Review Reports - Soil Fungal Communities’ Characteristics of the <i>Lamiophlomis rotata</i> Root-Zone to Altitude and Their Relationship with Environmental Factors

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is an interesting and informative study of the soil fungal-plant associations in the root zone and non-root zone soil. I found only some recommended corrections in the text and figures. As mentioned below, the authors need to carefully inspect their figures, because they have Chinese text inserted in the labels of some figures.

Line. Comment

Throughout the manuscript, all genus and species names must be italicized.

Likewise in the manuscript, the species name “rotate” must be corrected to “rotata”. Apparently, the automatic spell checker has changed the species name ‘rotata’ to ‘rotate’ at places.

94-102. All of the description of the methods must be in past tense, not present tense.

126-127 With increasing altitude, the Chao1of root-zone soil fungus appeared first decreasing and then increasing. This does not appear to be the case (?) As best I can see, it is always decreasing. Please check this.

140-141 Fig 1A is not fully spelled out, also there is no label A on Figure 1.

Also, the subsections of Fig. 1 have Chinese text inserted in portions of the labels on the graphs.

159-160 This sentence makes no sense “The dominant species was Dactylonectria sp. in 3,600m, while the Clavaria falcata was the dominant species in 3,600m.” It appears that the authors have accidentally duplicated the altitude 3,600 twice in this sentence ( ?).

167-168 Correct wording of sentence “Through LEfSe analysis, fungal taxa exhibiting significant differences in the root-zone of Lamiophlomis rotata at different altitudes were identified (Fig. 2)."

186 Figure 3 has no A or B labelling to discriminate the two portions of the graph (?)

203-204 Reword “An RDA analysis was performed on the soil fungal community characteristic indices in relation to soil environmental factors. ”

204-205 Reword “Fig.4A shows the root-zone soil fungal community indices as the first and second principal components.”

208-209 Reword “Fig.4B, shows the non-root-zone soil fungal community indices in the first and second principal components.”

212-214 Reword “Using Monte Carlo verification, it was found that, 8-O-Acetylgardenin methyl ester was a major factor influencing the fungal community in the root-zone soil (Fig.4C), but the difference was not significant (P＞0.05).”

224-226 Reword “It has been reported that altitude, as a comprehensive topographical factor, influences climatic variables (precipitation, temperature, insolation, etc.), through its variations, indirectly affects the soil fungal communities’ structure (Li et al., 2022).”

226-229 Reword “The root-zone serves as a vital mediator between the soil and root microecosystems, influencing plant growth and development through processes such as material transformation and the secretion of trace elements (He et al., 2015).”

240-244 Reword “Li et al.(2022) found that, the Chao1 and Shannon indexes of fungal communities in alpine meadow soils at first increased, and then decreased as altitude increased, with the Chao1 and Shannon indices in shaded locations generally higher than those in sunny locations. Hou et al. (2024) and Wu et al. (2024) found that, soil fungal diversity indices gradually increased with increasing altitude.”

245-246 Reword “Our research found that, with the increasing altitude, the Shannon Index and Chao1 of soil fungi first decreased then increased.”

255-257. Reword “Conversely, Liu et al. (2024) found no significant differences in soil fungal diversity indices or abundance indices at small-scale altitudinal gradients in alpine grasslands.

258-269 Reword “--- with relative abundances of 78.87%, 84.14%, 45.20%, and 81.25%.”

Comments on the Quality of English Language

Overall, this is an informative study that provides some useful evidence of differences in the root-zone and non root-zone soil fungi associated with the plant Lamiophlomis rotata. I have made some suggestions in my comments to the authors to improve portions of the English in the text, as well as some compositional corrections (for example, there is Chinese text inserted in the labels of some figures, etc.).

Author Response

For research article

Response to Reviewer 1 Comments
1. Summary
Thank you very much for taking the time to review this manuscript. The comments were all valuable and very helpful in revising and improving our paper; these comments are also important guidance that is significant to our research. We studied the comments carefully and made corrections that we hope meet with your approval. We tried our best to improve the manuscript and made changes to the manuscript accordingly. These changes do not influence the content or framework of the paper. Once again, thank you very much for your comments and suggestions. The amendments were marked in red.
2. Questions for General Evaluation	Reviewer’s Evaluation	Response and Revisions
Does the introduction provide sufficient background and include all relevant references?	Yes/Can be improved/Must be improved/Not applicable	[We agree with this comment. Or you can also give your corresponding response in the point-by-point response letter. The same as below]
Are all the cited references relevant to the research?	Yes	[We agree with this comment]
Is the research design appropriate?	Yes	[We agree with this comment]
Are the methods adequately described?	Yes	[We agree with this comment]
Are the results clearly presented?	Can be improved	[We agree with this comment]
Are the conclusions supported by the results?	Yes	[We agree with this comment]
3. Point-by-point response to Comments and Suggestions for Authors
Comments 1: [Throughout the manuscript, all genus and species names must be italicized. Likewise in the manuscript, the species name “rotate” must be corrected to “rotata”. Apparently, the automatic spell checker has changed the species name ‘rotata’ to ‘rotate’ at places.] Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we had modified in the manuscript. Comments 2: [94-102. All of the description of the methods must be in past tense, not present tense.] Response 2: Thank you for pointing this out. We agree with this comment. Therefore, we had modified in line 84-98. Comments 3: [126-127 With increasing altitude, the Chao1of root-zone soil fungus appeared first decreasing and then increasing. This does not appear to be the case (?) As best I can see, it is always decreasing. Please check this.] Response 3: Thank you for pointing this out. We agree with this comment. Therefore, we had modified in line 121-124. Comments 4: [140-141 Fig 1A is not fully spelled out, also there is no label A on Figure 1. Also, the subsections of Fig. 1 have Chinese text inserted in portions of the labels on the graphs.] Response 4: Thank you for pointing this out. We agree with this comment. Therefore, we had modified in line 135-146. Comments 5: [159-160 This sentence makes no sense “The dominant species was Dactylonectria sp. in 3,600m, while the Clavaria falcata was the dominant species in 3,600m.” It appears that the authors have accidentally duplicated the altitude 3,600 twice in this sentence ( ?).] Response 5: Thank you for pointing this out. We agree with this comment. Therefore, We had checked the source data and found that this was a typographical error. The correct answer was” The dominant species was Dactylonectria sp. in 3,600m, while the Clavaria falcata was the dominant species in 4,200m”. we had modified in line 152-155. Comments 6: [167-168 Correct wording of sentence “Through LEfSe analysis, fungal taxa exhibiting significant differences in the root-zone of Lamiophlomis rotata at different altitudes were identified (Fig. 2)."] Response 6: Thank you for pointing this out. We agree with this comment. Therefore, We had modified in line 161-162. Comments 7: [186 Figure 3 has no A or B labelling to discriminate the two portions of the graph (?)] Response 7: Thank you for pointing this out. We agree with this comment. Therefore, We had modified in line 178. Comments 8: [203-204 Reword “An RDA analysis was performed on the soil fungal community characteristic indices in relation to soil environmental factors. ”] Response 8: Thank you for pointing this out. We agree with this comment. Therefore, We had modified in line 196-197. Comments 8: [204-205 Reword “Fig.4A shows the root-zone soil fungal community indices as the first and second principal components.”] Response 8: Thank you for pointing this out. We agree with this comment. Therefore, We had modified in line 197-198. Comments 9: [208-209 Reword “Fig.4B, shows the non-root-zone soil fungal community indices in the first and second principal components.”] Response 9: Thank you for pointing this out. We agree with this comment. Therefore, We had modified in line 200-201. Comments 10: [212-214 Reword “Using Monte Carlo verification, it was found that, 8-O-Acetylgardenin methyl ester was a major factor influencing the fungal community in the root-zone soil (Fig.4C), but the difference was not significant (P＞0.05).””] Response 10: Thank you for pointing this out. We agree with this comment. Therefore, We had modified in line 204-205. Comments 11: [224-226 Reword “It has been reported that altitude, as a comprehensive topographical factor, influences climatic variables (precipitation, temperature, insolation, etc.), through its variations, indirectly affects the soil fungal communities’ structure (Li et al., 2022).”] Response 11: Thank you for pointing this out. We agree with this comment. Therefore, We had modified in line 215-217. Comments 12: [226-229 Reword “The root-zone serves as a vital mediator between the soil and root microecosystems, influencing plant growth and development through processes such as material transformation and the secretion of trace elements (He et al., 2015).”] Response 12: Thank you for pointing this out. We agree with this comment. Therefore, We had modified in line 218-220. Comments 13: [240-244 Reword “Li et al.(2022) found that, the Chao1 and Shannon indexes of fungal communities in alpine meadow soils at first increased, and then decreased as altitude increased, with the Chao1 and Shannon indices in shaded locations generally higher than those in sunny locations. Hou et al. (2024) and Wu et al. (2024) found that, soil fungal diversity indices gradually increased with increasing altitude.”] Response 13: Thank you for pointing this out. We agree with this comment. Therefore, We had modified in line 231-235. Comments 14: [245-246 Reword “Our research found that, with the increasing altitude, the Shannon Index and Chao1 of soil fungi first decreased then increased.”] Response 14: Thank you for pointing this out. We agree with this comment. Therefore, We had modified in line 236-237. Comments 15: [255-257. Reword “Conversely, Liu et al. (2024) found no significant differences in soil fungal diversity indices or abundance indices at small-scale altitudinal gradients in alpine grasslands.] Response 15: Thank you for pointing this out. We agree with this comment. Therefore, We had modified in line 246-247. Comments 16: [258-269 Reword “--- with relative abundances of 78.87%, 84.14%, 45.20%, and 81.25%.”] Response 16: Thank you for pointing this out. We agree with this comment. Therefore, We had modified in line258-259.
4. Response to Comments on the Quality of English Language
Point 1: Overall, this is an informative study that provides some useful evidence of differences in the root-zone and non root-zone soil fungi associated with the plant Lamiophlomis rotata. I have made some suggestions in my comments to the authors to improve portions of the English in the text, as well as some compositional corrections (for example, there is Chinese text inserted in the labels of some figures, etc.).
Response 1: Thank you very much for your suggestions and feedback on the article; they have helped to improve the quality of the paper. (The changes have been highlighted in red)
5. Additional clarifications
[no]

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This study examines soil fungal communities in the root-zone and non-root-zone of Lamiophlomis rotata across four altitudes in the Qinghai-Tibet Plateau. The topic is relevant, and the field work appears substantial. However, the manuscript suffers from serious language problems, taxonomic confusion, statistical inconsistencies, and overgeneralized conclusions. A major revision is needed before publication.

Abstract

The abstract is poorly written and grammatically incorrect. The conclusion claims “data support for the conservation” missing “provide”. Also, the keywords include “LEFse analyse” which should be “LEfSe analysis”.

Introduction

The introduction is overly long and repetitive. It fails to clearly state why Lamiophlomis rotata is important beyond being “endangered” and a “toxic weed” – these are contradictory roles. The two scientific hypotheses are stated clearly, which is good. However, the authors cite themselves excessively (Li et al., 2025 appears multiple times before publication). The claim that “livestock refuse to graze on L. rotata” is presented without a reference.

Materials and Methods

Species name confusion: The plant is called Lamiophlomis rotata in the title and abstract, but the text uses Lamioiphilus rotata, Lamioiphloris rotate, Dictamnus dasycarpus (completely different genus!), and Lamio phlomis rotata. This is unacceptable. The authors must verify the correct binomial.
Sampling design: “3 sampling plots exhibiting typical topography and Dictamnus dasycarpus growth patterns” – Dictamnus is not the study species. Likely a copy-paste error from another paper.
Root-zone definition: 2 cm distance is arbitrary. No justification is given for this cutoff.
PCR details: Primers ITS5-1737F and ITS2-2043R – the numbers 1737F and 2043R are non-standard for fungal ITS. Likely typographical errors.
Statistical methods: Very brief. No mention of normality tests, transformation, or multiple comparison corrections. RDA Monte Carlo test is mentioned but the p-value is given as “P > 0.05” in results – that means non-significant, yet they still call it a “key factor”.

Results

Table 2: Uppercase/lowercase letter notation is confusing. The legend says “uppercase letters denote differences at 0.05 level for root-zone at same altitudes” but the table shows letters A and B comparing C and D within the same altitude – that’s fine. However, at 3800 m, the non-root-zone Shannon index is 3.54±1.17 with SE larger than the mean – highly suspicious data.
Figure 1: The phylum-level figure (1A) is difficult to read. The genus-level list (1E) includes names like “unidentified” which should be clarified. Species-level analysis (1F) claims Clavaria falcata as dominant at 3600 m, but this is a macrofungus – unlikely to be a dominant soil OTU without verification.
Figure 2 (LEfSe): The figure is unreadable in this PDF (tiny text, overlapping labels). The authors claim “distinct species number” but LEfSe identifies differential taxa, not just counts.
Figure 3 (FunGuild): “Saprotroph” and “Pathotroph” are broad categories – the figure should show more detailed functional groups. Also, FunGuild annotations are predictive, not confirmatory.

Discussion

The discussion is largely a restatement of results. The authors compare their findings to previous studies (Hou et al., Wu et al.) and note discrepancies, but the explanation “due to the particular nature of the subjects” is vague and repeated verbatim twice. There is no mechanistic discussion of why 8-O-acetylgardenoside methyl ester (a compound with a misspelled name – should be “gardenoside”?) would influence fungal communities. The statement that “findings contribute to understanding assembly mechanisms” is an overclaim – correlation does not imply causation.

Language and Presentation

English quality: Very poor. Sentence fragments, misplaced commas, subject-verb agreement errors throughout. Examples: “the findings of this study were inconsistent with those of previous research” appears three times nearly identically.
Species name formatting: Genus and species should be italicized throughout – inconsistent.
References: Incomplete citations (e.g., reference 7 has “322(Pt” and stops mid-sentence). Reference 1 (Bahram 2018) is Nature – correct but page numbers missing.

Specific Critical Issues

Taxonomic inconsistency: Same plant called by at least four different names. Unacceptable.
Statistical overinterpretation: RDA factor with p>0.05 is called “key factor” – misleading.
Missing negative controls: No mention of sequencing blanks or positive controls.
Ecological functional groups: FunGuild predictions are not validated by any metabolic or enzymatic assay.
Sample size ambiguity: “3 replicates” per altitude – but how many plants pooled per replicate? Unclear.

Recommendations

Correct the plant species name throughout and verify with a taxonomic database.
Rewrite the abstract and introduction for clarity and grammar.
Provide raw sequencing data accession numbers (mentioned as “will be uploaded” – do it before resubmission).
Re-run statistical analyses and report effect sizes, not just p-values.
Remove or rephrase the claim about “key factor” when p>0.05.
Have the manuscript edited by a native English speaker or a professional service.

Recommendation

Major revision required. The study has merit, but in its current form, the taxonomic errors, poor English, and statistical issues make the conclusions unreliable. Without thorough revision, the paper should not be accepted.

Author Response

For review article

Response to Reviewer 2 Comments
1. Summary
Thank you very much for taking the time to review this manuscript. The comments were all valuable and very helpful in revising and improving our paper; these comments are also important guidance that is significant to our research. We studied the comments carefully and made corrections that we hope meet with your approval. We tried our best to improve the manuscript and made changes to the manuscript accordingly. These changes do not influence the content or framework of the paper. Once again, thank you very much for your comments and suggestions. The amendments were marked in red.

2. Questions for General Evaluation	Reviewer’s Evaluation	Response and Revisions
Does the introduction provide sufficient background and include all relevant references?	Yes/Can be improved/Must be improved/Not applicable	[We agree with this comment. Or you can also give your corresponding response in the point-by-point response letter. The same as below]
Does the introduction provide sufficient background and include all relevant references?	Can be improved	[We agree with this comment]
Is the research design appropriate?	Can be improved	[We agree with this comment]
Are the methods adequately described?	Can be improved	[We agree with this comment]
Are the results clearly presented?	Must be improved	[We agree with this comment]
Are the conclusions supported by the results?	Must be improved	[We agree with this comment]
Are all figures and tables clear and well-presented?	Must be improved	[We agree with this comment]
3. Point-by-point response to Comments and Suggestions for Authors			[We agree with this comment]
Comments 1: [Abstract The abstract is poorly written and grammatically incorrect. The conclusion claims “data support for the conservation” missing “provide”. Also, the keywords include “LEFse analyse” which should be “LEfSe analysis”.] Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we had modified “LEFse analyse” which should be “LEfSe analysis”, and checking carefully found that “conclusion claims “data support for the conservation” no missing “provide”” in line 28-30. To address poorly written and grammatically incorrect text, we had engaged the services of a professional language editing service. Comments 2: [Introduction The introduction is overly long and repetitive. It fails to clearly state why Lamiophlomis rotata is important beyond being “endangered” and a “toxic weed” – these are contradictory roles. The two scientific hypotheses are stated clearly, which is good. However, the authors cite themselves excessively (Li et al., 2025 appears multiple times before publication). The claim that “livestock refuse to graze on L. rotata” is presented without a reference.] Response 2: Thank you for pointing this out. We agree with this part comment. Therefore, 1）Perhaps due to language barriers, important of Lamiophlomis rotate was not to express this clearly. 2) Lamiophlomis rotata as “toxic weed” had deleted.3) the authors cite themselves excessively (Li et al., 2025 appears multiple times before publication) responded as” Li Q, Zhang Y, Liu X N, et al. 2025. Characteristics of the Bacterial Community in Alpine Meadows in Response to Altitude and Aspect in the Qilian Mountains, Northwest China [J]. Ecology and Evolution, 15:e70769 . Li Q, Fan M. 2025. Characteristics of Soil Nutrients and Stoichiometric Ratios in the Rhizosphere of Tibetan Medicine Lami-ophlomis rotata at Different Altitudes and Their Relationship with the Active Components [J]. Chinese Wild Plant Resources, 44(06): 30-35+48. (in Chinese). Comments 3: [Materials and Methods Species name confusion: The plant is called Lamiophlomis rotata in the title and abstract, but the text uses Lamioiphilus rotata, Lamioiphloris rotate, Dictamnus dasycarpus (completely different genus!), and Lamio phlomis rotata. This is unacceptable. The authors must verify the correct binomial.] Response 3: Thank you for pointing this out. We agree with this comment. Therefore, we had modified in the manuscript. Comments 4: [Sampling design: “3 sampling plots exhibiting typical topography and Dictamnus dasycarpus growth patterns” – Dictamnus is not the study species. Likely a copy-paste error from another paper.] Response 4: Thank you for pointing this out. We agree with this comment. Therefore, we had modified in line 86-87. Comments 5: [Root-zone definition: 2 cm distance is arbitrary. No justification is given for this cutoff.] Response 5: Thank you for pointing this out. We agree with this comment. Cite References in line 101 (Li Q, Fan M. 2025. Characteristics of Soil Nutrients and Stoichiometric Ratios in the Rhizosphere of Tibetan Medicine Lami-ophlomis rotata at Different Altitudes and Their Relationship with the Active Components [J]. Chinese Wild Plant Resources, 44(06): 30-35+48. (in Chinese) doi: 10.3969/j.issn.1006-9690.2025.06.005) Comments 6: [PCR details: Primers ITS5-1737F and ITS2-2043R – the numbers 1737F and 2043R are non-standard for fungal ITS. Likely typographical errors.] Response 6: Thank you for pointing this out. We agree with this comment. Therefore, We had modified in line 101-102. Comments 7: [Statistical methods: Very brief. No mention of normality tests, transformation, or multiple comparison corrections. RDA Monte Carlo test is mentioned but the p-value is given as “P > 0.05” in results – that means non-significant, yet they still call it a “key factor”.] Response 7: Thank you for pointing this out. We agree with part this comment. Therefore, we had modified in line 112-116. Comments 8: [Results Table 2: Uppercase/lowercase letter notation is confusing. The legend says “uppercase letters denote differences at 0.05 level for root-zone at same altitudes” but the table shows letters A and B comparing C and D within the same altitude – that’s fine. However, at 3800 m, the non-root-zone Shannon index is 3.54±1.17 with SE larger than the mean – highly suspicious data.] Response 8: Thank you for pointing this out. We understand with this comment. Uppercase and lowercase letters are widely used in two-factor ecological experiments. And checking carefully raw data found that “3.54±1.17 “should be “3.54±0.17”, we had modified Comments 8: [Figure 1: The phylum-level figure (1A) is difficult to read. The genus-level list (1E) includes names like “unidentified” which should be clarified. Species-level analysis (1F) claims Clavaria falcata as dominant at 3600 m, but this is a macrofungus – unlikely to be a dominant soil OTU without verification.] Response 8: Thank you for pointing this out. We agree with this comment. Therefore, we had modified. “unidentified” was included in “other”. Comments 9: [Figure 2 (LEfSe): The figure is unreadable in this PDF (tiny text, overlapping labels). The authors claim “distinct species number” but LEfSe identifies differential taxa, not just counts.] Response 9: Thank you for pointing this out. We agree with this comment. Therefore, we had modified in line167-168 and 177. Comments 10: [Figure 3 (FunGuild): “Saprotroph” and “Pathotroph” are broad categories – the figure should show more detailed functional groups. Also, FunGuild annotations are predictive, not confirmatory.] Response 10: Thank you for pointing this out. We agree with this comment. Therefore, we had modified in line 169-170. Comments 11: [Discussion The discussion is largely a restatement of results. The authors compare their findings to previous studies (Hou et al., Wu et al.) and note discrepancies, but the explanation “due to the particular nature of the subjects” is vague and repeated verbatim twice. There is no mechanistic discussion of why 8-O-acetylgardenoside methyl ester (a compound with a misspelled name – should be “gardenoside”?) would influence fungal communities. The statement that “findings contribute to understanding assembly mechanisms” is an overclaim – correlation does not imply causation.] Response 11: Thank you for pointing this out. We agree with this comment. Therefore, We had modified in discussion. 8-O-acetylgardenoside methyl ester was the sole medicinal ingredient specified in the Chinese Pharmacopoeia (Chinese Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China (Volume 1) [M]. Beijing: China Medical Science Press, 2020: 262.). Comments 12: [Language and Presentation English quality: Very poor. Sentence fragments, misplaced commas, subject-verb agreement errors throughout. Examples: “the findings of this study were inconsistent with those of previous research” appears three times nearly identically. Species name formatting: Genus and species should be italicized throughout – inconsistent. References: Incomplete citations (e.g., reference 7 has “322(Pt” and stops mid-sentence). Reference 1 (Bahram 2018) is Nature – correct but page numbers missing.] Response 12: Thank you for pointing this out. We agree with this comment. Therefore, we had modified. Comments 13: Specific Critical Issues 1)Taxonomic inconsistency: Same plant called by at least four different names. Unacceptable. 2)Statistical overinterpretation: RDA factor with p>0.05 is called “key factor” – misleading. 3)Missing negative controls: No mention of sequencing blanks or positive controls. 4)Ecological functional groups: FunGuild predictions are not validated by any metabolic or enzymatic assay. 5)Sample size ambiguity: “3 replicates” per altitude – but how many plants pooled per replicate? Unclear.] Response 13: Thank you for pointing this out. We agree with this comment. 1) We had modified in manuscript.2) We had modified. 3) This study is not a controlled trial and therefore cannot provide a blanks or positive controls.4) We had modified. 5) We had modified. Comments 14: [Recommendations Correct the plant species name throughout and verify with a taxonomic database. Rewrite the abstract and introduction for clarity and grammar. Provide raw sequencing data accession numbers (mentioned as “will be uploaded” – do it before resubmission). Re-run statistical analyses and report effect sizes, not just p-values. Remove or rephrase the claim about “key factor” when p>0.05. Have the manuscript edited by a native English speaker or a professional service.] Response 14: Thank you for pointing this out. We agree with this comment. Therefore, we had modified.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

I have attached the paper with some of my input.

some important points

You report correlations between fungal taxa with chemical factors like 8-O-acetyl compounds, geniposide derivatives, berberine derivatives without experimental validation.

The study reads more exploratory than hypothesis-driven. It require clear hypotheses or research question that must integrated in results or in discussion

Comments for author File: Comments.pdf

Author Response

For review article

Response to Reviewer 3 Comments
1. Summary
Thank you very much for taking the time to review this manuscript. The comments were all valuable and very helpful in revising and improving our paper; these comments are also important guidance that is significant to our research. We studied the comments carefully and made corrections that we hope meet with your approval. We tried our best to improve the manuscript and made changes to the manuscript accordingly. These changes do not influence the content or framework of the paper. Once again, thank you very much for your comments and suggestions. The amendments were marked in red.

2. Questions for General Evaluation	Reviewer’s Evaluation	Response and Revisions
Does the introduction provide sufficient background and include all relevant references?	Yes/Can be improved/Must be improved/Not applicable	[We agree with this comment. Or you can also give your corresponding response in the point-by-point response letter. The same as below]
Does the introduction provide sufficient background and include all relevant references?	Can be improved	[We agree with this comment]
Is the research design appropriate?	Can be improved	[We agree with this comment]
Are the methods adequately described?	Yes	[We agree with this comment]
Are the results clearly presented?	Yes	[We agree with this comment]
Are the conclusions supported by the results?	Can be improved	[We agree with this comment]
Are all figures and tables clear and well-presented?	Yes	[We agree with this comment]
3. Point-by-point response to Comments and Suggestions for Authors			[We agree with this comment]
Comments 1: [I have attached the paper with some of my input. some important points You report correlations between fungal taxa with chemical factors like 8-O-acetyl compounds, geniposide derivatives, berberine derivatives without experimental validation. The study reads more exploratory than hypothesis-driven. It require clear hypotheses or research question that must integrated in results or in discussion peer-review-55231126.v1.pdf] Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we had modified.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Despite being a second revision, several significant issues remain ranging from incomplete methodological details to statistical inconsistencies and presentation flaws. The authors have made some improvements, but the paper is not yet ready for publication.

Section-by-Section Issues

Title & Abstract

Title is overly long and grammatically clunky. "Tibetan Unique Medicine of the Lamiophlomis rotata Root-Zone to Altitude" is not standard English. Consider rephrasing.
Abstract lacks quantitative key results (e.g., actual p-values, effect sizes). It states "Shannon and Chao1 initially decreased and subsequently increased" but doesn’t mention at which altitude the shift occurs.
The phrase "available phosphorus was the primary factor influencing fungal communities in the non-root-zone soil" is clear, but the abstract does not explain why root-zone communities were not significantly influenced by any measured soil factor (only by a non-significant trend with 8-O-acetylberberine). This weakens the conclusion.

Introduction

The scientific hypotheses are stated clearly, which is good. However, the justification for comparing root-zone vs. non-root-zone soil is superficial. Why is non-root-zone soil relevant here? The authors mention it in hypotheses but never fully justify its inclusion in the study design.
Several citations are from 2024-2025, which is fine, but some seem forced (e.g., Fernandez et al. 2025 on pH and ectomycorrhiza is only loosely related to the current study’s focus on altitudinal gradients).
The claim that L. rotata is "Grade I endangered" and "Grade II Endangered Protected Plants" is confusing two different grades? Please clarify or unify.

Materials and Methods

3.1 Study Area

Coordinates are given as "N 31°39'36'19', E 89°45'102'23" these are impossible (seconds cannot exceed 59). Clearly a typo. This should have been caught in the first revision.
Table 1: Units for EC are "um s-1" likely a typo for µS/cm. Also, "Lamiphloiis rotata" misspelled in table title.
Soil pH ranges from 6.95 to 8.00 across altitudes, but no statistical comparison is provided. This is important because pH strongly affects fungal communities.

3.2 Sample Collection

Definition of root-zone ( 2 cm from root) and non-root-zone ( 2 cm) is arbitrary. Was there any prior validation (e.g., root density profiling) to support this cutoff? Without it, the distinction may introduce bias.
Only 10 plants per plot, 3 plots per altitude that’s only 30 root-zone samples and 30 non-root-zone samples total. This is acceptable but low for robust fungal diversity analysis, especially given the high variability shown in standard errors (e.g., Chao1 SD up to ±142).

3.3 & 3.4 Molecular and Statistical Methods

PCR conditions are given, but no mention of negative controls, number of replicates per sample, or how sequencing depth was normalized across samples.
"One-way ANOVA" is mentioned, but it is not explicitly stated which comparisons were post-hoc tested. Table 2 uses uppercase and lowercase letters for significance, but the method for correcting multiple comparisons (e.g., Tukey HSD, Bonferroni) is missing.
RDA was performed, but the authors did not state whether data were transformed or centered. Also, the Monte Carlo permutation test (Figure 4C, D) is mentioned, but the number of permutations is not given.

Results

3.1 Diversity Indices (Table 2)

At 3800 m, the non-root-zone (D) shows dramatically lower Pielou (0.33 vs 0.65-0.68 elsewhere) and Shannon (3.54 vs ~6 elsewhere). This is a huge drop, but the authors offer no explanation. Is this real or an artifact (e.g., contamination, low sequencing depth)?
Uppercase/lowercase notation in Table 2 is confusing. Uppercase compares root vs non-root within the same altitude (ok), lowercase compares same zone across altitudes, but the letters are repeated inconsistently (e.g., at 4200 m, root-zone Chao1 "Ab" and non-root-zone "Abc" what does that mean exactly?).

3.2 Composition (Figure 1)

Figure 1 is overcrowded. Panel A (phylum level) shows 8 phyla, but panels C and D only show Ascomycota, Basidiomycota, Mortierellomycota; why not show all?
At the genus level, Pseudosperma shows extreme variation: 75.6% at 3800 m in root-zone, but only 11.81% at 4000 m. This variation is ecologically interesting, but the authors do not discuss possible reasons (e.g., host root exudates, soil moisture differences).
Species-level identification: "Dactylonectria sp." is not a full species name. Was it only identified to genus? If so, say "unclassified Dactylonectria".

3.3 LEfSe & FunGuild (Figures 2 & 3)

LEfSe: Figure 2 is difficult to read, many taxa names are cut off or too small. Also, the authors report "number of distinct fungal species" per treatment, but LEfSe detects biomarkers, not total distinct species. The wording is misleading.
FunGuild: The authors state "dominant predictive ecological functional groups were saprotrophs and pathogens", but what about symbiotrophs? They seem underrepresented. Also, the cluster analysis (Figure 3B) is not clearly explained in the text.

3.4 Correlations and RDA (Table 3, Figure 4)

Table 3 is massive and difficult to interpret. Many correlations are extremely high (e.g., root-zone Pseudosperma with 8-O-Y = 0.977, with TN = 0.965) but p-values are not shown only asterisks for a few. Why not report p-values for all?
In Figure 4C, the variable "8-O-Acetylgardenin methyl ester" is said to have the greatest effect on root-zone fungal communities but P > 0.05. The authors conclude it is "not significant" then why highlight it? This undermines the finding.
For non-root-zone, AP is significant (P < 0.05), good. But the RDA axes explain 98.85% of variation, suspiciously high. This often indicates overfitting or too few samples relative to variables. Please check.

Discussion

The discussion is too short relative to the complexity of the data. It largely restates results without deep mechanistic insight.
The authors mention altitude effects but do not separate direct effects (temperature, UV, barometric pressure) from indirect effects (soil properties, plant physiology). This is a missed opportunity.
No comparison with similar studies on other alpine medicinal plants (e.g., Rhodiola, Gentiana). This would strengthen the generalizability.
The claim "findings enhance our understanding of mechanisms governing assembly of soil fungal communities" is overreaching; no mechanistic data (e.g., network analysis, functional gene profiling) are provided.

Conclusions

Conclusions are mostly a restatement of results, which is fine, but they should also include limitations. None are mentioned.
The statement "AP was identified as the key factor influencing fungal communities in non-root zone soil" is supported, but the root-zone remains unexplained this should be acknowledged as a key limitation.

References

Reference 5 (He & Wang 2015) is in Chinese with an English DOI, ok, but the journal name "Acta Ecologica Sinica" should be italicized.
Reference 10 (Li et al. 2025) has a typo in DOI: "Org/10.1002/Ece3.70769" should be "https://doi.org/10.1002/ece3.70769".
Several 2025 references are "in press" or very recent acceptable but check that all are actually published or DOI-valid.

Recommendation: Major revision (third round) or rejection with encouragement to resubmit after addressing these issues. The core data are potentially valuable, but the current presentation and analysis are not yet publication-ready.

Author Response

For review article

Response to Reviewer 2 Comments
1. Summary
Thank you very much for taking the time to review this manuscript. Thank you very much for your comments and suggestions regarding sample collection, experimental design and in-depth data analysis. Although some comments cannot be amended now that the experiment has already been carried out. The comments were all valuable and very helpful in revising and improving our paper; these comments are also important guidance that is significant to our research. We studied the comments carefully and made corrections that we hope meet with your approval. We tried our best to improve the manuscript and made changes to the manuscript accordingly. These changes do not influence the content or framework of the paper. Once again, thank you very much for your comments and suggestions. The amendments were marked in red.

2. Questions for General Evaluation	Reviewer’s Evaluation	Response and Revisions
Does the introduction provide sufficient background and include all relevant references?	Yes/Can be improved/Must be improved/Not applicable	[We agree with this comment. Or you can also give your corresponding response in the point-by-point response letter. The same as below]
Does the introduction provide sufficient background and include all relevant references?	Can be improved	[We agree with this comment]
Is the research design appropriate?	Can be improved	[We agree with this comment]
Are the methods adequately described?	Can be improved	[We agree with this comment]
Are the results clearly presented?	Must be improved	[We agree with this comment]
Are the conclusions supported by the results?	Must be improved	[We agree with this comment]
Are all figures and tables clear and well-presented?	Must be improved	[We agree with this comment]
3. Point-by-point response to Comments and Suggestions for Authors			[We agree with this comment]
Comments 1: [Title & Abstract Title is overly long and grammatically clunky. "Tibetan Unique Medicine of the Lamiophlomis rotata Root-Zone to Altitude" is not standard English. Consider rephrasing.] Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we had modified in line 1-2. Comments 2: [Abstract lacks quantitative key results (e.g., actual p-values, effect sizes). It states "Shannon and Chao1 initially decreased and subsequently increased" but doesn’t mention at which altitude the shift occurs.] Response 2: Thank you for pointing this out. We agree with part this comment. The “(e.g., actual p-values, effect sizes) ”, following discussion amongst all authors, it was decided that these should be presented in the Results section rather than the Abstract. The statement ‘Shannon and Chao1 initially decreased and subsequently increased’ is incorrect, as it fails to specify at what altitude the shift occurs, whereas the text mentions that this occurs with increasing altitude. Therefore, we had modified in line 19-20. Comments 3: [The phrase "available phosphorus was the primary factor influencing fungal communities in the non-root-zone soil" is clear, but the abstract does not explain why root-zone communities were not significantly influenced by any measured soil factor (only by a non-significant trend with 8-O-acetylberberine). This weakens the conclusion.] Response 3: Thank you for pointing this out. We agree with this comment. This weakens the conclusion is the result of amendments made in accordance with your feedback, and also reflects the current data. Comments 4: [[Introduction The scientific hypotheses are stated clearly, which is good. However, the justification for comparing root-zone vs. non-root-zone soil is superficial. Why is non-root-zone soil relevant here? The authors mention it in hypotheses but never fully justify its inclusion in the study design.] Response 4: Thank you for pointing this out. We agree with this comment. Therefore, we had modified in line 60-62. Comments 5: [Several citations are from 2024-2025, which is fine, but some seem forced (e.g., Fernandez et al. 2025 on pH and ectomycorrhiza is only loosely related to the current study’s focus on altitudinal gradients). The claim that L. rotata is "Grade I endangered" and "Grade II Endangered Protected Plants" is confusing two different grades? Please clarify or unify.] Response 5: Thank you for pointing this out. We agree with this comment. Therefore, We had modified in line 51-52. The L. rotata is "Grade I endangered" in Qinghai Provincial Botanical Society, L. rotata is"Grade II Endangered Protected Plants" in China Provincial Botanical Society. In the article, retain the Chinese Botanical Society.) Comments 6: [3.1 Study Area Coordinates are given as "N 31°39'36'19', E 89°45'102'23" these are impossible (seconds cannot exceed 59). Clearly a typo. This should have been caught in the first revision. Table 1: Units for EC are "um s-1" likely a typo for µS/cm. Also, "Lamiphloiis rotata" misspelled in table title.] Response 6: Thank you for pointing this out. We agree with this comment. Therefore, We had modified in line 71-72, 79. Comments 7: [Soil pH ranges from 6.95 to 8.00 across altitudes, but no statistical comparison is provided. This is important because pH strongly affects fungal communities.] Response 7: Thank you for pointing this out. We agree with part this comment. Therefore, Table 1 presents baseline data; no statistical comparisons were performed. Comments 8: [3.2 Sample Collection Definition of root-zone ( 2 cm from root) and non-root-zone ( 2 cm) is arbitrary. Was there any prior validation (e.g., root density profiling) to support this cutoff? Without it, the distinction may introduce bias.] Response 8: Thank you for pointing this out. We understand with this comment. There was no prior validation (e.g., root density profiling) to support this cutoff (The average diameter of the root of the L. rotata was 0.7 cm; samples were taken at three-fold intervals, but there was no basis for this classification.) Comments 9: [Only 10 plants per plot, 3 plots per altitude that’s only 30 root-zone samples and 30 non-root-zone samples total. This is acceptable but low for robust fungal diversity analysis, especially given the high variability shown in standard errors (e.g., Chao1 SD up to ±142).] Response 9: Thank you for pointing this out. We understand with this comment. Therefore, The sample test cannot be changed. Comments 10: [3.3 & 3.4 Molecular and Statistical Methods PCR conditions are given, but no mention of negative controls, number of replicates per sample, or how sequencing depth was normalized across samples.] Response 10: Thank you for pointing this out. We agree with this comment. Therefore, we have been in contact with the sequencing company, but the issue remains unresolved. We deeply regret this. Comments 11: ["One-way ANOVA" is mentioned, but it is not explicitly stated which comparisons were post-hoc tested. Table 2 uses uppercase and lowercase letters for significance, but the method for correcting multiple comparisons (e.g., Tukey HSD, Bonferroni) is missing. RDA was performed, but the authors did not state whether data were transformed or centered. Also, the Monte Carlo permutation test (Figure 4C, D) is mentioned, but the number of permutations is not given.] Response 11: Thank you for pointing this out. understand with this comment. Therefore, 1) the method for correcting multiple comparisons (e.g., Tukey HSD, Bonferroni) had no ecological significance. 2) RDA was performed, the data were not transformed or centered. 3) number of permutations of Monte Carlo permutation test was 999. Comments 12: [3.1 Diversity Indices (Table 2) At 3800 m, the non-root-zone (D) shows dramatically lower Pielou (0.33 vs 0.65-0.68 elsewhere) and Shannon (3.54 vs ~6 elsewhere). This is a huge drop, but the authors offer no explanation. Is this real or an artifact (e.g., contamination, low sequencing depth)? Uppercase/lowercase notation in Table 2 is confusing. Uppercase compares root vs non-root within the same altitude (ok), lowercase compares same zone across altitudes, but the letters are repeated inconsistently (e.g., at 4200 m, root-zone Chao1 "Ab" and non-root-zone "Abc" what does that mean exactly?)] Response 12: Thank you for pointing this out. We understand with this comment. Therefore, 1) At 3800 m, the non-root-zone (D) shows dramatically lower Pielou (0.33 vs 0.65-0.68 elsewhere) and Shannon (3.54 vs ~6 elsewhere) was result. 2) e.g., at 4200 m, root-zone Chao1 "Ab" and non-root-zone "Abc" what does that mean exactly?)], uppercase letters denote differences at the 0.05 significance level for root-zone at the same altitudes, lowercase letters denote differences in root-zone or non- root-zone at different elevations. Comments 13: [3.2 Composition (Figure 1) Figure 1 is overcrowded. Panel A (phylum level) shows 8 phyla, but panels C and D only show Ascomycota, Basidiomycota, Mortierellomycota; why not show all? At the genus level, Pseudosperma shows extreme variation: 75.6% at 3800 m in root-zone, but only 11.81% at 4000 m. This variation is ecologically interesting, but the authors do not discuss possible reasons (e.g., host root exudates, soil moisture differences). Species-level identification: "Dactylonectria sp." is not a full species name. Was it only identified to genus? If so, say "unclassified Dactylonectria".] Response 13: Thank you for pointing this out. We agree with this comment. Therefore, we had modified Figure 1. Comments 14: 3.3 LEfSe & FunGuild (Figures 2 & 3) LEfSe: Figure 2 is difficult to read, many taxa names are cut off or too small. Also, the authors report "number of distinct fungal species" per treatment, but LEfSe detects biomarkers, not total distinct species. The wording is misleading. FunGuild: The authors state "dominant predictive ecological functional groups were saprotrophs and pathogens", but what about symbiotrophs? They seem underrepresented. Also, the cluster analysis (Figure 3B) is not clearly explained in the text.] Response 14: Thank you for pointing this out. We agree with this comment. Therefore, we had modified. Comments 15: [Recommendations Correct the plant species name throughout and verify with a taxonomic database. Rewrite the abstract and introduction for clarity and grammar. Provide raw sequencing data accession numbers (mentioned as “will be uploaded” – do it before resubmission). Re-run statistical analyses and report effect sizes, not just p-values. Remove or rephrase the claim about “key factor” when p>0.05. Have the manuscript edited by a native English speaker or a professional service.] Response 15: Thank you for pointing this out. We agree part this comment. Therefore, we had modified”*Indicates a significance level of 0.01, Indicates a significance level of 0.05” in table 3.

Comments 16: [Discussion

The discussion is too short relative to the complexity of the data. It largely restates results without deep mechanistic insight.

The authors mention altitude effects but do not separate direct effects (temperature, UV, barometric pressure) from indirect effects (soil properties, plant physiology). This is a missed opportunity.

No comparison with similar studies on other alpine medicinal plants (e.g., Rhodiola, Gentiana). This would strengthen the generalizability.

The claim "findings enhance our understanding of mechanisms governing assembly of soil fungal communities" is overreaching; no mechanistic data (e.g., network analysis, functional gene profiling) are provided]

Response 16: Thank you for pointing this out. We agree part this comment. Therefore, we had modified Discussion .

Comments 17: [Conclusions

Conclusions are mostly a restatement of results, which is fine, but they should also include limitations. None are mentioned.

The statement "AP was identified as the key factor influencing fungal communities in non-root zone soil" is supported, but the root-zone remains unexplained this should be acknowledged as a key limitation.]

Response 17: Thank you for pointing this out. We agree part this comment. Therefore, we had modified.

Comments 17: [Reference 5 (He & Wang 2015) is in Chinese with an English DOI, ok, but the journal name "Acta Ecologica Sinica" should be italicized.

Reference 10 (Li et al. 2025) has a typo in DOI: "Org/10.1002/Ece3.70769" should be "https://doi.org/10.1002/ece3.70769".

Several 2025 references are "in press" or very recent acceptable but check that all are actually published or DOI-valid.]

Response 17: Thank you for pointing this out. We agree part this comment. Therefore, we had modified.

Author Response File: Author Response.pdf