-->PONE-D-26-03344-->-->Tree species influence carbon quality but not total storage across soil horizons: a comparison of European beech and Norway spruce-->-->PLOS One

Dear Dr. Mareckova,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

ACADEMIC EDITOR: -->-->The manuscript is methodologically rich and contains valuable data. However, due to concerns related to statistical testing strategy, independence of samples, interpretation of functional genes, and confounding of tree species and soil type, I recommend a major revision.-->-->The revised manuscript should:

- Clarify experimental design and replication structure

- Strengthen and possibly simplify statistical analyses

- Address multiple testing and model assumptions

- Moderate speculative interpretations

- Improve integration between microbial and SOC quality data-->-->==============================-->-->

Please submit your revised manuscript by Apr 13 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:-->

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We look forward to receiving your revised manuscript.

Kind regards,

Michal Bosela, Ph.D.

Academic Editor

PLOS One

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The funding was obtained from the Technology Agency of the Czech Republic project SS05010039 and Ministry of Agriculture of the Czech Republic, Institutional support MZE-RO0425. The project was also supported by COST Action CA21138 - CLEANFOREST.

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Additional Editor Comments :

The manuscript presents a detailed comparison of soil carbon distribution, organic matter quality, microbial community structure, and enzyme activities across complete soil profiles under European beech and Norway spruce at one study site. The study combines several analytical methods (soil chemistry, DRIFT spectroscopy, amplicon sequencing, ddPCR, enzyme measurements), which makes the dataset extensive and technically strong.

The main result, that total SOC stocks are similar between the two forest types, but carbon quality, vertical distribution, and microbial characteristics differ, is relevant for forest soil ecology and carbon cycling research. The inclusion of the full soil profile is an important strength of the study.

However, there are several issues that need to be addressed before publication:

1) The comparison is between two different soil types (Cambisol and Podzol), therefore the effects of tree species are partly confounded with soil type. In several parts, the manuscript attributes differences mainly to tree species without sufficiently discussing pedogenic effects.

2) Hypotheses are vague and not formally tested. Your three research questions are broad and exploratory.

3) The research was conducted at a single locality, which limits generalization of the conclusions. Some statements are formulated too broadly.

4) Some interpretations, especially regarding secondary metabolites and microbial competition, are rather speculative and should be presented more cautiously.

5) The Discussion is relatively long and descriptive, and would benefit from a clearer structure and a stronger synthesis of the main findings.

6) The manuscript relies on multiple Wilcoxon–Mann–Whitney tests applied separately for each horizon and variable which may raise some concerns.

Considering the quality of the data and the relevance of the topic, I recommend major revision. The revised version should:

1) More clearly distinguish tree species effects from soil-type effects

2) Rewrite aims as explicit hypotheses

3) Moderate causal and general conclusions

4) Improve hypothesis-driven interpretation

5) Shorten and better structure the Discussion

6) Using Wilcoxon–Mann–Whitney tests applied separately for each horizon and variable may raise concerns about:

- Multiple testing inflation (Type I error)

- Fragmented interpretation

- Lack of integrated multivariate analysis

The authors should:

- Indicate whether any correction for multiple comparisons (e.g. FDR) was applied.

- Consider multivariate approaches (e.g. PERMANOVA for chemistry, dbRDA linking SOC indices to microbial composition).

- Clarify the structure of the linear mixed models: What exactly was treated as random effect? Were horizons treated as nested within plots? Were assumptions of normality and homoscedasticity checked?

The mixed-model results are currently presented briefly and need clearer explanation of model structure, fixed effects, and interaction terms.

In addition to Methods:

Seven sampling sites per forest type are mentioned. It should be clearly stated:

- Whether horizons within one soil pit were treated as independent samples in statistical tests.

- How spatial independence between pits was ensured.

- Whether forest type is replicated at the landscape level (it appears not).

The manuscript should clarify the true level of replication to avoid pseudoreplication.

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Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Yes

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-->2. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

**********

-->3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

**********

-->4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

Reviewer #1: Yes

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-->5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: Dear Authors,

Thank you for the interesting manuscript. The submitted manuscript presents important findings that elucidate the differing effects of deciduous and coniferous litter on selected soil properties.

All my comments, questions, and suggestions are provided in the attached file.

Kind regards,

Reviewer

**********

-->6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.-->

Reviewer #1: No

**********

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Attachments

Attachment

Submitted filename: review_of_PONE-D-26-03344.pdf

-->PONE-D-26-03344R1-->-->Tree species influence carbon quality but not total storage across soil horizons: a comparison of European beech and Norway spruce-->-->PLOS One

Dear Dr. Mareckova,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.-->-->

Please submit your revised manuscript by Jun 22 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:-->

• A letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

-->

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

As the corresponding author, your ORCID iD is verified in the submission system and will appear in the published article. PLOS supports the use of ORCID, and we encourage all coauthors to register for an ORCID iD and use it as well. Please encourage your coauthors to verify their ORCID iD within the submission system before final acceptance, as unverified ORCID iDs will not appear in the published article. Only the individual author can complete the verification step; PLOS staff cannot verify ORCID iDs on behalf of authors.

We look forward to receiving your revised manuscript.

Kind regards,

Michal Bosela, Ph.D.

Academic Editor

PLOS One

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

The revision improved the manuscript substantially. In particular, the manuscript now better acknowledges that the comparison is between beech on Dystric Cambisol and spruce on Entic Podzol, and not only between two tree species. The hypotheses are clearer, the single-site design is described more openly, the B horizon sampling depth is now specified, and the Discussion is shorter and more focused.

However, before the manuscript can be accepted, I would like to ask you for a further minor revision. Please address the following points carefully:

Statistical methods:

Please describe more precisely how the Hotelling’s two-sample test was applied, how the permutation correction for multiple testing was done, and which tests were corrected. Also, please make the terminology consistent. In the response letter you mention PERMANOVA, but in the manuscript the methods refer mainly to Hotelling’s test and AMOVA.

Linear mixed models:

Please clearly describe the model structure: what was the response variable, what were the fixed effects, what was the random effect, and how horizons and sampling sites were treated. Please also briefly state how model assumptions were checked.

Please keep the conclusions cautious:

Because the study was done at one locality and the forest type was not replicated, some conclusions should be clearly limited to this study site. Please avoid broad general statements about tree species effects, forest management, water retention, or ecosystem resilience unless they are directly supported by the data.

Please reduce speculative wording:

Some interpretations related to secondary metabolites, microbial competition, and antagonistic mechanisms should be written more cautiously. Please use wording such as “may indicate”, “suggests”, or “is consistent with”, where appropriate.

In my view, the manuscript is close to being acceptable, but these points should be corrected before final acceptance.

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

-->

Tree species influence soil carbon quality but not total storage across horizons: European beech on Dystric Cambisol and Norway spruce on Entic Podzol

PONE-D-26-03344R2

Dear Dr. Mareckova,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Michal Bosela, Ph.D.

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments: