General comments:

Mai et al. investigated diazotroph abundance and activity in the East China Sea using qPCR and in situ incubations, and attempted to elucidate niche zonation with a maximum-entropy ecological model. While the data collection effort is acknowledged, the data analysis and discussion are regrettably inadequate. The modeling framework is introduced without sufficient explanation or validation, and the many results diverge from established patterns (e.g., this study suggests Trichodesmium favors low temperature and high nutrients). Inconsistent results with previous knowledge can be valuable, but the authors do not examine them critically and instead cite mostly only studies that support their observations. Consequently, the discussion remains largely descriptive and does not elucidate or discuss the physio-ecological mechanisms underlying diazotroph niche partitioning. Because reports of diazotrophy around the Kuroshio region and importance of Kuroshio intrusion are no longer novel, the modeling approach could have been the key contribution of this study. However, the absence of careful interpretation and the lack of clear validation leave the novelty and significance of the work unclear.

Major Comments:

1. Model justification and validation

The rationale for using the maximum-entropy model is not sufficiently explained, nor is its validity demonstrated. The authors should describe the model in more detail and clarify why it is appropriate for their dataset. Also I suppose that such models are not necessarily valid for all  dataset, and thus model significance validation should be done carefully. For example, Brun et al. (2015) evaluated model performance/validity using the AUC of the ROC curve and a student’s t-test with randomizations to exclude invalid models. A comparable validation is needed here. Without it, the model results cannot be considered reliable.

2. Seemingly selective and sometimes inappropriate citations  

References often appear cherry-picked to match the authors’ results. For instance, in discussing phylotype-specific niche partitioning, they cite only studies whose outcomes align with their data, ignoring meta-analyses and culture experiments that already provide robust estimates of optimal temperature and nutrient ranges (detail are mentioned in the specific comments). They should revise the citation thoroughly. 

3. RDA analysis

The RDA explains little of the variance of dataset (autumn: ~12 %, spring: ~34 %), yet the discussion relies on such weak relationship. RDA compresses relationships among multiple variables, but authors use them to claim direct correlations between specific taxa and single environmental parameters from RDA results. Also, collinearity among variables such as SRP, NOx, and depth is not tested or reported, VIF threshold should be set and reported. The authors should (i) report overall R² and significance of their RDA, (ii) test the significance of each variable’s significance and contribution in RDA. More simple analysis such as simple correlation and multiple-variable linear regression such as GLM may be better for understanding controlling factor on each diazotroph.

Specific comments:

Introduction

L69–70: Note that Shiozaki et al. (2018) also examined diazotroph abundance using qPCR.

L68–73: Because authors study phylotype-specific niches, the introduction should briefly describe the major unicellular diazotrophs rather than grouping them simply as “unicellular.”

L77: The term "ecotype" generally refers to distinct strains within a species. 

L81-84. Because most readers should not be familiar with maximum entropy method, authors should explain the method more detail.

L89-90: Brun et al. studied Trichodesmium and Richelia realized niche. It should be mentioned here and in the discussion.

Materials and Methods

L103: “summer” should be “autumn.”

L114: Anderson and Sarmiento (1994) did not introduce P*. To my knowledge, Deutsch et al. (2007) is the first.

L120: 200 µm mesh was used for RNA sample as well? 

L122–123: Fig. 6 here is inappropriate, considering the order of figure.

L147: Please provide the detection limit of the qPCR.

L148–150: Recent studies show that UCYN-C (Schvarcz et al. 2022, 2024) and γ-24774A11 (Tschitschko et al. 2024) are also likely diatom symbionts, almost organelle-like. This simplification should be describe more carefully.

L167: For transparency as suggested in (White et al. 2020), report the minimum quantifiable N2-fixation rate for each data and the value for each replicate as supplementary material.

L179–183: Many readers will not be familiar with the MaxEnt model; provide a detailed description. Past studies sometimes exclude absence data, did this study do the same?

L179: Define “breadth (σ).”, what does this mean?

L180: After integration, how many observations (n) were included?

L181: All parameter settings of MaxEnt should be clearly stated. Is it default setting?

L182: The GAM parameters is not explained and GAM result does not appear here after.

L191: Z-score-scaling applied to PCA, as well?

L193–194: Please clarify the VIF threshold used to avoid multicollinearity.

Results

L214–216: Provide more specific spatiotemporal deviations from the Redfield ratio (16).

L222: Because nifH was not comprehensively amplified, “nifH gene pool of targeted diazotrophs” is more appropriate.

L223–225: Present quantitative numerical values.

L238: Transects A and B should be mentioned in Methods and figures 1.

L242–243: The term “broader” is vague; specify how many of how many stations, or give quantitative metrics.

L243: UCYN-A, UCYN-C, and γ-24774A11 are now recognized as likely symbionts/organelles; grouping them as “unicellular diazotrophs” is questionable.

L243–245: This description conflicts with Fig. 5. Het-2 peaks in the subsurface, and UCYN-A2/A3/A4 show profiles similar to Trichodesmium. 

L246: Sampling was not performed at 60 m. If values are interpolated, please state that and use the actual sampled depth (e.g., 50 m).

L251: The phrase “were the most abundant among” is unclear; perhaps “were detected as the most abundant.”?

L269, L272: “CDW/CW-affected regions” should be defined quantitatively, e.g., by proportion of water masses.

L270: Figure 6 does not have station 1.

L271: “About 60%” is more appropriate.

L276–277: To support this, I recommend to perform multivariable regression for each phylotype and report the coefficients of determination, so that importance of each variable can be quantified.

L281: Figure S6 should be moved into the main text; otherwise readers cannot follow the discussion of μ and σ. Also, please evaluate model validity as mentioned in major comments.

L281: From Fig. S7, n = 3 seems most appropriate; consider adding a hierarchical cluster tree to illustrate the threshold objectively.

L290: “Relatively high” should be described with quantitative data.

Discussion

L295–297: Current ecological model (Fig. S6) shows Trichodesmium preferring low temperature, low salinity, and high nitrate waters. This contradicts both claim here and previous studies.

L298: growth was not measured in this study.

L300–301: Relationships inferred from RDA are weak and not checked stastically.

L303: “Below detection limit” does not mean complete absence.

L307–309: Conclusions based solely on weak RDA correlations are too qualitative. 

L309–311: To demonstrate major N2 fixers, statistically test relationships between gene (or transcript) abundance and N2-fixation rate. High nifH abundance alone is insufficient.

L314–315: Many studies detected Trichodesmium colonies in the ECS (e. g.  Marumo and Asaoka 1974, Jiang et al. 2023). Richelia colonies are also expected to removed by this mesh filtering. Was microscopy performed in this study? They should provide more solid evidence of not-underestimating, otherwise it is not convincing.

L319: Jiang et al. (2018, 2019) reported highest densities at the surface and 10–50 m, consistent with your results. Carpenter et al. (2004) also found maxima at ~20 m. Discussion here seems incorrect.

L323: If eddy is important, provide and check the SSH and its anomaly information during study period.

L324: Only UCYN-C and γ-24774A11 peak at 50 m; others peak above 30 m. Thus filamentous and unicellular diazotrophs are not “in contrast.”

L329: Lu et al. 2018 does not seem to provide the information of light adaptation of unicellular cyanobacterial diazotrophs?

L332: ~22℃ for optimal temp. of UCYN-B is not common understanding. Also, Webb et al 2009 report 26-30℃ for its optimal temperature. Other studies also reported ~25–30 °C (Tang and Cassar 2019; Mauda 2024).

L334–338: Tschitschko et al. 2024 in Nature strongly (almost conclusively) indicate that γ-24774A11 is a diatom symbiont. Include this information.

L346: Koening et al. (2009) only speculated about an r-strategy for Trichodesmium; they did not conclude it. Citing this paper as proof in current manner is inappropriate.

L351–353: This statement seems contradict the Maxent based discussion.

L360: Please quantitatively compare your data values with those of Jiang et al. 2023 with actual value.

L364–367: Only one phylotype of non-cyanobacterial diazotroph (NCD) was quantified. Many other NCDs likely exist; please acknowledge this limitation.

L367–368: A simple correlation analysis between N2-fixation rate and nifH transcript abundance is needed to support this argument.

L370: Recent studies report cell-specific rates for UCYN-C (Schvarcz et al. 2022) and γ-24774A11 (Tschitschko et al. 2024). Also, please provide standard deviations.

L375: The spring value of 58.5 seems incorrect; re-check Table S3.

L376: Because this study directly measured surface N2-fixation rates as well, authour should verify consistency with values inferred from nifH copies and per-cell rates and measured rates by incubations; otherwise the subsequent quantitative discussion can be  not-reliable.

L399: Is this broad niche of Trichodesmium consistent with the earlier claim of an r-strategy for Trichodesmium? 

L399–404: Of course there is intra- and inter-specific metabolic variability; explain how such variability could produce the observed differences.

L411–412: Zehr et al. (2007) did not study genotype-specific optimal temperatures. Webb et al. (2009) reported an optimum of 26–30 °C, which is not as broad and is inconsistently higher than current result (<24 °C).

L414: Brum et al. 2015 analyzed diatom-associated diazotrophs, please discuss it ?

K420-425: Please refer Tschitschko et al. 2024 which report impotant ecology and physiology of γ-24774A11.

L432: To my knowledge, Cheung et al. 2019 did not mention ecological niche similarity between UCYN-C and Hets.

L438–439: Cabello et al. (2016) did not report a distinct “reproductive strategy” for UCYN-A.

L440: Cabello et al. (2016) speculated on such a strategy, but many opposing studies also exist: UCYN-A2 is considered coastal ecotype, whereas UCYN-A1 is open-ocean ecotype (e.g., Turk-Kubo et al. 2017, 2021; Henke et al. 2018). Cite a more objective literature rather than only studies consistent with your results.

Figures

Figure 4: Clarify whether data represent an average across multiple stations and include error bars if so.

Figures 5 and 6: The numbers near the pie/bar charts appear to be station numbers—please clarify in the captions.

Figure 3: If station numbers are important here, add them for consistency.

General Comments:

This manuscript presents a comprehensive dataset on nifH gene copy numbers, expression levels of major diazotroph groups, and surface nitrogen fixation rates in the East China Sea (ECS). By applying maximum entropy methods and generalized additive models, the study delineates the realized ecological niches of these diazotrophic groups. These findings enhance our understanding of diazotrophic community composition and nitrogen fixation in the ECS. Overall, this study represents a rigorous and valuable contribution. However, certain aspects of the presentation require improvement. I recommend considering this manuscript for publication pending minor revisions.

1. The Kuroshio region is a recognized hotspot for nitrogen fixation, which forms a key premise for this study. However, the survey design did not cover the Kuroshio mainstream but was confined to its intruded waters. Strictly speaking, therefore, the study area is more accurately defined as the East China Sea (ECS) continental shelf. Given the potential for significant variations in nifH gene copy numbers, expression, and nitrogen fixation rates across different water masses, I recommend redefining the study area throughout the manuscript to "ECS continental shelf" to ensure precise interpretation. Correspondingly, the title should be updated to "Spatiotemporal heterogeneity in diazotrophic communities reveals novel niche zonation on the continental shelf of the East China Sea" to accurately reflect the spatial scope of the work.
2. The authors state: "Arguably, given the lack of observed Trichodesmium colonies in our samples, the pre-filtration through a 200-μm pore-size nylon mesh did not underestimate its abundance." This rationale requires clarification. How were Trichodesmium colonies determined – via microscopic examination or visual inspection? Pre-filtration through a 200-μm mesh is intended to remove large zooplankton but may also inadvertently remove Trichodesmium, including both free and colonial trichomes, potentially leading to an underestimation of its abundance. This potential limitation should be acknowledged and discussed in the manuscript.
3. The study did not clarify how the surface nitrogen fixation rate is related to diazotrophic biomass and physicochemical conditions.
4. The analysis of mean and breadth realized niches for diazotrophs is constrained by the lack of seasonal survey data. The ECS, influenced by the Kuroshio and Changjiang Diluted Water, exhibits strong seasonal variability in its physical and chemical environment (e.g., temperature, salinity, nutrient availability). The current niche modeling, which lacks data from summer and winter, may therefore not fully capture the annual ecological dynamics, potentially affecting the robustness of the conclusions. I therefore recommend that the authors include a clarification on this matter in their Discussion or Conclusions.
5. The manuscript would be significantly strengthened by the addition of a dedicated Conclusions section. This section should succinctly summarize the key findings, explicitly state the scientific innovations of the work, and thoughtfully discuss the study's limitations alongside suggestions for future research directions.

Specific Comments:

Line 11: Please replace "in the ECS" with "on the ECS shelf".

Lines 17-19: Please describe the key spatial distribution patterns and their primary Line 56: Please replace "nutrient-rich" with "phosphorus-rich" for accuracy.

Line 70: Please replace "Lee Chen et al." with "Chen et al.".

Line 103: The period October 13–30 should be explicitly referred to as autumn.

Line 164: Please specify whether this refers to particulate organic nitrogen or total particulate nitrogen.

Line 166: Please provide the limits of detection for the nitrogen fixation rates, as per the methodology of Montoya et al. (1996).

Lines 174-176: Since the Taiwan Warm Current is a mixture of Kuroshio Subsurface Water and water from the Taiwan Strait, using the Taiwan Strait water as an end-member, rather than the Taiwan Warm Current itself, might provide a better framework for interpreting the influence of Kuroshio intrusion on diazotrophs.

Lines 209-211: Please provide clear definitions for "nearshore waters" and "offshore regions" as used in this context.

Lines 212-214: Please replace "content" with "concentration". Was there any notable climatic anomaly during the survey period? The reported high SRP concentrations associated with Kuroshio Surface Water seem inconsistent with its typical oligotrophic character, as described in Line 277. Furthermore, Figures S3A and S3E suggest a slight upwelling at transect A; including data on the mixed layer depth and SRP profiles for this transect would be informative.

Line 223: Please briefly specify which diatoms Het1 and Het2 are associated with and highlight their key ecological differences.

Line 275: Please clarify whether "water depth" refers to the sampling depth or the station depth.

Lines 313-315: The manuscript does not detail a microscopic examination protocol. Please clarify how Trichodesmium colonies were observed and identified.

Line 347: Please replace the general term "nutrients" with the specific "SRP and iron".

Lines 350-353: Please elaborate on the proposed mechanism by which the northeast monsoon enhances Trichodesmium abundance in the southern ECS during spring.

Line 360: "similar to" or "comparable to" would be more appropriate phrasing.

Lines 377-378: Please rephrase for clarity, e.g., "In spring, the nitrogen fixation efficiency of Trichodesmium was likely suppressed by low temperatures."

Lines 379-381: Please specify the exact stations used to estimate NFRs in the high-salinity water during autumn and spring, as these cannot be readily identified in Fig. 3 or Table S3.

Lines 385-388: If the high nifH gene copy number in Trichodesmium is attributed to cell cycle-dependent polyploidy, please explain why this would lead to an overestimation of its relative contribution specifically in spring and not in other seasons.

Figure and Table Comments:

Figure 1: The path of the Kuroshio Subsurface Water (KSSW) shown appears to be based on its summer pattern (Yang et al., 2012). According to Yang et al. (2018), the branch of KSSW intruding off the Zhejiang coast typically occurs in April and recedes by July. Please ensure the path depicted is accurate for the study period. Additionally, please label the two transects (A and B) on the map.

Figure 7: Please describe in the Materials and Methods section how the environmental factors related to KSW, TWC, and CDW/CW were quantified for the RDA analysis.

Figure S2: The finding of nearly 100% contribution from Taiwan Warm Current Water in the coastal waters of Zhejiang (Panel B) seems unusually high. As the Taiwan Warm Current flows northward, it typically mixes with coastal water, diluted water, and shelf mixed water, making a 100% contribution improbable. Please review this data or provide further justification.

Table S3: The estimation of Trichodesmium NFRs based solely on qPCR data might be inflated, as nitrogen fixation does not occur in every single cell. This potential source of overestimation should be acknowledged as a limitation of the methodology.