Dear Dr. Daniel Robledo,

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.

The reviewers recognize the scientific merit and relevance of your work, which provides important insights into the reproductive biology, biochemical composition, and cultivation potential of a tropical carrageenophyte.

After careful evaluation, the editorial board has concluded that your manuscript requires major revision before it can be considered for publication. The reviewers have provided constructive comments focusing on statistical clarity, consistency in data presentation, and overall structural refinement to enhance readability and scientific impact.

We encourage you to carefully address all reviewer suggestions and resubmit a thoroughly revised version, highlighting the changes made and providing a detailed response to each comment.

We appreciate your contribution to the field and look forward to receiving your revised manuscript.

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Kind regards,

Inbakandan Dhinakarasamy, Ph.D

Academic Editor

PLOS ONE

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2. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why.

3. Thank you for stating the following financial disclosure:

“This study was funded by the project ARPA-e DE-AR0000912 - Sub-award 54336 Development of Techniques for the Cultivation of Tropical Algae.”

Please state what role the funders took in the study.  If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

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4. Thank you for stating the following in the Funding Section of your manuscript:

“This study was funded by the project ARPA-e DE-AR0000912 - Sub-award 54336 Development of Techniques for the Cultivation of Tropical Algae.”

We note that you have provided funding information that is currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“This study was funded by the project ARPA-e DE-AR0000912 - Sub-award 54336 Development of Techniques for the Cultivation of Tropical Algae.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

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.

Additional Editor Comments (if provided):

The manuscript presents valuable baseline information on the reproductive biology, biochemical composition, and cultivation potential of a native tropical carrageenophyte, offering important insights into its role in sustainable aquaculture. The study is scientifically relevant and timely, and it successfully integrates morphological, molecular, and biochemical approaches. However, the manuscript requires substantial revision before it can be considered for publication.

The statistical analysis needs improvement through the inclusion of variability measures such as mean ± SD or SE and clear indication of significance among treatments. The rationale for using non-parametric tests should be clarified by specifying which datasets failed normality and reporting corresponding test results. The organization of the text should be refined with appropriate subheadings in the Methods and Results sections to enhance readability and logical flow. Simplifying long sentences and reducing repetitive numerical details will make the manuscript more concise and engaging. Figures and tables need to consistently indicate the number of replicates, statistical notations, and appropriate salinity units (PSU or ppt), with improved legends that summarize the main findings. Consistency in terminology, species naming, abbreviations, and reference formatting is also necessary.

The introduction should be condensed to focus on the rationale and novelty of the study, highlighting how it differs from previous work on related species. Incorporating recent literature (2023–2024) would strengthen its scientific depth. The discussion could be improved by emphasizing the biological and aquacultural implications of the results, particularly how morphological variation, carrageenan characteristics, and induced sporulation relate to environmental adaptation and cultivation potential.

In summary, the manuscript has strong scientific potential and aligns well with the scope of the journal. With careful attention to structure, statistical rigor, and clarity in interpretation, it could become a valuable contribution to the field.

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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?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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.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??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: This manuscript has potential to publish, and, no doubt that the manuscript and the topic is interesting, significant and fits very well in the core topics covered by the journal. However, my opinion is, there are still major improvements needed to accept this manuscript for publication.

General Comments:

1. Please explain. how do environmental conditions in the season (temperature, light, nutrient availability) influence reproductive phenology and carrageenan biochemical composition of E. isiformis?

2. Do the morphotypes in the collection site adapt to different microhabitats and is possibly the reason of morphological differences but not molecular divergence?

3. How does the change of the carrageenan sulphate content of the morphotypes have any functional value, especially under the conditions of use in the industry?

Specific comments:

Abstract suggest to reduce and emphasize the key findings for clarity.

Line 15 and 16: “biomass fitness” → “biomass decline,” “lack of genetic exchange” → “limited genetic exchange.” Suggested word changes:

Line 38- 49: Repetition of “genetic diversification.” Please check once.

Line 49- 56: Some sentences appear long and complex, which can short sentences to improve clarity.

Proofread the entire paper and maintain abbreviations (e.g., Eucheumatopsis isiformis abbreviated to E. isiformis).

Line 114: The manuscript alternates between referring to the species as Eucheumatopsis isiformis and its synonym Eucheuma isiforme. Consistent usage throughout would avoid confusion.

Line 81 to 211: Detailed technical protocols are included, but use subheadings to break down the methods into separate sections (e.g., DNA extraction, PCR, carrageenan extraction) to improve readability.

Table 3 contains dense data, but lacks descriptive legends that clarify significance.

Sections sometimes jump suddenly from one topic to another (e.g., molecular analysis to carrageenan extraction).

Reviewer #2: I have carefully reviewed the manuscript entitled

“Studies on the tropical carrageenophyte Eucheumatopsis isiformis (Solieriaceae, Rhodophyta) from Yucatán, Mexico: cultivation potential based on morphological, chemical, and reproductive features.” This manuscript presents valuable baseline information on the reproductive biology, biochemical composition, and cultivation potential of the red alga Eucheumatopsis isiformis collected from Yucatán, Mexico. The topic is both relevant and timely, as it contributes to the understanding of native eucheumatoid species and their potential applications in sustainable aquaculture, an area of increasing scientific and economic importance.

The study integrates multiple aspects including field sampling, morpho-anatomical characterization, molecular analysis, and carrageenan composition, offering a comprehensive foundation for further research on E. isiformis.

However, while the study is scientifically significant, the manuscript requires structural refinement, stronger statistical justification, and improved consistency in data presentation to meet publication standards. My detailed comments are summarized below for your consideration.

Major Comments

1. Lack of variability reporting in polyamine-induced sporulation experiment

In the results of the polyamine-induced sporulation experiment (Page 378–382; Fig. 6), only the carpospore counts are presented (e.g., “363 carpospores per 5 mL…”), without reporting standard deviation (SD) values or error bars in the figure. Although the “Materials and Methods” section (Page 10, Lines 195–199) indicates that each treatment was performed in triplicate (n = 3), this information is not reflected in the results. The authors should include mean ± SD (or SE) values and, if applicable, significance indicators among treatments to ensure statistical completeness and data reliability.

2. Unclear justification for the use of non-parametric tests

The manuscript applies both parametric (ANOVA) and non-parametric (Kruskal–Wallis) tests, but the justification for using the non-parametric approach is unclear. Although the Statistical Analysis section (Page 10–11, Lines 208–221) mentions the use of Shapiro–Wilk and Levene’s tests to assess normality and homogeneity, the authors do not specify which dataset(s) failed these assumptions. Please clarify which data did not meet normality, report corresponding test results (e.g., p < 0.05), and explain the rationale for applying non-parametric analysis.

Minor Comments

1) Salinity units:

The unit of salinity should be expressed consistently throughout the manuscript according to the instrument used for measurement. Please use PSU (Practical Salinity Unit) or ppt (parts per thousand) instead of ppm, which is not appropriate for salinity reporting in marine studies.

2) Incomplete references:

Some references are missing publication years (e.g., “Refs. [37–38, 62–66]”). Please check all references carefully and complete missing bibliographic information in accordance with the journal’s reference style.

3) Statistical notation consistency:

Ensure consistent formatting of statistical terms (e.g., n = 3, p < 0.05, mean ± SD) throughout the manuscript, tables, and figure captions.

4) Figure legends:

Include the number of replicates (n) and clarify whether values are presented as mean or median in all figure legends.

5) Abbreviation clarity:

Check that all abbreviations (e.g., SPE, PUT, PES) are defined at first mention in both the text and figure captions.

In summary, the manuscript is scientifically promising and provides useful data for future studies on the reproductive biology and mariculture potential of E. isiformis. However, revisions are necessary to improve clarity, statistical transparency, and some issues that require correction before it can be considered for publication.

Reviewer #3: Studies on the tropical carrageenophyte Eucheumatopsis isiformis (Solieriaceae, Rhodophyta) from Yucatán, Mexico: cultivation potential based on morphological, chemical, and reproductive features”

1. Title is scientifically sound and informative, but it is quite long and descriptive.

May be considered

Cultivation potential of the tropical carrageenophyte Eucheumatopsis isiformis (Solieriaceae, Rhodophyta) from Yucatán, Mexico

2. The abstract effectively highlights the cultivation potential of Eucheumatopsis isiformis; it could be improved by briefly summarizing the key morphological and molecular differences among the analyzed morphotypes to strengthen the connection between diversity and cultivation potential.

3. The introduction is too lengthy, which may overwhelm readers.

4. Some background information especially on general seaweed life cycles and previously studied Eucheuma species—could be condensed to maintain focus on Eucheumatopsis isiformis and the specific research gap.

5. Although the section includes relevant citations up to 2022, it lacks very recent studies (2023–2024) on seaweed genetic improvement, reproductive biology, or carrageenan yield optimization. Including these would enhance the currency and scientific depth of the background.

6. The introduction should more clearly highlight why E. isiformis was chosen, how it differs from commercial Eucheuma species, and what specific research questions or hypotheses this study addresses.

7. The section is well-structured and provides comprehensive methodological details for replication. Provide subheadings with clearer separation (e.g., Sample processing, Microscopy and staining, Data analysis) to enhance readability, as the current format is text-heavy and occasionally dense.

8. The sporulation induction and carrageenan extraction procedures are well explained, but the rationale for selecting specific concentrations of polyamines and irradiance levels could be briefly justified with relevant citations or preliminary data to strengthen methodological reasoning.

9. The section demonstrates appropriate use of statistical tests; however, the choice of parametric vs. non-parametric methods could be better justified. Additionally, information on software used for analysis (e.g., SPSS, R, or GraphPad) should be explicitly mentioned for transparency and reproducibility.

10. Some sentences are overly long and could be simplified. There are minor typographical errors (e.g., double periods in “PCR conditions.” and “residual Cetavlon.”). Consistent use of units (e.g., “μmol·m⁻²·s⁻¹”) and spacing around symbols should be checked for formatting accuracy.

11. The result section provides rich data but could benefit from clearer subheadings for each main result (e.g., Morphoanatomical characterization, Carrageenan content, Molecular analysis, Reproductive phases, Sporulation experiments). This would enhance readability and help the reader follow the logical sequence of findings.

12. The text contains excessive numeric detail (e.g., every mean and standard deviation is listed in sentences as well as in tables). This makes the narrative dense and repetitive. Consider focusing the text on key trends, comparisons, and significant findings, while leaving raw data to tables and supplementary files.

13. The integration of figures and tables is appropriate, but figure legends and table references could be clearer. Some figure legends (e.g., Figs. 6–7) could briefly summarize the key outcome (e.g., “Carpospore release enhanced under 10⁻³ M PUT treatment compared with control”).

14. While some statistical results (e.g., F=9.57, p<0.01; H=75.74, p<0.01) are reported, interpretation of their biological meaning is limited. It would strengthen the result section to discuss what these differences imply e.g., whether morphotype differences correlate with environmental adaptation, or whether the yield reduction post-alkaline treatment is significant for industrial use.

15. The molecular analysis is concise and clear, but lacks a visual reference (e.g., phylogenetic tree figure or haplotype network). The paragraph would be stronger if you explicitly noted whether the lack of genetic differentiation despite morphological variability suggests phenotypic plasticity or cryptic speciation.

16. The experiment is well-detailed, but results and interpretation are somewhat blended with methods. For instance, repeated mentions of acclimatization periods, temperature, and irradiance fit better in the Methods. Here, focus on outcome patterns e.g., “Putrescine (10⁻³ M) significantly enhanced carpospore release at lower irradiance, although variability across days was noted” to clarify the biological relevance.

17. The discussion contains a lot of detailed data and literature references, which demonstrate strong experimental support. However, it would benefit from more concise synthesis clearly highlighting how your findings advance understanding of E. isiformis biology or cultivation compared to previous studies. Some paragraphs could be shortened by summarizing numerical data instead of repeating all values.

18. While the text notes that no genetic differences were observed among morphotypes, it could be strengthened by discussing the implications of this finding for example, whether this morphological variability is due to environmental plasticity or phenotypic adaptation.

19. The discussion ends with good experimental observations on polyamine-induced sporulation, but it should also clearly state the broader implications such as how these findings contribute to large-scale cultivation strategies or seedbank development for tropical carrageenophytes.

20. Adding 2–3 lines on how this work supports future selective breeding or aquaculture resilience would make the conclusion stronger and more impactful.

21. The conclusion clearly outlines future research directions but could be more concise and focused on the main findings such as species identification, morphotype characterization, and successful sporulation induction before discussing future perspectives.

22. It would be beneficial to separate experimental details from general recommendations, presenting a clearer summary of key outcomes followed by suggested future studies for better readability and impact.

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what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Rajasekar ThirunavukkarasuRajasekar ThirunavukkarasuRajasekar ThirunavukkarasuRajasekar Thirunavukkarasu

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Submitted filename: Review-Final.docx

Cultivation potential of the tropical carrageenophyte Eucheumatopsisisiformis (Solieriaceae, Rhodophyta) from Yucatán, Mexico

PONE-D-25-36873R1

Dear Dr. Daniel Robledo

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,

Inbakandan Dhinakarasamy, Ph.D

Academic Editor

PLOS One

Additional Editor Comments (optional):

Thank you for your careful revision and for satisfactorily addressing the reviewers’ comments. The manuscript is now considered suitable for acceptance; however, we kindly request that a few minor issues be addressed prior to final acceptance. Specifically, please ensure that the references are presented in a consistent format throughout, that all figures (particularly photographs) include clearly visible scale bars, and that a small number of recent and relevant references are incorporated while keeping the overall reference list concise. We would appreciate it if you could make these final adjustments and resubmit the revised manuscript at your earliest convenience to facilitate acceptance without further review.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

Reviewer #3: All comments have been addressed

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

Reviewer #1: (No Response)

Reviewer #3: Partly

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

Reviewer #1: (No Response)

Reviewer #3: 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.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: (No Response)

Reviewer #3: Yes

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

Reviewer #1: (No Response)

Reviewer #3: Yes

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Reviewer #1: The authors answered to satisfy the comments; however, there are a few minor corrections that should be done before acceptance of this manuscript; particularly, the references should be uniform. Figures (photos) should show the scale clearly visible. Latest references should be added and number of references should be limited.

Reviewer #3: 1. What are the challenging processes to extract carrageenan from seaweeds?

2. What percentage of carrageenan yield comes from Eucheumatopsis isiformis?

3. How do you differentiate carrageenan from E. isiformis compared with Kappaphycus alvarezii?

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what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). 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 For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy..-->

Reviewer #1: No

Reviewer #3: No

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