Dear Editor,

In this document we explain how we have revised the manuscript in response to the comments made by the editor and the reviewers. Our responses start with RESPONSE >. Line numbers correspond to the files with track changes.

The reviews were very constructive and helped to improve the manuscript.

Best wishes,

on behalf of all authors

PONE-D-25-16547

Translocated populations are genetically similar to natural populations and populations resulting from natural colonizations

PLOS ONE

Dear Dr. Meléndez,

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.

RESPONSE > Thank you very much for your comments. We are happy to continue improving this paper. Below, we describe all of the changes we have made in response to your comments and those of the reviewer.

Reviewer 1 made some valuable suggestions, especially regarding the statistical analyses, which should be carefully considered during the revision. Please also make sure that the figures are displayed as intended. In the current version, many of the figures are distorted or incomplete and therefore were not easily reviewed.

RESPONSE > Thank you for the comments. We explain below how we revised the manuscript in response to the comments. Here we would just like to point out that we reran the statistical analysis with Fis as suggested and that we fixed the problem with the figures.

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf.

RESPONSE > We have made the required changes following the suggested templates to fulfil the journal requirements.

2. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering.

RESPONSE > We added the request information to the manuscript (from line 97 to 99 and from 132 to 137). Please note that we had an animal welfare permit to conduct the study. In this permit, all animal welfare issues are described and specified.

3. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process.

RESPONSE > The data used for this study is already available on Zenodo with DOI: https://doi.org/10.5281/zenodo.15052694

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

“This project was made possible through the funds of the University of Zurich, Department of Evolutionary Biology and Environmental Studies and the Consejo Nacional de Humanidades Ciencias y Tecnologías, Mexico (CONAHCYT).”

We note that you have provided additional information within the Acknowledgements Section that is not currently declared in your Funding Statement. Please note that 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.

RESPONSE > We have removed the funding information from the Acknowledgements Section and from the manuscript.

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 project was made possible through the funds of the University of Zurich, Department of Evolutionary Biology and Environmental Studies and the Consejo Nacional de Humanidades Ciencias y Tecnologías, Mexico (CONAHCYT).”

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

RESPONSE > The Funding Statement we have provided is correct. We don’t wish to do any amendments.

5. We note that Figure 1 in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

a. You may seek permission from the original copyright holder of Figure 1 to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

RESPONSE > We made a new map using data which is all free to use. Specifically, we used map data which only uses publicly available data. The data is available in the R package “rnaturalearth” and “rnaturalearthdata”.

b. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

RESPONSE > We made a new map for which we do not need a permit.

The following resources for replacing copyrighted map figures may be helpful:

USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/

The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/

Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

Natural Earth (public domain): http://www.naturalearthdata.com/

RESPONSE > Thank you for the suggested resources. However, we used a different package in R for which we do not need a permit.

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

________________________________________

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

Reviewer #1: I Don't Know

________________________________________

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

________________________________________

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: This paper provides a valuable contribution to understanding the genomic patterns of naturally and artificially colonized ponds, and compares them to original source populations. I appreciate the authors' use of existing opportunities to assess the genomic status of an organism of conservation concern, a commendable approach to monitoring these populations. The manuscript employs a range of population genetic tools and statistical approaches to assess and project genetic diversity across population types. Overall, the methods appear thoughtfully selected and generally appropriate for the study's objectives. I offer the following comments and suggestions to help improve clarity and robustness:

RESPONSE > Thank you for your positive assessment of our manuscript.

Introduction:

The statement, “Beatrice Lüscher made sure that no translocations took place in these naturally colonised populations,” is mentioned. However, without additional context regarding who Beatrice Lüscher is outside the author section and how they ensured no translocations, it is difficult to fully understand the significance of this statement.

RESPONSE > We added the information to the manuscript that Beatrice Lüscher is the local amphibian conservation officer.

Sampling:

The study uses a good narrow sampling timeframe, which is especially relevant for a species that reproduces annually. This strengthens the validity of their results.

One point of clarification: I am unsure if the tadpoles sampled survived the sampling process. If the organisms do not survive, it would be helpful to specify this to avoid any potential confusion.

RESPONSE > It is a threatened species. Therefore, there was no lethal sampling. This is now explained in the section “Research permits and ethical consideration”.

Methods:

The laboratory methods used are sound, and bioinformatics techniques for assessing linkage disequilibrium (LD), Hardy-Weinberg equilibrium (HWE), null alleles, and percent missing data appear appropriate and well-executed. For genetic diversity and bottleneck detection, AR and private alleles are strongly associated with sample size. Larger sample sizes typically contain more alleles compared to smaller samples. It seems in your results that this pattern doesn't obscure your results, which I think makes them even more meaningful. The authors use mean Fis to assess whether it significantly differs from zero. However, Fis values are often not normally distributed across microsatellites. Given this, I suggest using the median Fis to summarize the data, as it may offer a more robust estimate, or demonstrate normality.

RESPONSE > We used the median Fis values as suggested by the reviewer to assess whether it significantly differs from zero. Only the result for Lucerne natural populations changed from no significant to significantly different from zero (line 157; from line 259 to 264; line 362; Table S1).

Statistical Analyses:

The use of linear models to compare genetic diversity indicators across regions and population types is valid for continuous response variables. However, I recommend clarifying whether the model assumptions (e.g., normality) were checked, particularly for metrics like Fst and Fis. These metrics may require transformations before fitting a linear model, and this should be addressed in the methods section.

RESPONSE > The model assumption of normality of residuals was checked for each model. For all models, the null hypothesis of normality was not rejected (From line 172 to 175).

On line 160, the authors suggest that the Ne values reaching infinity could be due to sampling error. I encourage the authors to review the literature for other possible explanations of this phenomenon, as it may not be solely a result of sampling error.

RESPONSE > We have added two possible more explanations in accord to our findings (from line 162 to 166).

The use of BOTTLENECK is appropriate for detecting recent bottlenecks, and the authors selected models suited to microsatellite data. However, I recommend reporting the specific significant p-values, rather than just using the asterisk notation, to allow for clearer interpretation. Additionally, given the weight placed on bottlenecks in the discussion, the results section could benefit from more detailed reporting of the bottleneck results, including data from the supplemental section.

RESPONSE > We have added the p-values in Table S4 for the models SMM and TPM. For the allele frequency distribution there is not p-values but frequencies across loci and tends to follow an L-shaped distribution a shifted mode. After a recent bottleneck rare alleles are lost more rapidly than intermediate-frequency alleles. This shifts the allele frequency distribution. This is known as shifted mode. So, we can only report if the frequencies of alleles across loci have a L-shape distribution or a shifted mode (presence of bottleneck). This all is explained in the references we cited in the manuscript

Attachments

Attachment

Submitted filename: ResponseToReviewers.docx

Dear Editor,

Thank you for giving us another opportunity to revise our manuscript. In this document we explain how we have revised the manuscript in response to the comments made by the editor and the reviewers. Our responses start with RESPONSE >. Line numbers correspond to the files with track changes.

We deleted text from the previous decision letter that is not relevant here.

The reviews were very constructive and helped to improve the manuscript.

Best wishes,

on behalf of all authors

Subject: PLOS ONE Decision: Revision required [PONE-D-25-16547R1]

PONE-D-25-16547R1

Translocated populations are genetically similar to natural populations and populations resulting from natural colonizations

PLOS ONE

Dear Dr. Meléndez-Cal-y-Mayor,

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.

I agree with the new reviewer that the manuscript did greatly improve and that many of the previous comment have been addressed but there are still a few comment that have not been answered thoroughly enough.

RESPONSE > Thank you. Below we explain how we improved the manuscript. In the last revision, we gave the most consideration to those comments that served to improve the answers to our scientific questions.

Please submit your revised manuscript by Dec 06 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'.

We look forward to receiving your revised manuscript.

Kind regards,

Sven Winter

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.

RESPONSE > We have checked our reference list, and they are complete and correct.

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: I have carefully examined the authors’ point-by-point responses to the previous review as well as the revised version of the manuscript. Overall, it is clear that the authors have made a substantial effort to address the earlier concerns, and many of the revisions have improved the clarity, methodological transparency, and overall quality of the paper. In particular, the handling of the statistical comments appears sound: the use of median Fis values was implemented as suggested, model assumptions were tested and reported, and the treatment of bottleneck results has been improved. Ethical aspects related to animal handling have been clarified with commendable precision, and the data availability now meets the journal’s requirements. These are all meaningful improvements.

The manuscript itself presents a well-defined and relevant study that addresses the important question of genetic similarity of translocated, colonized, and natural populations. The research is well framed and generally methodologically solid. The use of multiple analytical approaches and the integration of empirical data with forward simulations are notable strengths that make this study potentially valuable to the conservation genetics community.

RESPONSE > Thank you for the positive assessment of the manuscript and the revision.

However, some of the responses remain rather superficial. This is especially true for the interpretation of infinite Ne values, which is acknowledged but not convincingly discussed,

RESPONSE > We added more specific explanations of the potential reasons why we found infinite Ne values based on previous works and on our data (from line 160 to 165). We explain that we think that infinite Ne are most likely an artefact.

and for the section on bottleneck events, where additional depth and contextualization would strengthen the argument

RESPONSE > We added more additional information to strengthen our argument (from line 372 to 385). We explain why we believe that there were bottlenecks in the recently colonized populations (simply because colonization caused the bottleneck). In one of the “old” natural populations in Lucerne area, we believe it was due to maintenance of the well.

(please also check all references again, some are missing, e.g., l. 180 "residuals were tested for each model using the function jarqueberaTest () and dwtest ()").

RESPONSE > The citation brackets in PlosOne are []. The brackets that the reviewer mentioned as lacking a citation correspond to the functions jarqueberaTest and dwtest (it is R language). These brackets indicate that both are functions, and inside the brackets, the parameters are specified. The functions jarqueberaTest and dwtest do not require a citation because they are included in the fBasics package, which is already cited (lines 176-177).

The adjustments to figures and tables still fall short of making the results fully accessible at a glance. For example, the revised map is likely clearer than the original, but the description suggests that the overall visualization strategy remains basic rather than genuinely improved, and the whole map (still?) appears broken to me. Please check your visualization tool.

RESPONSE > It is a simple map which shows the location of the adjacent study areas and the populations. We used different colours for the two study areas. We believe that the map serves its purpose. We double-checked all files during the submission process to make sure that the maps are displayed correctly.

Moreover, all figures and tables need a proper caption, which is self-explanatory; e.g., table 1 lacks a conclusive caption.

RESPONSE > We have included more details in the captions of all figures and tables.

A general pattern throughout the responses is that several replies address the comments formally, but without fully exploiting the opportunity to clarify or deepen the presentation of the results. For example, the discussion of the infinite Ne values just lists two additional explanations without critically evaluating their likelihood in the specific study context or providing supporting literature.

RESPONSE > We added some additional explanations for Ne and bottlenecks. We don’t think that a further discussion is useful because it would be speculative. Regarding the infinite Ne values, we added additional explanations and supporting literature in the manuscript (from line 160 to 165). This new information explains the possible reasons why the Ne value was infinite for each population.

The response to the bottleneck-related comment adds some details but remains descriptive and lacks an interpretation of how these results inform the study’s main conclusions.

RESPONSE > We added in the manuscript more interpretation on the presence of bottleneck in 3 colonized populations and in a natural population (from line 374 to 385). This connects with the discussion we do on the presence of bottleneck from line 424 to 430. The main conclusion is that the three population types do not differ much despite the bottlenecks. We added some text in the discussion which clarifies this.

Likewise, the comment on improving figure clarity was addressed by creating a new map, but without any clear rationale for how the new visualization strategy better communicates the underlying patterns.

RESPONSE > We believe that the new maps convey that necessary information. Fig 1 shows the two study areas and all the populations while Fig 3 shows that spatial genetic structure. The maps are simple, but all the necessary information is there. There is no additional information in the maps that may distract (e.g. Hill shade).

While the core analyses seem sound and the technical points were largely addressed, the interpretation and communication of the findings could still be more thorough.

RESPONSE > We added additional explanations in the discussion in response to the previous comments and hope that this made the interpretation and discussion of the results more thorough.

In sum, the authors have clearly engaged with the reviewers’ feedback and improved the manuscript in several important respects.

RESPONSE > Thank you.

Yet some areas would benefit from more thoughtful revision, particularly regarding interpretation of key genetic results (biological context) and data presentation. I would therefore recommend minor revisions, with a focus on strengthening the clarity and depth of interpretation as well as improving the accessibility of figures and tables.

RESPONSE > We explain in the replies to the previous comments how we improved the manuscript.

Attachments

Attachment

Submitted filename: ResponseToReviewers_auresp_2.docx