Critical thresholds for co-benefits of carbon accumulation and biodiversity conservation under global nitrogen enrichment

Domain: biology
Published: 2026-01-08
Editorial outcome: minor_revision

Abstract
Global nitrogen (N) enrichment promotes soil organic carbon (SOC) accumulation but often causes biodiversity loss in plants and soil microbes, creating a central challenge for achieving co-benefits of carbon accumulation and biodiversity conservation. The extent to which biodiversity contributes to SOC accumulation, and how these trade-offs can be mitigated under N enrichment remain poorly understood. Here, we conduct a global meta-analysis of N enrichment experiments encompassing 2141 observations from 275 studies to evaluate the contributions of plant, bacterial, and fungal diversity to SOC accumulation and to determine associated ecological thresholds. Our findings indicate that biodiversity, especially in plant communities, is the most important factor for SOC accumulation. We identify three distinct N thresholds characterized by different ecological responses: biodiversity loss accelerates at 50 kg N ha⁻¹ yr⁻¹, soil degradation persists at 200 kg N ha⁻¹ yr⁻¹, and SOC accumulation declines beyond this point. Given that the promoting effects of bacterial, fungal, and plant diversity on SOC diminish or even reverse at 50, 67, and 74 kg N ha⁻¹ yr⁻¹, respectively, we suggest limiting fertilization to around 50 kg N ha⁻¹ yr⁻¹, as the trade-off between biodiversity loss and SOC accumulation under N enrichment is potentially minimized.

Introduction
Soil is the largest carbon (C) reservoir in terrestrial ecosystems, playing a critical role in regulating the global C cycle. Even small changes in the soil organic carbon (SOC) pool can have profound impacts on ecosystem services and land-climate feedbacks1,2. Soil N availability significantly influences storage capacity of SOC, since N is a key component of SOC3,4. While increased N input has alleviated constraints on global food production, it has also elevated ecosystem N availability, leading to eutrophication and altered ecosystem processes5–7. Previous studies have demonstrated that N enrichment can enhance SOC storage by promoting the formation of larger soil aggregates, improving soil fractions, and increasing N use efficiency4,8–10. However, these effects are not always consistent, as some studies have documented neutral or even negative outcomes11,12, potentially due to differences in substrate quantity, mineral associations, environmental conditions, and biodiversity13. Biodiversity, in particular, is widely recognized to regulate soil C cycling14,15, and its loss drives large-scale changes in ecosystem energy flow and nutrient cycling16,17. Higher plant diversity often increases biomass inputs and root exudation18, while soil microbial diversity governs the balance between C catabolism (decomposition) and anabolism (biomass and extracellular production)19–21, thereby shaping SOC dynamics. N enrichment generally reduces biodiversity22–25, primarily through mechanisms such as competitive exclusion, soil acidification, and cation toxicity24,26. Yet microbial diversity exhibits inconsistent responses27,28. For example, N enrichment significantly reduced overall microbial diversity, but the decline was mainly observed for bacteria, whereas fungal diversity remained unchanged27. This highlights the complex and taxon-specific nature of microbial responses to N enrichment. Therefore, such divergent responses between plant and microbial diversity may weaken the positive biodiversity effects on SOC sequestration29,30. Moreover, the potential erosion of functional diversity could further decrease resource-use efficiency and ecosystem productivity, posing a major challenge for sustaining SOC accumulation while mitigating biodiversity loss14,19,22,31–33. Despite increasing evidence of these complexities4,9,27,34, few studies have jointly assessed the relative contributions of major drivers, including plant and microbial diversity and biomass, soil physicochemical attributes (e.g., pH, moisture, nutrient availability), and climatic variables (e.g., mean annual precipitation (MAP), mean annual temperature (MAT)), to SOC responses under N enrichment. Therefore, a global assessment of the factors underpinning SOC dynamics is critical for reducing uncertainty in biogeochemical models of soil C turnover and advancing our understanding of how biodiversity mediates soil C dynamics in an N-enriched world35.

Biodiversity and soil C conservation are dual priorities in the global effort to mitigate climate change36,37. However, N enrichment can disrupt this co-benefit over time, decoupling biodiversity and C goals22. For example, a 27-year field experiment in temperate grassland reported SOC gains under high N input, but at the cost of substantial plant diversity loss38. Similarly, a global meta-analysis revealed that soil bacterial and fungal diversity are strongly negatively associated with soil C content, and their linkages are influenced by N fertilization rate and duration22. In contrast, tree species mixtures with high diversity can enhance SOC accumulation under elevated N availability39. These findings suggest that biodiversity-SOC relationships under N enrichment are context-dependent, and sustained N inputs may trigger trade-offs under specific environmental conditions. Therefore, identifying ecological thresholds along N gradients is essential to anticipate when biodiversity losses may begin to undermine soil C gains40,41. Recognizing and quantifying such thresholds is crucial for ecosystem-based management and policy formulation, as they offer valuable benchmarks for designing effective management interventions and conservation targets42–45. While prior studies have observed nonlinear or saturating responses of biodiversity and SOC to N inputs, systematic identification of critical thresholds remains limited. Given their high metabolic rates and short generation times46, soil microbial communities are more sensitive and responsive to environmental change than plants47,48 and are therefore expected to exhibit abrupt shifts at lower N levels than plants. However, these microbial shifts may not immediately translate into changes in SOC, particularly for mineral-associated organic carbon (MAOC), which is resistant to rapid turnover49. This temporal lag suggests that biodiversity thresholds, particularly for soil microbes, may precede those for C, potentially delaying detectable SOC destabilization under N enrichment. Understanding how biodiversity and SOC in terrestrial ecosystems respond to increasing N inputs is critical for advancing ecological threshold theory and informing estimates of N input levels that agroecosystems and natural ecosystems can tolerate before facing heightened risks of biodiversity loss and soil C destabilization.

To address this knowledge gap, we compile a global database containing 2,141 observations from 275 in situ N enrichment experiments (Fig. 1a, b), spanning a wide range of climates (MAT from −9 to 30 °C and MAP from 100 to 4500 mm), vegetation types (farmlands, grasslands, and forests), and soil properties (pH from 3.5 to 9.0, SOC from 1.6 to 203.8 g C/kg soil). We examine how plant and microbial diversity and biomass jointly affect SOC accumulation under N enrichment. Additionally, we quantify the critical thresholds of N inputs at which shifts in plant and microbial communities and SOC accumulation occur, and identify a defined window where soil C gains can be achieved with minimal biodiversity loss. This framework allows us to comprehensively assess the role of biodiversity in mediating SOC and to reveal the critical N thresholds that enable co-benefits of biodiversity conservation and soil C accumulation globally in an N-enriched world.

Results
N enrichment consistently reduced diversity across plant and soil microbial communities, with declines of 12.5% in plants (95% confidence interval (CI): −17.0% to −7.8%, not applicable to farmland; P<0.001), 5.5% in bacteria (95% CI: −7.7% to −3.1%; P<0.001), and 0.98% in fungi (95% CI: −4.6% to 2.8%; P=0.604) (Fig. 1c–e), aligning with findings from recent studies22,50. The weak negative effect of N enrichment on fungal diversity likely reflects heterogeneous responses among functional guilds51, as N reduces the diversity and abundance of arbuscular mycorrhizal fungi (AMF) while simultaneously increasing the relative proportion of saprotrophic fungi52,53. This functional reorganization may obscure declines in total fungal diversity. In addition, the compensatory dominance of tolerant fungal taxa may buffer the loss of more sensitive species under global change conditions54. Despite receiving frequent N inputs, farmland exhibited the largest declines in soil microbial diversity compared to natural ecosystems, with decreases of 6.9% in bacterial diversity (95% CI: −11.7% to −1.8%; P=0.009) and 5.6% in fungal diversity (95% CI: −13.4% to 3.0%; P=0.194). This suggests that agricultural soil microbial diversity may continue to decline under future N enrichment. Additionally, plant diversity declined mainly in temperate regions (−16.4%, 95% CI: −22.0% to −10.3%; P<0.001), while bacterial diversity decreased in both temperate (−5.7%, 95% CI: −8.3% to −3.1%; P<0.001) and tropical regions (−5.5%, 95% CI: −10.0% to −0.8%; P=0.021). In contrast, fungal diversity showed no significant change within any climatic region, but shifted from negative to positive responses with decreasing temperature (−1.2%, 95% CI: −5.1% to 2.9%, in temperate regions; 12.9%, 95% CI: −1.7% to 29.7%, in polar regions; all P>0.05). The greater decline in plant and bacterial diversity in warm regions may be explained by the amplification of aboveground biomass responses to N addition under higher temperature and precipitation, which intensifies light competition and leads to the exclusion of rare species55. Fungi may be more resistant because of their higher tolerance to osmotic and environmental stress56. These patterns highlight the influence of regional climatic conditions on the responses of plant and microbial communities to N inputs. Declines in plant and soil microbial diversity intensified with increasing N rates and prolonged exposure, likely driven by continued soil acidification24,57. These contrasting responses between plant and microbial communities underscore the complexity of ecosystem and climate dynamics under N enrichment, emphasize the importance of a nuanced understanding of N-driven linkages between above- and belowground processes in terrestrial ecosystems.

Beyond biodiversity loss, N enrichment also significantly reshaped biomass dynamics in both above- and belowground compartments. In contrast with reductions in diversity, N enrichment significantly increased plant biomass by 32.8% (95% CI: 24.8% to 41.3%; P<0.001) while reducing soil microbial biomass carbon (MBC) by 5.8% (95% CI: −10.6% to −0.7%; P=0.027), which was more pronounced in grassland ecosystems (Fig. 1f, g). The stronger responses may be linked to greater increases in photosynthetic rate, stomatal conductance, and water use efficiency under N addition in herbaceous species in grasslands, compared to woody species in forests58. Within each climatic region, plant biomass showed a significant increase in tropical regions (46.4%, 95% CI: 28.9% to 66.4%; P<0.001), whereas MBC showed greater reductions in temperate regions (−8.9%, 95% CI: −14.5% to −2.8%; P=0.004). Both trends intensified under higher N rates and longer exposure durations. Additionally, plant biomass showed a non-significant increase under organic fertilizer application (17.9%, 95% CI: −16.0% to 65.5%; P=0.342), suggesting that organic amendments may partially mitigate the trade-off between diversity loss and biomass gain40. As expected, SOC increased under N enrichment (Fig. 1h), particularly in grassland ecosystems (6.6%, 95% CI: 3.0% to 10.2%; P<0.001), reinforcing their role as C sinks in the context of global N enrichment59. Across climatic regions, SOC accumulation tended to decline from cold (9.5%, 95% CI: −5.3% to 26.7%; P=0.219) to warm regions (0.9%, 95% CI: −3.6% to 5.5%; P>0.5). This pattern is consistent with the metabolic theory of ecology, which predicts that increasing temperature enhances microbial metabolic activity and accelerates organic matter decomposition, thereby limiting net SOC storage9,60. Unlike other variables, SOC accumulation showed no consistent increase with rising N rates or durations, indicating its complex and the uncertainty of SOC stock predictions under future N enrichment scenarios. These findings highlight the complexity of ecosystem responses to N enrichment and the need for targeted management strategies to balance soil C storage and biodiversity conservation.

In addition to its biological effects, N enrichment also altered key soil chemical properties. N enrichment significantly reduced soil pH by 27.5% (95% CI: −32.2% to −22.5%; P<0.001), driven primarily by increases in total N (TN) (11.0%, 95% CI: 6.9% to 15.2%; P<0.001), nitrate nitrogen (NO₃⁻-N) (111.1%, 95% CI: 82.7% to 144.0%; P<0.001), and ammonium nitrogen (NH₄⁺-N) (50.1%, 95% CI: 33.4% to 68.8%; P<0.001) (Supplementary Fig. 1a–d). Despite these changes, soil phosphorus (P), including total P (TP) and available P (AP), remained largely unaffected (all P>0.05), indicating that our study excluded scenarios involving co-addition of N and P (Supplementary Fig. 1e, f). Unexpectedly, the reduction in soil pH in grassland was nearly identical to that in farmland (−30.0% vs. −30.6%; Supplementary Fig. 1d). This may be attributed to their low-stature plant communities reaching N saturation more readily, leaving more unassimilated H⁺ and reactive N in the soil61. Additionally, limited precipitation in many grassland systems may constrain plant uptake and buffering capacity, further exacerbating acidification under elevated N62. Regional variations in pH reduction were also evident, with the smallest declines observed in polar regions (−19.9%, 95% CI: −38.4% to 4.0%; P=0.10), followed by tropical regions (−22.7%, 95% CI: −30.7% to −13.8%; P<0.001), while temperate regions exhibited the greatest declines (−30.0%, 95% CI: −35.1% to −24.4%; P<0.001) (Supplementary Fig. 1d). Temperate soils experienced the strongest pH decline, likely due to their weak buffering capacity and widespread podzolization, which facilitates Al³⁺ release and amplifies acidification63. In contrast, tropical soils form stable Al oxides that immobilize Al³⁺, while polar soils show slight change due to low biological activity and thick organic horizons buffering acidity63,64. Furthermore, the reduction in soil pH was amplified by increasing N rates and longer durations, suggesting that the current quantification of N enrichment effects may underestimate its long-term impacts on soil health and ecosystem functioning. Notably, the application of organic fertilizers significantly mitigated acidification compared to synthetic N fertilizers, likely due to their buffering effects through enhanced cation exchange capacity40. Given the pivotal role of soil pH in maintaining ecosystem diversity and multifunctionality, sustainable N management strategies should prioritize organic fertilizers and limit excessive use of synthetic fertilizers in agriculture.

Given that plant and microbial biomass constitute the immediate foundation for SOC formation and turnover, we independently modeled the global responses of plant biomass, microbial biomass, and SOC under N enrichment to better elucidate ecosystem C dynamics (Fig. 2). Across regions, plant biomass exhibited consistently positive responses, especially in temperate and boreal regions of North America, Europe and Asia (Fig. 2a), reflecting N-stimulated productivity to N inputs65. In contrast, MBC exhibited greater spatial heterogeneity. Positive responses were prevalent in tropical and polar regions, while declines were widespread across mid-latitude temperate ecosystems such as central North America, Europe, and East Asia (Fig. 2b). This divergence could be attributed to regional disparities in baseline N deposition and soil sensitivity66. In tropical and polar regions with low ambient N, moderate enrichment promotes microbial growth. However, excess N in high-background areas induces acidification and nutrient imbalance, ultimately suppressing microbial biomass67,68. Similarly, SOC responses were largely positive across temperate regions, particularly in Europe and northern Asia. However, negative responses prevailed in parts of the Southern Hemisphere and tropical regions, including southern Africa, South Asia and Southeast Asia (Fig. 2c). The contrasting trends are likely shaped by nutrient limitation and turnover dynamics that differ across ecosystems. In temperate regions, N addition stimulates plant inputs while simultaneously suppressing decomposition, thereby favoring soil C accumulation69. In contrast, phosphorus limitation and high decomposition rates dominate in many tropical regions, where added N often fails to enhance biomass input and may even accelerate C loss70. Together, these spatial patterns illustrate how N enrichment reshapes C cycling across climatic gradients and emphasize the importance of region-specific N management aligned with soil C accumulation objectives.

To understand how biodiversity and SOC respond to N enrichment, we first examined the main environmental predictors associated with diversity and SOC patterns, respectively. Using the random forest approach, we found that the response of plant diversity was strongly linked to N availability (TN, NH₄⁺-N and NO₃⁻-N), MAT, and soil moisture (Fig. 3a; all P<0.05), reflecting their critical roles in supporting plant growth. In contrast, bacterial diversity was primarily associated with NH₄⁺-N and pH (Fig. 3b; all P<0.05), whereas fungal diversity responded most strongly to NO₃⁻-N, MAT, and soil moisture (Fig. 3c; all P<0.05). These findings are consistent with previous research, which highlights N-induced soil acidification as a primary predictor of biodiversity loss in terrestrial ecosystems71,72, while also underscoring the modulatory role of climatic factors in shaping biodiversity responses under N enrichment73. Given the direct contribution of plant and microbial biomass to the formation of SOC, we further identified the main predictors of these two biomass components. Our random forest and meta-regression analyses revealed that the responses of plant and microbial diversity are associated with changes in MBC under N enrichment (Supplementary Figs. 2 and 5), supporting that biodiversity contributes to belowground C turnover under N enrichment74. After controlling for N addition level in the meta-regression, we found that several pairwise associations remained significant (Supplementary Figs. 3 and 4). Importantly, by integrating all abiotic and biotic factors (e.g., diversity and biomass of plants and soil microbial communities), our random forest analysis revealed that the responses of TN and plant diversity were the strongest predictors of SOC changes, with plant diversity playing a pivotal role in regulating SOC accumulation under N enrichment (Fig. 3d; P<0.05). Our work supports the fundamental theory that biodiversity acts as a key regulator of ecosystem C dynamics33,75–77, suggesting the urgent need to incorporate the response of biodiversity to revisit SOC accumulation effect under N enrichment. Despite being much discussed, we provided the global evidence that biodiversity mediates SOC responses in the context of N enrichment.

Beyond identifying the key predictors of biodiversity and SOC, it is essential to assess whether and how biodiversity and SOC responses are interlinked under N enrichment. Mixed-effects meta-regression models revealed that N enrichment significantly altered the associations between bacterial, fungal, and plant diversity and SOC. Under control conditions, all three diversity metrics showed a significant positive correlation with SOC (Fig. 3e; all P<0.05). Following N enrichment, plant and fungal diversity turned negative, whereas bacterial diversity remained positively linked to SOC (Fig. 3f; all P<0.05). This shift suggests a transformation in ecosystem C cycling from synergistic to trade-off dynamics under N enrichment. The observed trade-off between plant diversity and SOC aligns with the competitive exclusion hypothesis, as SOC accumulation under N enrichment is primarily driven by increased plant biomass inputs3. The parallel responses of fungi and plant to N enrichment are likely rooted in the close functional integration between mycorrhizal fungi and their plant hosts51. Given that N enrichment affects both biodiversity and soil processes through multiple interacting pathways, the detected biodiversity-SOC associations may partly reflect shared environmental responses rather than direct causal relationships. Overall, these findings reveal a decoupling of soil C allocation between above- and belowground components under N enrichment29,78, and highlight the importance of incorporating synergistic plant–microbe responses into biogeochemical cycle models under future global change.

We analyzed the responses of plant and soil microbes, as well as SOC to increasing N rate and identified the thresholds at which these responses exhibited abrupt shifts. All measured variables showed nonlinear responses to rising N inputs (Fig. 4a and Supplementary Figs. 6, 7, all P<0.001). These shifts occurred in three distinct phases: accelerated biodiversity loss at 50 kg N ha⁻¹ yr⁻¹, persistent soil degradation at 200 kg N ha⁻¹ yr⁻¹, and a decline in SOC accumulation at higher N levels (Fig. 4a). In the first phase, plant and soil microbial diversity consistently declined with N enrichment, with sharp decreases observed at thresholds of 34, 48, and 50 kg N ha⁻¹ yr⁻¹ for fungi, bacteria, and plants, respectively. Soil fungi appeared more tolerant to N enrichment than bacteria, yet exhibited earlier threshold responses. This earlier threshold response of fungi may be attributed to their dual ecological roles as both decomposers and plant symbionts, which expose them to multiple forms of N-induced stress79. For instance, fungi must maintain functional symbiosis with host plants under altered nutrient conditions while also coping with increasing cation toxicity from acidification79. These simultaneous demands may trigger earlier physiological adjustments, even if their overall tolerance to high N levels remains greater than that of bacteria. Plant diversity exhibited a higher N threshold, likely due to plants’ longer life cycles and slower population dynamics47, which delay their responses to environmental changes. The early thresholds observed for biological communities underscore the importance of biodiversity monitoring as an early-warning indicator for significant ecosystem transformations45, advocating for strategic conservation efforts to safeguard terrestrial ecosystems and their services. During the soil degradation phase, we observed a significant decline in soil pH occurring at 66 kg N ha⁻¹ yr⁻¹. MBC remained stable until a threshold of 87 kg N ha⁻¹ yr⁻¹, after which it declined significantly. Although a threshold was identified for soil moisture, changes in soil water availability were minimal. Soil NH₄⁺-N, NO₃⁻-N, and TN levels increased rapidly with N enrichment initially but stabilized after surpassing their respective thresholds. In the last phase, SOC accumulation and plant biomass both showed positive correlations with N input before reaching their thresholds but shifted to negative correlations beyond these points. This indicates that continued microbial diversity loss and persistent soil acidification could offset the SOC gains from increased plant biomass observed at earlier stages of N enrichment, potentially destabilizing long-term soil C sequestration.

Moreover, we performed a moving-window analysis to evaluate the potential shifts in the associations between the responses of diversity and SOC with increasing N (Fig. 4b–d and Supplementary Fig. 8; all P<0.001). The responses of bacterial diversity and SOC showed a slightly negative correlation below ~50 kg N ha⁻¹ yr⁻¹ (Fig. 4b), indicating a transient non-causal association. At this stage, the increase in SOC may directly benefit from aboveground productivity (e.g., leaf litter and root exudates)80,81, while the decline in bacterial species may be functionally compensated by redundant taxa82. As N input exceeded this threshold, the association progressively became positive (reaching ~200 kg N ha⁻¹ yr⁻¹), marking a co-decline phase where intensified N enrichment suppressed both biodiversity and SOC. The response of fungal diversity fluctuated around ~67 kg N ha⁻¹ yr⁻¹ but maintained a positive association with SOC across the N gradient (Fig. 4c), suggesting that fungi play a continuous and important role in SOC accumulation83. For example, certain ectomycorrhizal (ECM) fungi are capable of expressing oxidative enzymes that decompose recalcitrant organic matter84, while arbuscular mycorrhizal (AM) fungi may enhance MAOM formation through efficient hyphal turnover and microbial-derived pathways85,86. At low N inputs (<50 kg N ha⁻¹ yr⁻¹), plant diversity and SOC responses were negatively associated (Fig. 4d), reflecting a decoupled pattern where diversity loss driven by light competition and cation toxicity co-occurred with ongoing SOC accumulation24. Between 50 and 74 kg N ha⁻¹ yr⁻¹, the association became strongly positive, indicating a shift toward synchronized biodiversity loss and slower or declining SOC gains. Above ~74 kg N ha⁻¹ yr⁻¹, the positive association weakened, likely reflecting reduced plant-derived inputs as severe plant diversity loss constrained new C incorporation into soils17. Collectively, these results delineate a three-phase trajectory: low-N decoupling, mid-N synergy, and high-N functional breakdown. This trajectory underscores a mechanistic shift from N-stimulated C gains to biodiversity-governed constraints under saturation. Importantly, our results showed that the intermediate range (~50–74 kg N ha⁻¹ yr⁻¹) emerges as a potential co-benefits threshold for maintaining SOC accumulation without severe biodiversity loss. Overall, this context-dependent transition from synergy to trade-off underscores the complex and multifaceted consequences of biodiversity loss for SOC accumulation. Limiting N inputs to around 50 kg N ha⁻¹ yr⁻¹ could help mitigate biodiversity loss and its negative effects on SOC storage, supporting long-term ecosystem stability.

Although we were able to collect a substantial dataset for this meta-analysis, research into joint effects of aboveground and belowground components on soil C dynamics is still in its infancy. First, SOC accumulation does not directly reflect long-term C accumulation potential, which is also influenced by decomposition rates, microbial metabolism, and environmental conditions. Second, while our soil microbes only included decomposers (bacteria and fungi), the potential influence of primary predators (nematodes and protists) and vertebrate control through trophic cascades on ecosystem C cycling under N enrichment have been overlooked, despite its recent identification as a significant factor in global C turnover87. Third, our study underrepresents Africa, South America, and Australia, potentially limiting the generalizability of the observed joint effects of N enrichment on biomass and soil C dynamics across biomes. Future studies should adopt factorial designs that independently manipulate biodiversity and N input (e.g., long-term crossed treatments), helping to disentangle causal effects from environmental confounding. Notwithstanding these challenges, our study provides robust global evidence that biodiversity, both aboveground and belowground, plays a pivotal role in regulating soil C stocks under N enrichment. Furthermore, we identified critical N thresholds that elucidate how above- and belowground systems alongside SOC respond simultaneously to N enrichment. These findings reveal key N input levels that balance biodiversity conservation and SOC accumulation, offering valuable evidence toward achieving a win-win scenario under N enrichment. Overall, our study develops a series of N enrichment thresholds that identify the critical inflection points for biodiversity and SOC responses under N enrichment. By jointly analyzing how above- and belowground community diversity mediates soil carbon dynamics, our findings demonstrate that intensified N inputs can induce nonlinear shifts in biodiversity-carbon relationships from synergy to decoupling and eventual collapse. In particular, once the threshold (e.g., ~50 kg N ha⁻¹ yr⁻¹) is exceeded, the sharp decline in biodiversity may offset the potential benefits of SOC accumulation. This global meta-analysis simultaneously evaluates plant and microbial diversity in relation to SOC across diverse ecosystems, offering an integrated perspective on how multi-trophic biodiversity regulates biogeochemical responses to N enrichment. This knowledge enables more precise prediction of ecosystem carbon dynamics across diverse terrestrial biomes under future global changes, as aboveground and belowground responses are critical to maintaining and conserving ecosystem service. In doing so, the identification of actionable N input thresholds can inform sustainable N management strategies, delivering co-benefits for biodiversity conservation and long-term carbon retention.

Methods
Data collection
We constructed a global database to jointly investigate how SOC responses are influenced by changes in plant and soil microbial diversity to N enrichment, adhering to the PRISMA guidelines88 (Preferred Reporting Items for systematic reviews and meta-analyses) (Supplementary Fig. 9). Peer-reviewed publications on the impacts of N enrichment on plant and soil microbial diversity in our study were searched using the Web of Science (https://webofscience.clarivate.cn/wos/woscc/basic-search) and China National Knowledge Infrastructure (CNKI, https://www.cnki.net), including all research up to April 1, 2023 (a full list is provided in Supplementary Data 1). Keywords used were: “(nitrogen fertilization OR N deposition OR nitrogen addition OR N enrichment) AND (plant OR soil microbial) AND (richness) AND (SOC OR MBC OR plant biomass)”. We focused on diversity and biomass of plant and soil microbes as: (1) there is a significant knowledge gap regarding how their responses affect carbon allocation between plant and soil microbial biomass, and thus soil C storage under N enrichment; and (2) to provide a balanced dataset that considers both plant and soil microbes, as these variables are rarely measured simultaneously in studies.

Relevant publications were selected based on the following criteria: (1) N fertilizer was directly applied to terrestrial ecosystems under field conditions using paired treatment-control plots, excluding laboratory incubation studies. (2) At least one soil microbial phylotype related to diversity (richness), including bacteria and fungi, or biomass (MBC) was reported. (3) N application rate and experiment duration were recorded to standardize the effect of N enrichment across studies. (4) Means and sample sizes for the selected variables were available or could be calculated from the publications. Our search yielded a dataset of 2141 observations from 275 peer-reviewed studies (Fig. 1a, b), encompassing plant diversity and biomass, soil microbial diversity and biomass, SOC content, and associated climate and soil variables (Supplementary Table 1).

Extracted data
We collected plant and multiple soil variables, including plant species richness and biomass, soil microbial diversity and biomass, soil properties, and SOC from the selected publications. Numerical data were extracted from text, tables, figures, and appendices, whereas graphical data were digitized using GetData Graph Digitizer (http://getdata-graph-digitizer.com). Mean values, standard deviations (SD), and sample sizes (n) were recorded or calculated. When studies reported standard error (SE), it was converted to SD: SD=SE×√n. Climatic variables, including MAT and MAP, were obtained from Worldclim 2.0 (http://www.worldclim.org) based on study coordinates. Ecosystem types were categorized as farmland, forest, and grassland, and climatic regions as tropical (tropical and subtropical), temperate, and polar (polar and boreal), following the 2010 Global Ecological Zones dataset (https://data.apps.fao.org/). Here, we used richness (that is, the number of species) as a metric of diversity, as richness is the most used and simplest89,90. Accordingly, only studies reporting microbial richness were included. Due to prevalent monoculture practices in farmlands, land diversity data were largely unavailable and therefore not included. Soil microbial biomass was represented by MBC91. Soil properties (e.g., pH and moisture), nutrients (e.g., total N, total P, available N, and available P) and SOC were collected in the dataset. Detailed descriptions of all variables are provided in Supplementary Table 1. Since the analytical data were transformed into response ratios for subsequent analyses, any method-induced differences are considered negligible.

Data analyses
In this study, the natural logarithmic response ratio (lnRR), an effect size metric widely applied in meta-analysis, was used to evaluate the effect of N enrichment on each variable. The lnRR of an observation was calculated as follows:

lnRR=ln(XT/XC) (1)

Where XT and XC represent the mean values of response variables in the N treatment (T) and control (C), respectively. The N effect on soil pH was calculated as the difference between the treatment and control groups, as soil pH uses a logarithmic scale.

The weighted mean response ratio (lnRR) for each group (e.g., ecosystem types, climatic regions) was calculated as follows:

lnRR=∑(wi* ×lnRRi)/∑wi* (2)

where m represents the number of observations in group, and wi* is the weighting factor of the ith observation in the group. The wi* was calculated as follows:

wi* = 1/vi* (3)

where vi* is the variance of study (i) in the group. The vi* was calculated as follows:

vi* = vi + T2 (4)

where vi is the within-study variance of study (i), and T2 is the between-studies variance. The vi was calculated as follows:

vi = (SDT2 / (nT XT2)) + (SDC2 / (nC XC2)) (5)

where SDT and SDC are the SD of the variables in the treatment and control treatments, respectively; nT and nC are the sample size in the treatment and control, respectively.

The SE of lnRR was calculated as follows:

s(lnRR)=√1/∑wi* (6)

The 95% CI for lnRR was calculated as follows:

95% CI=lnRR±1.96s(lnRR) (7)

If the 95% CI does not overlap zero, the overall effect size of the N enrichment was significant92. For easier interpretation, the lnRR was back-transformed and expressed as a percentage change (%):

Effect size (%) = (exp(lnRR)−1)×100% (8)

A mixed-effects model was employed to calculate the weighted mean of lnRR using the rma.mv function of the R package metafor93, with lnRR ID nested within study ID as the random effect. The distributions of lnRR for each variable analyzed in this study are presented in Supplementary Figs. 10 and 11. All statistical analyses were performed in R version 4.2.1.

Random forest model
We used random forest analysis to identify the primary predictors of plant and soil microbial diversity, biomass, and SOC responses94. Initially, we analyzed various environmental and biological variables to determine the key drivers of richness in plants, soil bacteria, and fungi. Given the pivotal role of diversity in influencing biomass, we incorporated diversity metrics into the original variables to identify the strongest predictors of plant and microbial biomass responses. Finally, we combined all environmental and biological variables, as well as diversity and biomass metrics from plants and soil microbial communities, to identify the main predictors of SOC response. The random forest models were implemented using the R package spatialRF95, which effectively reduces multicollinearity, captures key variable interactions, and evaluates model robustness through spatial cross-validation94. Before model fitting, we minimized multicollinearity by performing two steps: (1) correlation-based filtering (auto_cor function, |r|>0.6) to remove highly correlated predictors, and (2) variance inflation factor filtering (auto_vif function, VIF>2.5) to further exclude N-induced collinear variables. Predictor importance was assessed by measuring the average decrease in prediction accuracy (mean square error) when the data for a given predictor were permuted across trees.

Due to the uneven distribution of data when incorporating both aboveground and belowground variables, and the inability of the random forest model to handle missing values, we included observations with at least seven available variables in the dataset used to identify key predictors96. This approach ensured robust representation and analytical reliability across all study observations (Supplementary Figs. 12 and 13). To accommodate missing data, values were imputed using the missForest function from the missForest package with the parameter ntree set to 99997.

Mixed-effects meta regression
Mixed-effects meta-regression models were used to examine the relationships between climatic and soil factors (identified through the random forest model) and plant, soil microbial communities, and SOC, using the rma.mv function in the R package metafor92. All models were fitted using inverse-variance weighting to account for differential contributions of each study98. Study ID was included as a random factor, and to account for potential non-independence among response variables arising from shared variance related to N enrichment, N rate level was also included as a random effect.

Threshold identification
In this study, we applied a two-step procedure based on the Akaike Information Criterion (AIC) to identify the most suitable model describing the relationship between the response of soil factors, diversity and biomass or SOC (y) and N enrichment rates (x), followed by previous studies42,43. First, we fitted linear and nonlinear models, including quadratic regressions (simple nonlinear trends) and generalized additive models (GAM, flexible trends using smoothing parameters)42,43. The linear model served as the null hypothesis, representing a gradual x-y relationship. Lower AIC values for nonlinear models indicated a threshold point, suggesting a departure from linear trends. In the second step, thresholds were tested only when nonlinear models demonstrated a better fit. Thresholds were categorized as either continuous or discontinuous. A continuous threshold marks a point of gradual slope change, often identified using GAM or segmented regressions, which capture points of maximum curvature. In contrast, discontinuous thresholds involve abrupt changes in both slope and intercept and are best modeled using step regressions or stegmented regressions (a combination of step and segmented approaches). Among segmented, step, and stegmented regressions, the model with the lowest AIC value was selected. If GAM was the best model, the segmented regression threshold was reported to highlight the point of maximum curvature. To ensure robustness, we removed outliers using the Mahalanobis distance method (cutoff > 12) and restricted threshold searches to the 0.05–0.95 quantile range of x43. Each case underwent 1000 bootstrap resamplings to estimate plausible thresholds, with the mean threshold reported as the final value. We tested the significance of thresholds by bootstrapping linear regressions on either side of the threshold and comparing slopes using a two-sided Mann–Whitney U-test. Threshold detection revealed significant differences in slopes in nearly all cases where nonlinear regressions provided a better fit. All models were implemented using the chngpt99 and gam100 packages in R.

Moving-window analysis
We evaluated how the relationships between diversity and SOC in plant and soil microbial communities change along gradients of N enrichment using a moving-window approach. Linear mixed-effects models were applied to successive subsets of paired diversity–SOC observations, beginning with the lowest N enrichment values and iteratively shifting the window by one observation until the window limit was reached, with study ID included as a random factor101. Following previous studies102,103, the window size was set to 20 observations. For each subset, standardized coefficients of diversity with N rates were bootstrapped and aligned with the average N rate value of the subset101. Linear and nonlinear regressions were then used to identify N rate thresholds, with their validity tested using the methods outlined above.

Global distribution of above- and belowground biomass under N enrichment
Global data collection. Using the important predictors identified by the random forest models, we generated global spatial predictions for the responses of plant biomass, MBC, and SOC under N enrichment. In the absence of global N enrichment data, we substituted N deposition rates from Ackerman et al. for the global map values to represent N availability66. Plant diversity data were sourced from Cai et al. (https://gift.uni-goettingen.de/shiny/predictions)104. Soil biodiversity data from the project of Global Soil Biodiversity Atlas (https://esdac.jrc.ec.europa.eu/content/global-soil-biodiversity-atlas) were used as a proxy for soil microbial diversity. Climatic variables were obtained from Worldclim 2.0 (http://www.worldclim.org) based on location data. Soil properties such as pH and total N (TN) were extracted from SoilGrids (http://www.isric.org/explore/soilgrid). All covariate map layers were resampled and reprojected to a unified 0.25°-pixel grid in EPSG:4326 (WGS84). Higher-resolution layers were down-sampled using mean aggregation, while lower-resolution layers were up-sampled without interpolation to match the grid.

Prediction validation. We applied a random forest model to upscale site-level responses of plant biomass, MBC, and SOC under N enrichment to the regional scale, considering contemporary climate scenarios. Each tree in the model was trained on a randomly selected subset of data, with out-of-bag data used to estimate prediction error and variable importance105. Validation against observed data yielded Pearson’s r values of 0.54, 0.58, and 0.52 for plant biomass, MBC, and SOC, respectively (P<0.001) (Supplementary Fig. 14). The random forest training was repeated 1000 times, and the average predictions from these iterations were taken as the final variable estimates. All random forest computations were performed using the RandomForestRegressor function in the scikit-learn package in Python version 3.7106. The potential effects of global N deposition on these variables were then spatially extrapolated for each pixel using model parameters and the selected variables. Extrapolation and geospatial analyses were conducted in Python version 3.7 and ArcGIS version 10.4.1107,108. Prediction uncertainty for the global estimates was quantified as the 95% CIs (Supplementary Fig. 15).

Publication bias
To assess potential bias in our collected dataset, we employed three independent approaches. First, we utilized a funnel plot to visually examine the possibility of bias (Supplementary Fig. 16). To quantitatively test for asymmetry in the funnel plot, we applied Egger’s regression test109 (Supplementary Table 2). In cases where asymmetry was detected, we used the Rosenberg fail-safe number (Nfs) to determine the extent to which unpublished studies might influence our conclusions110. If the Nfs is larger than 5k+10 (k, number of study observations) (Supplementary Table 2), the results are robust regardless of any potential publication bias. Lastly, we evaluated the robustness of our results by performing a leave-one-out analysis using the leave1out function111 (Supplementary Fig. 17). These findings indicate that publication bias is unlikely to affect the interpretation of our results.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Pan et al. is a meta-analysis on the impacts of anthropogenic N enrichment of soils and its impact on plant and soil microbial diversity and their relationship to soil organic carbon (SOC). Importantly the study shows the links between N enrichment, SOC and soil biodiversity along a gradient of climatic and other factors to show where thresholds occur in the rate of N enrichment that alter the relationships between SOC and plant and soil microbial diversity. The manuscript is well written and the data are thoroughly analyzed for idiosyncrasies. The results show as expected the results that are well established that N enrichment increases plant biomass production, while decreasing plant and soil microbial diversity. This higher N leads to an increased plant derived increase in SOC. BUT that these relationships are conditional on the climate and the that the relationships between biodiversity and SOC have thresholds with the level of N enrichment. These latter points are the highlights of the meta-analysis of the conditional / threshold intricacies of plant and soil biodiversity and soil C dynamics and that these relationships across all these gradients show a nonlinear responses to rising N inputs across environments.\nI have read the manuscript line by line, considered the analyses and SI, and in general I feel this is a very timely and important paper that I have little criticisms of or feel I have many comments to improve the manuscript. My only caution is that SOC might not represent soil C sequestration it gets broken down and metabolized differently under the various ecosystem types and environmental conditions.\nDetailed comments that need to be addressed:\nL414: Plant diversity were sourced from\nL150 : ‘Despite much discussed,’ seems like a word is missing here.\nL643: figure caption “size in each variable is given at the bottom.” I think should be ‘given on the right’.\nFigure 2. Bottom legend labels soil bacterial richness twice. Second should be fungal richness.", "reviewer_2": "Dear Editors,\nI have reviewed the manuscript titled \"Critical Thresholds for Co-Benefits of Carbon Sequestration and Biodiversity Conservation Under Global Nitrogen Enrichment (NC-574816).\" The authors conducted a meta-analysis to identify the threshold point of nitrogen addition that significantly reduces plant, bacterial, and fungal diversity on a global scale. They also aim to determine the critical point for soil organic carbon (SOC) accumulation under nitrogen enrichment along the nitrogen gradient. By examining the relationship between diversity and SOC responses along this gradient, the authors seek to identify critical thresholds for the co-benefits of carbon sequestration and biodiversity conservation under global nitrogen enrichment.\nSeveral issues within the text require clarification. First, it is important to note that there may not always be a trade-off between diversity response and SOC response; at certain points, both parameters can exhibit either positive or negative responses to nitrogen enrichment. Second, the interpretation of the data presented in the main figures is challenging. In some cases, the authors find a negative correlation between plant/bacterial diversity and SOC, but this negative correlation appears to stem from the nitrogen-induced divergent responses of these parameters rather than reflecting biological significance. Although significant correlations are identified, their implications may not be fully clear.\nLastly, I believe the authors should exercise greater caution with their citations. In multiple instances, incorrect papers are cited, or the cited papers do not align with the authors’ claims. I have listed a few examples below for your check.\nI have provided detailed comments below:\nFig. 1: the sample size in each variable is given at the right. The closed symbols indicate significant effects, what is the significant level? What is compound N mean? The figure must be self-explained. Is urea N belonging to Organic N? What exactly organic N means here? Mean effect size of N enrichment (%). The (%) should add after effect size rather than enrichment. For panel g, the number of sample size is not algin on the right.\nFig. 2: Biodiversity drivers SOC accumulation under N enrichment. The legend reads like a results or conclusion, rarely see this in the caption. You need to illustrate what the figure means but not over-interpretation. Study IDs or study id, please consistent. It is wired that for the x axis label, sometimes you use text and some time you use picture. It is hard to understand.\nThe relationship between plant and soil biota? The sentence is confusing. Moreover, all parameters respond to nitrogen addition. When correlating these parameters, such as plant diversity versus soil organic carbon (SOC), how do you account for the effect of nitrogen addition, given that both parameters are influenced by it?\nFig. 3: The results are difficult to interpret. In panel b, if we examine the absolute values for most cases, most relationships fall below the zero line, suggesting that higher bacterial diversity leads to lower SOC. If this is the case, why should we prioritize the protection of bacterial diversity? Similarly, in panel d, if I understand correctly, most relationships are negative below 40 kg N, indicating that a higher response in plant diversity corresponds to a lower response in SOC. In such cases, why is it important to protect plant diversity?\nFig 4: Fig 4 seems out of the logic flow of the main story. All other figs target on the diversity effect on SOC, why Figure 4 focus on biomass and SOC?\nAbstract:\nLine 12: Global nitrogen enrichment does not always enhance soil organic carbon (SOC) accumulation; otherwise, threshold analysis would not be feasible. As your data indicate, in some cases, such as forest systems, nitrogen enrichment tends to reduce SOC accumulation.\nLine 24-26: The units \"kg N\" are incorrect. Per hectare per year?\nIntroduction:\nLines 35-42: These statements suggest that there must be a trade-off between SOC accumulation and biodiversity loss under nitrogen addition; however, this may not be accurate. What if, at certain points, both SOC and biodiversity increase with nitrogen addition? In such cases, no trade-offs would exist.\nLine 59: Why include croplands? If your aim is to examine how SOC accumulation and biodiversity loss change along the nitrogen addition gradient, croplands typically exhibit low plant biodiversity due to prevalent monoculture practices.\nLine 59: What about the mean annual precipitation?\nOverall, the introduction lacks sufficient information on how plant and microbial biodiversity respond to nitrogen addition and what expectations exist regarding which thresholds might be reached earlier and why. Additionally, the response of SOC to nitrogen addition is more complex than the background information currently presented by the authors. What factors contribute to the increased or decreased SOC response to nitrogen addition? Therefore, further elaboration is needed in this section.\nResults and Discussion:\nLine 70: Is it \"farmland\" same with \"cropland\"? If same, just use one for consistence.\nLine 76: Citation 34 is incorrect; it only studies arbuscular mycorrhizal fungi (AMF) responses and does not test how saprotrophic fungi respond to nitrogen addition. The authors should exercise greater caution with their citations. The same issue applies to citation 35; I do not believe the main findings from that paper support your claim that \"this functional reorganization may obscure substantial declines in total fungal diversity.\"\nLine 79: Additionally, plant and bacterial diversity were more strongly affected by nitrogen enrichment in warmer regions, while fungal diversity exhibited a greater response in colder regions (Fig. 1c-e). I do not see evidence for this. Are you referring to tropical versus temperate regions or temperate versus polar regions?\nLine 93: Soil biota also includes organisms other than microbes, such as nematodes. You do not present data on these organisms. Am I missing something? If not, the entire paragraph needs revision.\nLine 100: The use of \"however\" appears illogical here, as the two sentences address different topics, or at least emphasize different points. Please reassess the logic behind this.\nLine 106: What is the metabolic ecology theory, and why is it consistent with the metabolic theory of ecology (MTE)? The statement that elevated temperatures accelerate microbial catabolism does not clarify why nitrogen-induced SOC accumulation decreases from cold regions to the tropics. BTW, be careful, researchers normally use the metabolic theory of ecology, rather than metabolic ecology theory.\nLine 107: The assertion that SOC accumulation did not increase with rising nitrogen application rates or durations raises concerns about the predictability of SOC stocks under future nitrogen enrichment scenarios. I find this statement confusing.\nLine 122: This can be attributed to enhanced nitrification and ammonium oxidation driven by higher microbial diversity, processes that generate increased H⁺ concentrations and contribute to soil acidification in temperate regions. However, please check citation 47; the abstract states that \"soil nitrification rate tended to decrease with increasing latitude, especially in the Northern Hemisphere, and varied largely with ecosystem types. The soil nitrification rate significantly increased with mean annual temperature (MAT).\" After reading this, I would infer that nitrification should be higher in lower latitudes or warmer areas, meaning that tropical regions should exhibit higher nitrification rates compared to temperate regions. This does not support your findings. I strongly recommend that the authors verify the cited paper accordingly.\nLine 135: This is not true for fungi; temperature is more important than pH.\nLine 138: Are you referring to plant biodiversity or fungal diversity here? The text seems mismatched again. I suspect the labels in the figures may be incorrect.\nLine 146: Is plant diversity the most important factor? How does the model account for the collinearity of plant diversity and SOC along the nitrogen addition gradient? Furthermore, could this result conflict with the findings presented in panel f? The same question applies to the following paragraph (lines 152-167).\nLine 177: Does \"earlier\" refer to time or duration?\nLines 186-195: I do not understand why you refer to these parameters as part of the \"soil acidification phase.\" Particularly for soil moisture and microbial biomass carbon (MBC), there seems to be no relationship with acidification.\nLines 196-220: You discuss only the strength of correlations, but the direction and underlying biological significance are also crucial for interpretation. I am unclear about what the authors or the figures are attempting to convey.\nLines 222-260: This section appears to diverge from the main theme of the paper, shifting from a diversity-driven narrative to a biomass-driven one.\nLine 236: The term \"some temperate regions\" is vague; please be more specific.\nMethods:\nLine 287: What is meant by \"paired site design\"?\nLines 309-310: From the figure, I only see data for tropical, temperate, and polar regions.\nLine 310: Given the global decline in biodiversity due to nitrogen enrichment, are you suggesting that nitrogen enrichment is declining globally? This sentence needs revision.\nLine 347: You present figures S11 and S12 first, but Figure S9 appears later, and Figure S10 is not mentioned in the text. Please verify this.\nReferences:\nPlease review the references carefully, especially the abbreviations of journal names. Line 487 is missing a journal name, and this issue occurs in several other instances.\nThank you for considering my feedback."}, "author_response": "We thank the reviewer for this very positive evaluation of our study. We are very pleased to hear that the reviewer finds the study is very timely and important. We have carefully considered reviewer’s comments and suggestions, and have made substantial revisions, particularly in the criticisms of conclusions.\nComment 1: My only caution is that SOC might not represent soil C sequestration it gets broken down and metabolized differently under the various ecosystem types and environmental conditions.\nResponse: We sincerely thank the reviewer for this valuable caution. We fully agree that while SOC is widely used as an indicator of ecosystem carbon dynamics, it does not directly represent long-term carbon sequestration, as decomposition and turnover processes vary significantly across ecosystems and environmental conditions. Given that our primary focus was evaluating SOC responses to nitrogen enrichment and biodiversity shifts, rather than quantifying net carbon sequestration explicitly, we have replaced the term “soil C sequestration” with “carbon accumulation” or “SOC accumulation” throughout the manuscript (specifically in Title; Line 15, 19 and 31 of the Abstract; Line 86 of Introduction; Line 176, 261, 273, 290 of the Results and Discussion; Line 295 and 301 of the Conclusion sections) to avoid potential misinterpretation. Additionally, we added a clarifying limitation in the revised Results and Discussion section: “First, SOC accumulation does not directly reflect long-term carbon sequestration potential, as this is influenced by decomposition rates, microbial metabolism, and environmental conditions” (Lines 277–279).\nDetailed comments that need to be addressed:\nComment 2: L414: Plant diversity were sourced from.\nResponse: Thank you for pointing out the error. We have revised as “Plant diversity data were sourced from…” (Line 452)\nComment 3: L150: ‘Despite much discussed,’ seems like a word is missing here.\nResponse: We have revised the sentence as: “Despite being much discussed, …”. (Line 198)\nComment 4: L643: figure caption “size in each variable is given at the bottom.” I think should be ‘given on the right’.\nResponse: Thank you for pointing this out. We have corrected the figure caption from “size in each variable is given at the bottom” to “size in each variable is given on the right” in the legend of revised Fig. 1 (Line 701). Additionally, we have made the same correction in the legend of Fig. S1 to ensure consistency.\nComment 5: Figure 2. Bottom legend labels soil bacterial richness twice. Second should be fungal richness.\nResponse: Thank you for catching this oversight. We have corrected the bottom legend in Fig. 2 by replacing the second instance of “soil bacterial richness” with “fungal richness” as intended. The revised Fig. 2 as below:\nFig. 2 (revised) | Relative importance of different factors driving biodiversity and SOC responses under N enrichment. Results from random forest analysis, which shows the relative importance of the different drivers in predicting the response of plant richness (a), soil bacterial richness (b), fungal richness (c) and SOC (d) under N enrichment. Variable importance represents the increase in mean error (computed on the out-of-bag data) across trees when a predictor is permuted. MAT, mean annual temperature; TN, total N; SM, soil moisture; total nitrogen, TN; NH , ammonia nitrogen; NO3, nitrate nitrogen. The relationships between environmental factors (e.g., climatic variables and soil responses) and plant, soil microbial communities and SOC were analyzed using mixed-effects meta-regression with maximum likelihood estimation (ML). Study id was included as random factors to account for variability across studies. The meta-regressions of these variables are presented in Fig. S3-S5. e, The relationship between the diversity of plant and soil microbial communities and SOC under ambient by mixed-linear model, study id as random factors. f, The relationship between the responses of diversity of plant and soil microbial communities and the response of SOC under N enrichment by mixed-effects meta-regression with maximum likelihood estimation (ML), study id as random factors. *P ≤ 0.05; ***P ≤ 0.001.\nReviewer #2 (Remarks to the Author):\nI have reviewed the manuscript titled \"Critical Thresholds for Co-Benefits of Carbon Sequestration and Biodiversity Conservation Under Global Nitrogen Enrichment (NC-574816).\" The authors conducted a meta-analysis to identify the threshold point of nitrogen addition that significantly reduces plant, bacterial, and fungal diversity on a global scale. They also aim to determine the critical point for soil organic carbon (SOC) accumulation under nitrogen enrichment along the nitrogen gradient. By examining the relationship between diversity and SOC responses along this gradient, the authors seek to identify critical thresholds for the co-benefits of carbon sequestration and biodiversity conservation under global nitrogen enrichment. Several issues within the text require clarification.\nResponse: We sincerely thank the reviewer for their thorough and thoughtful assessment of our manuscript and are grateful for the constructive comments. We have carefully considered all the reviewer’s suggestions and have made substantial revisions accordingly. Below, we provide detailed point-by-point responses.\nComment 1: First, it is important to note that there may not always be a trade-off between diversity response and SOC response; at certain points, both parameters can exhibit either positive or negative responses to nitrogen enrichment.\nResponse: We appreciate the reviewer’s insightful comment. We fully agree with the reviewer that the trade-off between diversity responses and SOC responses does not always apply universally, and that both parameters can, in some cases, exhibit either positive or negative responses to nitrogen enrichment. We have rewritten paragraphs 1 and 2 of the Introduction section to better capture the variability and context-dependency in the response of diversity and SOC to N enrichment, as following: “Soil is the largest carbon (C) reservoir in terrestrial ecosystems, playing a critical role in regulating the global C cycle. Even small changes in the soil organic carbon (SOC) pool can have profound impacts on ecosystem services and land C–climate feedbacks1,2. Soil N availability significantly influences storage capacity of SOC, since N is a key component of SOC 3,4. While increased N input has alleviated constraints on global food production, it has also elevated ecosystem N availability, leading to eutrophication and altered ecosystem processes5-7. Numerous studies have demonstrated that N enrichment can enhance SOC storage by promoting the formation of larger soil aggregates, improving soil fractions, and increasing N use efficiency8-11. However, N addition does not consistently promote SOC accumulation, with some studies reporting neutral or negative effects12,13. These inconsistencies arise from the interplay of multiple drivers, such as plant and microbial biomass inputs, mineral associations, environmental conditions, and biodiversity-driven catabolism and anabolism, which collectively shape SOC formation and turnover14-17. Biodiversity is well-known in supporting SOC accumulation18,19, thus its responses to N enrichment could profoundly reshape SOC dynamics. N enrichment has been shown to reduce biodiversity of both above- and belowground communities20,21, primarily through mechanisms such as competitive exclusion, soil acidification and cation toxicity22-24. However, some studies have reported that N addition exerts only minor effects on soil microbial diversity, particularly within fungal communities25. These shifts can disrupt the balance between C and nutrient demands in plants and soil microbes, potentially decoupling their interactions or generating context-dependent responses that alter ecosystem C fluxes between above- and belowground compartments, ultimately affecting SOC dynamics14,18,26-30. Despite increasing evidence of such complexities9,11,31,32, few studies have simultaneously evaluated the relative contributions of fundamental drivers to SOC response under N enrichment, particularly regarding whether and how biodiversity mediates SOC dynamics. Therefore, a global assessment of the factors underpinning SOC dynamics is critical for reducing the uncertainty in biogeochemical models of soil C turnover and advancing our understanding of how biodiversity supports soil carbon stocks in an N-enriched world33.\nBiodiversity and carbon conservation are dual priorities in the global effort to mitigate climate change34. However, the divergent responses of biodiversity and SOC under N enrichment pose a challenge to achieving both goals simultaneously, as their co-occurrence may ultimately undermine long-term synergies30. This tension underscores the need to identify critical thresholds along N enrichment gradients, beyond which biodiversity losses may accelerate even as carbon stocks continue to rise35,36. Recognizing and quantifying these ecological thresholds is increasingly pivotal for ecosystem-based management and policy formulation, as they offer valuable benchmarks for designing effective management interventions and conservation targets37-40. Although previous studies have documented nonlinear or saturating responses of biodiversity and SOC to N enrichment, critical thresholds along N gradients have yet to be systematically quantified. Given their higher sensitivity to environmental change41, soil microbial communities are likely to exhibit detectable threshold responses earlier than plants along N gradients. Such shifts in microbial and plant communities may modify soil properties in ways that delay the manifestation of SOC thresholds relative to biodiversity thresholds. Understanding how biodiversity and SOC in terrestrial ecosystems respond to increasing N inputs is critical for advancing ecological threshold theory and informing estimates of N input levels that agroecosystems and natural ecosystems can tolerate before facing heightened risks of biodiversity loss, carbon destabilization, and eutrophication.”. (Line 34-76)\nComment 2: Second, the interpretation of the data presented in the main figures is challenging. In some cases, the authors find a negative correlation between plant/bacterial diversity and SOC, but this negative correlation appears to stem from the nitrogen-induced divergent responses of these parameters rather than reflecting biological significance. Although significant correlations are identified, their implications may not be fully clear.\nResponse: We fully thank the reviewer this valuable comments. We recognize that the original version lacked sufficient clarity in describing and discussing the results (as raised in Comment 6) and their ecological significance (as raised in Comment 27). To improve the interpretation of the origin Fig. 3, we have enhanced this section in two ways: (1) providing a clear interpretive framework to guide readers in understanding the ecological meaning of the point distributions and fitted trends, and (2) revising the Results and Discussion sections to offer a more detailed and structured interpretation of the observed patterns. The details are provided below:\n1. Interpretive framework. We first clarify the interpretive framework for understanding Fig. 3b-d. Each dot in the moving-window analysis represents the correlation between biodiversity (lnRR) and SOC (lnRR) sliding N addition window. A complete ecological interpretation of these correlations requires consideration of two dimensions: (1) The sign of the correlation coefficient (relative to the zero line, where correlation > 0 indicates a positive relationship and < 0 indicates a negative one), indicating whether biodiversity and SOC change in the same direction (i.e., co-increase or co-decline, positive correlation), or in opposite directions (negative correlation); (2) The trend of correlation strength across the nitrogen gradient, where a positive slope indicates strengthening biodiversity-SOC coupling with increasing N, while a negative slope suggests weakening or decoupling. Additionally, the final ecological interpretation integrates not only the overall effect sizes but also their directional shifts across the N enrichment threshold, providing a more comprehensive understanding of biodiversity and SOC dynamics. Based on this framework, we reorganized the text to more clearly delineate zones of biodiversity-SOC synergy and decoupling along the nitrogen gradient. This framework was used to reorganize the text and better delineate zones of biodiversity-SOC synergy or decoupling along the N gradient.\n2. Detailed results and interpretation. In Fig. 3b, bacterial diversity and SOC showed a slightly negative correlation below ~50 kg N ha⁻¹ yr⁻¹, reflecting a decoupled pattern where bacterial diversity declined slightly while SOC continued to increase under low-N inputs. As N input exceeded this threshold, the correlation gradually shifted toward positive (reaching ~200 kg N ha⁻¹ yr⁻¹), suggesting the onset of a co-decline phase where intensified N enrichment simultaneously undermines both biodiversity and carbon accumulation. In Fig. 3c, the response of fungal diversity fluctuated around ~67 kg N ha⁻¹ yr⁻¹ but maintained a generally positive correlation with SOC across the gradient, likely because its weak negative response to N enrichment did not disrupt its sustained functional role in carbon stabilization (e.g., via aggregate formation). In Fig. 3d, the plant diversity–SOC relationship followed a stronger nonlinear pattern. At low N inputs (<50 kg N ha⁻¹ yr⁻¹), we observed strong negative correlations, reflecting a decoupled pattern where diversity loss driven by light competition and cation toxicity occurred alongside continued SOC accumulation. Between 50–74 kg N ha⁻¹ yr⁻¹, the correlation became strongly positive, indicating a phase where accelerating biodiversity decline was accompanied by weakening or even reversing SOC accumulation, marking the onset of co-decline dynamics. However, beyond ~74 kg N ha⁻¹ yr⁻¹, although the correlation between plant diversity and SOC responses remained positive, the slope turned negative, signaling a weakening biodiversity–SOC coupling. This likely reflects that severe species loss and functional homogenization increasingly uncouple plant diversity from SOC dynamics, potentially undermining future soil carbon gains. These findings consistently demonstrate a trajectory of low-N decoupling, mid-N synergy, and high-N functional breakdown across microbial and plant communities, marking a shift from indirect N-driven SOC gains, to biodiversity-mediated regulation, and ultimately to diversity-imposed constraints under N saturation. Overall, this gradient-dependent transition from synergy to trade-off highlights the complex and multifaceted impacts of biodiversity loss on SOC storage capacity.\nThe revised explanation in the legend of Fig. 3, as following: “Moving-window correlations between the response of diversity and the response of SOC along N gradients for bacteria (b), fungi (c), and plants (d). The dots indicate the bootstrapped coefficients of the fixed terms shown for each subset window. The sign of each dot (relative to the zero line, where values > 0 indicate positive associations and values < 0 indicate negative associations) reflects whether biodiversity and SOC respond in the same (co-increase or co-decline) or in opposite directions. The slope of the fitted line indicates how biodiversity-SOC associations change across the N gradient: a positive slope suggests strengthening associations with increasing N input, whereas a negative slope suggests weakening or reversal.”. (Line 731-739)\nThe revised Results and Discussion for the Fig. 3, as following: “Moreover, we performed a moving-window analysis to evaluate the potential shifts in the relationships between diversity and SOC with increasing N (Fig. 4b-d and Fig. S8). The correlation between bacterial diversity and SOC initially exhibited a short-term negative relationship below the threshold, while fungal diversity maintained a positive correlation both before and after the threshold, though the strength of the positive correlation weakened beyond it. In contrast, the relationship between plant diversity and SOC shifted dramatically, transitioning from a positive to a negative correlation once the threshold was exceeded. The responses of bacterial diversity and SOC showed a slightly negative correlation below ~50 kg N ha⁻¹ yr⁻¹ (Fig. 4b), reflecting a decoupled pattern where bacterial diversity declined slightly while SOC continued to increase under low-N inputs. As N input exceeded this threshold, the correlation gradually shifted toward positive (reaching ~200 kg N ha⁻¹ yr⁻¹), suggesting the onset of a co-decline phase where intensified N enrichment simultaneously undermines both biodiversity and carbon accumulation. The response of fungal diversity fluctuated around ~67 kg N ha⁻¹ yr⁻¹ but maintained a generally positive correlation with SOC across the N gradient (Fig. 4c), likely because its weak negative response to N enrichment did not disrupt its sustained functional role in carbon stabilization70. At low N inputs (<50 kg N ha⁻¹ yr⁻¹), strong negative correlations emerged between the responses of plant diversity and SOC (Fig. 4d), reflecting a decoupled pattern where diversity loss driven by light competition and cation toxicity occurred alongside continued SOC accumulation22. Between 50-74 kg N ha⁻¹ yr⁻¹, the correlation became strongly positive, indicating a phase where accelerating biodiversity decline was accompanied by weakening or even reversing SOC accumulation, marking the onset of co-decline dynamics. However, beyond ~74 kg N ha⁻¹ yr⁻¹, although the correlation between plant diversity and SOC responses remained positive, the slope of this relationship turned negative, signaling a weakening biodiversity-SOC coupling. This pattern likely reflects that severe species loss and functional homogenization increasingly uncouple plant diversity from SOC dynamics, potentially undermining future soil carbon gains30. These findings consistently reveal a trajectory of low-N decoupling, mid-N synergy, and high-N functional breakdown across microbial and plant communities, marking a shift from indirect N-driven SOC gains, to biodiversity-mediated regulation, and ultimately to diversity-imposed constraints under N saturation. Overall, this gradient-dependent transition from synergy to trade-off underscores the complex and multifaceted consequences of biodiversity loss for SOC accumulation. Limiting N inputs to below 50 kg N ha⁻¹·yr⁻¹ could help mitigate biodiversity loss and its negative effects on SOC storage, supporting long-term ecosystem stability.”. (Line 244-276)\nComment 3: Lastly, I believe the authors should exercise greater caution with their citations. In multiple instances, incorrect papers are cited, or the cited papers do not align with the authors’ claims. I have listed a few examples below for your check.\nResponse: Thank you for your thoughtful feedback regarding the citations in our manuscript. We acknowledge the importance of accurate and contextually appropriate references.\nTo address this concern, we have carefully checked the wrong citation you pointed (i.e., Comment 15 and 21) and have replaced them with the correct and more appropriate references. Specifically, for the incorrect citations 34 and 35 raised in Comment 15, we have added a suitable reference (ref. 1) to support the point that N enrichment can simultaneously increase the relative proportion of saprotrophic fungi, and we have removed citation 35 accordingly. In addition, we have added another mechanism to explain the week response of fungal diversity under N enrichment in the study that is tolerant species could compensate for the species being reduced by global change factors, thereby maintaining overall ecosystem biodiversity at the same level (ref. 2). The revised sentence as following: “The weak negative effect of N enrichment on fungal diversity likely reflects heterogeneous responses among functional guilds44, as N reduces the diversity and abundance of arbuscular mycorrhizal fungi (AMF) while simultaneously increasing the relative proportion of saprotrophic fungi45,46, with this functional reorganization may obscure declines in total fungal diversity. In addition, the compensatory dominance of tolerant fungal taxa may further buffer the loss of more sensitive species under global change conditions47.”. (Line 93-98).\nFor the incorrect citations 47 raised in Comment 21, we have therefore removed this citation and now clarify that this is primarily due to the inherently lower base saturation and weaker buffering capacity of temperate soils, which increase their susceptibility to acidification when exposed to nitrogen inputs. For example, Xu et al. (ref. 3) reported that temperate forest soils in China had significantly lower base saturation than tropical soils under low N deposition, indicating limited buffering capacity and heightened acidification risk. Lu et al. (ref. 4) further observed that nitrogen addition elevated H⁺ concentrations and shifted the exchangeable cation pool from Al³⁺ to H⁺ and Fe³⁺, consistent with a more advanced acidification state in temperate soils. We have removed the citation 47 and revised the sentence as: “This may be attributed to the inherently lower base saturation and weaker buffering capacity of temperate soils, which amplifies the acidifying effects of N-induced nitrification8,57,58.”. (Line 140-142)\nAdditionally, we carefully revised the reference formatting throughout the manuscript (as raised in Comment 34) one by one, ensuring that both the in-text citations and the references used in the meta-analysis dataset now fully comply with the guidelines. Please refer to the revised manuscript and supplementary materials for these updates.\nReferences\n1. Morrison, E.W., Frey, S.D., Sadowsky, J.J., van Diepen, L.T., Thomas, W.K. and Pringle, A., 2016. Chronic nitrogen additions fundamentally restructure the soil fungal community in a temperate forest. Fungal Ecology, 23, pp.48-57.\n2. Zhou, G., Eisenhauer, N., Terrer, C., Eldridge, D.J., Duan, H., Guirado, E., Berdugo, M., Zhou, L., Liu, S., Zhou, X. and Delgado-Baquerizo, M., 2024. Resistance of ecosystem services to global change weakened by increasing number of environmental stressors. Nature Geoscience, 17(9), pp.882-888.\n3. Xu, R.K., Zhao, A.Z., Yuan, J.H. and Jiang, J., 2012. pH buffering capacity of acid soils from tropical and subtropical regions of China as influenced by incorporation of crop straw biochars. Journal of Soils and Sediments, 12, pp.494-502.\n4. Lu, X., Hou, E., Guo, J., Gilliam, F.S., Li, J., Tang, S. and Kuang, Y., 2021. Nitrogen addition stimulates soil aggregation and enhances carbon storage in terrestrial ecosystems of China: A meta‐analysis. Global Change Biology, 27(12), pp.2780-2792.\nI have provided detailed comments below:\nComment 4: Fig. 1: the sample size in each variable is given at the right. The closed symbols indicate significant effects, what is the significant level? What is compound N mean? The figure must be self-explained. Is urea N belonging to Organic N? What exactly organic N means here? Mean effect size of N enrichment (%). The (%) should add after effect size rather than enrichment. For panel g, the number of sample size is not algin on the right.\nResponse: We thank the reviewer for these detailed and constructive comments. In response:\n1. We have clarified in the revised figure caption that closed symbols indicate statistically significant effect sizes at P < 0.05, based on 95% confidence intervals that do not overlap zero. We have added this information in the legend of Fig. 1 as: “The closed symbols indicate significant effects using a significance level (α) of 0.05, and the open symbols indicate nonsignificant effects.”.\n2. “Compound N” refers to fertilizers composed of multiple nitrogen forms, such as mixtures of nitrate, ammonium, and/or urea. We have added this definition in the legend of Fig. 1: “Compound N refers to fertilizers composed of multiple nitrogen forms (e.g., mixtures of nitrate, ammonium, and/or urea)”.\n3. Our classification, urea (CO(NH₂)₂) was treated as a separate category (“Urea N”) rather than being grouped under “Organic N”. Although urea is chemically an organic compound, its biogeochemical behavior in soils aligns more closely with inorganic nitrogen sources due to its rapid hydrolysis into ammonium (NH₄⁺) in soils, bypassing the slow mineralization of traditional organic amendments (e.g., compost, manure). This dynamic behavior necessitates its separate classification as \"Urea N\" to avoid conflating it with stable organic nitrogen pools. Therefore, given the ecological relevance and consistency with previous research (ref. 1-2), we categorized urea separately to better capture its unique dynamics in the nitrogen cycle. We have revised the manuscript to clarify this rationale in Line 705-707.\n4. “Organic N” refers specifically to nitrogen inputs derived from organic sources, including industrial organic fertilizers, livestock manure, and compost. We have added this definition in the legend of Fig. 1: “Organic N refers to industrial organic fertilizers, livestock manure, and compost.”.\n5. We have revised the x-axis label from “Mean effect size of N enrichment (%)” to “Mean effect size (%) of N enrichment” in the revised Fig. 1.\n6. Alignment of sample size in panel g. We have corrected the formatting so that all sample size labels are now properly aligned on the right side of panel g.\nAll of these changes have been implemented in the revised Fig. 1 and its caption:\nFig. 1 (revised) | Effect of N enrichment on plants, soil microbial communities and SOC. a-b, Distribution (a) of study sites shown both globally (a) and across biomes (b). c-e, Effects of N enrichment on diversity in plants (c), soil bacteria (d) and fungi (e). f-g, Effects of N enrichment on biomass in plants (f) and soil microbial communities (g). h, Effects of N enrichment on SOC. Values are mean effect size ± 95% CI. The sample size in each variable is given at the bottom. The closed symbols indicate significant effects using a significance level (α) of 0.05, and the open symbols indicate nonsignificant effects. Compound N refers to fertilizers composed of multiple nitrogen forms (e.g., mixtures of nitrate, ammonium, and/or urea). Organic N refers to industrial organic fertilizers, livestock manure and compost. Urea (CO(NH₂)₂) was classified separately (“Urea N”) because its rapid hydrolysis to ammonium renders it functionally distinct from organic N sources like manure or compost.\nReferences\n1. Zhang, Y., Zhang, F., Abalos, D., Luo, Y., Hui, D., Hungate, B.A., García‐Palacios, P., Kuzyakov, Y., Olesen, J.E., Jørgensen, U. and Chen, J., 2022. Stimulation of ammonia oxidizer and denitrifier abundances by nitrogen loading: Poor predictability for increased soil N O emission. 2 Global Change Biology, 28(6), pp.2158-2168.\n2. Shi, T.S., Collins, S.L., Yu, K., Peñuelas, J., Sardans, J., Li, H. and Ye, J.S., 2024. A global meta-analysis on the effects of organic and inorganic fertilization on grasslands and croplands. Nature Communications, 15(1), p.3411.\nComment 5: Fig. 2: Biodiversity drivers SOC accumulation under N enrichment. The legend reads like a results or conclusion, rarely see this in the caption. You need to illustrate what the figure means but not over-interpretation. Study IDs or study id, please consistent. It is wired that for the x axis label, sometimes you use text and some time you use picture. It is hard to understand.\nThe relationship between plant and soil biota? The sentence is confusing. Moreover, all parameters respond to nitrogen addition. When correlating these parameters, such as plant diversity versus soil organic carbon (SOC), how do you account for the effect of nitrogen addition, given that both parameters are influenced by it?\nResponse: We thank the reviewer for these thoughtful comments. We have addressed each of the concerns as follows:\n1. We fully agree with reviewer that the original title was overly conclusive. To improve clarity and neutrality, we have revised the title as: “Relative importance of different factors driving biodiversity and SOC responses under N enrichment.”\n2. We have reviewed the entire manuscript and standardized all instances of “study id” for consistency in Lines 409, 441, 721, 724 and 727 of the revised manuscript.\n3. We have revised the x-axis labels to include both icons and their corresponding text terms. Additionally, we have made the same correction of Fig. S8 to ensure consistency.\n4. We acknowledge that the original wording may have mistakenly suggested a relationship between plant and microbial communities, whereas our intended focus was to present the relationships between biodiversity and SOC. We have revised this sentence more clearly as: “The relationship between diversity (plant, soil bacterial and fungal communities) and SOC…”. (Line 723-724)\n5. We fully agree with the reviewer that shared variance to N enrichment could introduce potential spurious correlations. To address this concern, we conducted an additional meta-regression analysis to account for potential co-variation between effect sizes (lnRRs), with N rate included as a random effect to control for shared variation associated with N treatments. The results showed that all diversity-SOC relationships remained statistically significant, suggesting that these patterns likely reflect true ecological couplings or trade-offs under N enrichment, rather than correlations merely driven by a common external driver. We have added the statistical and methodological description in the Methods section (Mixed-effects meta-regression) as: “…To account for potential non-independence among response variables arising from shared variance to nitrogen enrichment, N rate was also included as a random effect.”. (Line 410-411)\nAdditionally, we re-conducted all lnRR-to-lnRR meta-regression analyses in the study to control for potential spurious correlations arising from shared variance to N enrichment. The revised Fig. S3–S5 have been updated in the revision.\nThe revised Fig. 2 as below:\nFig. 2 (revised) | Relative importance of different factors driving biodiversity and SOC responses under N enrichment. Results from random forest analysis, which shows the relative importance of the different drivers in predicting the response of plant richness (a), soil bacterial richness (b), fungal richness (c) and SOC (d) under N enrichment. Variable importance represents the increase in mean error (computed on the out-of-bag data) across trees when a predictor is permuted. MAT, mean annual temperature; TN, total N; SM, soil moisture; total nitrogen, TN; NH , ammonia nitrogen; NO3, nitrate nitrogen. The relationships between environmental factors (e.g., climatic variables and soil responses) and plant, soil microbial communities and SOC were analyzed using mixed-effects meta-regression with maximum likelihood estimation (ML). Study id was included as random factors to account for variability across studies. The meta-regressions of these variables are presented in Fig. S3-S5. e, The relationship between the diversity of plant and soil microbial communities and SOC under ambient by mixed-linear model, study id as random factors. f, The relationship between the responses of diversity of plant and soil microbial communities and the response of SOC under N enrichment by mixed-effects meta-regression with maximum likelihood estimation (ML), study id as random factors. *P ≤ 0.05; ***P ≤ 0.001.\nFig. S3 (revised) | Relationships between the responses of diversity in plant and soil microbial communities, and environmental factors by mixed-effects meta-regression with maximum likelihood estimation (ML), study id as random factors. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.\nFig. S4 (revised) | Relationships between the responses of biomass in plant and soil microbial communities, environmental factors by mixed-effects meta-regression with maximum likelihood estimation (ML), study id as random factors. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.\nFig. S5 (revised) | Relationships between the responses of diversity and biomass in plant and soil microbial communities by mixed-effects meta-regression with maximum likelihood estimation (ML), study id as random factors. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.\nComment 6: Fig. 3: The results are difficult to interpret. In panel b, if we examine the absolute values for most cases, most relationships fall below the zero line, suggesting that higher bacterial diversity leads to lower SOC. If this is the case, why should we prioritize the protection of bacterial diversity? Similarly, in panel d, if I understand correctly, most relationships are negative below 40 kg N, indicating that a higher response in plant diversity corresponds to a lower response in SOC. In such cases, why is it important to protect plant diversity?\nResponse: We sincerely thank the reviewer for this valuable comment. In Fig. 3b and 3d, each data point represents the correlation between the lnRR of diversity and that of SOC within a sliding N addition window. This approach captures shifts in co-response patterns along the nitrogen gradient, rather than indicating static or one-directional causal relationships.\nTo address the concern regarding the difficulty of interpreting Fig. 3b-d, we have enhanced this section in two ways: (1) by providing a clear interpretive framework to guide readers in understanding the ecological meaning of the point distributions and fitted trends, and (2) by revising the Results and Discussion sections to offer a more detailed and structured interpretation of the observed patterns. The details are provided below:\n1. We first clarify the interpretive framework for understanding Fig. 3b-d. Each dot in the moving-window analysis represents the correlation between biodiversity (lnRR) and SOC (lnRR) sliding N addition window. A complete ecological interpretation of these correlations requires consideration of two dimensions: (1) The sign of the correlation coefficient (relative to the zero line, where correlation > 0 indicates a positive relationship and < 0 indicates a negative one), indicating whether biodiversity and SOC change in the same direction (i.e., co-increase or co-decline, positive correlation), or in opposite directions (negative correlation); (2) The trend of correlation strength across the nitrogen gradient, where a positive slope indicates strengthening biodiversity-SOC coupling with increasing N, while a negative slope suggests weakening or decoupling. Additionally, the final ecological interpretation integrates not only the overall effect sizes but also their directional shifts across the N enrichment threshold, providing a more comprehensive understanding of biodiversity and SOC dynamics. Based on this framework, we reorganized the text to more clearly delineate zones of biodiversity-SOC synergy and decoupling along the nitrogen gradient. This framework was used to reorganize the text and better delineate zones of biodiversity-SOC synergy or decoupling along the N gradient.\nWe have also included a concise explanation in the legend of Fig. 3 to enhance the readability and aid interpretation as: “Moving-window correlations between the response of diversity and the response of SOC along N gradients for bacteria (b), fungi (c), and plants (d). The dots indicate the bootstrapped coefficients of the fixed terms shown for each subset window. The sign of each dot (relative to the zero line, where values > 0 indicate positive associations and values < 0 indicate negative associations) reflects whether biodiversity and SOC respond in the same (co-increase or co-decline) or in opposite directions. The slope of the fitted line indicates how biodiversity-SOC associations change across the N gradient: a positive slope suggests strengthening associations with increasing N input, whereas a negative slope suggests weakening or reversal.”. (Line 731-739)\n2. In Fig. 3b, bacterial diversity and SOC showed a slightly negative correlation below ~50 kg N ha⁻¹ yr⁻¹, reflecting a decoupled pattern where bacterial diversity declined slightly while SOC continued to increase under low-N inputs. As N input exceeded this threshold, the correlation gradually shifted toward positive (reaching ~200 kg N ha⁻¹ yr⁻¹), suggesting the onset of a co-decline phase where intensified N enrichment simultaneously undermines both biodiversity and carbon accumulation. In Fig. 3c, the response of fungal diversity fluctuated around ~67 kg N ha⁻¹ yr⁻¹ but maintained a generally positive correlation with SOC across the gradient, likely because its weak negative response to N enrichment did not disrupt its sustained functional role in carbon stabilization (e.g., via aggregate formation). In Fig. 3d, the plant diversity–SOC relationship followed a stronger nonlinear pattern. At low N inputs (<50 kg N ha⁻¹ yr⁻¹), we observed strong negative correlations, reflecting a decoupled pattern where diversity loss driven by light competition and cation toxicity occurred alongside continued SOC accumulation. Between 50–74 kg N ha⁻¹ yr⁻¹, the correlation became strongly positive, indicating a phase where accelerating biodiversity decline was accompanied by weakening or even reversing SOC accumulation, marking the onset of co-decline dynamics. However, beyond ~74 kg N ha⁻¹ yr⁻¹, although the correlation between plant diversity and SOC responses remained positive, the slope turned negative, signaling a weakening biodiversity–SOC coupling. This likely reflects that severe species loss and functional homogenization increasingly uncouple plant diversity from SOC dynamics, potentially undermining future soil carbon gains. These findings consistently demonstrate a trajectory of low-N decoupling, mid-N synergy, and high-N functional breakdown across microbial and plant communities, marking a shift from indirect N-driven SOC gains, to biodiversity-mediated regulation, and ultimately to diversity-imposed constraints under N saturation. Overall, this gradient-dependent transition from synergy to trade-off highlights the complex and multifaceted impacts of biodiversity loss on SOC storage capacity.\nWe have re-written this paragraph and updated this clearly version in Results and Discussion in the revision as: “Moreover, we performed a moving-window analysis to evaluate the potential shifts in the relationships between diversity and SOC with increasing N (Fig. 4b-d and Fig. S8). The correlation between bacterial diversity and SOC initially exhibited a short-term negative relationship below the threshold, while fungal diversity maintained a positive correlation both before and after the threshold, though the strength of the positive correlation weakened beyond it. In contrast, the relationship between plant diversity and SOC shifted dramatically, transitioning from a positive to a negative correlation once the threshold was exceeded. The responses of bacterial diversity and SOC showed a slightly negative correlation below ~50 kg N ha⁻¹ yr⁻¹ (Fig. 4b), reflecting a decoupled pattern where bacterial diversity declined slightly while SOC continued to increase under low-N inputs. As N input exceeded this threshold, the correlation gradually shifted toward positive (reaching ~200 kg N ha⁻¹ yr⁻¹), suggesting the onset of a co-decline phase where intensified N enrichment simultaneously undermines both biodiversity and carbon accumulation. The response of fungal diversity fluctuated around ~67 kg N ha⁻¹ yr⁻¹ but maintained a generally positive correlation with SOC across the N gradient (Fig. 4c), likely because its weak negative response to N enrichment did not disrupt its sustained functional role in carbon stabilization70. At low N inputs (<50 kg N ha⁻¹ yr⁻¹), strong negative correlations emerged between the responses of plant diversity and SOC (Fig. 4d), reflecting a decoupled pattern where diversity loss driven by light competition and cation toxicity occurred alongside continued SOC accumulation22. Between 50-74 kg N ha⁻¹ yr⁻¹, the correlation became strongly positive, indicating a phase where accelerating biodiversity decline was accompanied by weakening or even reversing SOC accumulation, marking the onset of co-decline dynamics. However, beyond ~74 kg N ha⁻¹ yr⁻¹, although the correlation between plant diversity and SOC responses remained positive, the slope of this relationship turned negative, signaling a weakening biodiversity-SOC coupling. This pattern likely reflects that severe species loss and functional homogenization increasingly uncouple plant diversity from SOC dynamics, potentially undermining future soil carbon gains30. These findings consistently reveal a trajectory of low-N decoupling, mid-N synergy, and high-N functional breakdown across microbial and plant communities, marking a shift from indirect N-driven SOC gains, to biodiversity-mediated regulation, and ultimately to diversity-imposed constraints under N saturation. Overall, this gradient-dependent transition from synergy to trade-off underscores the complex and multifaceted consequences of biodiversity loss for SOC accumulation. Limiting N inputs to below 50 kg N ha⁻¹·yr⁻¹ could help mitigate biodiversity loss and its negative effects on SOC storage, supporting long-term ecosystem stability.”. (Line 244-275)\nComment 7: Fig 4: Fig 4 seems out of the logic flow of the main story. All other figs target on the diversity effect on SOC, why Figure 4 focus on biomass and SOC?\nResponse: We thank the reviewer for raising this important point. We would like to clarify that the global distributions presented in Fig. 4 were not intended to investigate the causal effects of biomass on SOC. Instead, the distributions of plant biomass, microbial biomass (MBC), and SOC were modeled independently, after accounting for climatic factors, soil properties, and diversity effects, to provide parallel and comprehensive insights into ecosystem carbon dynamics under N enrichment. Given that plant and microbial biomass constitute the immediate material basis for SOC formation and turnover, we believe presenting these distributions may help provide broader context for the observed SOC responses and illustrate the decoupled and complex above- and belowground processes under N enrichment. Importantly, we recognize that the ecological pathways linking biodiversity, biomass, and SOC differ between microbial and plant communities: in microbial communities, changes in biodiversity can directly influence microbial biomass and stabilization processes, whereas in plant communities, N-induced biomass accumulation may precede and contribute to biodiversity loss, with both processes jointly modifying soil carbon inputs and stabilization pathways.\nTherefore, we have retained Fig. 4 and repositioned it earlier in the manuscript (now Fig. 2), following the random forest analysis (origin Fig. 2) and before the diversity-SOC coupling section, to better align with the main narrative. This repositioning, together with the clarification that biomass and SOC projections were modeled independently, aims to strengthen the logical flow and avoid potential misinterpretations about causal pathways. Here, we would briefly highlight that Fig. 4 presents global-scale distributions of plant and microbial biomass, as well as SOC responses under N enrichment. Panel a shows widespread increases in plant biomass across most regions, consistent with nitrogen-stimulated productivity. Panel b reveals a more heterogeneous pattern for microbial biomass (MBC): while low-latitude (tropical) and high-latitude (boreal/polar) regions generally show positive responses, strong negative responses are evident in many mid-latitude areas, particularly in temperate ecosystems such as central North America, Europe, and parts of East Asia. This spatial asymmetry likely reflects regional differences in baseline nutrient availability, microbial community sensitivity, and climatic constraints on microbial turnover. Panel c indicates that SOC generally exhibits positive responses across much of the temperate Northern Hemisphere, particularly in regions such as Europe and central to northern Asia, where both plant carbon inputs and microbial transformation potential may be relatively high. In contrast, negative responses are more prevalent in many parts of the Southern Hemisphere and tropical zones, including southern Africa and parts of South and Southeast Asia. These patterns may reflect combined limitations such as reduced microbial activity or accelerated carbon mineralization under warmer and more nutrient-enriched conditions. Together, these spatial patterns provide complementary insights into the complex ways in which nitrogen enrichment reshapes ecosystem carbon dynamics across different climatic regions.\nTo further improve the logical flow, we have then added an introductory sentence at the beginning of the paragraph to better clarify how the global biomass projections relate to biodiversity-mediated regulation of SOC dynamics. In addition, we have substantially shortened this section and no longer present these global distributions as a standalone subsection. In addition, the figure is now briefly incorporated into a broader discussion of N-mediated ecosystem-level shifts, serving as a further extension of the the global patterns of N enrichment effects on biodiversity and ecosystem carbon stocks. The revised paragraph now reads as follows: “Given that plant and microbial biomass constitute the immediate foundation for SOC formation and turnover, we independently modelled the global responses of plant biomass, microbial biomass, and SOC under N enrichment to better elucidate ecosystem carbon dynamics (Fig. 2). Positive plant biomass responses largely dominated across the globe, particularly in temperate and boreal regions of North America, Europe, and Asia, reflecting N-stimulated productivity60 (Fig. 2a). In contrast, microbial biomass (MBC) exhibited greater regional heterogeneity, with positive responses concentrated in tropical and boreal zones but widespread declines across mid-latitude temperate ecosystems, including central North America, Europe, and East Asia (Fig. 2b). This spatial divergence likely reflects regional differences in background N deposition and soil sensitivity: in tropical and boreal zones with low ambient N, moderate enrichment promotes microbial growth, whereas in temperate regions with higher baseline N inputs, further enrichment induces acidification and nutrient imbalance, suppressing biomass61,62. Similarly, SOC responses were largely positive across temperate regions, particularly in Europe and northern Asia, whereas negative responses prevailed in parts of the Southern Hemisphere and tropical zones, including southern Africa, South Asia, and Southeast Asia (Fig. 2c). This likely reflects differences in nutrient limitations and ecosystem processes: in temperate systems, nitrogen (N) addition tends to stimulate plant growth and inputs while simultaneously reducing decomposition rates, thereby promoting SOC accumulation; in tropical and Southern Hemisphere regions, where phosphorus limitation and rapid organic matter turnover are more common, N addition often fails to enhance plant inputs and may even accelerate SOC loss63,64. Together, these spatial patterns illustrate how N enrichment reshapes carbon cycling across climatic gradients and emphasize the importance of region-specific N management aligned with carbon accumulation objectives.”. (Line 155-176)\nNonetheless, if the revised presentation still does not fully resolve the concern, we are happy to remove the origin Fig. 4 along with the associated text in the final version, and we also remain fully open to any further suggestions from the reviewer.\nAbstract:\nComment 8: Line 12: Global nitrogen enrichment does not always enhance soil organic carbon (SOC) accumulation; otherwise, threshold analysis would not be feasible. As your data indicate, in some cases, such as forest systems, nitrogen enrichment tends to reduce SOC accumulation.\nResponse: We thank the reviewer for this valuable comment. We fully agree that the original statement was overly absolute and did not adequately capture the context-dependent nature of SOC responses to nitrogen enrichment. To address this, we have revised the sentence as: “Global nitrogen (N) enrichment has a significant impact on soil organic carbon (SOC) accumulation, generally exerting a positive influence but it often leads to biodiversity loss in both plant and soil microbes.” (Line 13-15)\nComment 9: Line 24-26: The units \"kg N\" are incorrect. Per hectare per year?\nResponse: We thank the reviewer for this helpful comment. We confirm that “kg N” refers to “kg N ha⁻¹ yr⁻¹”, and we have now clarified this unit explicitly in the Abstract (Line 25-28) and Conclusion (Line 297) consistently across the revised manuscript.\nIntroduction:\nComment 10: Lines 35-42: These statements suggest that there must be a trade-off between SOC accumulation and biodiversity loss under nitrogen addition; however, this may not be accurate. What if, at certain points, both SOC and biodiversity increase with nitrogen addition? In such cases, no trade-offs would exist.\nResponse: We fully agree with the reviewer that a trade-off between SOC accumulation and biodiversity loss is not a universal outcome under N enrichment. For example, bacterial richness has been shown to increase under low levels of nitrogen input (ref. 1), and SOC accumulation was also observed under similar conditions (ref. 2). In such cases, biodiversity and SOC may respond in parallel, suggesting a coupled or synergistic relationship rather than a trade-off. We acknowledge that the original tone could be interpreted as overly categorical, and we have revised the text accordingly to better reflect the variability and context-dependency of biodiversity-SOC relationships under N enrichment.\nThe revised sentences of paragraph 1 in Introduction as: “While increased N input has alleviated constraints on global food production, it has also elevated ecosystem N availability, leading to eutrophication and altered ecosystem processes5-7. Numerous studies have demonstrated that N enrichment can enhance SOC storage by promoting the formation of larger soil aggregates, improving soil fractions, and increasing N use efficiency8-11. However, N addition does not consistently promote SOC accumulation, with some studies reporting neutral or negative effects12,13. These inconsistencies arise from the interplay of multiple drivers, such as plant and microbial biomass inputs, mineral associations, environmental conditions, and biodiversity-driven catabolism and anabolism, which collectively shape SOC formation and turnover14-17. Biodiversity is well-known in supporting SOC accumulation18,19, thus its responses to N enrichment could profoundly reshape SOC dynamics. N enrichment has been shown to reduce biodiversity of both above- and belowground communities20,21, primarily through mechanisms such as competitive exclusion, soil acidification and cation toxicity22-24. However, some studies have reported that N addition exerts only minor effects on soil microbial diversity, particularly within fungal communities25. These shifts can disrupt the balance between C and nutrient demands in plants and soil microbes, potentially decoupling their interactions or generating context-dependent responses that alter ecosystem C fluxes between above- and belowground compartments, ultimately affecting SOC dynamics14,18,26-30.”. (Line 37-54)\nAdditionally, we have also revised the first sentence of paragraph 2 in Introduction “The trade-off between biodiversity loss and SOC accumulation highlights the necessity of balanced N management strategies” to a more moderate tone. The revised sentence as: “Biodiversity and carbon conservation are dual priorities in the global effort to mitigate climate change34. However, the divergent responses of biodiversity and SOC under N enrichment pose a challenge to achieving both goals simultaneously, as their co-occurrence may ultimately undermine long-term synergies30.” (Line 60-63)\nReferences\n1. Liu, W., Liu, L., Yang, X., Deng, M., Wang, Z., Wang, P., Yang, S., Li, P., Peng, Z., Yang, L. and Jiang, L., 2021. Long‐term nitrogen input alters plant and soil bacterial, but not fungal beta diversity in a semiarid grassland. Global Change Biology, 27(16), pp.3939-3950.\n2. Xu, C., Xu, X., Ju, C., Chen, H.Y., Wilsey, B.J., Luo, Y. and Fan, W., 2021. Long‐term, amplified responses of soil organic carbon to nitrogen addition worldwide. Global Change Biology, 27(6), pp.1170-1180.\nComment 11: Line 59: Why include croplands? If your aim is to examine how SOC accumulation and biodiversity loss change along the nitrogen addition gradient, croplands typically exhibit low plant biodiversity due to prevalent monoculture practices.\nResponse: We appreciate the reviewer’s insightful comment and fully agree that croplands often support lower plant diversity due to monoculture practices, which is an important consideration when evaluating biodiversity patterns. In our analysis, we did not include plant diversity data from cropland sites, and accordingly, the effect of nitrogen enrichment on plant diversity in croplands is not represented in Fig. 1c. This exclusion is explicitly noted in Line 71 of the original manuscript, which states: “N enrichment consistently reduced diversity across plant and soil microbial communities, with declines of 12.5% (-17.0% to -7.8%, not applicable to farmland) in plants, …”.\nTo further improve clarity, we have added the following statement in the revision:\n“Due to prevalent monoculture practices in farmlands, we excluded farmlands from the plant diversity analysis.” (Line 348-349)\nComment 12: Line 59: What about the mean annual precipitation?\nResponse: Thank you for the comment. We agree that mean annual precipitation (MAP) is another important climatic variable. In our study, MAP ranges from 100 to 4500 mm across all sites. We have added this information following the description of mean annual temperature in the revised manuscript. The revised sentence as: “…spanning a wide range of climates (mean annual temperatures from -9°C to 30°C and mean annual precipitation from 100 to 4500 mm), …”. (Line 79)\nComment 13: Overall, the introduction lacks sufficient information on how plant and microbial biodiversity respond to nitrogen addition and what expectations exist regarding which thresholds might be reached earlier and why. Additionally, the response of SOC to nitrogen addition is more complex than the background information currently presented by the authors. What factors contribute to the increased or decreased SOC response to nitrogen addition? Therefore, further elaboration is needed in this section.\nResponse: We have carefully revised the Introduction section as suggested, and greatly appreciate the reviewer’s insightful feedback, which inspired us to more clearly highlight how nitrogen thresholds may influence the coupling or decoupling between biodiversity and soil carbon dynamics, thus raising an important perspective that was not sufficiently emphasized in the original version. Moreover, we believe that the conceptual framework refined through this revision may offer broader insights for identifying ecological thresholds under other global change drivers, particularly when synthesizing complex biodiversity and ecosystem function responses through large-scale meta-analyses. Specifically, we addressed the following four aspects, organized according to the logical flow of the Introduction:\n1. We added more detailed background information on the differential responses of plant and soil microbial communities to N enrichment, acknowledging that some microbial groups exhibit minor or inconsistent diversity changes.\n2. We clarified the rationale for threshold analyses and more explicitly discussed ecological expectations regarding which thresholds (plant vs. microbial) might be reached earlier and why.\n3. We revised the description of SOC responses to nitrogen enrichment to include not only positive but also neutral and negative outcomes reported in previous studies.\n4. We emphasized that SOC dynamics are shaped by the interplay of multiple factors, including plant and microbial biomass inputs, catabolic and anabolic processes, soil physicochemical properties, and biodiversity shifts.\nThe revised first and second paragraph of Introduction as: “Soil is the largest carbon (C) reservoir in terrestrial ecosystems, playing a critical role in regulating the global C cycle. Even small changes in the soil organic carbon (SOC) pool can have profound impacts on ecosystem services and land C–climate feedbacks1,2. Soil N availability significantly influences storage capacity of SOC, since N is a key component of SOC 3,4. While increased N input has alleviated constraints on global food production, it has also elevated ecosystem N availability, leading to eutrophication and altered ecosystem processes5-7. Numerous studies have demonstrated that N enrichment can enhance SOC storage by promoting the formation of larger soil aggregates, improving soil fractions, and increasing N use efficiency8-11. However, N addition does not consistently promote SOC accumulation, with some studies reporting neutral or negative effects12,13. These inconsistencies arise from the interplay of multiple drivers, such as plant and microbial biomass inputs, mineral associations, environmental conditions, and biodiversity-driven catabolism and anabolism, which collectively shape SOC formation and turnover14-17. Biodiversity is well-known in supporting SOC accumulation18,19, thus its responses to N enrichment could profoundly reshape SOC dynamics. N enrichment has been shown to reduce biodiversity of both above- and belowground communities20,21, primarily through mechanisms such as competitive exclusion, soil acidification and cation toxicity22-24. However, some studies have reported that N addition exerts only minor effects on soil microbial diversity, particularly within fungal communities25. These shifts can disrupt the balance between C and nutrient demands in plants and soil microbes, potentially decoupling their interactions or generating context-dependent responses that alter ecosystem C fluxes between above- and belowground compartments, ultimately affecting SOC dynamics14,18,26-30. Despite increasing evidence of such complexities9,11,31,32, few studies have simultaneously evaluated the relative contributions of fundamental drivers to SOC response under N enrichment, particularly regarding whether and how biodiversity mediates SOC dynamics. Therefore, a global assessment of the factors underpinning SOC dynamics is critical for reducing the uncertainty in biogeochemical models of soil C turnover and advancing our understanding of how biodiversity supports soil carbon stocks in an N-enriched world33.\nBiodiversity and carbon conservation are dual priorities in the global effort to mitigate climate change34. However, the divergent responses of biodiversity and SOC under N enrichment pose a challenge to achieving both goals simultaneously, as their co-occurrence may ultimately undermine long-term synergies30. This tension underscores the need to identify critical thresholds along N enrichment gradients, beyond which biodiversity losses may accelerate even as carbon stocks continue to rise35,36. Recognizing and quantifying these ecological thresholds is increasingly pivotal for ecosystem-based management and policy formulation, as they offer valuable benchmarks for designing effective management interventions and conservation targets37-40. Although previous studies have documented nonlinear or saturating responses of biodiversity and SOC to N enrichment, critical thresholds along N gradients have yet to be systematically quantified. Given their higher sensitivity to environmental change41, soil microbial communities are likely to exhibit detectable threshold responses earlier than plants along N gradients. Such shifts in microbial and plant communities may modify soil properties in ways that delay the manifestation of SOC thresholds relative to biodiversity thresholds. Understanding how biodiversity and SOC in terrestrial ecosystems respond to increasing N inputs is critical for advancing ecological threshold theory and informing estimates of N input levels that agroecosystems and natural ecosystems can tolerate before facing heightened risks of biodiversity loss, carbon destabilization, and eutrophication.”. (Line 34-76)\nResults and Discussion:\nComment 14: Line 70: Is it \"farmland\" same with \"cropland\"? If same, just use one for consistence.\nResponse: Yes, the term “farmland” used in this study is equivalent to “cropland.” Thank you for pointing out this inconsistency. We have carefully reviewed the manuscript and standardized the terminology by replacing “cropland” with “farmland” throughout the text, including in Line 80 of the Introduction.\nComment 15: Line 76: Citation 34 is incorrect; it only studies arbuscular mycorrhizal fungi (AMF) responses and does not test how saprotrophic fungi respond to nitrogen addition. The authors should exercise greater caution with their citations. The same issue applies to citation 35; I do not believe the main findings from that paper support your claim that \"this functional reorganization may obscure substantial declines in total fungal diversity.\"\nResponse: We thank the reviewer for this important comment. Upon re-examination, we acknowledge that citation 34 only investigated arbuscular mycorrhizal fungi (AMF), and does not include saprotrophic fungi. We have added another literature to support the view that N enrichment could simultaneously increase the relative proportion of saprotrophic fungi (ref. 1).\nFor the citation 35, we sincerely thank the reviewer for raising this important point regarding this citation and acknowledge that this reference does not directly support the mechanistic claim that functional reorganization obscures total fungal diversity loss. Our aim was not to overinterpret the reference, but rather to illustrate a possible explanation for the weak net response observed at the whole-community level, namely divergent responses among fungal guilds (e.g., decreases in arbuscular mycorrhizal fungi and potential increases in saprotrophs) may offset one another. To avoid misrepresentation, we have revised the sentence and removed citation 35. The revised sentence as: “The weak negative effect of N enrichment on fungal diversity likely reflects heterogeneous responses among functional guilds44, as N reduces the diversity and abundance of arbuscular mycorrhizal fungi (AMF) while simultaneously increasing the relative proportion of saprotrophic fungi45,46, with this functional reorganization may obscure declines in total fungal diversity.”. (Line 93-97)\nAdditionally, we have added another mechanism to explain the week response of fungal diversity under N enrichment, that is tolerant species could compensate for the species being reduced by global change factors, thereby maintaining overall ecosystem biodiversity at the same level (ref. 2). The added sentence as: “In addition, the compensatory dominance of tolerant fungal taxa may further buffer the loss of more sensitive species under global change conditions47.”. (Line 97-98)\nReferences\n1. Morrison, E.W., Frey, S.D., Sadowsky, J.J., van Diepen, L.T., Thomas, W.K. and Pringle, A., 2016. Chronic nitrogen additions fundamentally restructure the soil fungal community in a temperate forest. Fungal Ecology, 23, pp.48-57.\n2. Zhou, G., Eisenhauer, N., Terrer, C., Eldridge, D.J., Duan, H., Guirado, E., Berdugo, M., Zhou, L., Liu, S., Zhou, X. and Delgado-Baquerizo, M., 2024. Resistance of ecosystem services to global change weakened by increasing number of environmental stressors. Nature Geoscience, 17(9), pp.882-888.\nComment 16: Line 79: Additionally, plant and bacterial diversity were more strongly affected by nitrogen enrichment in warmer regions, while fungal diversity exhibited a greater response in colder regions (Fig. 1c-e). I do not see evidence for this. Are you referring to tropical versus temperate regions or temperate versus polar regions?\nResponse: We fully agree with the reviewer that our original description was overly generalized and lacked precision in reflecting the observed regional patterns. Specifically, the statement did not clearly distinguish between climate zones and may have conveyed an inaccurate interpretation of the trends presented in Fig. 1c-e. Upon closer examination, we found that plant diversity declined most significantly in temperate regions, while bacterial diversity declined in both tropical and temperate regions but showed little change in polar areas. Fungal diversity did not respond significantly in any region, but its effect sizes shifted from negative to positive along the temperature gradient, indicating a directional trend rather than a stronger response in colder regions.\nTo improve accuracy and clarity, we have revised the sentence and associated discussion in the main text as: “Additionally, plant diversity declined mainly in temperate regions, bacterial diversity decreased in both temperate and tropical regions, while fungal diversity showed no significant change across climatic regions but instead shifted from negative to positive responses with decreasing temperature (Fig. 1c-e).”. (Line 100-103)\nComment 17: Line 93: Soil biota also includes organisms other than microbes, such as nematodes. You do not present data on these organisms. Am I missing something? If not, the entire paragraph needs revision.\nResponse: We fully agree with the reviewer that “soil biota” is a broader term that encompasses diverse organisms including nematodes and mesofauna, which were not assessed in our dataset. In this study, we specifically focused on bacteria and fungi as representative components of the soil microbial community. To address this concern, we have revised the text by replacing “soil biota” with “soil microbial biomass” or “soil microbial communities” or “soil microbes” as appropriate throughout the manuscript in Lines of 15, 20 in Abstract; Lines 51, 83 in Introduction; Lines 90, 100, 109, 117, 119, 189, 192, 218, and 280 in Results and Discussion; Lines 306, 317, 326, 345, 384, 389, 408, 438 and 456 in Methods and Lines 705, 708, 728, 732, 734 and 737 in Figure legend. Additionally, Corrections have also been made to the legends of the supplementary figures.\nComment 18: Line 100: The use of \"however\" appears illogical here, as the two sentences address different topics, or at least emphasize different points. Please reassess the logic behind this.\nResponse: We fully agree that the use of “however” in this context was inappropriate, as the preceding and following sentences refer to results from different subgroups and are not logically contrasting. To improve clarity, we have replaced “however” with “Additionally” to better reflect that the two findings are parallel rather than contrasting. (Line 122)\nComment 19: Line 106: What is the metabolic ecology theory, and why is it consistent with the metabolic theory of ecology (MTE)? The statement that elevated temperatures accelerate microbial catabolism does not clarify why nitrogen-induced SOC accumulation decreases from cold regions to the tropics. BTW, be careful, researchers normally use the metabolic theory of ecology, rather than metabolic ecology theory.\nResponse: Thank you for this valuable comment. The Metabolic Theory of Ecology (MTE) states that individual metabolic rates scale predictably with body size and temperature, such that elevated temperatures inherently accelerate microbial metabolic processes and enhance organic matter decomposition (ref). We acknowledge the incorrect terminology and have replaced “metabolic ecology theory” with the widely accepted term “metabolic theory of ecology (MTE).” In tropical regions, where baseline temperatures are already high, this thermal acceleration effect can lead to faster turnover of soil organic carbon and thus lower net SOC accumulation under N enrichment. We have revised the sentence to more clearly reflect this mechanism. The revised sentence as: “SOC accumulation gradually decreased from cold regions to the tropics, consistent with metabolic ecology theory, which posits that higher temperatures accelerate microbial catabolism, thereby accelerating organic matter turnover and reducing net SOC storage54-56.” (Line 126-129)\nReferences\nBrown, J.H., Gillooly, J.F., Allen, A.P., Savage, V.M. and West, G.B., 2004. Toward a metabolic theory of ecology. Ecology, 85(7), pp.1771-1789.\nComment 20: Line 107: The assertion that SOC accumulation did not increase with rising nitrogen application rates or durations raises concerns about the predictability of SOC stocks under future nitrogen enrichment scenarios. I find this statement confusing.\nResponse: Thank you for this valuable comment. We agree with the reviewer that the original statement lacked sufficient clarity and may have caused confusion. Here, the statement was not intended to imply that N enrichment fails to promote SOC accumulation, but rather to emphasize that SOC responses do not follow a consistent, monotonic increase with increasing N rates or durations. Such complex, nonlinear patterns constrains our ability to predicting SOC accumulation under future N-rich scenarios.\nTo improve clarity, we have revised the sentence as: “Unlike other variables, SOC accumulation showed no consistent increase with rising N rates or durations, underscoring its complex and the uncertainty of SOC stock predictions under future N enrichment scenarios53.”. (Line 129-131)\nComment 21: Line 122: This can be attributed to enhanced nitrification and ammonium oxidation driven by higher microbial diversity, processes that generate increased H⁺ concentrations and contribute to soil acidification in temperate regions. However, please check citation 47; the abstract states that \"soil nitrification rate tended to decrease with increasing latitude, especially in the Northern Hemisphere, and varied largely with ecosystem types. The soil nitrification rate significantly increased with mean annual temperature (MAT).\" After reading this, I would infer that nitrification should be higher in lower latitudes or warmer areas, meaning that tropical regions should exhibit higher nitrification rates compared to temperate regions. This does not support your findings. I strongly recommend that the authors verify the cited paper accordingly.\nResponse: Thank you for this valuable comment. After carefully reviewing citation 47, we acknowledge that the cited study reports higher nitrification rates in warmer, lower-latitude regions, which contradicts our previous attribution of stronger acidification in temperate soils to enhanced nitrification. We have therefore removed this citation and revised the explanation accordingly. Our intention was not to suggest that temperate regions have higher baseline nitrification rates, but rather to explain why pH declines more markedly in temperate soils under nitrogen enrichment. We now clarify that this is primarily due to the inherently lower base saturation and weaker buffering capacity of temperate soils, which increase their susceptibility to acidification when exposed to nitrogen inputs. Xu et al. (2012) reported that temperate forest soils in China had significantly lower base saturation than tropical soils under low nitrogen deposition, indicating limited buffering capacity and heightened acidification risk. Lu et al. (2013) further observed that nitrogen addition elevated H⁺ concentrations and shifted the exchangeable cation pool from Al³⁺ to H⁺ and Fe³⁺, consistent with a more advanced acidification state in temperate soils.\nWe have removed the citation 47 and revised the sentence as: “This may be attributed to the inherently lower base saturation and weaker buffering capacity of temperate soils, which amplifies the acidifying effects of N-induced nitrification8,57,58.”. (Line 140-142)\nReferences\n1. Xu, R.K., Zhao, A.Z., Yuan, J.H. and Jiang, J., 2012. pH buffering capacity of acid soils from tropical and subtropical regions of China as influenced by incorporation of crop straw biochars. Journal of Soils and Sediments, 12, pp.494-502.\n2. Lu, X., Hou, E., Guo, J., Gilliam, F.S., Li, J., Tang, S. and Kuang, Y., 2021. Nitrogen addition stimulates soil aggregation and enhances carbon storage in terrestrial ecosystems of China: A meta‐analysis. Global Change Biology, 27(12), pp.2780-2792.\nComment 22: Line 135: This is not true for fungi; temperature is more important than pH.\nResponse: Apologies for the incorrect descriptions. We have revised the text accordingly to reflect the correct ranking of predictor importance as shown in origin Fig. 2 (now Fig. 3). The revised sentence now reads: “Using a random forest approach, we found that the diversity response of plants was strongly linked to N availability (TN, NH₄⁺ and NO₃⁻), MAT, and soil moisture (Fig. 3a), reflecting their critical roles in supporting plant growth. In contrast to bacterial diversity, which was primarily associated with NH₄⁺ and pH (Fig. 3b), fungal diversity responded most strongly to NO₃⁻, MAT, and soil moisture (Fig. 3c).”. (Line 179-183)\nComment 23: Line 138: Are you referring to plant biodiversity or fungal diversity here? The text seems mismatched again. I suspect the labels in the figures may be incorrect.\nResponse: Thank you for pointing this incorrect citation of Figures. Specifically, the origin Fig. 2a displays plant diversity, while the Figs. 2b and 2c correspond to bacterial and fungal diversity, respectively. We have corrected the figure labels and revised the text to ensure consistency between the description and the figure. The revised description as: “Using a random forest approach, we found that the diversity response of plants was strongly linked to N availability (TN, NH₄⁺ and NO₃⁻), MAT, and soil moisture (Fig. 3a), reflecting their critical roles in supporting plant growth. In contrast to bacterial diversity, which was primarily associated with NH₄⁺ and pH (Fig. 3b), fungal diversity responded most strongly to NO₃⁻, MAT, and soil moisture (Fig. 3c). These findings are consistent with previous research, which highlights N-induced soil acidification as a primary driver of biodiversity loss in terrestrial ecosystems, while also underscoring the modulatory role of climate factors in shaping biodiversity responses under nitrogen enrichment, as further supported by our meta-regression analysis (Fig. S3)”. (Line 179-186)\nComment 24: Line 146: Is plant diversity the most important factor? How does the model account for the collinearity of plant diversity and SOC along the nitrogen addition gradient? Furthermore, could this result conflict with the findings presented in panel f? The same question applies to the following paragraph (lines 152-167).\nResponse: We sincerely thank the reviewer for raising these important points. Firstly, we acknowledge that in the original wording, our aim is to highlight the important role of plant diversity in driving SOC responses under nitrogen enrichment by stating “except for TN”. However, we realize that this phrasing may have led to confusion during quick reading, potentially obscuring the actual ranking of predictor importance. We now revised this sentence as: “…, our analysis revealed that the responses of TN and plant diversity were the strongest predictors of SOC changes, with plant diversity playing a pivotal role in regulating SOC accumulation under N enrichment (Fig. 3d and Fig. S5).”. (Line 192-195)\nSecond, we would like to clarify that potential collinearity between plant diversity and SOC was already carefully considered during our original analysis. Specifically, prior to model fitting, we applied a two-step variable selection procedure using the spatialRF package (Benito et al., 2021) followed by Sáez-Sandino et.al (Nat. Clim. Chang., 2023): (1) correlation-based filtering (auto_cor function, r > 0.6) to remove highly correlated predictors, and (2) variance inflation factor filtering (auto_vif function, VIF > 2.5) to further exclude N-induced collinear variables. This procedure ensured that only predictors with low intercorrelations and acceptable multicollinearity levels were retained for model construction, thereby preserving the ecological interpretability of our Random Forest results. Additionally, predictor importance was assessed by measuring the mean decrease in prediction accuracy (mean squared error) when each predictor was permuted across trees, and model performance was evaluated using out-of-bag error estimates, providing an internal, unbiased measure of predictive accuracy. We have now added this methodological detail more explicitly in the revised Methods section as: “…Before model fitting, we minimized multicollinearity by performing two steps: (1) correlation-based filtering (auto_cor function, r > 0.6) to remove highly correlated predictors, and (2) variance inflation factor filtering (auto_vif function, VIF > 2.5) to further exclude N-induced collinear variables…”. (Line 392-395)\nThird, we agree that the results from the Random Forest analysis and the meta-regression presented in panel f may initially appear different; however, they reflect different analytical perspectives rather than a contradiction. Specifically, the Random Forest analysis evaluated the relative contribution of multiple predictors to SOC responses across N enrichment gradients, identifying plant diversity response as a stronger predictor compared to microbial diversity in a multivariate modeling context. This reflects how plant diversity changes, alongside other variables, jointly contribute to SOC variation when interactions among predictors are considered. In contrast, the meta-regression analysis in panel f examined the pairwise correlation between diversity responses and SOC responses individually, without controlling for other variables. In this framework, microbial diversity (particularly fungal diversity) showed a slightly stronger direct association with SOC change than plant diversity. Thus, the two analyses offer complementary insights: Random Forest identifies the key predictors influencing SOC when multiple factors interact; Meta-regression captures the strength of individual diversity-SOC relationships. We believe that together, these approaches provide a more comprehensive understanding of the pathways through which biodiversity changes influence SOC dynamics under N enrichment.\nReferences\nBenito, B.M., 2021. spatialRF: Easy spatial regression with random forest. R package version, 1(0), p.1.\nSáez-Sandino, T., García-Palacios, P., Maestre, F.T., Plaza, C., Guirado, E., Singh, B.K., Wang, J., Cano-Díaz, C., Eisenhauer, N., Gallardo, A. and Delgado-Baquerizo, M., 2023. The soil microbiome governs the response of microbial respiration to warming across the globe. Nature Climate Change, 13(12), pp.1382-1387.\nComment 25: Line 177: Does \"earlier\" refer to time or duration?\nResponse: Thank you for this valuable comment. In this sentence, “earlier” refers to the timing of the response, indicating that fungi reacted to N enrichment at lower levels or earlier stages compared to bacteria. To improve clarity, we have revised the sentence as follows: “Soil fungi appeared more tolerant to N enrichment than bacteria, but they responded at earlier stages.” (Line 225-226)\nComment 26: Lines 186-195: I do not understand why you refer to these parameters as part of the \"soil acidification phase.\" Particularly for soil moisture and microbial biomass carbon (MBC), there seems to be no relationship with acidification.\nResponse: We sincerely thank the reviewer for this important and insightful comment. We fully agree that soil moisture and microbial biomass carbon (MBC) are not direct indicators of soil acidification processes, unlike soil pH, ammonium (NH ⁺-N), and 4 nitrate (NO ⁻-N). Our original intention was not to imply mechanistic causality (i.e., 3 that acidification directly drives changes in moisture or MBC), but rather to illustrate the clustering of ecological thresholds for multiple soil properties along the nitrogen gradient. Specifically, we aimed to highlight that critical transitions for soil pH, soil moisture, MBC, and inorganic nitrogen concentrations occur within a broadly similar N range (66-199 kg N ha⁻¹ yr⁻¹), reflecting a phase of overall soil functional degradation following initial biodiversity losses. To avoid the potential confusion, we now describe it as the “soil degradation persist”, which more accurately reflects the synchronous threshold shifts across soil physical, chemical, and other biological properties under N enrichment. The revised Fig. 3 (now Fig. 4) as below:\nFig. 3 (revised, now Fig. 4) | Trade-offs in the response of plant and soil microbial diversity and SOC under N enrichment. a, Critical thresholds of above- and below-ground compartments and SOC. The regressions of each variable are presented in Supplementary Fig. S6-S7. b-d, Moving-window correlations between the response of diversity and the response of SOC along N gradients for bacteria (b), fungi (c), and plants (d). The dots indicate the bootstrapped coefficients of the fixed terms shown for each subset window. The sign of each dot (relative to the zero line, where values > 0 indicate positive associations and values < 0 indicate negative associations) reflects whether biodiversity and SOC respond in the same (co-increase or co-decline) or in opposite directions. The slope of the fitted line indicates how biodiversity-SOC associations change across the N gradient: a positive slope suggests strengthening associations with increasing N input, whereas a negative slope suggests weakening or reversal. The dashed lines denote the nonlinear trend fitted by GAMs. The vertical dashed lines and inset numbers represent the N enrichment rates thresholds identified, and the solid lines represent the linear fits at both sides of each threshold.\nAdditionally, this correction has been applied to Line 25 of the Abstract and Line 221 of the Results and Discussion sections in the revision.\nComment 27: Lines 196-220: You discuss only the strength of correlations, but the direction and underlying biological significance are also crucial for interpretation. I am unclear about what the authors or the figures are attempting to convey.\nResponse: We sincerely thank the reviewer for highlighting the need to clarify not only the strength but also the direction and biological significance of the observed correlations. In the revised manuscript, we now explicitly detail how the relationships between biodiversity (bacterial, fungal, and plant) and SOC shift across the N gradient, capturing both the correlation direction and the underlying ecological dynamics. Our findings consistently reveal a clear trajectory: a phase of low-N decoupling where biodiversity and SOC respond independently, followed by mid-N synergy where diversity loss begins constraining SOC accumulation, and ultimately high-N functional breakdown, where intensified N saturation simultaneously undermines biodiversity and carbon storage. This gradient-dependent transition from synergy to trade-off highlights the urgent need to recognize biodiversity as a critical regulator of carbon cycling under global N enrichment. We conclude that limiting N inputs to below ~50 kg N ha⁻¹·yr⁻¹ may help safeguard both biodiversity and long-term soil carbon storage, offering crucial benchmarks for ecosystem management and policy.\nPlease refer to our response to Comment 6 for more details.\nComment 28: Lines 222-260: This section appears to diverge from the main theme of the paper, shifting from a diversity-driven narrative to a biomass-driven one.\nResponse: We sincerely thank the reviewer for raising this important point. We would like to clarify that the global projections presented in Fig. 4 were not intended to investigate the causal effects of biomass on SOC. Instead, we independently modelled the responses of plant biomass, microbial biomass, and SOC under nitrogen enrichment to provide a broader understanding of ecosystem carbon dynamics. Given that plant and microbial biomass constitute the immediate material basis for SOC formation and turnover, we believe that presenting these projections may help provide broader context for the observed SOC responses and illustrate the decoupled and complex above- and belowground processes under nitrogen enrichment. Importantly, we recognize that the ecological pathways linking biodiversity, biomass, and SOC differ between microbial and plant communities: in microbial communities, changes in biodiversity can directly influence microbial biomass and stabilization processes, whereas in plant communities, nitrogen-induced biomass accumulation may precede and contribute to biodiversity loss, with both processes jointly modifying soil carbon inputs and stabilization pathways.\nTherefore, we have retained Fig. 4 and repositioned it earlier in the manuscript (now Fig. 2), following the random forest analysis (origin Fig. 2) and before the diversity-SOC coupling section, to better align with the main narrative. This repositioning, together with the clarification that biomass and SOC projections were modeled independently, aims to strengthen the logical flow and avoid potential misinterpretations about causal pathways.\nTo improve the logical flow, we first added an introductory sentence at the beginning of the paragraph to better clarify how the global biomass projections relate to biodiversity-mediated regulation of SOC dynamics, as following: “Given that plant and microbial biomass constitute the immediate foundation for SOC formation and turnover, we independently modelled the global responses of plant biomass, microbial biomass, and SOC under N enrichment to better elucidate ecosystem carbon dynamics (Fig. 2).” (Line 155-157). In addition, to address the reviewer’s concern and to ensure a more coherent focus on biodiversity-SOC relationships, we have substantially shortened this section and no longer present the global biomass-SOC projections as a standalone subsection. Instead, the figure is now briefly incorporated into a broader discussion of nitrogen-mediated ecosystem-level shifts in the revision. Please refer to our response to Comment 7 for more details.\nComment 29: Line 236: The term \"some temperate regions\" is vague; please be more specific.\nResponse: Thank you for pointing out this ambiguity. have revised this sentence to make it clearer as: “Similarly, SOC responses were largely positive across temperate regions, particularly in Europe and northern Asia, whereas negative responses prevailed in parts of the Southern Hemisphere and tropical zones, including southern Africa, South Asia, and Southeast Asia (Fig. 2c). This likely reflects differences in nutrient limitations and ecosystem processes: in temperate systems, nitrogen (N) addition tends to stimulate plant growth and inputs while simultaneously reducing decomposition rates, thereby promoting SOC accumulation; in tropical and Southern Hemisphere regions, where phosphorus limitation and rapid organic matter turnover are more common, N addition often fails to enhance plant inputs and may even accelerate SOC loss63,64.”. (Line 166-174)\nMethods:\nComment 30: Line 287: What is meant by \"paired site design\"?\nResponse: We thank the reviewer for pointing out this important clarification. In our study, “paired-site design” refers to field experiments in which both nitrogen-enriched (treatment) and control conditions were established at the same site, typically as paired plots located within a common experimental area. This design minimizes environmental heterogeneity and enables robust within-site comparisons by controlling for confounding site-level variables. To improve clarity and align with commonly used terminology in high-impact ecological studies, we have revised the manuscript text as follows: “…1) N fertilizer was directly applied to terrestrial ecosystems under field conditions using paired treatment-control plots, excluding laboratory incubation studies.” (Line 318-320)\nComment 31: Lines 309-310: From the figure, I only see data for tropical, temperate, and polar regions.\nResponse: We thank the reviewer for this insightful comment. In our study, we grouped the climate zones based on the 2010 Global Ecological Zones mapping to ensure consistency across datasets (a map of the climate zones is provided below for reference). Specifically, the “Tropical” category includes both tropical and subtropical zones, while the “Polar” category encompasses both boreal and polar zones, consistent with previous classifications (ref.1). It provides a simplified yet ecologically relevant framework for aggregating complex climate gradients, ensuring comparability across studies. Additionally, the relatively small sample size for boreal zones alone further supported combining it with polar regions. For clarity and simplicity, the figure uses abbreviated labels: “Tropical”, “Temperate”, and “Polar” correspond to “Tropical & Subtropical”, “Temperate”, and “Boreal & Polar”, respectively in the origin version.\nTo address this concern, we have clarified this sentence in the Method section to avoid any potential misunderstanding. The revised sentence as follows: “The final dataset categorized three ecosystem types (farmland, forest, and grassland) and three climate regions, tropical (tropical and subtropical regions), temperate (temperate regions) and polar (polar and boreal regions), based on the 2010 Global Ecological Zones mapping (https://data.apps.fao.org/). (Line 340-343)\nThe FAO Global Ecological Zones recognize five major domains and 20 ecological zones. Reprinted with permission from © FAO (2012) (ref. 2).\nReferences\n1. Zeng, X.M., Berdugo, M., Saez-Sandino, T., Tao, D., Ren, T., Zhou, G., Liu, Y.R., Terrer, C., Reich, P.B. and Delgado-Baquerizo, M., 2025. Temperature thresholds induce abrupt shifts in biodiversity and ecosystem services in montane ecosystems worldwide. Proceedings of the National Academy of Sciences, 122(16), p.e2413981122.\n2. FAO. Global Ecological Zones for FAO Forest Reporting: 2010 Update. Working Paper 179. FAO, Rome, Italy (2012).\nComment 32: Line 310: Given the global decline in biodiversity due to nitrogen enrichment, are you suggesting that nitrogen enrichment is declining globally? This sentence needs revision.\nResponse: Thank you for pointing out this ambiguity. We agree with the reviewer’s comment and have revised the sentence to clarify that nitrogen enrichment remains a major driver of biodiversity loss, rather than suggesting a global decline in nitrogen inputs. The revised sentence now reads: “Given the global biodiversity decline driven by N enrichment, we aimed to….”. (Line 343-344)\nComment 33: Line 347: You present figures S11 and S12 first, but Figure S9 appears later, and Figure S10 is not mentioned in the text. Please verify this.\nResponse: Thank you for pointing out this issue. Fig. S9 was intentionally introduced earlier to support specific results in the legend of origin Fig. 4, which may have contributed to a perceived disruption in the citation sequence. To address this and maintain consistency with the order of figures as they appear in the main text, we have slightly adjusted the description in the legend of Fig. 4 to ensure that supplementary figures are cited in sequential order. In addition, Fig. S10 is indeed cited in the main text, though its reference appears in a later section and may have been less immediately visible. We have carefully reviewed the order and placement of all supplementary figure citations and confirmed that they are now clearly and appropriately referenced throughout the revision.\nReferences:\nComment 34: Please review the references carefully, especially the abbreviations of journal names. Line 487 is missing a journal name, and this issue occurs in several other instances.\nResponse: We sincerely thank the reviewer for pointing out this important issue. Following the reviewer’s suggestion, we have carefully reviewed and corrected the reference formatting throughout the manuscript, including both the in-text citations and the references used in the meta-analysis dataset. All references have now been standardized according to the guidelines, and missing journal names (e.g., Line 487) have been added to ensure consistency and compliance. Please refer to the revised manuscript and supplementary materials for details.\nComment 35: Thank you for considering my feedback.\nResponse: Thank you very much for your thorough review and constructive feedback on our manuscript, covering aspects ranging from figure clarity and textual logic to the accurate citation of references and beyond. We greatly appreciate the time and effort you invested in evaluating our work. We have carefully considered all of your suggestions and have made substantial revisions in response. We sincerely hope that the changes we have made address your concerns and meet your expectations.\nReviewer #2 (Remarks to the Author):\nI have reviewed the revised manuscript titled \"Critical Thresholds for Co-Benefits of Carbon Sequestration and Biodiversity Conservation Under Global Nitrogen Enrichment (NC-574816).\" The authors have made revisions in response to my previous comments; however, several issues remain unaddressed. Additionally, the authors have acknowledged that many references I raised were incorrect, which raises concerns about the overall quality of the manuscript. Below are my specific comments:\nResponse: We sincerely thank you for your time and effort in continuing the evaluation of our manuscript, and for recognizing the revisions made in response to your previous round of comments. Your earlier feedback was instrumental in helping us substantially improve the manuscript, particularly in terms of logical structure, the meta-regression analyses, textual clarity, discussion, and figure presentation. We also acknowledge that in some cases, our revisions may not have fully conveyed our intended rationale or supporting evidence. We greatly appreciate your additional suggestions, which have enabled us to undertake a more comprehensive round of revisions aimed at addressing all outstanding issues. Regarding the references, in our previous response we noted and corrected several cases where references were either misaligned with the context or outdated. These issues arose primarily from formatting inconsistencies or unintentional misattributions. We have carefully re-checked all references, verified their relevance and accuracy, and corrected or replaced any that were outdated or misaligned. We provide detailed, point-by-point responses below and look forward to your further comments.\nComment 1 (Line 45): The term “biodiversity-driven catabolism and anabolism” is unclear. Please provide a definition or explanation of these concepts and how they relate to the study's objectives.\nResponse: We appreciate the reviewer’s comment. The previously used term “biodiversity-driven catabolism and anabolism” refers to microbial diversity-mediated processes of organic matter decomposition (catabolism) and microbial biomass and extracellular product formation (anabolism). These processes are jointly regulated by microbial carbon use efficiency (CUE), which is shaped by microbial diversity. In parallel, plant diversity contributes to C cycling by diversifying organic inputs (e.g., litter and root exudates), while microbial diversity determines how efficiently these inputs are processed and transformed. Together, these mechanisms align with theories of functional trait diversity and niche complementarity, thereby supporting our study’s objective of elucidating how biodiversity regulates SOC dynamics under N enrichment. We have revised these descriptions to elaborate on the mechanisms through which plant and soil microbial diversity influence soil carbon (C) dynamics to better link biodiversity with SOC outcomes, as: “The inconsistencies may arise from differences in substrate quantity, mineral associations, environmental conditions and biodiversity13. Biodiversity, in particular, is widely recognized to regulate soil C cycling14,15, with its loss driving large-scale changes in ecosystem energy flow and nutrient cycling16,17. Higher plant diversity often increases biomass inputs and root exudation18, while soil microbial diversity governs the balance between C catabolism (decomposition) and anabolism (biomass and extracellular production)19-21, thereby shaping SOC dynamics.”. (Line 40-46)\nComment 2 (Lines 49-51): The logical flow is confusing. You state that nitrogen (N) addition reduces plant diversity but not microbial diversity. Subsequently, you claim that these shifts can disrupt the balance. What specific shifts are you referring to, and how do they disrupt the ecological balance? Please clarify.\nResponse: We thank the reviewer for highlighting this point and for the opportunity to clarify the logical flow in our original text. Our intention was to emphasize the complex and often divergent responses of plant and soil microbial diversity to N enrichment, and how such differential responses may influence SOC dynamics. It is well established that N enrichment typically reduces plant diversity, primarily through mechanisms such as competitive exclusion, light limitation due to increased canopy dominance, and changes in soil chemistry. In contrast, microbial responses to N addition are more variable and taxon-dependent. For instance, Wang et al. (2023, Soil Biology and Biochemistry) reported that while N enrichment significantly reduced overall microbial alpha diversity, this effect was primarily driven by declines in bacterial diversity, whereas fungal diversity did not show a statistically significant change. Importantly, other studies have similarly noted inconsistent or weak responses of fungal richness to N addition, underscoring the complex and taxon-specific nature of microbial responses to N enrichment. Such divergent responses between plant and microbial diversity may weaken the positive biodiversity effects on SOC sequestration. Moreover, the potential erosion of functional diversity could further decrease resource-use efficiency and ecosystem productivity, posing a major challenge for sustaining SOC accumulation while mitigating biodiversity loss.\nWe have made revision as following: “N enrichment generally reduces biodiversity22-25, primarily through mechanisms such as competitive exclusion, soil acidification and cation toxicity24,26. Yet microbial diversity exhibits inconsistent responses27,28. For example, while N enrichment significantly reduced overall microbial diversity, the decline was mainly observed for bacteria, whereas fungal diversity remained unchanged27. This highlights the complex and taxon-specific nature of microbial responses to N enrichment. Therefore, such divergent responses between plant and microbial diversity may weaken the positive biodiversity effects on SOC sequestration29,30. Moreover, the potential erosion of functional diversity could further decrease resource-use efficiency and ecosystem productivity, posing a major challenge for sustaining SOC accumulation while mitigating biodiversity loss14,19,22,31-33.” (Line 46-55)\nComment 3 (Line 55): The phrase “fundamental drivers” is vague. What specific factors are being referred to here? It is crucial in scientific writing to be precise and clear.\nResponse: We fully agree with the reviewer that “fundamental drivers” should be defined more explicitly. In our study, these drivers refer to key biotic, abiotic, and climatic factors that potentially regulate SOC dynamics under N enrichment. Specifically, they include above- and belowground biodiversity and biomass, soil physicochemical properties (e.g., pH, soil moisture and nutrients availability), and climatic variables (e.g., mean annual precipitation, mean annual temperature).\nWe have revised the sentence to clarify these components in the introduction as: “Despite increasing evidence of these complexities4,9,27,34, few studies have jointly assessed the relative contributions of major drivers, including plant and microbial diversity and biomass, soil physicochemical attributes (e.g., pH, moisture, nutrient availability), and climatic variables (e.g., mean annual precipitation, mean annual temperature), to SOC responses under N enrichment.”. (Line 55-59)\nComment 4 (Second Paragraph): The manuscript suggests a potential trade-off between diversity response and SOC response to N addition. However, this relationship is context-dependent and should not be presented as universally applicable. The writing needs further refinement to reflect this nuance.\nResponse: We sincerely appreciate the reviewer’s insightful comment. We fully agree that the potential trade-off between biodiversity and soil organic carbon (SOC) responses to nitrogen (N) enrichment is context-dependent and should not be generalized across all ecosystems or N input scenarios. Our original intent in the second paragraph of the Introduction was to highlight that such decoupling has been observed under specific conditions, rather than to imply a universal or inevitable pattern. We acknowledge that the initial wording lacked sufficient nuance and contextual qualifiers, such as ecosystem type, rate and duration of N addition, climate, and soil properties, that are critical in determining whether a trade-off emerges. To address this concern, we have revised the text to more explicitly frame the biodiversity-SOC relationship as conditionally mediated by ecological and experimental context. Moreover, we elaborated through three case studies to demonstrate that such trade-offs arise only under particular ecological contexts, in order to reduce the misconception that trade-offs are inevitable or universal.\nThe revised sentences in second paragraph as: “Biodiversity and soil C conservation are dual priorities in the global effort to mitigate climate change36,37. However, N enrichment can disrupt this synergy over time, decoupling biodiversity and C goals22. For example, a 27-year field experiment in temperate grassland reported SOC gains under high N input, but at the cost of substantial plant diversity loss38. Similarly, a global meta-analysis revealed that soil bacterial and fungal diversity are strongly negatively associated with soil C content, and their linkages are influenced by N fertilization rate and duration22. In contrast, tree species mixtures with high diversity can enhance SOC accumulation under elevated N availability39. These findings suggest that biodiversity-SOC relationships under N enrichment are context-dependent, and continued N inputs may trigger trade-offs under specific environmental conditions. Therefore, identifying ecological thresholds along N gradients is essential to anticipate when biodiversity losses may begin to undermine soil C gains40,41.”. (Line 63-73)\nComment 5: The statement that “soil microbial communities are likely to exhibit detectable threshold responses earlier than plants along N gradients” conflicts with your earlier assertion that plant diversity typically responds negatively to N addition, while microbial communities, particularly fungal diversity, show no response. Please resolve this inconsistency.\nResponse: We thank the reviewer for raising this important and insightful concern. We acknowledge that the original wording may have created an apparent inconsistency between two key points: (1) that plant diversity generally shows stronger and more consistent declines in response to N enrichment, and (2) that microbial communities may exhibit threshold responses earlier along N gradients. We agree that these statements require careful contextualization to avoid misinterpretation. While plant communities often show greater magnitude of diversity loss under N enrichment, particularly in terms of species richness and evenness, microbial communities may respond differently in terms of timing and sensitivity. Despite relatively stable alpha diversity (especially in fungi), soil microbes can undergo rapid and detectable shifts in community composition, functional gene expression, or metabolic activity in response to even low levels of N addition. This high sensitivity is attributed to their short generation times, high metabolic rates, and strong coupling to soil resource chemistry. For example, studies have shown that microbial community composition (e.g., shifts in dominant taxa or functional groups) can change abruptly at relatively low N inputs, before significant changes in plant diversity are observed. These early microbial shifts may serve as leading indicators of ecosystem change, even when overall microbial richness remains unchanged. Thus, while plants may exhibit larger net losses in diversity over time, microbes may reach critical threshold responses earlier along the N gradient due to their physiological and ecological traits.\nTo resolve this ambiguity, we have now revised the relevant sentences as in Line 77-79: “Given their high metabolic rates and short generation times46, soil microbial communities are more sensitive and responsive to environmental changes than plants47,48”, and in Line 48-50: “Yet microbial diversity exhibits inconsistent responses27,28. For example, while N enrichment significantly reduced overall microbial diversity, the decline was mainly observed for bacteria, whereas fungal diversity remained unchanged27. This highlights the complex and taxon-specific nature of microbial responses to N enrichment.”\nComment 6: The claim that “shifts in microbial and plant communities may modify soil properties in ways that delay the manifestation of SOC thresholds relative to biodiversity thresholds” requires further explanation. Why do these shifts delay the SOC threshold? Please clarify the underlying mechanisms.\nResponse: We thank the reviewer for this insightful question. We agree that our original statement regarding the delayed manifestation of SOC thresholds relative to biodiversity thresholds required more explicit mechanistic explanation. Our claim is based on the well-documented temporal decoupling between biotic community dynamics and soil carbon cycling. While plant and microbial communities can exhibit rapid compositional shifts in response to N enrichment, driven by changes in species dominance, root exudation, litter quality, and microbial metabolic strategies, these biotic changes do not immediately translate into detectable changes in SOC stocks. This lag arises because SOC accumulation and stabilization are governed by relatively slow, physically and chemically protected processes. In particular, a significant portion of SOC is stabilized through mechanisms such as aggregation, organo-mineral interactions, and chemical recalcitrance, especially in the case of mineral-associated organic carbon (MAOC). These pools are inherently resistant to decomposition and have turnover times ranging from decades to millennia. As a result, even when N-induced shifts in plant-microbe communities alter the quantity and quality of organic inputs (e.g., favoring more recalcitrant or labile compounds), the net impact on total SOC may remain undetectable over shorter timescales due to the buffering capacity and inertia of these stable carbon pools. Therefore, while biodiversity thresholds, such as abrupt changes in community composition or functional group dominance, may be detectable early along an N gradient, corresponding SOC thresholds are likely to emerge only after prolonged N exposure, once the system’s capacity to stabilize carbon is overwhelmed. This time-lag effect implies that biodiversity responses may serve as leading indicators of ecosystem change, preceding measurable SOC losses.\nTo address this issue, we have added further explanation to reflect the time-lagged relationship between community- and carbon-level responses. The revised sentences as:\n“However, these microbial shifts may not immediately translate into changes in SOC, particularly in the case of mineral-associated organic carbon (MAOC), which is resistant to rapid turnover49. This temporal lag suggests that biodiversity thresholds, particularly for soil microbes, may precede those of C, potentially delaying detectable SOC destabilization under N enrichment.”. (Line 80-84)\nComment 7: In my previous comment, I raised a question regarding the negative correlation between plant/bacterial diversity and SOC. The revised text provides explanations for the figures but does not address the core issue: why should we expect a negative correlation between microbial diversity and SOC? Does low bacterial diversity truly lead to higher SOC? This still needs further revision.\nResponse: We sincerely thank the reviewer for raising this important concern. We agree that the negative correlation observed between microbial diversity and SOC requires careful ecological interpretation. The slight negative correlation observed at low N inputs (~<50 kg N ha⁻¹ yr⁻¹) reflects a decoupled response of microbial and carbon dynamics rather than a causal relationship. Specifically, microbial diversity may decline due to the negative effects of N enrichment, such as soil acidification, cation toxicity, and nutrient imbalances, while SOC continues to increase primarily due to plant-mediated carbon inputs (e.g., leaf litter and root exudates) driven by competitive interactions among plant species. Thus, this negative correlation does not imply that low microbial diversity directly leads to higher SOC. Instead, it represents a transient, non-causal association where SOC accumulation is primarily governed by plant productivity, whereas microbial communities respond more rapidly to N enrichment due to their high turnover rates.\nTo clarify, we emphasize that the observed negative correlation between microbial diversity and SOC at low N levels reflects an asynchronous response, where microbial diversity declines due to the negative impacts of N enrichment, while SOC continues to increase due to plant-mediated carbon inputs. This represents a transient, non-causal relationship. The revied sentences as: “The responses of bacterial diversity and SOC showed a slightly negative correlation below ~50 kg N ha⁻¹ yr⁻¹ (Fig. 4b), indicating a transient non-causal association. At this stage, the increase in SOC may directly benefit from aboveground productivity (e.g., leaf litter and root exudates)81,82, while the decline in bacterial species may be functionally compensated by redundant taxa83.”. (Line 269-272)\nComment 8: The authors have admitted that many cited papers were incorrect. While these references have been removed, this raises concerns about adherence to proper writing and referencing standards throughout the manuscript.\nResponse: We sincerely thank the reviewer for this important observation. We regret that in earlier drafts, some citations were included with only partial contextual alignment, which may have undermined the manuscript’s scholarly precision. We clarify that these instances involved contextual mismatches rather than factual inaccuracies in the cited works themselves. Nevertheless, we fully acknowledge the importance of rigorous and contextually appropriate referencing.\nIn response, we have conducted a comprehensive, line-by-line review of all citations in the manuscript. Each reference has been carefully re-evaluated to ensure it is: (1) directly relevant to the claim it supports, (2) accurately interpreted, and (3) drawn from high-quality, peer-reviewed literature. Any citations that did not meet these criteria have been revised or removed. Furthermore, we have meticulously verified that all remaining references adhere strictly to the journal’s formatting guidelines, ensuring consistency and integrity in presentation.\nWe deeply value the standards of academic integrity and responsible scholarship, and we appreciate the reviewer’s vigilance in upholding them. This feedback has been instrumental in strengthening the overall quality and credibility of our work. We remain fully committed to maintaining the highest standards in scientific writing and citation practices in this submission and in all future research communications.\nComment 9: Regarding the additional meta-regression analysis, the authors claim to have accounted for potential co-variation between effect sizes (lnRRs) by including N rate as a random effect. However, upon reviewing Figure 2f, the results appear unchanged. Which I doubt the results is corrected. Although some results did show significant changes in Fig S3, S4, etc, particularly correlations becoming non-significant. But the authors said that results remained statistically significant. This discrepancy needs clarification, and results should be updated accordingly. Furthermore, it should be clearly indicated in the figure legend that N addition rate was included as a random factor rather than study ID. Overall, the new analysis support my comment that ‘The influence of N addition rates on co-correlations should be treated with caution.’\nResponse: Thank you for this important comment. As noted, we did incorporate N addition rate as a random effect in our meta-regression models to account for the potential co-variation among lnRRs for your previous comment. Upon closer examination of the results in the Fig. 2f, both the regression coefficient and its associated significance level did change after including N rate as a random effect, although the effect size remained in the same direction and the magnitude of the change was modest. However, we acknowledge that the visual appearance of Fig. 2f may not suggest a major shift, this may be due to the relatively small changes on the slopes after accounting for the N addition rate. To address this concern, we included a comparison table reporting the regression outputs with and without incorporating N addition rate as a random effect (see below). The inclusion of N rate resulted in minor changes in both slope estimates and associated P-values among the three associations. This suggests that although model outputs were slightly adjusted, the visual interpretation of overall trends in the figure would remain largely unaffected.\nAssociations Slope (w/o N rate) P Slope (with N rate) P\nBacteria diversity-SOC 0.1498 <.0001 0.1020 0.0007\nFungal diversity-SOC -0.0744 <.0001 -0.0795 <.0001\nPlant diversity-SOC -0.0628 0.0403 -0.0618 0.0460\nWe also reviewed and rechecked the results presented in Fig. S3-S5, as previously noted by the reviewer, and indeed observed some degree of change across a few associations after incorporating N addition rate as a random effect. For example, the slope between plant diversity (lnRR) and plant biomass (lnRR) shifted non-significant in Fig. S5, suggesting that N input levels indeed influence co-correlation outcomes, particularly among highly covarying variables. In this study, we aimed to investigate how plant and microbial diversity responses jointly regulate SOC dynamics under varying N enrichment, recognizing that these responses may diverge at certain N input levels and result in ecological trade-offs. However, examining associations between diversity and SOC is challenging, as both may respond to shared N gradients, as you rightly pointed out. While multilevel models can account for some sources of confounding, shared responsiveness to environmental gradients like N input may still introduce statistical co-variation between response variables. Despite this, several pairwise associations were affected by incorporating N addition rate as a random effect, the key analytical results such as the magnitude and direction of variable responses, variable rankings in the random forest model, and the identification of response thresholds along the N gradient, remained unaffected. These adjustments improve model accuracy, and the revised results do not compromise the validity of our main findings. Overall, these observations underscore your important point that shared environmental gradients like N enrichment must be carefully considered when interpreting observational co-correlations.\nTo address this concern, we have softened the tone of certainty and added a sentence to cautious conclusion involving meta-regression analyses in the Result and Discussion section, to reflect the potential influence of shared environmental gradients. We also recommend that future studies adopt better suited experimental designs to disentangle such covariation effects. For example, controlled multifactorial experiments that independently manipulate N input and plant/microbial diversity such as biodiversity-N factorial designs in long-term field experiments, would allow clearer attribution of causality and reduce confounding from shared environmental gradients. Specially, we revised the description of the meta-regression results to provide a more accurate representation as: “After controlling for N addition rate, several pairwise associations in meta-regression analysis remained supported (Fig. S3-S4).”. (Line 205-207). We then softened deterministic phrasing (e.g., “biodiversity drives SOC”) to more cautious alternatives such as “biodiversity is associated with SOC response” or “biodiversity mediates SOC response” in the subtitle “Biodiversity mediates SOC responses under global N enrichment” of the revised manuscript, as meta-analysis cannot support causal inference. We added the discussion following the meta-regression results to caution readers about potential confounding effects from shared environmental drivers (e.g., N rate) as: “Given that N enrichment affects both biodiversity and soil processes through multiple interacting pathways, the detected biodiversity-SOC associations may partly reflect shared environmental responses rather than direct causal relationships.”. (Line 231-233); We also propose that future studies improve causal inference by employing experimental designs that independently manipulate key variables to test hypothesized pathways, rather than relying solely on correlative patterns. The added sentences in as: “Future studies should adopt factorial designs that independently manipulate biodiversity and N input (e.g., long-term crossed treatments), helping to disentangle causal effects from environmental confounding.”. (Line 304-306).\nAdditionally, we thank the reviewer for the close reading and pointing out the issue in the figure legend. We would like to clarify that both Study id and N addition rate were included as random effects in all relevant meta-regression analyses, in order to account for the hierarchical structure and potential co-variation among lnRRs. Specifically, Study id was retained as a random effect to account for the non-independence of effect sizes extracted from the same publication, a standard practice in ecological meta-analyses to avoid pseudo replication. We acknowledge that the original figure legend did not clearly reflect this model structure, and we have now revised it accordingly in line 794-795 and the Fig. S3-S5 in the revision.\nLastly, we appreciate the reviewer’s critical insights, which have helped us identify and clarify analytical nuances that might have been previously overlooked. Your comment prompted us to revisit and revise our modeling approach, enhance the transparency of result presentation, and temper interpretive claims to better reflect the limitations of meta-analytic inference. We hope that the current revision has addressed your concerns in both substance and clarity. Thank you again for your thoughtful and constructive feedback, which has undoubtedly strengthened the overall quality and rigor of our study.\nComment 10: if the authors are correct, I would say that I should trust the results more from the random forest rather than pairwise correlation? Correct?\nResponse: Thank you for this important follow-up question. We appreciate the opportunity to clarify the distinct yet complementary roles and interpretative frameworks of the two approaches used in our study, meta-regression and random forest model. As noted in our response to Comment 9, meta-regression quantifies the direction and strength of associations between variables. However, these associations may be confounded by shared environmental gradients such as N rate, which can simultaneously influence both biodiversity and SOC. In our context, N enrichment may act as both an ecological filter (i.e., influencing species richness) and a biogeochemical driver (i.e., affecting carbon cycling), thereby inducing statistical covariation that does not necessarily reflect causality. In contrast, the random forest model does not rely on pairwise relationships but evaluates the relative importance of multiple predictors in shaping SOC responses. It is particularly well-suited for detecting nonlinearities and complex interactions, offering a system-level perspective on potential drivers. Rather than suggesting that one approach is more trustworthy than the other, we view them as complementary: meta-regression provides interpretable pairwise patterns, while random forest offers an integrative understanding of system-level drivers.\nReviewer #3 (Remarks to the Author):\nThank you for the opportunity to review this manuscript. This study presents an ambitious and timely global synthesis of how nitrogen (N) enrichment influences biodiversity, biomass, and soil organic carbon (SOC) across terrestrial ecosystems. The integration of meta-analysis, random forest modeling, threshold detection, and spatial analysis is both methodologically rigorous and conceptually innovative. The identification of critical ecological thresholds beyond which biodiversity declines amplify soil C responses to N additions represents a valuable contribution with strong implications for ecological theory and sustainable land management. The framing of trade-offs in biodiversity and carbon accumulation trade-offs under N enrichment is compelling, and your dataset is impressive in both spatial and habitat breadth. Figures are clear, well-designed, and effectively highlight key results. I have carefully reviewed the revised manuscript and response letter, and I commend the authors for their thoughtful and thorough responses. The changes effectively address prior reviewer concerns and improve the manuscript substantially. That said, I believe a few minor revisions are still warranted to enhance clarity, sharpen the narrative, and deepen interpretation. My specific comments are as follows:\nResponse: We sincerely thank the reviewer for participating in this round of review and for the positive and encouraging evaluation of our study. We are particularly grateful for the recognition of the quality of the revised manuscript and previous responses, as well as the acknowledgment of its methodological rigor, conceptual novelty, and global relevance. We also greatly appreciate the constructive suggestions to further improve clarity, sharpen the narrative, and deepen the interpretation of key findings, especially regarding biodiversity-SOC trade-offs, underlying mechanisms, and identified threshold effects. We have carefully considered all suggestions and revised the manuscript accordingly. Below, we provide detailed point-by-point responses.\nComment 1: The manuscript hints at trade-offs between biodiversity loss and SOC accumulation, but this idea could be introduced earlier and more explicitly, including in the Introduction. Currently, it appears for the first time in the abstract once and at the very end of the introduction, where it is only listed, without major explanation. “Additionally, we quantified the critical thresholds of N inputs for the shift of plant and soil microbial communities and SOC accumulation and identified the point at which their trade-off is minimized.” Providing some theoretical context would help frame the narrative and guide the reader through the results.\nResponse: Thank you for this valuable comment. We fully agree that the concept of trade-offs between biodiversity loss and SOC accumulation is central to our study and should be introduced earlier and with greater clarity. To address this concern, we now introduced this concept in the first paragraph in the revision, following the context of SOC and biodiversity. Specifically, we now added how biodiversity and SOC may respond divergently to N enrichment and underlying mechanisms, from plant to soil microbial. Specifically, we added several sentences to introduce N enrichment can reduce biodiversity and alter community composition, particularly among plants, leading to disrupted plant-microbe interactions and decreased functional complementarity. These shifts collectively lower resource use efficiency and may hinder SOC accumulation, highlighting a potential trade-off between biodiversity conservation and carbon storage in N-enriched ecosystems. This framing helps establish the conceptual foundation for a potential biodiversity-carbon trade-off, providing a clearer narrative that guides the reader from background to the concept.\nThe recognized relevant sentences in first paragraph of Introduction section as:\n“…The inconsistencies may arise from differences in substrate quantity, mineral associations, environmental conditions and biodiversity13. Biodiversity, in particular, is widely recognized to regulate soil C cycling14,15, with its loss driving large-scale changes in ecosystem energy flow and nutrient cycling16,17. Higher plant diversity often increases biomass inputs and root exudation18, while soil microbial diversity governs the balance between C catabolism (decomposition) and anabolism (biomass and extracellular production)19-21, thereby shaping SOC dynamics. N enrichment generally reduces biodiversity22-25, primarily through mechanisms such as competitive exclusion, soil acidification and cation toxicity24,26. Yet microbial diversity exhibits inconsistent responses27,28. For example, while N enrichment significantly reduced overall microbial diversity, the decline was mainly observed for bacteria, whereas fungal diversity remained unchanged27. This highlights the complex and taxon-specific nature of microbial responses to N enrichment. Therefore, such divergent responses between plant and microbial diversity may weaken the positive biodiversity effects on SOC sequestration29,30. Moreover, the potential erosion of functional diversity could further decrease resource-use efficiency and ecosystem productivity, posing a major challenge for sustaining SOC accumulation while mitigating biodiversity loss14,19,22,31-33.…”. (Line 40-55)\nComment 2: While the pattern of plant diversity-SOC trade-offs is related to the competitive exclusion hypothesis, the mechanisms underlying these trade-offs remain underdeveloped. For example, how does biodiversity loss disrupt carbon stabilization pathways (e.g., through microbial C use efficiency, functional trait diversity, or soil aggregation). Expanding a bit on this would strengthen the ecological interpretation.\nResponse: Thank you for this constructive suggestion. We agree that the ecological mechanisms underlying the observed trade-offs between biodiversity and SOC accumulation require further clarification. In the revised Introduction (paragraph 1), we have expanded the theoretical context to better explain how biodiversity loss may disrupt carbon stabilization pathways. Specifically, we incorporated the concept of biodiversity complementarity, highlighting that both plant and microbial diversity jointly contribute to SOC stabilization through distinct but interconnected pathways. Diverse plant communities supply heterogeneous inputs to soils (e.g., litter and root exudates), while diverse microbial communities regulate decomposition and anabolic processes through shifts in carbon use efficiency and functional metabolism. These synergistic effects promote efficient carbon cycling and storage. We further clarified that N enrichment typically exerts negative effects on both plant and microbial diversity, via mechanisms such as competitive exclusion and acidification. These losses in diversity may weaken the ecological interactions between plants and soil microbes, reduce their combined biomass and metabolic efficiency, and thereby limit their capacity to retain SOC. This expanded ecological framework strengthens the theoretical basis for interpreting the trade-offs and supports our identification of thresholds where SOC gains may come at the cost of biodiversity decline. The revised sentences were in Line 40-55. Please see details for our response for Comment 1.\nComment 3: The concept of \"co-benefits\" is useful (not sure to be honest), but could be more clearly defined. Co-benefits appear in the title and abstract, and at the end of the introduction once each (three times in total). However, it is never mentioned again in the manuscript, not in the discussion.\nResponse: We appreciate your insightful comment. We fully agree you that the concept of “co-benefits” requires clearer articulation and follow-through in the manuscript. In response, we have first defined its introduction to explicitly denote co-benefits as the concurrent achievement of enhanced soil carbon storage and minimized biodiversity loss under N enrichment. In response, we firstly added this definition before the last sentence in the Introduction section. The revised sentences as: “Additionally, we quantified the critical thresholds of N inputs at which shifts in plant and microbial communities and SOC accumulation occur, and identified a defined window where soil C gains can be achieved with minimal biodiversity loss. This framework allowed us to fully assess the role of biodiversity in mediating SOC and reveal the critical N thresholds for co-benefits of biodiversity conservation and soil C accumulation globally in a N-enriched world.”. (Line 93-98)\nWe then expanded the discussion after the moving-window analysis to explicitly demonstrate where and how these “co-benefits” manifest along the N gradient. Specifically, our results revealed a distinct mid-level N range (~50-74 kg N ha⁻¹·yr⁻¹) where biodiversity loss and SOC dynamics were positively coupled, corresponding to a transitional synergy phase before the onset of high-N co-decline. This window represents a critical point where both carbon storage and biodiversity conservation goals can be jointly realized, forming the empirical basis for our use of the term “co-benefits”. We now clearly highlight this phase in the concluding paragraph of the Results and Discussion section as: “Collectively, these results delineate a three-phase trajectory: low-N decoupling, mid-N synergy, and high-N functional breakdown. This trajectory underscores a mechanistic shift from N-stimulated C gains to biodiversity-governed constraints under saturation. Importantly, our results showed that the intermediate range (~50-74 kg N ha⁻¹ yr⁻¹) emerges as a potential co-benefits threshold for maintaining SOC accumulation without severe biodiversity loss. Overall, this context-dependent transition from synergy to trade-off underscores the complex and multifaceted consequences of biodiversity loss for SOC accumulation. Limiting N inputs to around 50 kg N ha⁻¹ yr⁻¹ could help mitigate biodiversity loss and its negative effects on SOC storage, supporting long-term ecosystem stability.”. (Line 286-294)\nLastly, we emphasized this concept in the Conclusion as: “…In doing so, the identification of actionable N input thresholds can inform sustainable N management strategies, delivering co-benefits for biodiversity conservation and long-term carbon retention.”. (Line 326-328)\nComment 4: Meta-analyses can’t infer causation, so the authors should soften deterministic phrasing (e.g., “biodiversity drives SOC” and reword as “biodiversity is associated with SOC changes” or “mediates SOC responses”).\nResponse: We fully agree with the reviewer that causal language should be avoided in a meta-analytical framework, where inference is correlational rather than mechanistic. Accordingly, we have revised the manuscript to remove deterministic terms such as “drives” and “driver”, replacing them with more accurate and neutral alternatives such as “is associated with”, “predictor”, or “mediates”, depending on the context.\nComment 5: The fungal diversity pattern is intriguing (positive association with SOC despite negative response to N). This could benefit from a clearer explanation.\nResponse: We thank the reviewer for this important point. We acknowledge that the observed pattern, declining fungal diversity under N enrichment but a sustained positive association with SOC merits clearer interpretation. Given the meta-regression analysis and moving-window analysis both involved with the pattern and mechanisms of positive association with SOC while avoiding redundancy, we provide a concise explanation when first reporting this result in Lines 229-230, suggesting that the parallel responses of fungi and plants to N enrichment could root in the close functional integration between mycorrhizal fungi and their plant hosts. A more in-depth mechanistic explanation is presented in the subsequent section (Lines 266-268), based on our moving-window analysis (Fig. 4c), where we show that fungal communities maintain their role in stabilizing SOC through contributions to aggregate formation and carbon protection even under elevated N.\nThe clearer explanation was added as: “As N input exceeded this threshold, the association progressively became positive (reaching ~200 kg N ha⁻¹ yr⁻¹), marking a co-decline phase where intensified N enrichment suppressed both biodiversity and SOC. The response of fungal diversity fluctuated around ~67 kg N ha⁻¹ yr⁻¹ but maintained positively association with SOC across the N gradient (Fig. 4c), suggesting that fungi play a continuous and important role in SOC accumulation84. For example, certain ectomycorrhizal (ECM) fungi are capable of expressing oxidative enzymes that decompose recalcitrant organic matter85, while arbuscular mycorrhizal (AM) fungi may enhance MAOM formation through efficient hyphal turnover and microbial-derived pathways86,87.”. (Line 272-280)\nComment 6: Expand on mechanisms by which biodiversity changes might influence SOC (e.g., changes in litter quality, root exudates, microbial turnover).\nResponse: We sincerely thank the reviewer for this valuable comment. In response, we have expanded our explanation of the mechanisms by which diversity changes in bacteria, fungi, and plants may influence soil organic carbon (SOC), respectively. Specifically, we highlight that bacterial diversity loss under low N inputs may not immediately affect SOC due to microbial functional redundancy; fungal diversity, by contrast, maintains SOC through its structural contributions to aggregate formation and carbon stabilization; while plant diversity loss reduces the quantity and biochemical quality of organic inputs, ultimately constraining SOC accumulation.\nThe revised sentence for bacteria as: “The responses of bacterial diversity and SOC showed a slightly negative correlation below ~50 kg N ha⁻¹ yr⁻¹ (Fig. 4b), indicating a transient non-causal association. At this stage, the increase in SOC may directly benefit from aboveground productivity (e.g., leaf litter and root exudates)81,82, while the decline in bacterial species may be functionally compensated by redundant taxa83.”. (Line 269-272)\nThe revised sentence for fungi as: “The response of fungal diversity fluctuated around ~67 kg N ha⁻¹ yr⁻¹ but maintained positively association with SOC across the N gradient (Fig. 4c), suggesting that fungi play a continuous and important role in SOC accumulation84. For example, certain ectomycorrhizal (ECM) fungi are capable of expressing oxidative enzymes that decompose recalcitrant organic matter85, while arbuscular mycorrhizal (AM) fungi may enhance MAOM formation through efficient hyphal turnover and microbial-derived pathways86,87.”. (Line 275-280)\nThe revised sentence for plant as: “At low N inputs (< 50 kg N ha⁻¹ yr⁻¹), plant diversity and SOC responses were negatively (Fig. 4d), reflecting a decoupled pattern where diversity loss driven by light competition and cation toxicity co-occurred with ongoing SOC accumulation24. Between 50-74 kg N ha⁻¹ yr⁻¹, the association became strongly positive, indicating a shift toward synchronized biodiversity loss and slower or declining SOC gains. Above ~74 kg N ha⁻¹ yr⁻¹, the positive association weakened, likely reflecting reduced plant-derived inputs as severe plant diversity loss constrained new C incorporation into soils88.”. (Line 280-286)\nComment 7: The conclusion reiterates major findings but could more strongly emphasize: (i) How this work advances ecological threshold theory; (ii) The novelty of jointly analyzing plant and microbial diversity with SOC under N inputs, at a global scale; and (iii) The practical application of N input thresholds for land managers.\nResponse: We sincerely thank the reviewer for their valuable feedback. Our study advances ecological threshold theory by identifying critical N input thresholds that demonstrate how biodiversity loss accelerates once these thresholds are exceeded, which significantly reduces SOC accumulation. This provides a practical framework to address real-world challenges, illustrating how N enrichment and biodiversity loss interact to affect soil carbon dynamics. Additionally, we emphasize the novelty of our work in providing a global-scale analysis that jointly examines the impacts of above- and belowground biodiversity on SOC accumulation under N enrichment. This comprehensive analysis improves our understanding of the complex interactions between biodiversity and soil carbon dynamics. We also highlight the practical implications of our findings for land management, specifically that managing N inputs below 50 kg N ha⁻¹ yr⁻¹ could mitigate biodiversity loss while supporting SOC accumulation, benefiting both food security and ecosystem stability.\nThe revised Conclusion section as: “Overall, our study firstly develops a series of N enrichment thresholds that identify the critical inflection point for biodiversity and SOC responses under N enrichment. By jointly analyzing how above- and belowground community diversity mediates soil carbon dynamics, our findings demonstrate that intensified N inputs can induce nonlinear shifts in biodiversity-carbon relationships from synergy to decoupling and eventual collapse. In particular, once the threshold (e.g., ~50 kg N ha⁻¹ yr⁻¹) is exceeded, the sharp decline in biodiversity may offset the potential benefits of SOC accumulation. This global meta-analysis is the first to simultaneously evaluate plant and microbial diversity in relation to SOC across diverse ecosystems, offering an integrated perspective on how multi-trophic biodiversity regulates biogeochemical responses to N enrichment. This knowledge enables more precise prediction of the ecosystem carbon dynamics across diverse terrestrial biomes to future global changes, as aboveground and belowground responses are critical to maintaining and conservation ecosystem service. In doing so, the identification of actionable N input thresholds can inform sustainable N management strategies, delivering co-benefits for biodiversity conservation and long-term carbon retention.” (Line 315-328)\nMinor Comments\nComment 8: Clarify in the Methods whether the meta-regressions were weighted by variance.\nResponse: We thank the reviewer for pointing out the need for clarification. All meta-regressions in this study were fitted using the rma.mv function in the metafor package follow by previous studies (refs), with inverse-variance weighting applied by specifying effect sizes (yi) and their corresponding sampling variances (vi) as the dependent variables (V = vi). Here, vi denotes the sampling variance for each effect size, and the model assigns weights equal to the inverse of these variances, so that studies with higher precision (smaller variance) contribute proportionally more to the estimation. We have now added this information in the Methods as: “All models were fitted using inverse-variance weighting to account for differential contribution of each study99.”. (Line 434-435)\nComment 9: Figures are excellent overall.\nResponse: We sincerely thank the reviewer for the positive evaluation of our figures. We are pleased that the visual design and clarity successfully highlight the main findings, as presenting complex meta-analytic results in an accessible manner was a key objective of our figure preparation. We will ensure that all figures in the final version remain at publication quality, with high resolution and consistent formatting.\nSome sentences are awkward or ungrammatical, particularly in the Results and Discussion. Examples:\nComment 10: Line 169 ...nutrient limitation, not limitations\nResponse: Done. We have revised as you suggested.\nComment 11: Line 195 remove “the”...from theory that the biodiversity…\nResponse: Done. We have revised as you suggested.\nComment 12: Line196 replace…suggesting is an urgent need…” with “...suggesting the urgent need…”\nResponse: Done. We have revised as you suggested.\nComment 13: Line198 …we provide global evidence instead “the global evidence”\nResponse: Done. We have revised as you suggested.\nComment 14: Line 298 …by leveraging this knowledge, we can precisely predict...” to “This knowledge enables more precise prediction of...”\nResponse: Done. We have revised as you suggested.\nComment 15: Add transition sentences at the beginning of paragraphs. Some transitions are abrupt.\nResponse: We thank the reviewer for this helpful comment. We acknowledge the need to improve logical flow and narrative continuity, particularly in the Results and Discussion section. In response, we carefully reviewed the paragraph’s structure and added appropriate transition sentences at the beginning of relevant paragraphs to enhance coherence. The specific revisions are detailed below:\nIn the subsection titled “Effects of N enrichment on biodiversity and SOC” in the Results and Discussion section: (1) To clarify the shift from biodiversity to biomass responses, we added the sentence at the beginning of the second paragraph as: “B Beyond biodiversity loss, N enrichment also significantly reshaped biomass dynamics in both above- and belowground compartments.” (Lines 127-128); (2) To facilitate the transition from biotic to soil chemical responses, we added the sentence at the beginning of the third paragraph: “In addition to its biological effects, N enrichment also altered key soil chemical properties.” (Lines 148)\nIn the subsection titled “Biodiversity mediates SOC responses under global N enrichment” in the Results and Discussion section: (1) to clarify the analytical objective of this section and to provide a smooth conceptual transition into the random forest and meta-regression analyses, we inserted a bridging sentence at the beginning of the first paragraph as: “To understand how biodiversity and SOC respond to N enrichment, we first examined the main environmental predictors associated with diversity and SOC patterns, respectively.”. (Line 197-198); (2) To improve logical coherence and guide the reader through the transition from identifying independent predictors to examining their potential interdependencies, we inserted a bridging sentence at the beginning of the second paragraph as: “Beyond identifying the key predictors of biodiversity and SOC, it is essential to assess whether and how biodiversity and SOC responses are interlinked under N enrichment.”. (Line 220-221)\nComment 16: Some figure interpretations (especially Fig. 2 and Fig. 4) are dense. Consider breaking them down more explicitly. For example, Lines 236 to 240 or 245 to 248 these are long sentences.\nResponse: We appreciate the reviewer’s suggestion and fully agree that some figure interpretations were overly dense. In response, we revised the descriptions of Fig. 2 and Fig. 4 by reorganizing sentence structure, simplifying complex phrasing, and enhancing logical flow.\nThe revised paragraph about Fig. 2 as: “Given that plant and microbial biomass constitute the immediate foundation for SOC formation and turnover, we independently modelled the global responses of plant biomass, microbial biomass, and SOC under N enrichment to better elucidate ecosystem C dynamics (Fig. 2). Across regions, plant biomass exhibited consistently positive responses, especially in temperate and boreal regions of North America, Europe and Asia (Fig. 2a), reflecting N-stimulated productivity by N imputs65. In contrast, microbial biomass (MBC) exhibited greater spatial heterogeneity. Positive responses were prevalent in tropical and boreal regions, while declines were widespread across mid-latitude temperate ecosystems such as central North America, Europe and East Asia (Fig. 2b). This divergence could be attributed to regional disparities in baseline N deposition and soil sensitivity66. In tropical and boreal zones with low ambient N, moderate enrichment promotes microbial growth. However, excess N in high-background areas induces acidification and nutrient imbalance, ultimately suppressing microbial biomass67,68. Similarly, SOC responses were largely positive across temperate regions, particularly in Europe and northern Asia. However, negative responses prevailed in parts of the Southern Hemisphere and tropical zones, including southern Africa, South Asia and Southeast Asia (Fig. 2c). The contrasting trends are likely shaped by nutrient limitation and turnover dynamics that differ across ecosystems. In temperate ecosystems, N addition stimulates plant inputs while simultaneously suppressing decomposition, thereby favoring soil C accumulation69. In contrast, phosphorus limitation and high decomposition rates dominate in many tropical systems, where added N often fails to enhance biomass input and may even accelerate C loss70. Together, these spatial patterns illustrate how N enrichment reshapes C cycling across climatic gradients and emphasize the importance of region-specific N management aligned with soil C accumulation objectives.”. (Line 173-194)\nThe revised paragraph about Fig. 4 as: “Moreover, we performed a moving-window analysis to evaluate the potential shifts in the associations between the responses of diversity and SOC with increasing N (Fig. 4b-d and Fig. S8). The responses of bacterial diversity and SOC showed a slightly negative correlation below ~50 kg N ha⁻¹ yr⁻¹ (Fig. 4b), indicating a transient non-causal association. At this stage, the increase in SOC may directly benefit from aboveground productivity (e.g., leaf litter and root exudates)81,82, while the decline in bacterial species may be functionally compensated by redundant taxa83. As N input exceeded this threshold, the association progressively became positive (reaching ~200 kg N ha⁻¹ yr⁻¹), marking a co-decline phase where intensified N enrichment suppressed both biodiversity and SOC. The response of fungal diversity fluctuated around ~67 kg N ha⁻¹ yr⁻¹ but maintained positively association with SOC across the N gradient (Fig. 4c), suggesting that fungi play a continuous and important role in SOC accumulation84. For example, certain ectomycorrhizal (ECM) fungi are capable of expressing oxidative enzymes that decompose recalcitrant organic matter85, while arbuscular mycorrhizal (AM) fungi may enhance MAOM formation through efficient hyphal turnover and microbial-derived pathways86,87. At low N inputs (< 50 kg N ha⁻¹ yr⁻¹), plant diversity and SOC responses were negatively (Fig. 4d), reflecting a decoupled pattern where diversity loss driven by light competition and cation toxicity co-occurred with ongoing SOC accumulation24. Between 50-74 kg N ha⁻¹ yr⁻¹, the association became strongly positive, indicating a shift toward synchronized biodiversity loss and slower or declining SOC gains. Above ~74 kg N ha⁻¹ yr⁻¹, the positive association weakened, likely reflecting reduced plant-derived inputs as severe plant diversity loss constrained new C incorporation into soils88. Collectively, these results delineate a three-phase trajectory: low-N decoupling, mid-N synergy, and high-N functional breakdown. This trajectory underscores a mechanistic shift from N-stimulated C gains to biodiversity-governed constraints under saturation. Importantly, our results showed that the intermediate range (~50-74 kg N ha⁻¹ yr⁻¹) emerges as a potential co-benefits threshold for maintaining SOC accumulation without severe biodiversity loss. Overall, this context-dependent transition from synergy to trade-off underscores the complex and multifaceted consequences of biodiversity loss for SOC accumulation. Limiting N inputs to around 50 kg N ha⁻¹ yr⁻¹ could help mitigate biodiversity loss and its negative effects on SOC storage, supporting long-term ecosystem stability.”. (Line 267-294)\nAdditionally, we also corrected similar issues in other parts of the text during the revision.\nComment 17: This is an important and well-executed study with significant potential for impact, especially given the intensifying nitrogen deposition projected under future global change scenarios. I commend the authors on their thoughtful synthesis and extensive effort that went into this work. I thoroughly enjoyed reading the manuscript.\nResponse: We sincerely thank you for your kind and encouraging words. We are pleased to hear that the study was well-received and that our synthesis and efforts were appreciated. We hope that the revised manuscript meets the high standards for publication and contributes meaningfully to understanding the complex relationships between biodiversity, N enrichment, and soil carbon dynamics."}, {"round": 2, "reviewer_comments": {"reviewer_2": "The authors have addressed all the issues I raised. I suggest that the manuscript be accepted after this round.", "reviewer_3": "I have carefully reviewed the revised manuscript. The authors have substantively and thoughtfully addressed my previous comments, clarifying key arguments, enhancing details on the method section, and strengthening the overall coherence and impact of the work. The revisions reflect rigorous scholarship and improve the paper's contribution to the field. I congratulate the authors on these substantial improvements. I am satisfied with this current version and have no further comments."}, "author_response": ""}]

Decision letter