PONE-D-23-06554Phenolic concentrations and carbon/nitrogen ratio in annual shoots of bilberry (Vaccinium myrtillus) after simulated herbivoryPLOS ONE

Dear Dr. Schrijvers-Gonlag,

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.

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The manuscript has been judged as too descriptive. The authors should put additional effort to reorganize it to gain scientific rigidity. Additional experimental setups and measurements might be needed to avoid speculations. Refocusing the manuscript to herbivory assessment only might be one of the major advices, as suggested by Reviewer #3.

The main editorial advice is to have the manuscript proofread by a senior researcher form the same field to diminish uninformative and redundant parts, by increasing its overall informativeness. Please be aware of the supplementary file provided by Reviewers #1 and #3.

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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

Reviewer #2: Partly

Reviewer #3: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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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

Reviewer #2: Yes

Reviewer #3: Yes

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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

Reviewer #2: Yes

Reviewer #3: No

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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: The manuscript entitled Phenolic concentrations and carbon/nitrogen ratio in annual shoots of bilberry (Vaccinium myrtillus) after simulated herbivory cover an interesting topic about the allocation of phenolic compounds during differents scenarios of herbivory. It is in general well written.

However there are some minor aspects that need to be taken to be ready to publish

Why no to use another statistic test such as GLM to test interactions between location and soil productivity

Don you think table 1 should be better in supplementary material?

I think is more informative to add the letters according to a post hoc test in table two. And why are you adding the column of all treatments together? It is useful??

Is it on the guidelines or why the figure legends are in the text?

I don’t think that is necessary to add SE and SD from you data every time you describe the results

I think first paragraph of discussion should be a summary of the results, why start with one particular compound?

When you make your comparisons with other systems, why do not you use related species instead of mountain birch and mopane? there are some interesting articles of other vaccinium species that can be more comparable to your study system

Reviewer #2: I reviewed the paper entitled ‘Phenolic concentrations and carbon/nitrogen ratio in annual shoots of bilberry (Vaccinium myrtillus) after simulated herbivory’. Authors identified several hypotheses related to phenolics in response to simulated herbivory. Principally, related to whether these defenses follow the C:nutrient balance hypothesis or Optimal Defense Theory. The actual experimental data was quite straightforward. Various phenolic compounds and C:N ratios were measured in bilberry shoots. That being said, in my view there is excessive text and lots of speculation that goes far beyond what was tested in this manuscript. The protein competition model is quite interesting, however not properly tested. I am not sure how you can test the protein competition model without measuring any protein…There are many reasons why phenolics would be reduced or unchanged. Did you see differences in protein quantity/quality under simulated herbivory? Further, the conclusion that this may be due to clonality is likewise interesting, but was not tested. Did you look at C:N ratios in clonal ramets connected to experimental ramets, for example, to see whether there may be a flux or allocation of resources to damaged plants. Additionally, why was the foliar content not measured? Especially considering you had a defoliation treatment, one might expect differences to be most apparent in leaves. Authors should justify why this was.

What was actually found, as I understand, is that, other than essentially complete removal of aboveground tissue, your treatments do not reduce specific constitutive phenolics, whereas under severe damage, shoots had lower phenolics, presumably because carbon is being used for regrowth of tissue growth and is limiting. Under ambient herbivory, carbon may not be limiting due to the fact that only minor additional growth is necessary to reach static conditions, and enough is C is thus available for phenolics biosynthesis. Did you have data for changes in biomass? Although the takeaway message is rather simple, I found the MS slightly challenging to read. And was distracted by a lot of possibilities and details that were not tested in the experiments. Perhaps if the paper was reframed and simplified to highlight what was tested, as opposed to what was not (all of these trade-off theories) it would be easier to follow. For example, your first prediction is not even possible to test, because you don’t detect any gallic acid-derived phenolics at all. As such, it doesn’t make much sense to include it in the MS. Your first prediction might instead be something like ‘determine whether bilberry has these compounds at all, and compare them with Phe-derived phenolics’. Then you might be able to contextualize this in the protein competition model. However, because they are not present, this is of course not possible.

I did not leave specific, detailed comments, as lines were not numbered. In subsequent drafts I would recommend adding line numbers.

Reviewer #3: After reading this study several times I can only conclude that to have a chance to be publishable, the manuscript needs to be completely rewritten to address two major problems: 1) a weak initial study design that prevents conclusions to be made with confidence followed by a modification that does little to improve the scientific integrity, and 2) a writing style that is difficult to follow due to a lack of flow and clarity arising from awkwardly constructed sentences and arguments, excessive repetition and inclusion of unnecessary text and information.

With respect strengthening the design of the study, I recommend removing the issue of high- and low productivity sites. The definition of productivity appears to be defined and supported data-wise with one variable, total nitrogen. This information comes from one composite sample per site (n=1). To merge your data and compare this to the one location which “behaved differently” also weakens your study in my opinion. I suggest you remove the idea of site productivity as a factor and rewrite the manuscript based on your “herbivory treatments”; something for which you have much better resolution and data from which to draw more confident conclusions. Present the plant chemistry data (phenols, tannins, N, C, and C/N) together in one Table or Figure, so that the reader can better visualize possible relationships among these variables (for example, see Nosko et al 2020, https://doi.org/10.1016/j.foreco.2019.117839). In your discussion, you can then focus on browsing treatments as they affect the relationships among plant chemistry variables. In the absence of such relationships, some inspiration might be obtained from Warbrick et al 2020 https://doi.org/10.1007/s11258-020-01027-y and Nosko and Embury 2018 https://doi.org/10.1007/s11258-018-0821-7.

The manuscript is very difficult to read and needs to be completely rewritten and shortened to improve readability, flow and clarity. Every element, including captions, needs to be edited and reorganized. Reading through comparisons among sites/treatments is quite cumbersome (see examples on the manuscript). Flow and clarity could be greatly improved by adopting a code or acronym for your site and treatment names (see suggestions in edited manuscript). Flow and length will also be improved by removing site productivity as a factor and presenting plant chemistry data together. I did not provide editorial suggestions for the entire manuscript (see attached), but did so in several sections. Hopefully this will give you an idea of my concerns.

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Reviewer #1: Yes: JHC

Reviewer #2: No

Reviewer #3: No

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Attachments

Attachment

Submitted filename: Plos OnePhenolics.pdf

Attachment

Submitted filename: Manuscript - MSG20230306 TRACK CHANGES VERSION.docx

PONE-D-23-06554R1Phenolic concentrations and carbon/nitrogen ratio in annual shoots of bilberry (Vaccinium myrtillus) after simulated herbivory.PLOS ONE

Dear Dr. Schrijvers-Gonlag,

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.

==============================Reviewer #2 raised several very important concerns regarding the tradeoff existing in changing herbivory regimes between defense and metabolic processes. Please take into a careful consideration their points, which can aid in further improvement of the manuscript quality. Furthermore, a visual presentation of the study setup would be much helpful.==============================

Please submit your revised manuscript by Jan 26 2024 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 rebuttal 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.

We look forward to receiving your revised manuscript.

Kind regards,

Branislav T. Šiler, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

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2. 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 #2: Partly

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

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4. 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 #2: Yes

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5. 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 #2: Yes

**********

6. 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 #2: This is my second time reviewing this paper, and while I appreciate that the authors have made substantial revisions to their MS, which have certainly increased the overall quality and clarity of the paper, I still have some concerns regarding the data presentation and the claims made. Namely, there is substantial mention of growth, for example, lines 60, 369 and 459. While I understand authors may want to use biomass data (which they have admittedly collected) for a second MS, I find that it is an important piece of data that would make their claims and discussions more robust and compelling. Especially if the aim is to, at least in part, understand the tradeoffs between defense and other metabolic processes across variable herbivory regimes. For example, you can not say for certain that plants are prioritizing growth more or less between different defoliation treatments because you have no metric of growth (total biomass, compensatory growth). To me this is extremely relevant. One scenario I could think of is that the loss of biomass from mild herbivory treatments is effectively inconsequential for plant fitness and thus responses are not obvious. When biomass loss becomes too great, plants must begin to divert resources from other pools to compensate for biomass losses. It would be interesting to see the differences in growth after herbivory treatments, which would determine if severely damaged plants actually put on more biomass then more moderately damaged plants, or if simply the amount of resources are so low that these severely damaged plants not only produce less phenolics, but also less growth and thus metabolize less, generally. Also, while the C and N concentrations may remain stable across treatments, the total pool of each that plants have access to is not consistent; in this case it is likely a function of biomass. So the amount of carbon severely damaged plants have access to should be less then mildly-damaged plants if there is a biomass reduction.

Additionally, I find the entire conclusion section quite difficult to follow. What exactly is the takeaway message?

Finally, perhaps a diagram of the plant treatments would be helpful. It is difficult to determine exactly how your treatments were conducted. As such, I can't help but wonder if some of your results could be an artifact of some concentration/dilution effect by sampling different numbers of stems/ramets between treatments, that is more severe with increasing herbivory. A clear outline (visual) could help to assuage these concerns.

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Reviewer #2: No

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Phenolic concentrations and carbon/nitrogen ratio in annual shoots of bilberry (Vaccinium myrtillus) after simulated herbivory.

PONE-D-23-06554R2

Dear Dr. Schrijvers-Gonlag,

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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Kind regards,

Branislav T. Šiler, Ph.D.

Academic Editor

PLOS ONE

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