Plant traits explain variation in symbiotic nitrogen fixation responses to global nitrogen enrichment: a meta-analysis

Domain: chemistry
Published: 2026-02-20
Editorial outcome: major_revision

Abstract
Anthropogenic nitrogen enrichment is widely expected to suppress symbiotic nitrogen fixation in terrestrial ecosystems. Nevertheless, observed symbiotic nitrogen fixation responses remain incompletely explained by exogenous nitrogen inputs, climate, and edaphic factors. In this meta-analysis, we integrate 908 globally distributed field measurements to identify the key predictors that improve simulation of symbiotic nitrogen fixation responses to nitrogen enrichment. On average, symbiotic nitrogen fixation declines by 33.0% upon nitrogen enrichment, with the reduction being more pronounced in non-croplands than croplands. Models considering only environmental factors overestimate symbiotic nitrogen fixation decline relative to observations. The better performance of plant traits like plant growth and biomass allocation (shoot:root ratio) partially buffers symbiotic nitrogen fixation suppression under nitrogen enrichment. Integrating both environmental factors and plant performance traits improves predictive accuracy of symbiotic nitrogen fixation responses by 42.7% and brings the simulated symbiotic nitrogen fixation reductions into close agreement with observations. The alterations in plant performance traits are thus critical for explaining variability in terrestrial symbiotic nitrogen fixation responses, and incorporating plant trait dynamics in Earth System Models can quantitatively partition the compensatory symbiotic nitrogen fixation supported by nitrogen-fixing plant growth from the direct negative impact of nitrogen inputs.

Introduction
Symbiotic nitrogen (N) fixation (SNF), carried out by plant-microbe symbionts (e.g., rhizobia and Frankia), sustains critical terrestrial ecosystem functions, including biogeochemical N cycling and gross primary production1,2. Globally, SNF replenishes 50-70 Tg N yr−1 in croplands3,4 and 31-66 Tg N yr−1 in non-croplands1,4. Anthropogenic N enrichment, especially via atmospheric deposition and synthetic fertilizer inputs, has markedly altered SNF dynamics5,6. Consistent with carbon-economic theory, the declines in SNF under exogenous N sufficiency are confirmed, particularly for facultative fixers7,8. Yet the observed magnitude of N-induced SNF suppression remains poorly predicted by environmental factors alone9. In particular, substantial taxon-specific variation persists under comparable conditions10–12, implicating unaccounted plant traits (e.g., performance traits) as key regulators. However, the role of plant performance traits in predicting SNF responses to N enrichment remains largely overlooked, particularly at large spatial scales.

A comprehensive understanding of SNF responses and their predictors is essential to project terrestrial N budgets accurately, thus informing ecosystem functionality assessments under anthropogenic change.

Environmental factors, including exogenous N inputs, climate, and edaphic conditions, have been extensively investigated for their roles in regulationg SNF responses9,13. Carbon-economic theory posits that exogenous N inputs (e.g., nitrate and ammonium enrichment) suppress legume SNF, as direct N uptake is energetically cheaper than maintaining N-fixing symbionts14,15. Accordingly, legumes in N-rich conditions preferentially assimilate soil bioavailable N while down-regulating SNF16,17, thereby weakening plant-microbe mutualism under N enrichment18. This suppression operates mechanistically through reduced rhizobia colonization, diminished rhizobia populations, and curtailed nodule biomass17,19,20. Climatic and edaphic factors can also modulate SNF sensitivity to N enrichment. For instance, SNF declines upon N enrichment were less pronounced at high mean annual temperatures9, while elevated soil carbon: N ratios and pH buffer diazotrophs against N inhibition13 and consequently attenuate SNF declines. Despite the importance of environmental factors, these factors explain only 2-33% of observed variation in SNF responses upon N enrichment9,13, leaving a large proportion of variability attributable to unidentified predictors.

Plant performance traits emerge as critical predictors of SNF responses to environmental change. Biomass allocation patterns substantially contribute to observed SNF variability21, e.g., in Glycine max, SNF increases by 35% as shoot biomass rises from 842.9 g m−2 to 1200.2 g m−2 22, while root biomass consistently correlates with SNF magnitude21. Higher shoot:root ratios often correlate with enhanced SNF, potentially through improved light interception, as light availability is a key factor regulating the symbiotic efficiency of legumes23,24. Given that N enrichment frequently enhances biomass accumulation and shoot:root ratios in N-fixing plants25–27, these trait adjustments may partially alleviate SNF declines by augmenting photosynthetic capacity and carbon supply to fuel energetically costly N fixation. Accordingly, we hypothesized that plant trait plasticity may explain a significant fraction of the residual heterogeneity in SNF responses to N enrichment (Hypothesis 1). Critically, the quantitative global-scale contribution of plant performance traits to SNF responses, beyond environmental predictors, has yet to be assessed.

Divergent baseline N conditions between non-croplands and croplands due to long-term anthropogenic management can fundamentally alter SNF responses to experimental N enrichment. Chronic fertilization in croplands elevates soil N availability while simultaneously disrupting legume-rhizobia mutualism and reshaping microbial communities28,29. Consequently, legumes in croplands may acclimatize to high exogenous N inputs and exhibit attenuated SNF sensitivity relative to non-croplands18,30,31. In contrast, N-fixing plants in non-croplands experience more marked declines in nodule biomass and SNF following N inputs due to limited microbial adaptation, abrupt symbiosis breakdown, and competitive exclusion pressure from non-fixing species7,32,33. Accordingly, we propose that N enrichment suppresses SNF more markedly in non-croplands than croplands (Hypothesis 2).

This study focuses on accurate SNF predictability rather than mechanistic causation, to identify plant predictors explaining heterogeneity in SNF responses to N enrichment across sites and taxa. Leveraging a global synthesis based on 908 field observations, we determine the dominant predictors of SNF responses to N enrichment. Two specific questions were addressed: (a) What proportion of variability in SNF responses is explained by environmental factors (exogenous N inputs, climate, and edaphic properties) versus plant performance traits? (b) What are the differences in SNF responses between croplands and non-croplands?

Results
Environmental forces are insufficient to explain SNF responses upon N enrichment

N enrichment reduced SNF (8.49 g N m−2 yr−1 of SNF upon N enrichment versus 9.51 g N m−2 yr−1 upon ambient conditions), partially attributable to decreased SNF per unit biomass (Fig. S1). On average, the weighted effect size of SNF was −0.40 (95% CI: –0.58 ~ −0.22), corresponding to a 33.0% global decline relative to controls (Fig. 1a). This suppression in SNF intensified poleward (slope = −0.01, p < 0.05) (Fig. S2), and SNF decline was aggravated with high levels of N enrichment rate (a slope of −0.16, p < 0.01) (Fig. 1b).

Environmental factors partially accounted for the global variation in SNF responses to N enrichment (Fig. S3). SNF decline attenuated in conditions with higher mean annual temperature (slope = 0.42, p < 0.05), while the effects of precipitation on SNF response were insignificant (p = 0.50; Fig. S3a, b). Edaphically, SNF decline amplified under lower soil pH (slope = 0.21, p < 0.05), greater carbon:N ratio (slope = −0.53, p < 0.05), and elevated available phosphorus (slope = −0.32, p < 0.05). Ambient soil ammonium (p = 0.17) and nitrate (p = 0.66) exerted negligible influences (Fig. S3c–g). Moreover, SNF decline was more pronounced in soils with higher microbial biomass carbon (slope = −0.33, p < 0.05) and microbial biomass N (slope = −0.41, p < 0.05) (Fig. S3h–i). Notably, N enrichment rate (relative importance = 37.4%) and soil carbon:N ratio (relative importance = 31.4%) emerged as dominant predictors of the variability of SNF responses at a global scale (Fig. 1). The combined factors (i.e., mean annual temperature, N enrichment rate, pH, soil carbon:N ratio, available phosphorus, the carbon and N contents in microbial biomass) explained approximately one third of the variability of SNF responses at a global scale (34.2%) (Fig. 1c).

Enhancement of plant performance traits ameliorated the SNF decline upon N enrichment

SNF response to N enrichment also diverged across plant taxa (ranging from −1.29 to 0.44; Fig. 2a). Plant trait plasticity buffers SNF declines upon N enrichment. The enhanced N-fixing plant biomass or shoot:root ratios upon N enrichment attenuated SNF decline, while changes in plant nutrient contents had little impact on SNF responses (Fig. 2). Specifically, SNF decline trended to neutralize with the enhancement of plant performance traits, with a slope of 0.47 for N-fixing plant biomass (p < 0.001) and 2.75 for the plant shoot:root ratios (p < 0.01), respectively (Fig. 2).

Integrating plant performance traits, soil properties, microbial traits, N enrichment rate, and climatic factors explained 48.8% of the variability in SNF responses to N enrichment (Fig. 3). This represents a substantial improvement of 42.7% (a relative increase in explanatory power) over the prediction considering merely environmental factors (34.2% explained variance in Fig. 1c), which was consistently confirmed by both linear mixed-effects model and structural equation model (SEM). In the SEM, soil carbon:N ratio was the crucial driver of the variability in SNF responses to N enrichment (relative importance = 35.8%), with a standardized coefficient of −0.43 (p < 0.001) (Fig. 3). Moreover, plant performance traits (relative importance = 24.6%) were also important for modulating SNF responses to N enrichment, with the standardized coefficient being 0.29 (p < 0.001) (Fig. 3). The N enrichment rate explained 11.7% of the relative importance in SNF responses with a standardized coefficient of −0.30 (p < 0.001) (Fig. 3). The remaining variables (i.e., mean annual temperature and microbial traits) collectively accounted for 24.6% of the relative importance in SNF responses (Fig. 3). Moreover, environmental factors indirectly mediated SNF responses upon N enrichment through plant performance trait modification, with a standardized coefficient of −0.56 for mean annual temperature (p < 0.001), 0.11 for N enrichment rate (p < 0.05), 0.41 for soil carbon:N ratio (p < 0.001), and 0.60 for microbial traits (p < 0.001), respectively (Fig. 3b).

Methods

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Comments on manuscript: NCOMMS-24-62012\nThe manuscript entitled \"Enhanced plant performance counteracts declines in symbiotic nitrogen fixation (SNF) upon\nnitrogen enrichment\" addressed an important topic of decrease in SNF under N fertilization using a meta-dataset of\npublished literatures. The main conclusion is that plant performance traits could be a previously neglected drivers of the\nSNF responses to N enrichment. Although the finding is interesting, there are quite a lot of flaws to draw this conclusion.\nPlease see my comments below.\nFirst of all, the knowledge gap of this study is a bit questionable. The study raised a seemly interesting but rarely\ndemonstrated topic of the SNF responses – SNF remains unexpectedly high in some N-rich ecosystems. Nevertheless,\nsuch knowledge gap, from this study, is mainly based on the observation from ‘free-living’ and ‘associated’ N\nfixation, but not ‘symbiotic’ N fixation. For this point, there are quite a few citations that are incorrect or misleading (e.g., Line 59-60).\nAs a previous N paradox hypothesized, free-living N fixers are active in many tropical forests due to N-homogeneous\ndistribution (Hedin et al. 2009, also shown in the citation list). This hypothesis has been well demonstrated by many field\nstudies. By contrast, symbiotic N fixation, performed by legume plants, are quite different. Although there are two N-fixing\nstrategies (obligate and facultative), both of them face the same fate of disadvantage of SNF under N-rich environment. For\nexample, facultative N fixers downregulate fixation rates, and obligate N fixers are substituted by non-N fixers. Based on the\nfact above, the starting point (knowledge gap) of the study should be re-organized or alternatively, more lines of evidence\nare needed for supporting this point.\nAdditionally, the conceptual framework of plant performance that regulates SNF responses are not persuasive enough.\nParticularly, in the Introduction, such possibility is based on relationships of some plant-growth variables, but why ‘Plant\nPerformance Does Alleviate the Negative N Impacts on SNF’? What is the driver of this observation? Besides, it is difficult to\ndemonstrate whether N addition/fixation affect plant performance or plant performance affects the responses of SNF. These\nissues should be clearly addressed, not only simply basing on certain relationships and observation.\nThe study aimed to distinguish the contribution from environmental factors and plant performance traits, but the size of the\ncurrent dataset is small. Only a few soil properties (e.g., pH, C, and N), microbial biomass and climatic factors were\nconsidered. Many other factors that are also important for supporting SNF process, such as micronutrients (Mo and V),\nphosphorus availability, soil moisture, and light availability, were not evaluated. Lack of evaluating these potential factors\ncould lead to draw a conclusion of ‘low’ contribution from environmental factors.\nImportantly, the selected ‘plant performance traits (biomass, N, P, and shoot/root)’ may not be the best predictors. These\nvariables are usually thought to be affected by N fertilization and SNF. We could expect that N input (from fertilization and\nfixation) change plant biomass, N, P, and shoot/root, but it is less thought that these plant-growth variables in turn affect SNF\n(or SNF responses). They look like the ‘results’ but not the ‘drivers’. This is particularly true, given that meta-analysis method\nis commonly based on relationships of the selective variables, and thus possibly leads to unexpected ‘high’ contribution\nobserved in this study.\nInstead, there are some truly ‘drivers’ like leaf photosynthesis, plant N demand (N pools), and evapotranspiration, which\ncould change the resource (e.g., carbohydrate) and energy investment of plants into root nodules and SNF. Unfortunately,\nsuch types of variables are not considered in this study.\nWhile different tree species have divergent capacity of SNF, I am happy to see ‘plant species’ being included as an\ninfluencing factor. Furthermore, I would like to see ‘age’ of plants as a predictor, because different ages/growth-phases of N-\nfixing plants also show different capacity of SNF. Thus, the age of N-fixing plants may affect SNF in response to N\nfertilization and soil N availability.\nMinor comments:\nL 100. It is unclear why one of the hypotheses is to examine the responses of SNF between cropland and non-cropland\nsystems? Is there any typical characteristic for cropland systems when you pointed to SNF?\nL 103-105. This hypothesis appears to compare by different N fertilization rates, but how it will happen if both types of\nsystems receive the same rates of N input?\nL 106. What does ‘legume populations’ mean? Legume plant biomass, diversity, or root nodule biomass?\nL 106-107. This inference is oddness. How to understand the croplands receiving high N fertilization and then additional N\ninputs?\nL133-134. This result is interesting, but SNF rates, in many previous researches, were usually measured in the labs. MAT\nshould be a poor predictor compared to soil temperature.\nL199. Again, it is NOT clear why make such comparison. Do all the other ecosystems (forests and grasslands) have the\nsame responses of SNF to N fertilization?\nL232. Compare with non-croplands, croplands are affected more by anthropogenic management than environmental factors.\nL271-275. As pointed above, N input could change plant biomass, but how to demonstrate that the change in plant biomass\ncan affect N input (SNF)?\nL276. So, photosynthesis is a more effective predictor than biomass.\nL292-295. Again, why increase in temperature could offset the declines in SNF? Does it happen in high-latitude areas?", "reviewer_2": "Synopsis\nThe manuscript by Yao et al. presents a global analysis assessing the importance of plant performance traits (biomass and\nshoot:root ratios) when estimating SNF in terrestrial ecosystems and how SNF responds to N enrichment. The authors use a\ndataset of 64 papers (908 observations) providing data on SNF rates and various aspects of the ecosystem (abiotic\ncharacteristics as well as plant performance traits). They conclude that while N enrichment decreases SNF under N\nenrichment, that including plant performance traits in the model mitigates this SNF decrease (and improves model\npredictions of SNF). The authors also note that the declines in SNF are stronger in non-agricultural systems than in\nagricultural systems. Overall, the idea of this analysis is a valuable one, but the text focuses so heavily on the statistical\n the statistical\nanalyses themselves that the biological interpretation for what these results mean in a real-world context is difficult to\ndiscern. Below are a set of general and specific comments that may help the authors improve their work.\nGeneral Comments\n• A major missing piece to the data/analyses you present is the delineation between different aspects of measuring SNF.\nYou looked at SNF exclusively in terms of “N fixed per m2 per year” which has N-fixer abundance/biomass “baked in” to the\nestimate. I think the crux of the phenomenon you are trying to convey is that even if individual SNF rates (N fixed per unit N-\nfixer biomass) decrease with N fertilization, as long as N-fixer biomass increases, this will mitigate those declines. Your\nargument as a whole would be much stronger if you could present data on individual SNF rate declines separately from N-\nfixer biomass/abundance responses, and then put those together to get the overall response of SNF at the scale of “N fixed\nper m2 per year.”\n• Is the “plant biomass” performance trait a metric of N-fixer biomass responses, or all plants in the plot? Particularly in non-\nagricultural systems, I would assume that some fertilization studies saw an increase in total plant biomass but an exclusion of N-\nfixing species under fertilization treatments.\n• More information on how you converted data from the papers you used into individual pairs of control-treatment plots is\nneeded. The discrepancy between the number of individual studies (68) and the number of data points (908) you used is\nvery large. It’s not uncommon to obtain multiple independent samples from a single study in a meta-analysis, but a >10x\nincrease in sample size from study to data point seems very large. I also think you should provide justification for using\nmultiple plots at the same site as independent observations.\n• The declines in SNF in response to N enrichment (such as those you report in lines 229-241) are reported in % decline.\nWhat is the unit of N enrichment for these declines? Given that your data points span a range of N enrichment levels, a\nsingle % decline is difficult to understand. In other words, does a 39% decline in SNF mean a 39% decline per X amount of\nN added, or simply SNF declines by 39% when N is added regardless of how much N was added?\n• It’s difficult to understand what you mean when you say the models with only environmental variables “overpredict the\ndecline in SNF due to N enrichment relative to the observed decline.” It seems what you’ve done is used the data on SNF\ndeclines and the site-level environmental data to build a statistical model, and then you’ve built a second set of statistical\nmodels that also incorporate plant performance. In the first set (environmental factors only), I can see how only adding in\nthese factors makes the model a poor predictor (i.e. low R2), but the model shouldn’t systematically over- or under-predict\nthe observations. The statistical model is made to slightly over-predict some observations and under-predict others in order\nto get a “best fit” approximation. I may be missing some fundamental aspect to how you conducted your statistics. If so,\nI apologize, but would suggest that this needs to be explained much more clearly.\n• The authors make an idle attempt to include an analysis of SNF responses in croplands vs. non-croplands. Not much effort\nis made in the introduction to identify the knowledge gap or importance of SNF responses across ecosystem types. At least\none paragraph in the introduction should be dedicated to the importance of SNF responses in differing ecosystem types and\nadditional text in the discussion focusing on why we might expect these different responses between agricultural and non-\nagricultural systems.\nSpecific Comments\nL51-52. This needs to be generalized. Legumes and rhizobia are not the only nodulating plant symbiosis that fixes nitrogen.\nActinorhizal N-fixers and Frankia bacteria also do this and are the dominant N-fixers in many ecosystems. (indeed, it looks\nlike you include several actinorhizal N-fixers in your analyses (Fig 2a)).\n- A follow up point to the delineation between legumes and actinorhizal N-fixers: The prevailing paradigm is that actinorhizal N\nfixers have an “obligate fixation strategy” where they fix N at the same rates per unit biomass regardless of ecosystem N\navailability. Your data in Fig 2a provide solid evidence that this is not the case (that old paradigm is progressively falling,\nwhich your data add to its demise). This is not particularly relevant to the main ideas of your paper, but an interesting side\nuse of these data.\nL53. There are many more appropriate citations than the Mason paper for the importance of SNF to GPP (SNF really isn’t a\nprimary focus of that paper). The Davies-Barnard paper (your current citation #4) or Weider et al. 2015 Environmental\nResearch Letters would be good alternatives.\nL55-56. This sentence implies that we have solid evidence for alterations to natural SNF. The Galloway paper certainly does\nnot discuss that and I’m not sure the rock weathering paper does either. This sentence needs better support, given how\ncritical it is to the foundation of the paper.\nL70. Citation 11 is for a lichen, which is typically considered asymbiotic N fixation (certainly not the legume-rhizobia\nsymbiosis that the text refers to).\nL112-113: Are these two SNF values significantly different from each other or are you describing a general trend?\nL115-116: This part of the sentence is a bit unclear\nL117: add “is” after SNF?\nL145: This may be more accurately described as “approximately one third of the variability”\nL150: Rewrite this sentence, the way it reads is a bit unclear\nL186: Use another word besides responsibility\nL201: Use of the word “minor” seems a bit too strong considering we are talking about global SNF responses (for specific\necosystem types), the environmental factors accounted for >30% of variation\nL209: “indispensable” is very value-based term. “most important” might be a better alternative.\nL210: delete or change the word “Besides” in this sentence\nFigure 4: Make it more clear which graphs represent croplands and non-croplands by including titles on your graph\nL231-235: This sentence needs to be re-worded\nL248: Change which to what\nL254: Delete yet\nL255: Add “a” after only and change “had” to “have”\nL261: Not sure where you get 40.3% from, and are you talking about improvements for overall SNF models or cropland/non-\ncropland models\nL273-275. Following up on the general comment on this topic, the plant biomass response to N enrichment could be very\ndifferent from the N-fixer biomass response to N enrichment. How did you verify that the response of the entire plant\npopulation is indicative of the biomass responses of N fixers?\nL278: Add “system” after “root”?\nL278. “locus” should be “loci” and “rhizobia” should be changed to “N-fixing bacterial symbionts” to include Frankia\nL299: Delete “the” after “prefer”, add an s on “neutral soil”, and replace “instead of the” with “over”\nL312-315: Expand on this idea more.\nL319-320: This part of the sentence is confusing and does not read well\nL325-326: This idea needs to be flushed out more, from the way it reads now it feels randomly thrown in the paragraph\nL326: This statement needs a citation\nL331-341: This paragraph feels a little unnecessary, I also found the section title to be misleading. With the current title, I\nthink you can start this section at ln 341. The first paragraph in this section does not provide much new insight\nL 337. This statement is too broad. ESMs vary widely in how they represent SNF, and many do allow for SNF to vary with\necosystem N availability or plant N demand, etc.\nL344-345: Even if with the lack of data on SNF strategy, it might be worth elaborating on incomplete downregulation for\nfacultative N-fixers. Even if you cannot\nL363-364: This language feels a bit strong. Conjectures about a mechanistic understanding of SNF model performance was\nonly provided in the discussion", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "I perused the entire manuscript entitled “Enhanced plant performance counteracts declines in symbiotic nitrogen fixation\nupon nitrogen enrichment” that has been submitted by Yao et al. It is an interesting work and well-presented, and has the\npotential for publication. I agree with the authors that plant performance is an indispensable predictor of SNF responses to N\nenrichment. However, I still have some comments and hope that these suggestions will be helpful in improving the\nmanuscript.\n(1) My major concern is that the authors try to establish a perspective that plant performance, like the environmental factors\ndirectly regulating the activities of rhizobia, is a potential determinant of SNF responses to N enrichment. Indeed, it has been\nwidely demonstrated that N enrichment can improve productivity and limit symbiotic nitrogen fixation, thereby impelling the\nN-fixing plants to uptake N from the soil relative to that from the SNF. Hence, there might raise a positive association\nbetween the increase in plant performance traits and decrease in SNF. Nevertheless, I do not think the SNF decline directly\nresults from the increase in plant performance traits. By contrast, the increase in plant performance traits might be due to the\ndecline of SNF which leads to plants obtaining N from soil by a cheap way rather than the energetically expensive process.\nTherefore, I suggest cautiously using some words (counteract, etc.) and providing more detailed information to illustrate how\nplant performance traits influence SNF under N enrichment conditions.\n(2) Line 53, The “biogeochemical N cycle” should be included in the ecosystem function besides the “gross primary\nproduction”, as the next sentence states the quantity of N introduced into the ecosystem.\n(3) Line 55, Not only synthetic N, but also atmospheric deposited N has affected the terrestrial ecosystems and altered the\ndynamics of SNF.\n(4) Line 66, Please add some references here.\n(5) Line 68, I am confused about the phrase “save photosynthates”. Theoretically, the enhanced N input reduces SNF and\nthe N-fixing plants tend to uptake N by a cheap process with root rather than the high energetic cost process through SNF,\nthus leading to the changes in the allocation of photosynthates between above- and below-ground growth.\n(6) Line 80, Delete the word “for”.\n(7) Line 81, It seems to talk about the same thing with the sentence mentioned above (see line 60-61). Please rephrase\nthese sentences.\n(8) Line 89-90, Many studies have shown that light is a key environmental factor regulating SNF, but the current analysis\ndoes not include light as an environmental factor. Does this reduce the explained proportion of environmental factors to SNF\ndecline?\n(9) Line 90-92, Please add more explanation on how plant performance counteracts the decline of SNF.\n(10) Line 106, Why did the authors use “population” instead of “species”, as numerous plant species are examined in this\nstudy?\n(11) Line 186-196, It is well known that the N enhancement has a direct effect on soil N availability and subsequently\ninfluence soil C/N. But in the SEM, I am confused about the absence of the relationship between soil C/N and N enrichment.\nBesides, please provide the parameters indicating the goodness of the SEM.\n(12) Line 271-280, Please provide more explanation on how shoot: root ratio influences SNF, although the authors have\nrespectively stated the effect of shoot biomass and root traits on SNF. This is important to reveal the mechanisms underlying\nthe decline of SNF with increasing in shoot: root ratio.\n(13) Line 293, The word “reduced” should be “increased”.\n(14) Line 314, This word “wild” should be in upright font. In addition, please see comment 11 about “population”.\n(15) Line 713, It should be “carbon to nitrogen ratio of soil”.\n(16) Line 712, In figure S4, this study found a weak relationship of SNF in response to MAP. I am wondering whether the\nauthors take irrigation conditions into account for each sampling site in the croplands when analyzing this relationship, as\nthere should be difference in SNF performance between irrigated and unirrigated plants in the croplands, which might\ninfluence the response of SNF to MAP."}, "author_response": "Responses to reviewers’ comments\nReviewer #1' comments:\nThe manuscript entitled \"Enhanced plant performance counteracts declines in\nsymbiotic nitrogen fixation (SNF) upon nitrogen enrichment\" addressed an important\ntopic of decrease in SNF under N fertilization using a meta-dataset of\npublished literatures. The main conclusion is that plant performance traits could be a previously\nneglected drivers of the SNF responses to N enrichment. Although the finding is\ninteresting, there are quite a lot of flaws to draw this conclusion. Please see my\ncomments below.\nResponse: We greatly appreciate the reviewer’s positive comments and valuable\nsuggestions. According to the suggestions, we have carefully improved the\nmanuscript. Please see our point-by-point responses below.\n1. First of all, the knowledge gap of this study is a bit questionable. The study raised a\nseemly interesting but rarely demonstrated topic of the SNF responses – SNF remains\nunexpectedly high in some N-rich ecosystems. Nevertheless, such knowledge gap,\nfrom this study, is mainly based on the observation from ‘free-living’ and ‘associated’ N\nfixation, but not ‘symbiotic’ N fixation. For this point, there are quite a few citations\nthat are incorrect or misleading (e.g., Line 59-60).\nResponse: Thank you very much for your valuable comment. We have carefully\nrevised the Introduction to clarify the central question we address is identifying\nthe predictors of observed variability in SNF responses among legumes, rather\nthan whether SNF remains high in N-rich ecosystems (please see lines 55-62 in\nrevised Manuscript).\nLines 55-62: “Consistent with carbon-economic theory, the decline in SNF\nunder exogenous N sufficiency are predicted for both obligate and facultative\nfixers 6,7. Yet observed magnitudes of N-induced SNF suppression remain poorly\npredicted by environmental factors alone 8. In particular, substantial taxon-\nspecific variation persists under comparable conditions 9,10, implicating\nunaccounted plant traits (e.g., performance traits) as key regulators. However, the\nrole of plant performance traits in predicting SNF responses to N enrichment\nremains largely overlooked, particularly at large spatial scales.”\nMoreover, we have doubled checked the references on SNF in revised\nManuscript (e.g., Lines 55-57 and 58-59). We hope these revisions have clearer\npresented the knowledge gap of this study and addressed the reviewer’s concern.\n2. As a previous N paradox hypothesized, free-living N fixers are active in many tropical\nforests due to N-homogeneous distribution (Hedin et al. 2009, also shown in the citation\nlist). This hypothesis has been well demonstrated by many field studies. By contrast,\nsymbiotic N fixation, performed by legume plants, are quite different. Although there\nare two N-fixing strategies (obligate and facultative), both of them face the same fate\nof disadvantage of SNF under N-rich environment. For example, facultative N fixers\ndownregulate fixation rates, and obligate N fixers are substituted by non-N fixers.\nBased on the fact above, the starting point (knowledge gap) of the study should be re-\norganized or alternatively, more lines of evidence are needed for supporting this point.\nResponse: Thanks to the insightful comment. We agree with the reviewer’s\nviewpoint that the mechanisms underlying the responses of free-living N fixation\nand SNF may differ under nitrogen enrichment. The “N paradox” hypothesis,\nproposed by Hedin et al. (2009), was used to explain the persistence of free-living\nN fixation in N-rich tropical ecosystems, which has not be directly applied to SNF.\nTo address this concern, we have removed potentially misleading phrases such as\n“SNF remains high” or “SNF is unexpectedly active,” which may have implied\nanomalous or counter-theoretical responses. Moreover, the purpose of this study\nis to improve predictive capability of SNF responses under N enrichment, rather\nthan to put forward a new mechanism (please see lines 55-62 in revised\nManuscript).\nLines 55-62: “Consistent with carbon-economic theory, the decline in SNF\nunder exogenous N sufficiency are predicted for both obligate and facultative\nfixers 6,7. Yet observed magnitudes of N-induced SNF suppression remain poorly\npredicted by environmental factors alone 8. In particular, substantial taxon-\nspecific variation persists under comparable conditions 9,10, implicating\nunaccounted plant traits (e.g., performance traits) as key regulators. However, the\nrole of plant performance traits in predicting SNF responses to N enrichment\nremains largely overlooked, particularly at large spatial scales.”\nWe hope that these revisions have clarified the study's rationale and\naddressed the reviewer’s concern regarding the starting point.\n3. Additionally, the conceptual framework of plant performance that regulates SNF\nresponses are not persuasive enough. Particularly, in the Introduction, such possibility\nis based on relationships of some plant-growth variables, but why ‘Plant Performance\nDoes Alleviate the Negative N Impacts on SNF’? What is the driver of this observation?\nBesides, it is difficult to demonstrate whether N addition/fixation affect plant\nperformance or plant performance affects the responses of SNF. These issues should be\nclearly addressed, not only simply basing on certain relationships and observation.\nResponse: Thank you very much for this critical and insightful comment. We fully\nagree that the original manuscript may have overemphasized the potential of plant\nperformance traits to \"alleviate\" the negative impact of nitrogen enrichment on\nSNF, which could be misinterpreted as a causal mechanism rather than a statistical\nassociation.\nTo address this issue, we have carefully revised both the Introduction and\nDiscussion to emphasize that our primary aim is to assess whether plant\nperformance traits can serve as predictors of SNF variability under N enrichment,\nwithout implying a direct cause-effect relationship (please see lines 85-86, 86-89\nand 94-97 in revised Manuscript).\nLines 85-86: “Plant performance traits emerge as critical predictors of SNF\nresponses to environmental change.”\nLines 86-89: “Biomass allocation patterns substantially contribute to\nobserved SNF variability 20, e.g., in Glycine max, SNF increases by 35% as shoot\nbiomass rises from 842.9 g m-2 to 1200.2 g m-2 21, while root biomass consistently\ncorrelates with SNF magnitude 20,22. Higher shoot:root ratios often associate with\nenhanced SNF”\nLines 94-97: “Accordingly, we hypothesized that plant trait plasticity may\nexplain significant heterogeneity in SNF responses (Hypothesis 1). Critically, the\nquantitative global-scale contribution of plant traits to SNF responses, beyond\nenvironmental predictors, remains unassessed.”\nTo avoid misunderstanding, we also have carefully revised the manuscript,\nand replaced phrases such as “regulate,” “drive,” or “alleviate” in the Discussion\nwith neutral terms like “associated with,” “linked to,” or “predict”.\nWe have also revised the sentences in the lines 273-277, 295-297 and 299-301\nof revised manuscript.\nLines 273-277: “Despite a declining trend in SNF upon N enrichment, SNF\nresponses exhibited considerable variability (Figure 1a), implicating factors\nbeyond exogenous N inputs may be also important to explain SNF responses\n(Figure 1b). Plant performance traits, particularly biomass and shoot:root ratios,\nexhibit strong associations with SNF responses upon N enrichment (Figures 3-4).”\nLines 295-297: “Overall, these trait-mediated pathways explain significant\nglobal heterogeneity in SNF responses upon N enrichment (Figures 1 and S3).”\nLines 299-301: “Nevertheless, these findings highlight that plant performance\ntraits can serve as potential statistical predictors to improve the predictive\naccuracy of SNF responses in earth system models.”\nFurthermore, we have added the statement in the Discussion (please see lines\n297-299 in revised Manuscript).\nLines 297-299: “The associations are not analogous to direct causation, and\nfuture experimental studies are needed to determine the causality between trait\nchanges and plant-microbe symbiont alterations.”\nWe therefore believe that the revised manuscript is more clear.\n4. The study aimed to distinguish the contribution from environmental factors and plant\nperformance traits, but the size of the current dataset is small. Only a few soil properties\n(e.g., pH, C, and N), microbial biomass and climatic factors were considered. Many\nother factors that are also important for supporting SNF process, such as\nmicronutrients (Mo and V), phosphorus availability, soil moisture, and light availability,\nwere not evaluated. Lack of evaluating these potential factors could lead to draw a\nconclusion of ‘low’ contribution from environmental factors.\nResponse: Thank you very much for your thoughtful and critical comment. The\nprimary objective of this study is not to determine which variables are more\nimportant, but rather to evaluate whether plant performance traits can serve as\nthe practical predictors of SNF variability.\nWe absolutely agree that several environmental factors - such as\nmicronutrients (e.g., Mo and V), phosphorus availability, soil moisture, and light\navailability - may play important roles in regulating SNF by influencing microbial\nactivity and plant–microbe interactions. Unfortunately, data of these variables in\nexisting field studies were scarcity, making it difficult to systematically include\nthem in our synthesis. We have added more information in Discussion in the\nrevised manuscript (please see lines 422-427 and 382-384 in revised Manuscript).\nLines 422-427: “The dataset encompasses variation in site conditions (e.g.,\nclimatic and edaphic factors), species identity, N treatment rate, and plant\nperformance traits (plant biomass, shoot:root ratios, N and phosphorus contents).\nThe data capture key sources of heterogeneity across studies, although some\ninfluential variables, such as micronutrients (Mo and V), soil moisture, and light\navailability, plant ontogeny, and finer-resolution physiological traits, were rarely\nreported across studies.”\nLines 382-384: “Secondly, predictors critical to SNF regulation, such as\nmicronutrients (Mo and V), soil moisture, light availability, plant ontogeny, and\nfiner-resolution physiological traits (e.g., photosynthesis, and internal N status),\ncould not be integrated due to data paucity.”\nNevertheless, we have incorporated available data on soil phosphorus and re-\nrun the mixed-effect models, and the core conclusion is robust: plant performance\ntraits still significantly enhanced the predictability of SNF responses to N\nenrichment (Figures 1-4).\nMoreover, we have revised the manuscript to further emphasize that our\nobjective is not to compare the relative importance of environmental variables\nversus plant performance traits, but rather to assess whether plant performance\ntraits can enhance the prediction of SNF responses beyond the meticulously\nfocused environmental variables (please see Title and lines 96-97 in revised\nManuscript).\nTitle: “Plant traits integration resolves systematic bias in symbiotic nitrogen\nfixation responses to global nitrogen enrichment”\nLines 96-97: “Critically, the quantitative global-scale contribution of plant\ntraits to SNF responses, beyond environmental predictors, remains unassessed.”\nWe hope these additional analyses and clarifications address the reviewer’s\nconcern and reinforce the robustness and interpretability of our findings under\nthe constraints of available global datasets.\n5. Importantly, the selected ‘plant performance traits (biomass, N, P, and shoot/root)’\nmay not be the best predictors. These variables are usually thought to be affected by N\nfertilization and SNF. We could expect that N input (from fertilization and fixation)\nchange plant biomass, N, P, and shoot/root, but it is less thought that these plant-growth\nvariables in turn affect SNF (or SNF responses). They look like the ‘results’ but not the\n‘drivers’. This is particularly true, given that meta-analysis method\nis commonly based on relationships of the selective variables, and thus possibly leads to unexpected ‘high’\ncontribution observed in this study.\nResponse: Thank you very much for this constructive advice. It is true that there\nare complex interactions between N addition, plant performance traits and SNF\nresponses. We acknowledge that variables such as biomass, plant N and\nphosphorus content and shoot:root ratios have often been considered as ‘outcomes’\nof SNF responses in some studies, and therefore need to be interpreted with extra\ncaution in terms of causality.\nNevertheless, we included these plant performance traits based on two main\nconsiderations:\n1. Predictive utility vs. mechanistic causality: The primary goal of this study\nis not to infer causality between SNF and environments, but rather to evaluate\nwhether plant performance traits can serve as the useful predictors of variability\nin SNF responses under nitrogen enrichment. In large-scale studies, plant\nperformances offer important insights for ecological modeling and projection.\n2. Ecological relevance and data availability: Plant traits such as biomass and\nshoot:root ratio reflects the integrated outcome of plant resource acquisition\nstrategies and are closely linked to carbon availability for SNF. They can act as\nproxies to indicate plant investment under changing nutrient conditions.\nFurthermore, these traits are among the most frequently reported variables in the\nliterature, making themselves available for parameters in modelling.\nTo avoid misunderstanding, we also have carefully revised the manuscript to\nremove language suggesting causality (e.g., “alleviate” or “drive”).\nWe also revised the relevant sentences to clarify that this study explores\npredictors of SNF variability rather than drivers of SNF change (please see Title,\nlines 57-60, 85-86, 94-97, 277-281 and 295-301 in revised Manuscript).\nTitle: “Plant traits integration resolves systematic bias in symbiotic nitrogen\nfixation responses to global nitrogen enrichment”\nLines 57-60: “Yet observed magnitudes of N-induced SNF suppression remain\npoorly predicted by environmental factors alone 8. In particular, substantial taxon-\nspecific variation persists under comparable conditions 9,10, implicating\nunaccounted plant traits (e.g., performance traits) as key regulators.”\nLines 85-86: “Plant performance traits emerge as critical predictors of SNF\nresponses to environmental change.”\nLines 94-97: “Accordingly, we hypothesized that plant trait plasticity may\nexplain significant heterogeneity in SNF responses (Hypothesis 1). Critically, the\nquantitative global-scale contribution of plant traits to SNF responses, beyond\nenvironmental predictors, remains unassessed.”\nLines 277-281: “Without direct physiological proxies, plant performance\ntraits are commonly reported and can reflect photosynthetic capacity,\ncarbohydrate availability, root architecture, and growth stage, serving as scalable\npredictors of symbiotic efficiency and SNF responses at spatial scales where\nmechanistic data are unavailable.”\nLines 295-301: “Overall, these trait-mediated pathways explain significant\nglobal heterogeneity in SNF responses upon N enrichment (Figures 1 and S3). The\nassociations are not analogous to direct causation, and future experimental studies\nare needed to determine the causality between trait changes and plant-microbe\nsymbiont alterations. Nevertheless, these findings highlight that plant\nperformance traits can serve as potential statistical predictors to improve the\npredictive accuracy of SNF responses in earth system models.”\n6. Instead, there are some truly ‘drivers’ like leaf photosynthesis, plant N demand (N\npools), and evapotranspiration, which could change the resource (e.g., carbohydrate)\nand energy investment of plants into root nodules and SNF. Unfortunately, such types\nof variables are not considered in this study.\nResponse: Thank you for this insightful comment. We fully agree that\nphysiological traits such as leaf photosynthetic rate, plant N demand, and\nevapotranspiration are drivers of SNF because they directly influence the carbon\nand energy allocation to sustain N-fixing symbioses. Ideally, such variables would\nbe included in large-scale analyses. However, one challenge in a global meta-\nanalysis is data availability. These finer-resolution physiological traits are rarely\nreported in field studies, making it difficult to incorporate them systematically into\nour cross-site synthesis (please see lines 425-427 in revised Manuscript).\nLines 425-427: “some influential variables, such as micronutrients (Mo and\nV), soil moisture, and light availability, plant ontogeny, and finer-resolution\nphysiological traits, were rarely reported across studies.”\nTo address this, we selected the commonly reported traits, e.g., plant biomass,\ntissue N content, and shoot:oot ratio, as the coarse proxies for physiological\nfunctions. Plant biomass can approximate the cumulative photosynthate;\nshoot:root ratio reflects resource allocation strategy; and tissue N content may\nindirectly represent nutrient demand. These traits are not mechanistic drivers per\nse, but they provide meaningful predictors of SNF variability at broad scales\nwithout physiological data. We have also clarified this rationale in the revised\nmanuscript and added more information (please see lines 277-281 and 381-384 in\nrevised Manuscript).\nLines 277-281: “Without direct physiological proxies, plant performance\ntraits are commonly reported and can reflect photosynthetic capacity,\ncarbohydrate availability, root architecture, and growth stage, serving as scalable\npredictors of symbiotic efficiency and SNF responses at spatial scales where\nmechanistic data are unavailable.”\nLine 381-384: “Secondly, predictors critical to SNF regulation, such as\nmicronutrients (Mo and V), soil moisture, light availability, plant ontogeny, and\nfiner-resolution physiological traits (e.g., photosynthesis, and internal N status),\ncould not be integrated due to data paucity.”\n7. While different tree species have divergent capacity of SNF, I am happy to see ‘plant\nspecies’ being included as an influencing factor. Furthermore, I would like to see ‘age’\nof plants as a predictor, because different ages/growth-phases of N-fixing plants also\nshow different capacity of SNF. Thus, the age of N-fixing plants may affect SNF in\nresponse to N fertilization and soil N availability.\nResponse: Thank you very much for this comment. We agree with the reviewer\nthat different developmental stages or ages of N-fixing plants may lead to\ndifferences in their SNF capacity. Unfortunately, the majority of studies did not\nreport explicit information on plant age, and plant age is not reliably inferred\nwithout a standard method across studies and ecosystems. As a result, we were\nunable to incorporate plant age as a predictor in the current analysis (please see\nlines 425-427 in revised Manuscript).\nLines 425-427: “some influential variables, such as micronutrients (Mo and\nV), soil moisture, and light availability, plant ontogeny, and finer-resolution\nphysiological traits, were rarely reported across studies.”\nWe also have added the statements (please see lines 277-281 and 381-384 in\nrevised Manuscript).\nLines 277-281: “Without direct physiological proxies, plant performance\ntraits are commonly reported and can reflect photosynthetic capacity,\ncarbohydrate availability, root architecture, and growth stage, serving as scalable\npredictors of symbiotic efficiency and SNF responses at spatial scales where\nmechanistic data are unavailable.”\nLine 381-384: “Secondly, predictors critical to SNF regulation, such as\nmicronutrients (Mo and V), soil moisture, light availability, plant ontogeny, and\nfiner-resolution physiological traits (e.g., photosynthesis, and internal N status),\ncould not be integrated due to data paucity.”\n8. Line 100. It is unclear why one of the hypotheses is to examine the responses of SNF\nbetween cropland and non-cropland systems? Is there any typical characteristic for\ncropland systems when you pointed to SNF?\nResponse: Thank you very much for your helpful comment. We agree that the\nreason for comparing cropland versus non-cropland needs to be more clearly\nexplained.\nSome characteristics in croplands due to long-term exposure to\nanthropogenic management (e.g., frequent fertilization) are distinct from non-\ncroplands, such as elevated soil inorganic N, altered microbial communities, and\nweakened rhizobia-legume symbioses (Fan et al. 2019; Sun et al. 2021). Under such\nconditions, SNF may be lowered.\nWe have clarified this rationale in the revised manuscript (please see lines 98-\n104 in revised Manuscript).\nLines 98-104: “Divergent baseline N conditions between non-croplands and\ncroplands due to long-term anthropogenic management can fundamentally alter\nSNF responses to experimental N enrichment. Chronic fertilization in croplands\nelevates soil N availability while simultaneously disrupting legume-rhizobia\nmutualism and reshaping microbial communities 27,28. Consequently, legumes in\ncroplands upon exogenous N inputs may lead to SNF acclimatization and yield\nattenuated SNF sensitivity to N enrichment compared to non-croplands 15,29,30.”\n9. Line 103-105. This hypothesis appears to compare by different N fertilization rates,\nbut how it will happen if both types of systems receive the same rates of N input?\nResponse: Thanks for this insightful question. We agree that different SNF\nresponses between ecosystems derived from distinct ecological contexts when\necosystems receive the same rate of N input.\nOur hypothesis is based on the premise that croplands exhibit higher\ninorganic N and microbial changes to chronic N inputs due to long-term\nanthropogenic management (e.g., fertilization, tillage, crop selection). These\nlegacy effects may lead to SNF acclimatization to exogenous N inputs in croplands.\nMore pronounced SNF declines under N enrichment may appear in non-croplands\ncharacterized by lower inorganic N. Thus, even under equivalent N input rates,\nSNF responses can differ between these ecosystems.\nWe have clarified this rationale in the revised manuscript (please see lines 98-\n108 in revised Manuscript).\nLines 98-108: “Divergent baseline N conditions between non-croplands and\ncroplands due to long-term anthropogenic management can fundamentally alter\nSNF responses to experimental N enrichment. Chronic fertilization in croplands\nelevates soil N availability while simultaneously disrupting legume-rhizobia\nmutualism and reshaping microbial communities 27,28. Consequently, legumes in\ncroplands upon exogenous N inputs may lead to SNF acclimatization and yield\nattenuated SNF sensitivity to N enrichment compared to non-croplands 15,29,30. In\ncontrast, N-fixing plants in non-croplands experience more marked declines in\nnodule biomass and SNF following N inputs due to limited microbial pre-\nadaptation, abrupt symbiosis breakdown, and competitive exclusion pressure\nfrom non-fixing species 6,31,32. Accordingly, we propose that N enrichment\nsuppresses SNF more markedly in non-croplands than croplands (Hypothesis 2).”\n10. Line 106. What does ‘legume populations’ mean? Legume plant biomass, diversity,\nor root nodule biomass?\nResponse: Thank you. We have corrected the “legumes population” into “nodule\nbiomass” (please see lines 105 of revised manuscript).\n11. Line 106-107. This inference is oddness. How to understand the croplands receiving\nhigh N fertilization and then additional N inputs?\nResponse: Thank you very much. We have clarified this logic in the revised\nmanuscript (please see lines 98-104 in revised Manuscript).\nLines 98-104: “Divergent baseline N conditions between non-croplands and\ncroplands due to long-term anthropogenic management can fundamentally alter\nSNF responses to experimental N enrichment. Chronic fertilization in croplands\nelevates soil N availability while simultaneously disrupting legume-rhizobia\nmutualism and reshaping microbial communities 27,28. Consequently, legumes in\ncroplands upon exogenous N inputs may lead to SNF acclimatization and yield\nattenuated SNF sensitivity to N enrichment compared to non-croplands 15,29,30.”\n12. Line 133-134. This result is interesting, but SNF rates, in many previous researches,\nwere usually measured in the labs. MAT should be a poor predictor compared to soil\ntemperature.\nResponse: Thank you. Soil temperature is better than air temperature to explore\nthe SNF changes, especially given that SNF is sensitive to micro-environmental\nconditions. However, soil temperature was not available across the field studies.\nHence, we used the site-level mean annual temperature as a coarse-scale proxy for\nthermal conditions. Despite of less precise, mean annual temperature was\nsignificantly associated with SNF responses (Figure S3) (please see lines 306-309\nin revised Manuscript). In general, the relationship between N process rate\nconducted in incubation and mean annual temperature was reported in previous\nstudies (Wang et al., 2018).\nLines 306-309: “Mean annual temperature was just as a predictor\nrepresenting coarse-grained climate conditions, soil temperature was rarely\nreported across studies even as microbial activity and rhizobia-legume symbiosis\nis closer to soil temperature.”\n13. Line 199. Again, it is NOT clear why make such comparison. Do all the other\necosystems (forests and grasslands) have the same responses of SNF to N fertilization?\nResponse: Thank you for your comment. Our hypothesis is based on the premise\nthat croplands exhibit higher inorganic N and microbial changes to chronic N\ninputs due to long-term anthropogenic management (e.g., fertilization, tillage,\ncrop selection). These legacy effects may lead to SNF acclimatization to exogenous\nN inputs in croplands. More pronounced SNF declines under N enrichment may\nappear in non-croplands characterized by lower inorganic N. Thus, even under\nequivalent N input rates, SNF responses can differ between these ecosystems.\nWe have now added more information to the text in the revised manuscript\n(please see lines 98-108 in revised Manuscript).\nLines 98-108: “Divergent baseline N conditions between non-croplands and\ncroplands due to long-term anthropogenic management can fundamentally alter\nSNF responses to experimental N enrichment. Chronic fertilization in croplands\nelevates soil N availability while simultaneously disrupting legume-rhizobia\nmutualism and reshaping microbial communities 27,28. Consequently, legumes in\ncroplands upon exogenous N inputs may lead to SNF acclimatization and yield\nattenuated SNF sensitivity to N enrichment compared to non-croplands 15,29,30. In\ncontrast, N-fixing plants in non-croplands experience more marked declines in\nnodule biomass and SNF following N inputs due to limited microbial pre-\nadaptation, abrupt symbiosis breakdown, and competitive exclusion pressure\nfrom non-fixing species 6,31,32. Accordingly, we propose that N enrichment\nsuppresses SNF more markedly in non-croplands than croplands (Hypothesis 2).”\n14. Line 232. Compare with non-croplands, croplands are affected more by\nanthropogenic management than environmental factors.\nResponse: Thank you very much for this insightful comment. We fully agree that\ncroplands are strongly influenced by long-term anthropogenic management\npractices, such as fertilization, tillage and crop rotation, which may weaken the\napparent contribution of environmental factors to SNF variability in these systems.\nHowever, we would like to clarify that our analysis was based on paired\ncomparisons between control and N-enriched plots within each study, under the\nsame site-level managements. Therefore, anthropogenic management practices\nwere effectively held constant between control and N-enriched plots.\n15. Line 271-275. As pointed above, N input could change plant biomass, but how to\ndemonstrate that the change in plant biomass can affect N input (SNF)?\nResponse: Thank you very much for this insightful comment. There were\nfeedbacks between plant biomass changes and SNF responses. We did not aim to\ntest their causality, but rather to evaluate whether plant performance traits are\nessential for predicting SNF responses across ecosystems.\nWe have revised the relevant section to avoid misunderstanding (please see\nTitle, lines 85-86, 275-281 and 295-301 in revised Manuscript).\nTitle: “Plant traits integration resolves systematic bias in symbiotic nitrogen\nfixation responses to global nitrogen enrichment”\nLines 85-86: “Plant performance traits emerge as critical predictors of SNF\nresponses to environmental change.”\nLines 275-281: “Plant performance traits, particularly biomass and\nshoot:root ratios, exhibit strong associations with SNF responses upon N\nenrichment (Figures 3-4). Without direct physiological proxies, plant performance\ntraits are commonly reported and can reflect photosynthetic capacity,\ncarbohydrate availability, root architecture, and growth stage, serving as scalable\npredictors of symbiotic efficiency and SNF responses at spatial scales where\nmechanistic data are unavailable.”\nLines 295-301: “Overall, these trait-mediated pathways explain significant\nglobal heterogeneity in SNF responses upon N enrichment (Figures 1 and S3). The\nassociations are not analogous to direct causation, and future experimental studies\nare needed to determine the causality between trait changes and plant-microbe\nsymbiont alterations. Nevertheless, these findings highlight that plant\nperformance traits can serve as potential statistical predictors to improve the\npredictive accuracy of SNF responses in earth system models.”\n16. Line 276. So, photosynthesis is a more effective predictor than biomass.\nResponse: Thank you very much for this advice. Indeed, photosynthesis rate is a\nmore effective predictor for SNF responses. However, such data are rarely\nreported in field experiments and were unavailable in the large-scale dataset.\nHence, we selected the widely reported traits (e.g., biomass and shoot:root\nratio) as the coarse proxies for photosynthetic capacity (please see lines 277-281 in\nrevised Manuscript).\nLines 277-281: “Without direct physiological proxies, plant performance\ntraits are commonly reported and can reflect photosynthetic capacity,\ncarbohydrate availability, root architecture, and growth stage, serving as scalable\npredictors of symbiotic efficiency and SNF responses at spatial scales where\nmechanistic data are unavailable.”\n17. Line 292-295. Again, why increase in temperature could offset the declines in SNF?\nDoes it happen in high-latitude areas?\nResponse: Thank you for this comment. We have revised the discussion to clarify\nthe potential mechanism underlying more pronounced SNF declines in higher\nlatitudes (please see lines 302-306 in revised Manuscript).\nLines 302-306: “In terms of environmental factors, SNF decline upon N\nenrichment attenuated with increasing temperature (Figure S3), implying that\nwarming can partly counteract the decline of SNF, likely through enhanced plant\ngrowth and resultant carbon supply 20,42. A latitudinal pattern of SNF responses\n(Figure S2) further supports the role of temperature in dictating SNF under N\nenrichment.”\nReviewer #2' comments:\nThe manuscript by Yao et al. presents a global analysis assessing the importance of\nplant performance traits (biomass and shoot:root ratios) when estimating SNF in\nterrestrial ecosystems and how SNF responds to N enrichment. The authors use a\ndataset of 64 papers (908 observations) providing data on SNF rates and various\naspects of the ecosystem (abiotic characteristics as well as plant performance traits).\nThey conclude that while N enrichment decreases SNF under N enrichment, that\nincluding plant performance traits in the model mitigates this SNF decrease (and\nimproves model predictions of SNF). The authors also note that the declines in SNF are\nstronger in non-agricultural systems than in agricultural systems. Overall, the idea of\nthis analysis is a valuable one, but the text focuses so heavily on the statistical\nanalyses themselves that the biological interpretation for what these results mean in a\nreal-world context is difficult to discern. Below are a set of general and specific comments that\nmay help the authors improve their work.\nResponse: We greatly appreciate the reviewer for the positive and insightful\ncomments. According to these comments, we have carefully improved the\nmanuscript.\n1. A major missing piece to the data/analyses you present is the delineation between\ndifferent aspects of measuring SNF. You looked at SNF exclusively in terms of “N fixed\nper m2 per year” which has N-fixer abundance/biomass “baked in” to the estimate. I\nthink the crux of the phenomenon you are trying to convey is that even if individual SNF\nrates (N fixed per unit N-fixer biomass) decrease with N fertilization, as long as N-fixer\nbiomass increases, this will mitigate those declines. Your argument as a whole would\nbe much stronger if you could present data on individual SNF rate declines separately\nfrom N-fixer biomass/abundance responses, and then put those together to get the\noverall response of SNF at the scale of “N fixed per m2 per year.”\nResponse: We fully agree with your comment: Total SNF at the ecosystem scale\n(e.g., per m² per year) can be decomposed into two components: (1) individual SNF\nrate per unit N-fixer biomass and (2) the biomass or abundance of N-fixing plants.\nThis decomposition would enrich the interpretation of mechanisms behind SNF\nresponses to N enrichment. In the current study, however, we focused on total SNF\nper unit area as the integrated response variable. This decision was made based\non two main considerations:\n1. The methods (e.g., 15N isotope techniques and acetylene reduction assay)\nused to measure individual SNF rates per unit biomass vary substantially among\nprimary studies, making meta-analytic synthesis of these values difficult. In\ncontrast, area-based SNF estimates (e.g., g N m⁻² year⁻¹) are more consistently\nreported and allow for higher comparability across sites.\n2. The primary goal of this study was to assess whether incorporating plant\nperformance traits could improve the predictive capacity for SNF responses to\nnitrogen enrichment. For this purpose, area-based SNF responses reflect the net\necological effect - including both physiological and structural changes of N-fixing\nplants - and are thus more relevant to large-scale projections and Earth system\nmodel applications.\nDespite of these, we appreciate the conceptual importance of decomposing\nSNF responses into component processes. We have added a new figure (Figure 5)\nin the Supplementary Information that displays the decline of SNF per unit\nbiomass under N enrichment, based on a subset of studies reporting both total SNF\nand biomass. This analysis demonstrates that, on average, N enrichment reduces\nthe SNF efficiency per unit biomass, supporting the notion that physiological\ndownregulation plays a role in the observed SNF decline (please see lines 121-123\nin revised manuscript).\nLines 121-123: “N enrichment slightly reduced SNF (8.49 g N m-2 year-1 of\nSNF upon N enrichment versus 9.51 g N m-2 year-1 upon ambient condition, p =\n0.12), partially attributable to decreased SNF per unit biomass (Figure S1).”\nWe have also added information in the Discussion (please see lines 385-387 in\nrevised manuscript).\nLine 385-387: “Additionally, areal SNF rates (e.g., g N m⁻² year⁻¹) integrate\nboth physiological efficiency and diazotroph biomass. Though SNF per unit\nbiomass declined in available subsets (Figure S1), future works are needed to\nsystematically partition these components.”\n2. Is the “plant biomass” performance trait a metric of N-fixer biomass responses, or\nall plants in the plot? Particularly in non-agricultural systems, I would assume that\nsome fertilization studies saw an increase in total plant biomass but an exclusion of N-\nfixing species under fertilization treatments.\nResponse: Thank you very much for your valuable comment. In non-croplands, N\nenrichment often suppresses N-fixing plants through increasing competition from\nnon-N-fixing species. Therefore, it is essential to clarify what is meant by “plant\nbiomass” in our study.\nIn this study, plant biomass specifically refers to the biomass of N-fixing\nplants, not the total biomass of the plant community. This distinction is crucial to\navoid conflating responses of non-fixing species, which could obscure the\ninterpretation of SNF responses. Studies that only provided community-level\nbiomass, or failed to isolate N-fixing plant responses, were excluded from our\nsynthesis.\nTo avoid ambiguity, we have revised the manuscript by (1) consistently\nreferring to “N-fixing plant biomass” throughout the text, and (2) explicitly stating\nthis definition (please see lines 427-430 in revised manuscript).\nLines 427-430: “Here, plant performance traits (plant biomass, shoot:root\nratios, plant N and phosphorus contents) refer exclusively to N-fixing species\nrather than community-level vegetation, ensuring SNF responses reflect\ndiazotroph physiology.”\n3. More information on how you converted data from the papers you used into\nindividual pairs of control-treatment plots is needed. The discrepancy between the\nnumber of individual studies (68) and the number of data points (908) you used is very\nlarge. It’s not uncommon to obtain multiple independent samples from a single study\nin a meta-analysis, but a >10x increase in sample size from study to data point seems\nvery large. I also think you should provide justification for using multiple plots at the\nsame site as independent observations.\nResponse: Thanks for this valuable comment. To clarify, each unique comparison\nbetween a nitrogen-enriched treatment and its corresponding control - regarding\nplant species, N levels, or experimental units - was treated as an independent\nobservation. Importantly, we retained multiple observations from the same study\nonly when they were clearly independent in experimental design - for example, if\nthey represented different species, different treatment intensities, or spatially\nseparated plots. The substantial variation in SNF responses among different N-\nfixing species, even under similar environmental conditions, supports this\napproach (please see lines 419-421 in revised manuscript).\nLine 419-421: “where each unique comparison between a treatment and its\ncorresponding control - defined by distinct species, N levels, or experimental units\n- constituted an independent observation.”\nFurthermore, to more rigorously address statistical dependencies introduced\nby shared control groups, we incorporated a variance–covariance matrix into the\nmeta-analysis. This matrix explicitly captures the non-independence among lnRR\nvalues derived from multiple treatment groups sharing the same control group\nwithin a study (please see lines 455-481 in revised manuscript).\nMoreover, we also used linear mixed-effect models with study ID as random\neffects to account for the variation among studies (please see lines 490-495 in\nrevised manuscript).\n4. The declines in SNF in response to N enrichment (such as those you report in lines\n229-241) are reported in % decline. What is the unit of N enrichment for these declines?\nGiven that your data points span a range of N enrichment levels, a single % decline is\ndifficult to understand. In other words, does a 39% decline in SNF mean a 39% decline\nper X amount of N added, or simply SNF declines by 39% when N is added regardless\nof how much N was added?\nResponse: Thank you very much for this insightful comment. The reported\ndeclines (e.g., 39%) were calculated from the weighted mean of lnRR values and\nrepresent average differences between N-enriched and control treatments. Thus,\nthese values reflect a categorical comparison of SNF under N enrichment versus\nambient conditions.\nIndeed, N enrichment levels vary across studies. We have also presented a\nregression analysis (Figure 1b) examining the relationship between SNF responses\nand N enrichment rate, and the result showed that SNF decline aggravated with\nincreasing N input levels. We have clarified this distinction in both the Results\n(please see lines 123-125, 126-127 and 134-137 in revised manuscript).\nLines 123-125: “On average, the weighted effect size of SNF was -0.40 (95%\nCI: -0.58 ~ -0.22), corresponding to a 33% global decline relative to controls\n(Figure 1a).”\nLines 126-127: “and SNF decline was aggravated with high levels of N\nenrichment rate (a slope of -0.16, p < 0.01) (Figure 1b).”\nLines 134-137: “In a, the blue and red dot stand for the individual effect size\nand average effect size, respectively, based on comparisons between N-enriched\nand control plots. The percentage declines reflect average categorical differences\nrather than per-unit N input effects.”\n5. It’s difficult to understand what you mean when you say the models with only\nenvironmental variables “overpredict the decline in SNF due to N enrichment relative\nto the observed decline.” It seems what you’ve done is used the data on SNF\ndeclines and the site-level environmental data to build a statistical model, and then you’ve built\na second set of statistical models that also incorporate plant performance. In the first\nset (environmental factors only), I can see how only adding in these factors makes the\nmodel a poor predictor (i.e. low R2), but the model shouldn’t systematically over- or\nunder-predict the observations. The statistical model is made to slightly over-predict\nsome observations and under-predict others in order to get a “best fit” approximation.\nI may be missing some fundamental aspect to how you conducted your statistics. If so,\nI apologize, but would suggest that this needs to be explained much more clearly.\nResponse: Thank you very much for this important comment. The confusion may\nbe caused by the term “overpredict,” and we appreciate the opportunity to clarify.\nWe constructed a full linear mixed-effects model including both environmental\nfactors and plant performance traits rather than two models, to evaluate the role\nof environmental factors and/or plant performance traits on SNF simulating.\nThe earlier use of the term “overpredict” was not intended to suggest model\nbias or misbehaviour, but rather to indicate that the simulated SNF decline (e.g., –\n40.1%) based solely on environmental variables was greater than the observed\nvalue across all sites (e.g., -33.0%). To avoid confusion, we have revised the Results\nand Methods sections to clarify the approach (please see lines 240-251 and 517-525\nin revised manuscript).\n6. The authors make an idle attempt to include an analysis of SNF responses in\ncroplands vs. non-croplands. Not much effort is made in the introduction to identify the\nknowledge gap or importance of SNF responses across ecosystem types. At least\none paragraph in the introduction should be dedicated to the importance of SNF responses\nin differing ecosystem types and additional text in the discussion focusing on why we\nmight expect these different responses between agricultural and non-agricultural\nsystems.\nResponse: Thanks. We have revised the Introduction to highlight the importance\nof SNF comparison between ecosystem types (please see lines 98-108 in revised\nManuscript).\nLines 98-108: “Divergent baseline N conditions between non-croplands and\ncroplands due to long-term anthropogenic management can fundamentally alter\nSNF responses to experimental N enrichment. Chronic fertilization in croplands\nelevates soil N availability while simultaneously disrupting legume-rhizobia\nmutualism and reshaping microbial communities 27,28. Consequently, legumes in\ncroplands upon exogenous N inputs may lead to SNF acclimatization and yield\nattenuated SNF sensitivity to N enrichment compared to non-croplands 15,29,30.”\nWe have also revised the Discussion to further interpret the observed\ndifferences in SNF responses between croplands and non-croplands, from the\nperspective of soil conditions and legume–microbe associations (please see lines\n328-334, 339-343 and 345-352 in revised manuscript).\n7. Line 51-52. This needs to be generalized. Legumes and rhizobia are not the only\nnodulating plant symbiosis that fixes nitrogen. Actinorhizal N-fixers and Frankia\nbacteria also do this and are the dominant N-fixers in many ecosystems. (indeed, it\nlooks like you include several actinorhizal N-fixers in your analyses (Fig 2a)).\nA follow up point to the delineation between legumes and actinorhizal N-fixers: The\nprevailing paradigm is that actinorhizal N fixers have an “obligate fixation strategy”\nwhere they fix N at the same rates per unit biomass regardless of ecosystem N\navailability. Your data in Fig 2a provide solid evidence that this is not the case (that\nold paradigm is progressively falling, which your data add to its demise). This is not\nparticularly relevant to the main ideas of your paper, but an interesting side\nuse of these data.\nResponse: Thank you very much for this insightful comment. We agree that\nrhizobia-legume symbioses are not the only forms of symbiotic nitrogen fixation\n(SNF), and that actinorhizal N-fixers forming associations with Frankia bacteria\nalso play key roles in many ecosystems. We have revised the sentence in the\nIntroduction to more accurately reflect the diversity of symbiotic systems involved\nin SNF (please see lines 50-52 in revised manuscript).\nLines 50-52: “Symbiotic nitrogen (N) fixation (SNF), performed by plant-\nmicrobe symbiont (e.g., rhizobia and Frankia), sustains critical terrestrial\necosystem functions, including biogeochemical N cycling and gross primary\nproduction 1,2.”\nAdditionally, we also acknowledge that that SNF in actinorhizal species also\nexhibits measurable variation in response to N enrichment (please see lines 377-\n379 in revised manuscript).\nLines 377-379: “Notably, some actinorhizal species (traditionally considered\nobligate) exhibited significant SNF declines under N enrichment (Figure 2a),\nsuggesting broader plasticity than theorized.”\n8. Line 53. There are many more appropriate citations than the Mason paper for the\nimportance of SNF to GPP (SNF really isn’t a primary focus of that paper). The Davies-\nBarnard paper (your current citation #4) or Weider et al. 2015 Environmental\nResearch Letters would be good alternatives.\nResponse: Thank you for this advice, we have revised the citations (please see line\n52 in revised manuscript).\n9. Line 55-56. This sentence implies that we have solid evidence for alterations to\nnatural SNF. The Galloway paper certainly does not discuss that and I’m not sure the\nrock weathering paper does either. This sentence needs better support, given how\ncritical it is to the foundation of the paper.\nResponse: Thank you for this advice, we have corrected the citations (please see\nline 55 in revised manuscript).\n10. Line 70. Citation 11 is for a lichen, which is typically considered asymbiotic N\nfixation (certainly not the legume-rhizobia symbiosis that the text refers to).\nResponse: Thank you very much, we have corrected the citations (please see line\n75 in revised manuscript).\n11. Line 112-113: Are these two SNF values significantly different from each other or\nare you describing a general trend?\nResponse: Thanks, we have compared two SNF values using an independent-\nsamples t-test, and revised the sentence (please see lines 121-123 in revised\nmanuscript).\nLine 121-123: “N enrichment slightly reduced SNF (8.49 g N m-2 year-1 of SNF\nupon N enrichment versus 9.51 g N m-2 year-1 upon ambient condition, p = 0.12),\npartially attributable to decreased SNF per unit biomass (Figure S1).”\n12. Line 115-116: This part of the sentence is a bit unclear\nResponse: Thank you. We have revised the sentence (please see lines 125-126 in\nrevised manuscript).\nLines 125-126: “This suppression in SNF intensified poleward (slope = -0.01,\np < 0.05) (Figure S2)”\n13. Line 117: add “is” after SNF?\nResponse: Thanks. We have added “was” after SNF (please see line 126 in revised\nmanuscript).\n14. Line 145: This may be more accurately described as “approximately one third of\nthe variability”\nResponse: Thanks. We have changed the “less than half variability” into\n“approximately one third of the variability” (please see line 157 in revised\nmanuscript).\n15. Line 150: Rewrite this sentence, the way it reads is a bit unclear\nResponse: Thank you. We have rewritten the sentence (please see lines 162-163 in\nrevised manuscript).\nLines 162-163: “SNF response to N enrichment also diverged across plant\ntaxa (ranging from -1.29 to 0.44; Figure 2a).”\n16. Line 186: Use another word besides responsibility\nResponse: Thanks. We have changed the “responsibility” into “relative\ncontributions” (please see line 197 in revised manuscript).\n17. Line 201: Use of the word “minor” seems a bit too strong considering we are\ntalking about global SNF responses (for specific ecosystem types), the environmental\nfactors accounted for >30% of variation\nResponse: Thanks. We have revised the sentence (please see lines 212-214 in\nrevised manuscript).\nLines 212-214: “Environmental factors alone poorly explained the variability\nof SNF responses in croplands (37.0%) and non-croplands (33.0%) (Figures. 4b-\nc).”\n18. 209: “indispensable” is very value-based term. “most important” might be a better\nalternative.\nResponse: Thanks. We have revised it (please see lines 219-221 in revised\nmanuscript).\n19. Line 210: delete or change the word “Besides” in this sentence\nResponse: Thanks. We have changed the “Besides” to “while” (please see line 221\nin revised manuscript).\n20. Figure 4: Make it more clear which graphs represent croplands and non-croplands\nby including titles on your graph\nResponse: Thanks. We have added the titles to represent croplands and non-\ncroplands in Figure 6.\n21. Line 231-235: This sentence needs to be re-worded\nResponse: Thanks. We have rewritten the sentence (please see lines 243-246 in\nrevised manuscript).\nLines 243-246: “Similarly, the predicted SNF decline across ecosystem types\nbased on environmental variables alone (-22.3% and -43.0% for croplands and\nnon-croplands, respectively) were substantially greater than the observed ones (-\n15.9 and -40.7% for croplands and non-croplands, respectively) (Table 1).”\n22. Line 248: Change which to what\nResponse: Thanks. We have changed the “which” into “what” (please see line 258\nin revised manuscript).\n23. Line 254: Delete yet\nResponse: Thanks. We have deleted “yet” (please see line 262 in revised\nmanuscript).\n24. Line 255: Add “a” after only and change “had” to “have”\nResponse: Thanks. We have deleted this sentence (please see line 263 in revised\nmanuscript).\n25. Line 261: Not sure where you get 40.3% from, and are you talking about\nimprovements for overall SNF models or cropland/non-cropland models.\nResponse: Thanks. We have revised the sentence (please see lines 266-268 in\nrevised manuscript).\nLines 266-268: “Models incorporating these plant traits improved predictive\naccuracy of SNF responses by 42.7% over the frameworks with environmental\nfactors alone at a global scale (Table 1 and Figure 3)”\n26. Line 273-275. Following up on the general comment on this topic, the plant biomass\nresponse to N enrichment could be very different from the N-fixer biomass response to\nN enrichment. How did you verify that the response of the entire plant population is\nindicative of the biomass responses of N fixers?\nResponse: Thanks for your valuable comment. The “plant biomass” refers\nspecifically to the biomass of individual N-fixing plant. The peer-reviewed studies\ncomposing the dataset focuses on the performance of N-fixing plants under N\nenrichment, and are not the response of the entire plant population.\nTo avoid confusion, we have revised the “plant biomass” into “N-fixing plant\nbiomass” in the manuscript, and checked it throughout the manuscript, and\nexplicitly stated this definition (please see lines 427-430 in revised manuscript).\nLines 427-430: “Here, plant performance traits (plant biomass, shoot:root\nratios, plant N and phosphorus contents) refer exclusively to N-fixing species\nrather than community-level vegetation, ensuring SNF responses reflect\ndiazotroph physiology.”\n27. Line 278: Add “system” after “root”?\nResponse: Thanks. We have added “system” after “root” (please see line 286 in\nrevised manuscript).\n28. Line 278. “locus” should be “loci” and “rhizobia” should be changed to “N-fixing\nbacterial symbionts” to include Frankia\nResponse: Thank you for this advice. We have changed “locus” and “rhizobia”\ninto “loci” and “N-fixing bacterial symbionts”, respectively. (please see lines 286-\n287 in revised manuscript).\n29. Line 299: Delete “the” after “prefer”, add an s on “neutral soil”, and replace\n“instead of the” with “over”.\nResponse: Thanks. Done as suggested (please see lines 318 in revised manuscript).\n30. Line 312-315: Expand on this idea more.\nResponse: Thanks for this valuable advice. We have expanded the idea (please see\nlines 329-355 in revised manuscript).\n31. Line 319-320: This part of the sentence is confusing and does not read well\nResponse: Thanks for this advice. We have revised the sentence (please see lines\n340-344 in revised manuscript).\n32. Line 325-326: This idea needs to be flushed out more, from the way it reads now it\nfeels randomly thrown in the paragraph.\nResponse: Thanks for this valuable advice. We have rewritten the idea (please see\nlines 344-354 in revised manuscript).\n33. Line 326: This statement needs a citation\nResponse: Thanks for this valuable advice. We have added the citations (please see\nlines 353 in revised manuscript).\n34. Line 331-341: This paragraph feels a little unnecessary, I also found the section\ntitle to be misleading. With the current title, I think you can start this section at ln 341.\nThe first paragraph in this section does not provide much new insight\nResponse: Thanks for this valuable advice. We have revised this section as follows\n(please see lines 357-372 in revised manuscript).\n35. Line 337: This statement is too broad. ESMs vary widely in how they represent SNF,\nand many do allow for SNF to vary with ecosystem N availability or plant N demand,\netc.\nResponse: Thanks for the comment. We have removed this statement (please see\nlines 367-372 in revised manuscript).\n36. Line 344-345: Even if with the lack of data on SNF strategy, it might be worth\nelaborating on incomplete downregulation for facultative N-fixers. Even if you cannot\nResponse: Thanks for this valuable advice. Accordingly, we have revised the\nstatement (please see lines 373-381 in revised manuscript).\n37. Line 363-364: This language feels a bit strong. Conjectures about a mechanistic\nunderstanding of SNF model performance was only provided in the discussion.\nResponse: Thanks for this valuable advice. Accordingly, we have revised this\nlanguage (please see lines 397-399 in revised manuscript).\nReviewer #3' comments:\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse: Thank you for your efforts in reviewing our manuscript, along with the\nother reviewers.\nReviewer #4' comments:\nI perused the entire manuscript entitled “Enhanced plant performance counteracts\ndeclines in symbiotic nitrogen fixation upon nitrogen enrichment” that has been\nsubmitted by Yao et al. It is an interesting work and well-presented, and has the\npotential for publication. I agree with the authors that plant performance is an\nindispensable predictor of SNF responses to N enrichment. However, I still have some\ncomments and hope that these suggestions will be helpful in improving the manuscript.\nResponse: We greatly appreciate the reviewer’s positive comments.\n1. My major concern is that the authors try to establish a perspective that plant\nperformance, like the environmental factors directly regulating the activities of\nrhizobia, is a potential determinant of SNF responses to N enrichment. Indeed, it has\nbeen widely demonstrated that N enrichment can improve productivity and limit\nsymbiotic nitrogen fixation, thereby impelling the N-fixing plants to uptake N from the\nsoil relative to that from the SNF. Hence, there might raise a positive association\nbetween the increase in plant performance traits and decrease in SNF. Nevertheless, I\ndo not think the SNF decline directly results from the increase in plant performance\ntraits. By contrast, the increase in plant performance traits might be due to the\ndecline of SNF which leads to plants obtaining N from soil by a cheap way rather than the\nenergetically expensive process. Therefore, I suggest cautiously using some words\n(counteract, etc.) and providing more detailed information to illustrate how plant\nperformance traits influence SNF under N enrichment conditions.\nResponse: Thank you very much for this thoughtful and constructive comment.\nWe have revised the manuscript to more clearly state that our purpose is to\nevaluate whether plant performance traits are helpful to better simulate SNF\nresponse, particularly at large spatial scales (please see lines 110-111 and 299-301\nin revised Manuscript).\nLines 110-111: “This study focuses on accurate SNF predictability rather\nthan mechanistic causation”\nLines 299-301: “Nevertheless, these findings highlight that plant performance\ntraits can serve as potential statistical predictors to improve the predictive\naccuracy of SNF responses in earth system models.”\nWe also have carefully revised both the Introduction and Discussion to\nemphasize that our primary aim is to assess whether plant performance traits can\nserve as predictors of SNF variability under N enrichment, without exploring their\ncausality (please see lines 55-62, 85-86, 88-89 and 94-97 in revised Manuscript).\nTo avoid misunderstanding, we also have replaced phrases such as “regulate,”\n“drive,” or “alleviate” in the Discussion with neutral terms like “associated with,”\n“linked to,” or “predict” throughout manuscript.\nWe also have remodified the sentences (please see lines 273-277 and 295-298\nin revised Manuscript).\n2. Line 53, The “biogeochemical N cycle” should be included in the ecosystem function\nbesides the “gross primary production”, as the next sentence states the quantity of N\nintroduced into the ecosystem.\nResponse: Thanks for this advice. According to the reviewer’s comment, we have\nadded the “biogeochemical N cycle” into ecosystem function (please see lines 50-\n52 in revised Manuscript).\n3. Line 55, Not only synthetic N, but also atmospheric deposited N has affected the\nterrestrial ecosystems and altered the dynamics of SNF.\nResponse: Thanks for this valuable advice. We have revised the sentence (please\nsee lines 54-55 in revised Manuscript).\n4. Line 66, Please add some references here.\nResponse: Thanks, we have added the references (please see lines 67 in revised\nManuscript).\n5. Line 68, I am confused about the phrase “save photosynthates”. Theoretically, the\nenhanced N input reduces SNF and the N-fixing plants tend to uptake N by a cheap\nprocess with root rather than the high energetic cost process through SNF, thus leading\nto the changes in the allocation of photosynthates between above- and below-ground\ngrowth.\nResponse: Thank you very much for your valuable suggestion. We have revised\nthe sentence to avoid any ambiguity (please see lines 68-71 in revised Manuscript).\n6. Line 80, Delete the word “for”.\nResponse: Thanks, we have deleted the word “for” (please see lines 83 in revised\nManuscript).\n7. Line 81, It seems to talk about the same thing with the sentence mentioned above (see\nline 60-61). Please rephrase these sentences.\nResponse: Thank you for this advice, we have rewritten this sentence (please see\nlines 82-84 in revised Manuscript).\n8. Line 89-90, Many studies have shown that light is a key environmental factor\nregulating SNF, but the current analysis does not include light as an environmental\nfactor. Does this reduce the explained proportion of environmental factors to SNF\ndecline?\nResponse: Thanks for your valuable comment. We fully agree with the you and\nReviewer #1 that light is a key environmental factor regulating SNF, as it directly\naffects photosynthesis and thus the carbon supply for SNF. However, in practice,\nlight intensity is rarely quantified in field experiments. Moreover, the scarcity of\nglobal datasets about light information makes us unable to incorporate light as a\npredictor in data synthesis.\nDue to these limitations, light was not included as an environmental predictor\nin our current analysis. Nonetheless, we acknowledge that the absence of light data\nmay partially reduce the explanatory power of environmental variables, and we\nnow highlight this limitation (please see lines 381-384 and 425-427 in revised\nManuscript).\n9. Line 90-92, Please add more explanation on how plant performance counteracts the\ndecline of SNF.\nResponse: Thank you very much for your helpful comment. We have revised the\ncorresponding paragraph in the Introduction (please see lines 86-96 in revised\nManuscript).\n10. Line 106, Why did the authors use “population” instead of “species”, as numerous\nplant species are examined in this study?\nResponse: Thank you. We have corrected the “legumes population” into “nodule\nbiomass” for avoiding the ambiguous (please see lines 105 in revised Manuscript).\n11. Line 186-196, It is well known that the N enhancement has a direct effect on soil N\navailability and subsequently influence soil C/N. But in the SEM, I am confused about\nthe absence of the relationship between soil C/N and N enrichment. Besides, please\nprovide the parameters indicating the goodness of the SEM.\nResponse: Thank you for this valuable comment. We have re-constructed the\nstructural equation model (SEM), incorporating the direct effect of N enrichment\non soil carbon to N ratio. Additionally, we have provided the SEM fit indices in\nthe Figure 7.\n12. Line 271-280, Please provide more explanation on how shoot: root ratio influences\nSNF, although the authors have respectively stated the effect of shoot biomass and root\ntraits on SNF. This is important to reveal the mechanisms underlying the decline of\nSNF with increasing in shoot: root ratio.\nResponse: Thank you for this valuable comment. We have accordingly revised the\nmanuscript to elaborate on the underlying mechanisms (please see lines 290-295\nin revised Manuscript).\n13. Line 293, The word “reduced” should be “increased”.\nResponse: Thanks, we have revised this sentence (please see lines 302-303 in\nrevised Manuscript).\n14. Line 314, This word “wild” should be in upright font. In addition, please see\ncomment 11 about “population”.\nResponse: Thank you. We have italicized the word \"wild\" and changed\n\"population\" to \"nodule biomass\" (please see lines 335 in revised Manuscript).\n15. Line 713, It should be “carbon to nitrogen ratio of soil”.\nResponse: Thank you. We have changed \"carbon to nitrogen of soil\" to \" carbon\nto nitrogen ratio of soil \" (please see lines 768 in revised Manuscript).\n16. Line 712, In figure S4, this study found a weak relationship of SNF in response to\nMAP. I am wondering whether the authors take irrigation conditions into account for\neach sampling site in the croplands when analyzing this relationship, as there should\nbe difference in SNF performance between irrigated and unirrigated plants in the\ncroplands, which might influence the response of SNF to MAP.\nResponse: Thank you very much for your insightful comment. We agree that\nirrigation could influence the relationship between MAP and SNF, especially in\ncroplands. However, in this study, MAP was included only as a background\nclimatic factor rather than a key predictor. Meanwhile, due to limited reporting\non irrigation conditions across the collected studies, we were unable to\nsystematically account for irrigation practices. We have revised the discussion to\nclarify the role of MAP and the limitations in interpreting its effects (please see\nlines 309-314 in revised Manuscript).\nResponses to reviewers’ comments\nReviewer #2' comments:\nThe manuscript by Yao et al. presents a global analysis assessing the importance of\nplant performance traits (biomass and shoot:root ratios) when estimating SNF in\nterrestrial ecosystems and how SNF responds to N enrichment. The authors have done a thorough job of addressing the comments from the initial set\nof reviews. In particular, there were substantial improvements in the language around\nthe direction of causality between plant trait responses and SNF responses to N\nenrichment. The description of how the authors got 908 observations from 68 studies\nalso improved substantially.\nResponse: We greatly appreciate the reviewer’s positive comments.\n1. However, with the clarity provided in the updated description, I am less convinced of\nthe fundamental value of the analysis. In the end, the main conclusion of the paper is\n“N additions decrease SNF, but N additions also make N fixers grow larger, which\ndampens the negative effect of N additions on SNF.” This is an interesting finding, but\nwithout the ability to link any mechanistic causality to the analyses, it is hard to envision\nhow this conclusion would be used in future endeavors such as Earth System Models.\nModel development typically relies on improved mechanistic understanding of the\nregulation of SNF rather than phenomenological correlations.\nResponse: Thank the reviewer for this insightful advice. We agree that a\nmechanistic understanding, rather than a purely phenomenological correlation, is\ncritical for the developing of Earth System Models. However, effectively\nincorporating the mechanistic understanding into Earth System Models is a\nfundamental challenge, as environmental heterogeneity complicates the direct\nextrapolation to macroscales. Scaling up is a crucial step in reconciling microscale\nmechanistic insights with macroscale predictive frameworks (Li et al., 2025,\naccepted by Frontiers of Earth Science). Global analyses, such as those presented\nin our study, allow scaling up the mechanistic understanding of the compensatory\nSNF supported by plant growth under N enrichment by accounting for both direct\nand indirect effects in Earth System Models. In the revised manuscript, we have\nexpanded the Discussion to explicitly bridge our findings with model parameters\nand their applicability for model predictions (please see lines 318-319, 321-323,\nand 385-388 in the revised manuscript).\nMoreover, our work enables the quantitative partitioning of the positive effect\nof N-fixer growth from the negative effect of N deposition on biological N fixation,\nalthough a complete reformulation of Earth System Models remains aspirational.\nImportantly, the relative contribution of these two components varies significantly\nacross ecosystems—legumes in croplands exhibit stronger compensatory gains\nthan those in non-croplands (Table 1), highlighting the need for ecosystem-specific\nparameterizations in model development. In our revised manuscript, we have\nelaborated on how these identified mechanisms can be incorporated and\nparameterized (Eqns 8 and 9) within Earth System Model frameworks (please see\nlines 364-368, 388-392, 392-396, and 396-398 in the revised manuscript).\n2. The authors also miss the opportunity to discuss how they reconcile their plant traits\nresponses with the common finding that N enrichment decreases N-fixer biomass and\ncover (e.g., Tognetti et al. 2021, PNAS,\nhttps://www.pnas.org/doi/full/10.1073/pnas.2023718118).\nResponse: We thank the reviewer for this insightful and important comment. We\nagree that reconciling our findings with the observation that N enrichment\ndecreased the biomass and cover of N-fixing species (e.g., Tognetti et al., 2021,\nPNAS) is essential for a comprehensive interpretation. Such decreases can occur\nwhen the increased competitive ability of non-fixing species under N fertilization\nsuppresses N-fixers, particularly at low background soil N availability. However,\nthese reductions are not exclusively driven by N enrichment; a spontaneous\ndecline in N-fixer biomass has also been observed in unfertilized plots, indicating\nthat other ecological pressures beyond N availability may contribute to reductions\nin N-fixer abundance. We have clarified it in the revised manuscript (please see\nlines 294-298 in the revised manuscript).\n3. The other main conclusion is that these effects are not as strong in agricultural as\nthey are in non-agricultural ecosystems, but this line of inquiry in the paper remains\npoorly developed.\nResponse: Thanks the reviewer for the advice. We have added more discussion on\nthis topic (please see lines 331-338, 342-346 and 364-368 in the revised manuscript).\n4. I have provided a set of suggestions that I hope will be useful to the authors as they\nwork to improve the manuscript. Lines 50-53. Since the original submission of this\nmanuscript, a new global synthesis of SNF has been published that is based on a much\nlarger data set than the Davies-Barnard synthesis. I would suggest keeping the Davies-\nBarnard reference here and adding Reis Ely et al. 2025, Nature\n(https://www.nature.com/articles/s41586-025-09201-w).\nResponse: We sincerely appreciate the above insightful suggestions; we have\ncarefully incorporated them and substantially revised our manuscript accordingly.\nIndeed, the topic of SNF is highly important and has garnered widespread\nattention. During our review period, Nature published a paper on SNF (Reis Ely\net al. 2025) that marks a critical milestone in global-scale SNF estimation, which\nfurther motivated us to enhance this work. We also hope to have the opportunity\nto collaborate with members of Reis Ely’s research team on our next SNF-related\nproject. We have added this citation (please see lines 45-46 and 342-346 in the\nrevised manuscript).\n5. Line 67. Double check that your reference numbers got updated during revisions.\nHere in line 67 you have reference 19 show up before refs 11-18. I also feel like several\nof the references seem out of place (e.g., ref 11 would seemingly fit better with refs 9\nand 10), so double check that there isn’t some systematic issue with the references.\nResponse: Thank the reviewer for the kind advice. We have conducted a\nsystematic check and correction of reference citations throughout the manuscript\nto address this issue.\n6. Lines 209-212. These statements seem incompatible with each other. The first\nsentence says that including plant traits improved model accuracy by 63% and 56% in\nnon-croplands and croplands, but then the next sentence says that total explanatory\npower in these two groups is 60% and 51% (a total that is lower than the amount the\nmodel improved by).\nResponse: Thanks for the valuable comment. The first percentage (e.g., 63.5% and\n43.3%) represents the relative improvement in the model’s explanatory power,\nwhereas the second percentage (e.g., 60.5% and 51.6%) denotes the final absolute\nexplanatory power of the model after integrating plant traits (i.e., R2). We have\ncorrected the calculation error (43.3% instead of 56.4%) and revised the sentence\nto avoid any ambiguity (please see lines 207-209 in the revised manuscript).\n7. Results: In general throughout the results it is very hard for the reader to make sense\nof the many percentages that are reported. Which set of results should the reader rely\non, the percent changes in the SEMs or those in the mixed effects models? It is also\noften unclear exactly what the percent changes you are reporting mean. In a\nhypothetical example, if SNF decline due to N enrichment was -30% with just\nenvironmental factors and -25% when you add in plant traits, would you report that as\na 5% change (the difference between 30% and 25%) or a 16.66% change (the 5%\ndifference divided by the original value – so the proportional change)? My comment on\nlines 209-212 above suggests it’s the later, but this needs to be made clear.\nResponse: Thanks for this valuable advice. We agree with the reviewer’s\nsuggestion and have standardized the expressions of all percentage values and\nclearly clarified their meanings both in the text and figure legends.\n8. Line 263. This statement that models improve by 42.7% when plant traits are\nincluded is what is referenced in both the abstract and conclusions, but it’s hard to tell\nwhere this figure comes from in the results. Between the SEM and mixed-effects model\napproaches, it is very hard to understand which percentage improvements the reader\nshould be focusing on, and nowhere in the results do we see that plant traits improve\nthe model accuracy by 42.7%.\nResponse: We thank the reviewer for this comment. We have clarified the origin\nof the 42.7% improvement in model performance by revising the relevant\ndescription (please see lines 32-34, 170-173, 262-265 and 426-428 in the revised\nmanuscript).\n9. Lines 288-289. It doesn’t seem like there is any support/evidence for the statement\nmade about legumes producing fewer more efficient nodules when they have high\nshoot:root ratios.\nResponse: Thanks for this helpful comment. We have deleted the sentence (please\nsee line 288 in the revised manuscript).\n10. Lines 373-375. A recently published study on the time lags of SNF regulation in\nresponse to N additions supports this statement and would be a useful reference here.\nBytnerowicz et al. 2025 New Phytologist\n(https://nph.onlinelibrary.wiley.com/doi/full/10.1111/nph.70295).\nResponse: Thanks for this valuable advice. We have rewritten the sentence (please\nsee lines 404-406 in the revised manuscript).\n11. Lines 394. I believe NF should be SNF.\nResponse: We have corrected “NF” to “SNF” (please see line 426 in the revised\nmanuscript).\n12. Lines 515-523. I don’t see any mention of z-score standardizing the many\nindependent variables for which you are trying to compare their importance. Simply\ncomparing effect sizes of relationships for climate factors and plant traits doesn’t work\nunless those climate factors and plant traits have all been z-scored so that they are all\nin the same units, right?\nResponse: Thanks for this advice. We used the R package “glmm.hp” to calculate\nthe relative importance of each driving factor. This package is based on the\nprinciple of hierarchical partitioning, which quantifies the independent\ncontribution of each predictor variable to the total explained variance (R²) of the\nmodel. Because this method decomposes a dimensionless R², the results are\ntheoretically unaffected by the original scales of the independent variables.\nAlthough the calculation method is scale-invariant, we still standardized all\ncontinuous independent variables using Z-score normalization to ensure that our\nanalysis adhered to best practices in statistical modeling. We have added the\ndescription (please see lines 542-545 and 564-565 in the revised manuscript).\n13. Line 533. Do you mean “the a priori models”?\nResponse: Thanks. We have revised the sentence (please see lines 552-554 in the\nrevised manuscript).\nReviewer #4' comments:\nThe authors have thoroughly addressed all may comments and I have no further\ncomments.\nResponse: We greatly thank the reviewer for the time and effort they devoted to\nimproving our manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_2": "General Comments\nThe authors have done a thorough job of addressing the comments from the initial set of reviews. In particular, there\nwere substantial improvements in the language around the direction of causality between plant trait responses and SNF\nresponses to N enrichment. The description of how the authors got 908 observations from 68 studies also improved\nsubstantially. However, with the clarity provided in the updated description, I am less convinced of the fundamental value of\nthe analysis. In the end, the main conclusion of the paper is “N additions decrease SNF, but N additions also make N fixers\ngrow larger, which dampens the negative effect of N additions on SNF.” This is an interesting finding, but without the ability\nto link any mechanistic causality to the analyses, it is hard to envision how this conclusion would be used in future\nendeavors such as Earth System Models. Model development typically relies on improved mechanistic understanding of the\nregulation of SNF rather than phenomenological correlations. The authors also miss the opportunity to discuss how they\nreconcile their plant traits responses with the common finding that N enrichment decreases N-fixer biomass and cover (e.g.,\nTognetti et al. 2021, PNAS, https://www.pnas.org/doi/full/10.1073/pnas.2023718118). The other main conclusion is that\nthese effects are not as strong in agricultural as they are in non-agricultural ecosystems, but this line of inquiry in the paper\nremains poorly developed. I have provided a set of suggestions that I hope will be useful to the authors as they work to\nimprove the manuscript.\nSpecific Comments\nL50-53. Since the original submission of this manuscript, a new global synthesis of SNF has been published that is based\non a much larger data set than the Davies-Barnard synthesis. I would suggest keeping the Davies-Barnard reference here\nand adding Reis Ely et al. 2025, Nature (https://www.nature.com/articles/s41586-025-09201-w).\nL67. Double check that your reference numbers got updated during revisions. Here in line 67 you have reference 19 show\nup before refs 11-18. I also feel like several of the references seem out of place (e.g., ref 11 would seemingly fit better with\nrefs 9 and 10), so double check that there isn’t some systematic issue with the references.\nL209-212. These statements seem incompatible with each other. The first sentence says that including plant traits improved\nmodel accuracy by 63% and 56% in non-croplands and croplands, but then the next sentence says that total explanatory\npower in these two groups is 60% and 51% (a total that is lower than the amount the model improved by).\nResults: In general throughout the results it is very hard for the reader to make sense of the many percentages that are\nreported. Which set of results should the reader rely on, the percent changes in the SEMs or those in the mixed effects\nmodels? It is also often unclear exactly what the percent changes you are reporting mean. In a hypothetical example, if SNF\ndecline due to N enrichment was -30% with just environmental factors and -25% when you add in plant traits, would you\nreport that as a 5% change (the difference between 30% and 25%) or a 16.66% change (the 5% difference divided by the\noriginal value – so the proportional change)? My comment on lines 209-212 above suggests it’s the later, but this needs to\nbe made clear.\nL263. This statement that models improve by 42.7% when plant traits are included is what is referenced in both the abstract\nand conclusions, but it’s hard to tell where this figure comes from in the results. Between the SEM and mixed-effects model\napproaches, it is very hard to understand which percentage improvements the reader should be focusing on, and nowhere in\nthe results do we see that plant traits improve the model accuracy by 42.7%.\nL288-289. It doesn’t seem like there is any support/evidence for the statement made about legumes producing fewer more\nefficient nodules when they have high shoot:root ratios.\nL373-375. A recently published study on the time lags of SNF regulation in response to N additions supports this statement\nand would be a useful reference here. Bytnerowicz et al. 2025 New Phytologist\n(https://nph.onlinelibrary.wiley.com/doi/full/10.1111/nph.70295).\nL394. I believe NF should be SNF.\nL515-523. I don’t see any mention of z-score standardizing the many independent variables for which you are trying to\ncompare their importance. Simply comparing effect sizes of relationships for climate factors and plant traits doesn’t work\nunless those climate factors and plant traits have all been z-scored so that they are all in the same units, right?\nL533. Do you mean “the a priori models”?", "reviewer_4": "The authors have thoroughly addressed all may comments and I have no further comments."}, "author_response": "Responses to reviewers’ comments\nReviewer #2' comments:\nThe authors have done a thorough job of addressing the comments from the initial set\nof reviews. In particular, there were substantial improvements in the language around\nthe direction of causality between plant trait responses and SNF responses to N\nenrichment. The description of how the authors got 908 observations from 68 studies\nalso improved substantially.\nResponse: We greatly appreciate the reviewer’s positive comments.\n1. However, with the clarity provided in the updated description, I am less convinced of\nthe fundamental value of the analysis. In the end, the main conclusion of the paper is\n“N additions decrease SNF, but N additions also make N fixers grow larger, which\ndampens the negative effect of N additions on SNF.” This is an interesting finding, but\nwithout the ability to link any mechanistic causality to the analyses, it is hard to envision\nhow this conclusion would be used in future endeavors such as Earth System Models.\nModel development typically relies on improved mechanistic understanding of the\nregulation of SNF rather than phenomenological correlations.\nResponse: Thank the reviewer for this insightful advice. We agree that a\nmechanistic understanding, rather than a purely phenomenological correlation, is\ncritical for the developing of Earth System Models. However, effectively\nincorporating the mechanistic understanding into Earth System Models is a\nfundamental challenge, as environmental heterogeneity complicates the direct\nClassified as Internal | Intern\nextrapolation to macroscales. Scaling up is a crucial step in reconciling microscale\nmechanistic insights with macroscale predictive frameworks (Li et al., 2025,\naccepted by Frontiers of Earth Science). Global analyses, such as those presented\nin our study, allow scaling up the mechanistic understanding of the compensatory\nSNF supported by plant growth under N enrichment by accounting for both direct\nand indirect effects in Earth System Models. In the revised manuscript, we have\nexpanded the Discussion to explicitly bridge our findings with model parameters\nand their applicability for model predictions (please see lines 318-319, 321-323,\nand 385-388 in the revised manuscript).\nLines 318-319: “Moreover, climatic and edaphic factors regulate SNF\nresponses, both directly and indirectly via plant performance traits (Figure 3b).”\nLines 321-323: “Embedding both direct and indirect effects in models enables\na mechanistic scaling-up of SNF predictions that accounts for both environmental\nconstraints and plant-mediated compensation.”\nLines 385-388: “Global analyses, such as those presented here, allow scaling\nup the mechanistic understanding of the compensatory SNF supported by plant\ngrowth under N enrichment by accounting for both direct and indirect effects in\nEarth System Models.”\nMoreover, our work enables the quantitative partitioning of the positive effect\nof N-fixer growth from the negative effect of N deposition on biological N fixation,\nalthough a complete reformulation of Earth System Models remains aspirational.\nImportantly, the relative contribution of these two components varies significantly\nClassified as Internal | Intern\nacross ecosystems—legumes in croplands exhibit stronger compensatory gains\nthan those in non-croplands (Table 1), highlighting the need for ecosystem-specific\nparameterizations in model development. In our revised manuscript, we have\nelaborated on how these identified mechanisms can be incorporated and\nparameterized (Eqns 8 and 9) within Earth System Model frameworks (please see\nlines 364-368, 388-392, 392-396, and 396-398 in the revised manuscript).\nLines 364-368: “Recognizing the differences in the importance of predictors\nin different ecosystems is an essential step towards refining N fixation\nparameterizations in Earth System models. Accordingly, these models should\nadopt ecosystem-specific schemes 62, assigning driver weights (e.g., N enrichment\nrate, soil carbon:N ratio and available phosphorus) that reflect the biogeochemical\ncontext of croplands versus non-croplands.”\nLines 388-392: “Specifically, the empirical relationships derived here (Eqns\n8 and 9) can be implemented within N fixation subroutines of Earth System\nModels, where environmental drivers define the baseline limitation of SNF, and\nplant performance traits act as dynamic modifiers that enable models to simulate\nhow shifts in plant growth or carbon allocation mediate SNF responses.”\nLines 392-396: “The observed SNF compensation, as supported by plant\ngrowth under N enrichment, provides the quantitative relationships and driver\nsensitivities needed to mechanistically parameterize the dynamic representation\nof SNF under N enrichment in current models 62,66,67.”\nLines 396-398: “In that way, this study provides Earth System Models with\nClassified as Internal | Intern\nthe quantitative basis (Eqns 8 and 9) to explicitly distinguish between SNF\ncompensation generated by N-fixer growth and direct SNF suppression induced\nby N enrichment.”\n2. You also miss the opportunity to discuss how you\nreconcile their plant traits responses with the common finding that N enrichment decreases N-fixer biomass and cover (e.g., Tognetti et al. 2021, PNAS, https://www.pnas.org/doi/full/10.1073/pnas.2023718118).\nResponse: We thank the reviewer for this insightful and important comment. We\nagree that reconciling our findings with the observation that N enrichment\ndecreased the biomass and cover of N-fixing species (e.g., Tognetti et al., 2021,\nPNAS) is essential for a comprehensive interpretation. Such decreases can occur\nwhen the increased competitive ability of non-fixing species under N fertilization\nsuppresses N-fixers, particularly at low background soil N availability. However,\nthese reductions are not exclusively driven by N enrichment; a spontaneous\ndecline in N-fixer biomass has also been observed in unfertilized plots, indicating\nthat other ecological pressures beyond N availability may contribute to reductions\nin N-fixer abundance. We have clarified it in the revised manuscript (please see\nlines 294-298 in the revised manuscript).\nLines 294-298: “Occasionally, the sharp increase in the competitive ability of\nnon-fixing species following external N addition can cause a pronounced reduction\nin N-fixer abundance and biomass at low soil N availability 32, masking this SNF\nClassified as Internal | Intern\ncompensation. Despite these exceptions, recent global syntheses align with our\nfindings that positive growth is a pervasive attribute of N-fixers under N\nenrichment 27,46.”\n3. The other main conclusion is that these effects are not as strong in agricultural as\nthey are in non-agricultural ecosystems, but this line of inquiry in the paper remains\npoorly developed.\nResponse: Thanks the reviewer for the advice. We have added more discussion on\nthis topic (please see lines 331-338, 342-346 and 364-368 in the revised manuscript).\nLines 331-338: “Non-croplands are commonly phosphorus-limited 56,57; N\nenrichment can exacerbate this deficiency, tightening phosphorus constraints on\nthe N fixation process and ultimately leading to more pronounced SNF\nsuppression (e.g., phosphorus content is a key driver of SNF variability in Figure\n4c) 58. Moreover, under sustained exogenous N inputs, leguminous crops may\nundergo adaptive domestication that prioritizes soil N acquisition and yield over\nSNF 30,31. Such long-term adaptations lessen crop dependence on SNF, thereby\nbuffering SNF decline even under N enrichment 30,59.”\nLines 342-346: “Given that the global SNF in croplands now approximates\nthat in non-croplands 4 and that the SNF in these two ecosystem types exhibits a\ndifferent sensitivity to N enrichment (Figure 4 and Table 1), Earth System Models\nshould explicitly address the contrasting SNF dynamics in croplands and non-\ncroplands under anthropogenic perturbation.”\nClassified as Internal | Intern\nLines 364-368: “Recognizing the differences in the importance of predictors\nin different ecosystems is an essential step towards refining N fixation\nparameterizations in Earth System models. Accordingly, these models should\nadopt ecosystem-specific schemes 62, assigning driver weights (e.g., N enrichment\nrate, soil carbon:N ratio and available phosphorus) that reflect the biogeochemical\ncontext of croplands versus non-croplands.”\n4. I have provided a set of suggestions that I hope will be useful to the authors as they\nwork to improve the manuscript. Lines 50-53. Since the original submission of this\nmanuscript, a new global synthesis of SNF has been published that is based on a much\nlarger data set than the Davies-Barnard synthesis. I would suggest keeping the Davies-\nBarnard reference here and adding Reis Ely et al. 2025, Nature\n(https://www.nature.com/articles/s41586-025-09201-w).\nResponse: We sincerely appreciate the above insightful suggestions; we have\ncarefully incorporated them and substantially revised our manuscript accordingly.\nIndeed, the topic of SNF is highly important and has garnered widespread\nattention. During our review period, Nature published a paper on SNF (Reis Ely\net al. 2025) that marks a critical milestone in global-scale SNF estimation, which\nfurther motivated us to enhance this work. We also hope to have the opportunity\nto collaborate with members of Reis Ely’s research team on our next SNF-related\nproject. We have added this citation (please see lines 45-46 and 342-346 in the\nrevised manuscript).\nClassified as Internal | Intern\nLines 45-46: “Globally, SNF replenishes 50 – 70 Tg N yr-1 in croplands 3,4 and\n31 – 66 Tg N yr-1 in non-croplands 1,4.”\nLines 342-346: “Given that the global SNF in croplands now approximates\nthat in non-croplands 4 and that the SNF in these two ecosystem types exhibits a\ndifferent sensitivity to N enrichment (Figure 4 and Table 1), Earth System Models\nshould explicitly address the contrasting SNF dynamics in croplands and non-\ncroplands under anthropogenic perturbation.”\n5. Line 67. Double check that your reference numbers got updated during revisions.\nHere in line 67 you have reference 19 show up before refs 11-18. I also feel like several\nof the references seem out of place (e.g., ref 11 would seemingly fit better with refs 9\nand 10), so double check that there isn’t some systematic issue with the references.\nResponse: Thank the reviewer for the kind advice. We have conducted a\nsystematic check and correction of reference citations throughout the manuscript\nto address this issue.\n6. Lines 209-212. These statements seem incompatible with each other. The first\nsentence says that including plant traits improved model accuracy by 63% and 56% in\nnon-croplands and croplands, but then the next sentence says that total explanatory\npower in these two groups is 60% and 51% (a total that is lower than the amount the\nmodel improved by).\nClassified as Internal | Intern\nResponse: Thanks for the valuable comment. The first percentage (e.g., 63.5% and\n43.3%) represents the relative improvement in the model’s explanatory power,\nwhereas the second percentage (e.g., 60.5% and 51.6%) denotes the final absolute\nexplanatory power of the model after integrating plant traits (i.e., R2). We have\ncorrected the calculation error (43.3% instead of 56.4%) and revised the sentence\nto avoid any ambiguity (please see lines 207-209 in the revised manuscript).\nLines 207-209: “Incorporating plant performance traits considerably\nimproved predictive accuracy, enhancing the explanatory power by 63.5% in\ncroplands and 43.3% in non-croplands (Figures 4d-e).”\n7. Results: In general throughout the results it is very hard for the reader to make sense\nof the many percentages that are reported. Which set of results should the reader rely\non, the percent changes in the SEMs or those in the mixed effects models? It is also\noften unclear exactly what the percent changes you are reporting mean. In a\nhypothetical example, if SNF decline due to N enrichment was -30% with just\nenvironmental factors and -25% when you add in plant traits, would you report that as\na 5% change (the difference between 30% and 25%) or a 16.66% change (the 5%\ndifference divided by the original value – so the proportional change)? My comment on\nlines 209-212 above suggests it’s the later, but this needs to be made clear.\nResponse: Thanks for this valuable advice. We agree with the reviewer’s\nsuggestion and have standardized the expressions of all percentage values and\nclearly clarified their meanings both in the text and figure legends.\nClassified as Internal | Intern\n8. Line 263. This statement that models improve by 42.7% when plant traits are\nincluded is what is referenced in both the abstract and conclusions, but it’s hard to tell\nwhere this figure comes from in the results. Between the SEM and mixed-effects model\napproaches, it is very hard to understand which percentage improvements the reader\nshould be focusing on, and nowhere in the results do we see that plant traits improve\nthe model accuracy by 42.7%.\nResponse: We thank the reviewer for this comment. We have clarified the origin\nof the 42.7% improvement in model performance by revising the relevant\ndescription (please see lines 32-34, 170-173, 262-265 and 426-428 in the revised\nmanuscript).\n9. Lines 288-289. It doesn’t seem like there is any support/evidence for the statement\nmade about legumes producing fewer more efficient nodules when they have high\nshoot:root ratios.\nResponse: Thanks for this helpful comment. We have deleted the sentence (please\nsee line 288 in the revised manuscript).\n10. Lines 373-375. A recently published study on the time lags of SNF regulation in\nresponse to N additions supports this statement and would be a useful reference here.\nBytnerowicz et al. 2025 New Phytologist\n(https://nph.onlinelibrary.wiley.com/doi/full/10.1111/nph.70295).\nResponse: Thanks for this valuable advice. We have rewritten the sentence (please\nsee lines 404-406 in the revised manuscript).\n11. Lines 394. I believe NF should be SNF.\nResponse: We have corrected “NF” to “SNF” (please see line 426 in the revised\nmanuscript).\n12. Lines 515-523. I don’t see any mention of z-score standardizing the many\nindependent variables for which you are trying to compare their importance. Simply\ncomparing effect sizes of relationships for climate factors and plant traits doesn’t work\nunless those climate factors and plant traits have all been z-scored so that they are all\nin the same units, right?\nResponse: Thanks for this advice. We used the R package “glmm.hp” to calculate\nthe relative importance of each driving factor. This package is based on the\nprinciple of hierarchical partitioning, which quantifies the independent\ncontribution of each predictor variable to the total explained variance (R²) of the\nmodel. Because this method decomposes a dimensionless R², the results are\ntheoretically unaffected by the original scales of the independent variables.\nAlthough the calculation method is scale-invariant, we still standardized all\ncontinuous independent variables using Z-score normalization to ensure that our\nanalysis adhered to best practices in statistical modeling. We have added the\ndescription (please see lines 542-545 and 564-565 in the revised manuscript).\n13. Line 533. Do you mean “the a priori models”?\nResponse: Thanks. We have revised the sentence (please see lines 552-554 in the\nrevised manuscript).\nReviewer #4' comments:\nThe authors have thoroughly addressed all may comments and I have no further\ncomments.\nResponse: We greatly thank the reviewer for the time and effort they devoted to\nimproving our manuscript."}]

Decision letter