PONE-D-24-25938Karst-environments of the southeastern Yucatan Peninsula: hotspots for modern freshwater microbialitesPLOS ONE

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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

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

Reviewer #1: Yes

Reviewer #2: 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.

Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: 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: The manuscript titled "High Connectivity of Microbial Communities in Karst Aquifers: A Study of Microbialites in the Yucatan Peninsula" presents a comprehensive study on the hydrogeochemical profile, mineralogy, elemental geochemistry, and microbial composition of microbialites from the Yucatan Peninsula. With some minor revisions to improve clarity and readability, this paper should make a significant contribution to the field of microbialite ecology and conservation. The authors should address the grammatical issues, consider revising some figures for clarity, and potentially expand on the implications of their findings for environmental monitoring and conservation efforts.

Ln 61-66 This sentence is too long. Consider to break it down.

Ln 141 Remove the comma “.”

Ln 174 hclust function?

Ln 189 Do not italic, keep the format consistent throughout

Provide high-resolution figures (e.g., figure 7) for better readability.

Ln 257 Is that common to see high levels of rubidium and niobium in the lake that forms microbialites? What are the potential implications of these trace elements for microbialite formation or ecology?

The manuscript suggested that microbialites could serve as biosensors for water quality. Could the authors elaborate on how this might be implemented in practice? What specific microbial community changes would indicate declining water quality?

The authors mentioned the need for conservation of microbialites. Based on their findings, what specific conservation measures would they recommend for the microbialite sites in the Yucatan Peninsula?

The study also mentioned the presence of eukaryotic organisms associated with microbialites. I wonder how might these eukaryotes influence microbialite formations and their stabilities.

Reviewer #2: This work is a significant contribution, and it should be published. However, as a comparative study, the authors have overlooked several facts occurring in this region. Some of the results from this study do not compare well with previous results published by them or other researchers. I will present a more detailed account of them in the following paragraphs.

Additionally, I detect some errors in their interpretations and affirmations. This region is unique in the world, and all papers published should establish a good comprehensive understanding of existing literature together with the results presented in this manuscript, to help in more integral actions for their conservation, not only for the microbialites, because they are part of complex and fragile ecosystems with many interactions not known. The region has been seriously affected by a disordered growth of population and tourism and, lately by a controversial train:

Martínez-Romero, E., G. Gasparello & M. A. Díaz-Perera, 2023. Territorios mayas en el paso del tren. Riesgos previsibles y posturas independientes sobre el tren maya. Bajo Tierra Ediciones, Mexico. 332 p.

Ortega, R. P. & I. G. Jaber, 2022. ENVIRONMENT A controversial train heads for the Maya forest. Science 375(6578):250-251.

The only way I envisage is using the data presented in the literature to support a better decision from the whole society to conserve this fragile region.

It's crucial to clearly distinguish between new and previously reported data to avoid confusion. For instance, in lines 521-524, the authors discuss the dominant species in Cenote Azul, but they reported in their citation [27] that 70% was Microcystaceae from Cenote Azul. They do not cite their previous study. This lack of distinction is not unique to this instance, making it difficult to discern new information from previously reported data in the whole paper. Accurate citation is a fundamental part of our academic community, and it's essential that we all play our part in maintaining its integrity. Additionally, Planktothrix is not mentioned in the citation [27] (see Line 527), at least not in my reading.

Some punctual remarks are as follows:

Line 63.- It is not the second, it is the deepest:

Alcerreca-Huerta, J. C., O. F. Reyes-Mendoza, J. A. Sanchez-Sanchez, T. Alvarez-Legorreta & L. Carrillo, 2024. Recent records of thermohaline profiles and water depth in the Taam ja’ Blue Hole (ChetumalBay, Mexico). Frontiers in Marine Science 11:1387235. doi:10.3389/fmars.2024.1387235.

Line 64.- What about Calakmul reserve, it is bigger than Sian Ka’an.

Line 65.- The authors should have mentioned Chichancanab in the introduction, where a unique flock of Cyprinodon speciates. It is the second largest lake from the Yucatan Peninsula and hosts microbialites, as they will report later in this manuscript. Moreover, it is part of the systems studied here, but at this point of the manuscript, I did not detect it.

Strecker, U. S. u.-h., de), 2004. The Cyprinodon species flock from Laguna Chichancanab, Mexico (Teleostei): sexual and

disruptive selection driving adaptive radiation. Mitteilungen aus dem Hamburgischen Zoologischen Museum und Institut 101:65-74.

In Fig. 1 authors should explain the studied sites with detail.

Line 97.- Please provide coordinates of the sampling sites; microbialites are not the same in large lakes as in Bacalar. Later, the authors said they sampled Bacalar North and South. Besides, the results presented in previous studies must be contrasted with those of this new study.

In addition, how did they sample five sites in small and deep systems such as Cenote Azul and large systems such as Bacalar, Chichancanab, or Muyil? At what deep? Cenote Azul is 65 m deep, and Bacalar averages 8.5 m. Moreover, on which dates did the authors take the sampling? A severe brownification event in Bacalar in 2020 lasted more than one year for a full recovery of the system. We do not know the effect of this event on the stromatolites:

Carrillo, L., M. Yescas, M. O. Nieto-Oropeza, M. Elías-Gutiérrez, J. C. Alcérreca-Huerta, E. Palacios-Hernández & O. F. Reyes-Mendoza, 2024. Investigating the Morphometry and Hydrometeorological Variability of a Fragile Tropical Karstic Lake of the Yucatán Peninsula: Bacalar Lagoon. Hydrology 11(68) doi:10.3390/hydrology11050068.

Line 105.- Maybe the largest in the world:

Gischler, E., M. A. Gibson & W. Oschmann, 2008. Giant Holocene freshwater microbialites, Laguna Bacalar, Quintana Roo, Mexico. Sedimentology 55(5):1293-1309.

Line 109.- It was a personal observation or it was published?

Perry, E., G. Velazquez-Oliman & L. Marin, 2002. The hydrogeochemistry of the karst aquifer system of the northern Yucatan Peninsula, Mexico. International Geology Review 44(3):191-221.

Line 110.- The maximum depth reported and detected is 65 m, not 100 m. How the authors find stromatolites at 70 m? In ther citation 27, same authors collected up to 30 m, and they do not report to 70 m, as well the citation number 30 does not say anything about stromatolites to 70 m. The most interesting point in the citation 30 is that Cenote Azul has almost an entirely different composition of zooplankton than Bacalar. Then, not only the water chemistry is different, also the communities, including the stromatolites.

Cervantes-Martínez, A., M. Mezeta-Barrera & M. Gutiérrez-Aguirre, 2009. Basic limnology of the karstic tourist lake Cenote Azul in Quintana Roo, Mexico. Hidrobiologica 19(2):177-180.

Line 114-122.- Apart of the points remarked by authors Chichancanab hosts a speciation of Cyprinodon (from marine origin) quite special, and they do not mention it. I mentioned this point previously.

Line 123-131.- Here the authors do not mention a comparison of Muyil with other lakes nearby

Valdez-Moreno, M., M. Mendoza-Carranza, E. Rendon-Hernandez, E. Alarcon-Chavira & M. Elias-Gutierrez, 2021. DNA Barcodes Applied to a Rapid Baseline Construction in Biodiversity Monitoring for the Conservation of Aquatic Ecosystems in the Sian Ka'an Reserve (Mexico) and Adjacent Areas. Diversity-Basel 13(7):22 doi:10.3390/d13070292.

Line 234.- Muyil lake is about 9 km from the shoreline, and is connected indirectly to the sea by a complex series of channels. I believe is not extrictely “coastal”. It does not show brackish conditions as any coastal lake.

Line 237.- NOx and ammonium values are too high. Did the authors some crosslab comparisons?

Line 239.- The authors should have mentioned that they sampled Bacalar North and South in the methods section. That is why they should detail how the sampling was conducted and on which dates. In their previous cited paper [12], they found differences between the north and south of Bacalar.

Line 316-317.- There is no explanation of sampling deeps for Cenote Azul in methods section.

Line 334-336.- The authors did not say the deeps of their samplings. Moreover, they found differences in the different deeps of Cenote Azul in a previous study (see reference 27).

Line 404.- The values presented in the previous work are quite different as far as I can see. That is the reason to know dates of sampling.

Line 405.- These systems are connected through a complex series of channels, but how they are connected should be considered: with Chile Verde, this connection is on the north side of Bacalar.

Line 417-418.- What about the fauna? There is some penetration of the marine fauna? It is documented.

Line 441.- I do not agree. Most (if not all) water inflows to Bacalar are also groundwaters. Some superficial sporadic inflows are present in rain season in the north.

Line 456-463.- Because of these reasons, it is important to know the sampling dates. Bacalar Lake and its associated systems passed an intense event of brownification and it should be established a before-after baseline.

It apparently caused also mortality in several of the communities from this site, for example (unfortunately, this report is gray literature):

Castro-Chan, R.A.; De Jesús Navarrete, A.; Zavala Mendoza, A. Estudio Para Establecer Las Causas de La Mortalidad Masiva Del Caracol de Agua Dulce Chivita Pomacea Flagellata; Consejo Quintanarroense de Ciencia y Tecnología: Chetumal, Mexico, 2021.

Line 492-497.- It is unclear if these are results from this study or the previous study because the authors do not cite it in this part, and the sampling they used is unclear. See citation 27.

Line 518.- What about citation 27, it compares different depths in Cenote Azul.

Line 530.- Please add some citations.

Line 562-565.- It is not at all accurate; the communities are realizing the relevance of microbialites, and some examples exist (maybe small or limited, but they are starting points):

O'Kelard, A., 2021. Don Lito habla desde Bacalar: Importancia de los estromatolitos en el origen de la vida en el planeta. Global Design. 121 p.

Line 582-585. The authors miss out on the fact that each system studied seems unique, with a particular bacteria association. The stromatolites are part of the whole picture of each system's biodiversity. Then, by the end of the study, it is unclear whether connectivity exists. As far as I can understand, all studied systems are different in composition in microbialites and turnover, with different chemistry of the waters. These results can be obvious if comparing, for example, Cenote Azul and Bacalar, which are about 200 m apart but also occur in more or less similar physiographically systems such as Chichancanab and Muyil or Bacalar.

Although the manuscript's scope is to highlight the Yucatan Peninsula as a hotspot for modern microbialites, the authors should mention the 2020 brownification event and the posterior recovery of some of these systems. They should also mention the actual challenges and dangers for these ecosystems. If the anthropogenic effects continue, we are approaching a non-returning point, with a possible severe consequence for the stromatolites and the other communities dwelling in these unique aquatic ecosystems.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-24-25938R1Karst-environments of the southeastern Yucatan Peninsula: hotspots for modern freshwater microbialitesPLOS ONE

Dear Dr. Falcón,

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 Apr 08 2025 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,

Yizhi Sheng

Academic Editor

PLOS ONE

Journal Requirements:

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.

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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 #1: (No Response)

Reviewer #2: All comments have been addressed

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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 #1: (No Response)

Reviewer #2: Yes

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

Reviewer #1: (No Response)

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 #1: (No Response)

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 #1: (No Response)

Reviewer #2: Yes

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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 #1: The authors addressed all questions and concerns, and I do not have additional comments on this manuscript.

Reviewer #2: The document was much improved, and I think it can be published. It is an important contribution to the knowledge of the microbialites in this region, highlighting its importance. Some minor corrections are needed for the style. I suggest an English native speaker for it.

The only point I want to make is that some conclusions are not based on the results presented in this study, for example, lines 557-564. They can be part of the introduction. Line 568 is an important point, but to my mind, it should be focused differently and based on this study's findings. Lines 571-575 also are out of the results presented here.

Throughout the manuscript, I have only some small observations.

Line 100.- In which season of the year were sampled Muyil, Chichankanaab, and Azul?

Line 391.- Citation 52 is correct for this paragraph?

Line 416-417.- Please give a citation for Alchichica Lake.

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Reviewer #1: No

Reviewer #2: No

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Karst-environments of the southeastern Yucatan Peninsula: hotspots for modern freshwater microbialites

PONE-D-24-25938R2

Dear Dr. Falcón,

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,

Yizhi Sheng

Academic Editor

PLOS ONE

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