be formulated differently.\nI congratulate the authors with their paper which is a nice contribution to the literature!\nWith best regards,\nHarry olde Venterink"}, "author_response": "Point-by-point response to the reviewers’ comments\nDear Reviewers:\nWe sincerely thank the three Reviewers for your constructive and insightful\ncomments on our manuscript of Global nitrogen enrichment impacts plant diversity\nmore than soil bacterial and fungal diversity: a meta-analysis (NCOMMS-25-\n30290A). We carefully considered all of the comments in our revision. Please find the\ndetailed revisions in the revised manuscript. The point-to-point responses to your\ncomments are shown in blue in the following, corresponding changes in the manuscript.\nReviewer #1\nThe authors have made good revisions to this manuscript. All the questions and\nconcerns I raised in the previous review have been fully addressed. In terms of scientific\nquality, the manuscript now meets the high standards required by Nature\nCommunications. I strongly the acceptance of this manuscript for publication.\n(Remarks on code availability)\nResponse: We sincerely thank you for the positive evaluation and recommendation for\nacceptance. In response to the remark on code availability, we have now provided all R\nscripts used for the meta-analysis, meta-regression, and figure generation in the\nSupplementary Data (Code file), ensuring full transparency and reproducibility of the\nanalyses.\nReviewer #2\nI have reviewed the revised version of the manuscript and the authors' response to the\nreviewers' comments. I find that all of my previous comments have been sufficiently\naddressed, and the manuscript has been significantly improved as a result.\nI have also reviewed the responses to the other reviewers' comments and am satisfied\nwith the changes made in the manuscript to address their concerns. The authors have\ndone an excellent job of incorporating the feedback and strengthening the paper's\narguments and presentation.\nBased on this comprehensive revision, I believe the manuscript is now ready for\npublication. I commend the authors on their thorough and professional response to the\nfeedback. (Remarks on code availability)\nResponse: We sincerely thank you for the encouraging comments and positive\nassessment of our revised manuscript.\nReviewer #3\nThe authors addressed my comments mostly very well, including the suggestion to skip\nthe global modal component. I feel sorry for the work that was related with it, but I\nthink you made a good decision to focus on the meta-analysis part. The paper now reads\nvery well, and I only have a few many points left.\nResponse: We thank you for the positive feedback and for recognizing the\nimprovements made in the revised manuscript. We glad that the decision to remove the\nglobal modeling component and focus on the meta-analysis was considered appropriate.\nThis change helped streamline the structure, clarify the methods, and highlight the\nempirical synthesis, which is the main strength of this study. We also appreciate your\nconstructive engagement, which greatly improved the coherence and readability of the\npaper.\nComment 1: Lines 123-124 and previous comments #11 and #20. As previously stated\nin my previous comment #20. I doubt that it can be concluded from the two cited papers\n(which mostly focus on N and not much on other elements) that boreal systems are\ngenerally N-limited. Also Supplementary Fig 2 does not provide evidence for that.\nHence, either provide better references for this or tune down this point and it’s\nimplications for the text.\nResponse: We thank you for revisiting this important point and for the helpful\ncomments provided earlier (comments #11 and #20). We understand the concern\nthat the previously cited studies do not directly demonstrate nitrogen limitation\nacross all boreal ecosystems. To address this, we have clarified both the empirical\nand literature bases for our interpretation and replaced the corresponding\nreferences. Our data indicate that N enrichment rates were lower in boreal regions\nthan in tropical and temperate regions (Supplementary Fig. 2), reflecting lower\nexperimental N inputs. In addition, it has been reported that high-latitude\necosystems are typically characterized by nitrogen limitation due to low\ntemperatures and slow nutrient cycling (McKane et al., 2002; Chapin et al., 2011).\nTogether, these findings suggest that in boreal regions with low N inputs and\nlimited N availability, plant communities are more responsive to N enrichment.\nAccordingly, we have revised the description and updated the references to ensure\nthat the statement is fully supported by the appropriate literature, the sentence in\nthe manuscript has been revised as follows:\nThis more negative effect in boreal climates may arise from intensified\ninterspecific competition induced by N enrichment, as boreal regions receive lower N\ninputs (Supplementary Fig. 2) and N limitation is common in cold high-latitude\necosystems (McKane et al., 2002; Chapin, Matson & Vitousek, 2011). (Lines 155-158)\nReferences cited in this response:\nMcKane, R. et al. Resource-based niches provide a basis for plant species diversity and\ndominance in arctic tundra. Nature 415, 68-71 (2002).\nChapin, F. S. III, Matson, P. A. & Vitousek, P. M. Principles of Terrestrial Ecosystem\nEcology (Springer, New York, 2nd edn, 2011).\nComment 2: Lines 197-182, and previous comment #13. I agree that N enrichment can\nstimulate phosphatase activity. I have experimentally demonstrated that myself for\ngrassland plants (Plant and Soil 347: 137-146). However, I think that generally it can\nnot be concluded that this only counts for forests and not for grasslands. See my\nmentioned paper for the grassland plants, and also your own response to this point in\nwhich you mention that the meta-analysis of Gou et al (2024 ) showed that the effect of\nN enrichment on acid phosphatase was clearly not different between forests and\ngrassland, and that only the alkaline phosphatase response differed. But in the latter\ncase it was even reduced upon N-enrichment in grasslands soils. Was that due to an N\neffect or to something else (e.g. pH). Anyway, I think the nuance made by the authors\nin their response is fine, but the single sentence in the manuscript (Line 179-182) should\nbe formulated differently.\nResponse: We thank you for the comment that N enrichment generally stimulates\nsoil phosphatase activity. We have now cited this experimental study (Plant and\nSoil 347: 137-146) to include direct evidence from grassland systems. Our intention\nwas to explain the mechanism underlying the significant increase in fungal\nShannon diversity observed in forest ecosystems, rather than to imply that this\nresponse is unique to forests. To clarify this point, we have reformulated the\nsentence to focus on the forest context and to reflect the quantitative evidence\nreported by Gou et al. (2024), which shows that N enrichment consistently\nincreases both acid and alkaline phosphatase activities in forest ecosystems. In the\nrevised version, we clarified this statement as below:\nNitrogen enrichment usually increases phosphorus availability by increasing\nphosphatase activities (Xu et al., 2022; Olde Venterink et al., 2011) and promoting the\ndissolution of immobile inorganic phosphorous to more available forms (Wang et al.,\n2022). This explanation is supported by meta-analytical evidence showing that N\nenrichment results in consistently increase of soil phosphatase activities in forests (Gou\net al., 2024).\nReferences cited in this response:\nOlde Venterink, H. Legumes have a higher root phosphatase activity than other forbs,\nparticularly under low inorganic P and N supply. Plant and Soil 347, 137-146\n(2011).\nXu, H. W. et al. Impact of nitrogen addition on plant-soil-enzyme C-N-P stoichiometry\nand 708 microbial nutrient limitation. Soil Biol. Biochem. 170, 108714 (2022).\nWang, R. Z. et al. Nitrogen enrichment buffers phosphorus limitation by mobilizing\nmineral- bound soil phosphorus in grasslands. Ecology 103, e3616 (2022).\nGou, X. M., Ren, Y. Q., Qin, X., Wei, X. R. & Wang, J. J. Global patterns of soil\nphosphatase responses to nitrogen and phosphorus fertilization. Pedosphere 34,\n200-210 (2024).\nComment 3: I congratulate the authors with their paper which is a nice contribution to\nthe literature!\nWith best regards,\nHarry olde Venterink\nResponse: Thank you for the positive evaluation of our work. We are delighted that the\nfinal version of the manuscript is viewed as a valuable contribution to the literature.\nYour constructive feedback throughout the review process greatly helped us refine the\nanalyses and strengthen the overall presentation of the study."}]

Decision letter