Grassland Degradation Changes the Complexity of Ant-Hemipteran-Plant Tritrophic Mutualisms
and Yingzhi Gao 1,4,*Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis study investigated the effects of grassland degradation on ant–plant networks and the three-trophic-level mutualistic relationships among ants, hemipterans, and plants through field and greenhouse controlled experiments conducted on the Songnen Plain in China. It also preliminarily revealed the mechanisms underlying these relationships in both aboveground and belowground components. Overall, the paper demonstrates some innovation, and the findings have practical significance for global research on the restoration of degraded grasslands. However, there are areas where the paper could be improved:
- In the abstract, the original text states: “ flavus promotes the establishment... by increasing the height..., reducing soil moisture content, and transporting... seeds.” The phrase “promoting the establishment of communities” is somewhat broad (line 25). Based on the following context, it appears that L. flavus enhances individual fitness (plant height) and population dispersal (seed dispersal) through these mechanisms, thereby promoting the dominance of A. scoparia in degraded habitats. This section requires more precise wording from the authors.
- Overall, the literature review is well written; however, in lines 57–60, the authors state that “there is currently a lack of information on how ant-plant network organizations respond to grassland degradation,” and subsequently, in lines 98–107, they propose a broad hypothesis regarding “mutualistic interactions enhancing community survival.” However, the core focus of your research is a three-trophic-level system involving Hemiptera insects. Therefore, please consider whether a transitional sentence should be added before stating the specific research objectives, emphasizing that “particularly within a three-trophic-level mutualistic framework, such network responses and specific changes in interaction patterns remain unknown,” thereby linking the research gap more directly to your research focus.
- Please provide a clearer explanation in Section 2.1 regarding the selection of these two insect species and the scientific rationale behind this choice.
- The Venn diagram in Figure 4A of Section 3.3 indicates that plant height, seed bank, and soil moisture content (SMC) together explain only 30% (4% + 13% + 13%) of the variation in the abundance of Artemisia scoparia. The remaining 62% is attributed to other unknown factors. Therefore, in the subsequent discussion, the authors should use more cautious language, such as “ flavus may have partially facilitated the establishment of the A. scoparia community through the following three pathways,” and consider exploring other potentially significant influencing factors.
- In the discussion of plant strategies in Section 4.3, the authors note that A. scoparia attracts ants and hemipteran insects by developing a well-developed, nutrient-rich root system. However, the results of the random forest model (Figures 4B and 4C) show that the key root system indicators influencing the abundance of L. flavus and aphids/mealybugs are not entirely the same. Does this also suggest that there are differences in the mechanisms by which A. scoparia’s root system attracts ants and insects?
- In the conclusion, the authors emphasize that this study provides valuable insights for the conservation and restoration of degraded grasslands. Therefore, based on the restoration model presented in this study, it is recommended that a more specific, keystone-species-based restoration strategy be proposed in the conclusion to guide future grassland restoration practices.
- Other points requiring attention include that “” in Latin names should not be italicized, and colloquial expressions such as “and so on” should be avoided.
Author Response
Comment 1: In the abstract, the original text states: “ flavus promotes the establishment... by increasing the height..., reducing soil moisture content, and transporting... seeds.” The phrase “promoting the establishment of communities” is somewhat broad (line 25). Based on the following context, it appears that L. flavus enhances individual fitness (plant height) and population dispersal (seed dispersal) through these mechanisms, thereby promoting the dominance of A. scoparia in degraded habitats. This section requires more precise wording from the authors.
Response 1: Thank you for your careful revision and valuable comment. We fully agree with your opinion. In the revised abstract, we have refined the inaccurate description and replaced the vague expression of “promoting the establishment of A. scoparia communities”. Revised description: L. flavus improves individual plant performance by increasing plant height, reducing soil moisture content, and facilitating seed transportation of A. scoparia. These advantages enhance plant fitness and population spread of A. scoparia, consequently boosting its dominance within degraded grassland habitats. (See Line 24-28)
Comment 2: Overall, the literature review is well written; however, in lines 57–60, the authors state that “there is currently a lack of information on how ant-plant network organizations respond to grassland degradation,” and subsequently, in lines 98–107, they propose a broad hypothesis regarding “mutualistic interactions enhancing community survival.” However, the core focus of your research is a three-trophic-level system involving Hemiptera insects. Therefore, please consider whether a transitional sentence should be added before stating the specific research objectives, emphasizing that “particularly within a three-trophic-level mutualistic framework, such network responses and specific changes in interaction patterns remain unknown,” thereby linking the research gap more directly to your research focus.
Response 2: We thank the reviewer for this thoughtful and valuable suggestion to polish the logical transition of our introduction. Following your advice, we have supplemented a transitional sentence between the literature gap description and research hypotheses in the revised manuscript. The added content highlights that previous knowledge gaps on ant-plant network responses to grassland degradation are even more prominent when extended to a tritrophic system including Hemipteran aphids and mealybugs, where the shifts in mutualistic networks and interaction patterns have rarely been documented. This revision closely connects the general research blank of ant-plant networks with our specific three-trophic study object, rationalizing the subsequent hypothesis on tripartite mutualism. (See Line 118-121)
Comment 3: Please provide a clearer explanation in Section 2.1 regarding the selection of these two insect species and the scientific rationale behind this choice.
Response 3: Thank you for your comments. We have supplemented detailed explanations on species selection in Section 2.1. The two insect species (aphids and mealybugs) were selected mainly based on our field observational findings: individuals of these two insects regularly distribute in clustered linear arrangements on the root systems of A. scoparia (Fig. S3). This distinctive aggregation pattern inspired us to explore the underlying mechanisms driving such spatial distribution and their associated tritrophic interactions with A. scoparia and L. flavus, which constitutes the core scientific rationale for selecting the two insect species in the present study. (See Line 194-200)
Comment 4: The Venn diagram in Figure 4A of Section 3.3 indicates that plant height, seed bank, and soil moisture content (SMC) together explain only 30% (4% + 13% + 13%) of the variation in the abundance of Artemisia scoparia. The remaining 62% is attributed to other unknown factors. Therefore, in the subsequent discussion, the authors should use more cautious language, such as “ flavus may have partially facilitated the establishment of the A. scoparia community through the following three pathways,” and consider exploring other potentially significant influencing factors.
Response 4: Thank you for this valuable comment and careful analysis of our statistical results. We fully agree with your suggestion. In the revised Discussion section, we have revised relevant statements and adopted more cautious expressions as recommended. We now describe that L. flavus may partially facilitate the establishment of A. scoparia communities via the three above-mentioned pathways. In addition, we have supplemented discussions on other potential influencing factors. We speculate that the remaining unexplained variation may be related to soil structure, microbial community composition, anthills temperature and other biotic or abiotic factors, which are worthy of further investigation in future studies. (See Line 500-511)
Comment 5: In the discussion of plant strategies in Section 4.3, the authors note that A. scoparia attracts ants and hemipteran insects by developing a well-developed, nutrient-rich root system. However, the results of the random forest model (Figures 4B and 4C) show that the key root system indicators influencing the abundance of L. flavus and aphids/mealybugs are not entirely the same. Does this also suggest that there are differences in the mechanisms by which A. scoparia’s root system attracts ants and insects?
Response 5: Thank you for this insightful question. We fully agree that the inconsistent key root traits identified by the random forest model indeed indicate divergent attraction mechanisms of Artemisia scoparia toward Lasius flavus and aphids/mealybugs. We have supplemented relevant explanations in the revised Discussion section. Specifically, different root morphological and physiological characteristics act as distinct driving factors: soluble sugars and root tip number mainly mediate the attraction to ants, while root water content, root volume and root length play a dominant role in recruiting aphids and mealybugs. This differentiation implies that the root system of A. scoparia adopts targeted strategies to associate with different organisms, forming differentiated interaction pathways in this tritrophic mutualistic system. We have clarified this inference and the underlying implications in the revised manuscript. (See Line 526-560)
Comment 6: In the conclusion, the authors emphasize that this study provides valuable insights for the conservation and restoration of degraded grasslands. Therefore, based on the restoration model presented in this study, it is recommended that a more specific, keystone-species-based restoration strategy be proposed in the conclusion to guide future grassland restoration practices.
Response 6: We appreciate this constructive suggestion. In the revised Conclusions section, we have supplemented a targeted restoration strategy centred on keystone species according to our tritrophic interaction findings. Based on our results that the tritrophic mutualism of L. flavus-A. scoparia-aphid/mealybug strengthens prominently in severely degraded grassland and improves ecosystem resilience, we put forward a keystone-species oriented restoration scheme: Take A. scoparia as the pioneer constructive plant species for degraded saline-alkali grassland restoration, artificially supplement its seeds to facilitate population establishment; Protect indigenous L. flavus ant populations instead of pesticide elimination, as this keystone ant can promote A. scoparia growth and form stable tritrophic symbiosis with root-feeding aphids and mealybugs. This concrete species-based restoration proposal has been added into the final paragraph of Conclusions to practically guide local field grassland restoration work. (See Line 571-578)
Comment 7: Other points requiring attention include that “” in Latin names should not be italicized, and colloquial expressions such as “and so on” should be avoided.
Response 7: Thank you for your comments. Contents inside quotation marks of Latin names have been formatted in accordance with standard specifications. After full manuscript inspection, all colloquial phrases throughout the text have been replaced with formal academic expressions compliant with journal standards.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe subject of tritrophic interactions is interesting from an ecological perspective. The MS presents some novel data and is worth publication. However, I have a feeling the authors were not fully aware of other studies on ants and aphids' interactions across various habitats. I would recommend that authors check Joanna Pętal papers on the vegetation of ant hills; some are available online. Additionally, there are papers by L. Godske (1991, 1992) and M.G. Nielsen (1976) on Lasius flavus populations and aphids. I also recommend becoming familiar with the papers by L. Depa (2011, 2024) and Bielecki et al. (2025), which may provide background for the presented results.
Please explain how the degree of degradation of vegetation was established? What were the reasons for degradation? Authors indicate it in general, but please state the reasons concerning these particular study sites and how it was measured.
Line 92 – remove space
Line 230: What was the method of excavation? A metal cylinder of some diameter, or what?
Line 237: the roots were preserved in ethanol, and later the fresh weight was measured.
Line 216: control groups mean randomly chosen places?
All this methodical part on roots needs to be rewritten, step by step, explaining what was done and how.
Line 272 – perhaps it would be written: plants were distributed in clusters (or aggregations)
Line 275 – ad space before 88%
Line 318 – 320 – the sentence must be fixed, as it is without meaning.
Line 459 onwards – not A. Scoparia but A. scoparia.
Author Response
Comment 1: The subject of tritrophic interactions is interesting from an ecological perspective. The MS presents some novel data and is worth publication. However, I have a feeling the authors were not fully aware of other studies on ants and aphids' interactions across various habitats. I would recommend that authors check Joanna Pętal papers on the vegetation of ant hills; some are available online. Additionally, there are papers by L. Godske (1991, 1992) and M.G. Nielsen (1976) on Lasius flavus populations and aphids. I also recommend becoming familiar with the papers by L. Depa (2011, 2024) and Bielecki et al. (2025), which may provide background for the presented results.
Response 1: We sincerely appreciate your valuable suggestions on supplementary references. Following your advice, we have thoroughly retrieved and reviewed all recommended publications. Key findings from these literature have been incorporated into the Introduction of the revised manuscript, and corresponding citations have been added to the reference list accordingly. (See Line 70-78)
Comment 2: Please explain how the degree of degradation of vegetation was established? What were the reasons for degradation? Authors indicate it in general, but please state the reasons concerning these particular study sites and how it was measured.
Response 2: Thank you for pointing out this.
- How the degree of degradation of vegetation was established?
Vegetation degradation was categorized into lightly degraded and severely degraded levels by three quantitative indicators: alkali spot area, vegetation coverage and above-ground biomass. Relevant classification criteria and measured data of all indices are listed in the attached table (Table S1), and detailed descriptions have been supplemented in the revised manuscript. (See Line 162-168)
- Site-specific driving factors of grassland degradation
In the revised manuscript, we have supplemented above site-specific degradation causes rather than generalized descriptions. Since the late 1970s, the Songnen Plain’s dominant Leymus chinensis grasslands, critical for grazing and mowing, have suffered widespread degradation. Two core site-specific drivers are clarified in the revised manuscript:
Anthropogenic factor: Rising population pressure leads to overgrazing and farmland reclamation, which directly degrade native grasslands.
Natural environmental factor: Long-term aeolian sand deposition modifies soil microbial community structure, limits soil carbon emission and carbon cycling, and further exacerbates regional land degradation. (See Line 38-46)
Comment 3: Line 92 – remove space
Response 3: Thank you for pointing out this. The extra space has been removed. (See Line 108)
Comment 4: Line 230: What was the method of excavation? A metal cylinder of some diameter, or what?
Response 4: Thank you for pointing this out. We did not employ a metal cylinder during root sampling. Instead, the excavation method was used to retrieve root systems as completely as possible. Detailed descriptions of the root excavation procedure have been supplemented in the revised manuscript. (See Line 265-268)
Comment 5: Line 237: the roots were preserved in ethanol, and later the fresh weight was measured.
Response 5: Thank you for the nice suggestion. Relevant supplementary information has been supplemented in the revised manuscript. (See Line 271-272)
Comment 6: Line 216: control groups mean randomly chosen places?
Response 6: We apologize for the unclear description. The control groups refer to anthill-free sampling sites randomly selected from lightly degraded and severely degraded experimental sites, and this clarification has been supplemented in the revised manuscript. (See Line 248-249)
Comment 7: All this methodical part on roots needs to be rewritten, step by step, explaining what was done and how.
Response 7: Thank you for your comments. The root-related experimental section has been fully restructured with stepwise descriptions of experimental operations and implementation details supplemented. (See Line 265-291)
Comment 8: Line 272 – perhaps it would be written: plants were distributed in clusters (or aggregations)
Response 8: Thank you for the nice suggestion. Revisions have been made in line with your suggestions. (See Line 315)
Comment 9: Line 275 – ad space before 88%
Response 9: We apologize for the oversight of the missing space, which has now been inserted in the revised manuscript. (See Line 318)
Comment 10: Line 318 – 320 – the sentence must be fixed, as it is without meaning.
Response 10: We thank the reviewer for pointing out this critical issue. After careful re-evaluation, we have completely rewritten this sentence to clearly convey our intended meaning. The changes have been marked in the revised manuscript. (See Line 363-365)
Comment 11: Line 459 onwards – not A. Scoparia but A. scoparia.
Response 11: Sorry for the mistake writing. A full-text check has been completed, and relevant contents have been revised to the correct format accordingly. (See Line 524)
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have addressed my comments, leading to a significant improvement in the manuscript, which I believe is now suitable for publication.
