PONE-D-19-19484

Geographical origin determines responses to salinity of Mediterranean caddisflies

PLOS ONE

Dear Dr. Ramos-Jiliberto,

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 minor revision of the manuscript that addresses the points raised during the review process.

In your re-submission, please carefully address each of the reviewer comments. The significant difference in responses between basins was striking, raising the question about the nature of population differentiation. Please address this point in revisions, as well as more minor comments regarding clarification of details.

We would appreciate receiving your revised manuscript by Dec 23 2019 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Rachel A Hovel

Academic Editor

PLOS ONE

Journal Requirements:

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

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

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

1. In your Methods section, please provide additional location information of the study areas, including geographic coordinates for the data set if available.

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. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

[Note: HTML markup is below. Please do not edit.]

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: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

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

Reviewer #1: No

Reviewer #2: 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

Reviewer #2: 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: According to the instructions to authors, the authors either need to place the data in a public repository, where URL, accession numbers or DOIs can be cited, or can be contained in Supporting Information files. The current statement says that the data will be available upon request.

The paper needs a little editing, but, per instructions, I did not note specific edits, except that in the second sentence of the Introduction, the verbs should be "disrupts" and "threatens".

The authors might consider discussing how a species might adapt to differences in salinity across its range. At higher taxonomic levels, adaptation to different salinities can involve small mutations to the genes for the ion transporter genes that affect influx and efflux rates. In anadromous or catadromous species, such as salmonids and eels, adaptation may involve up-regulation or down-regulation of these ion transporters or of entire ionocytes.

Reviewer #2: Carter, Flores, Ramos-Jiliberto

Geographical origin determines responses to salinity of Mediterranean caddisflies

This study looks the importance of source (river basin of origin) when assessing behavioral and survivorship responses of larval caddisflies (Smicridea annulicornis) when exposed to elevated salinity. This species appears to be widely distributed across Chile and Argentina, and expresses significant genetic differentiation within a basin, and across basins. Larvae were collected from three basins: Choapa (low conductivity), Maipo (medium conductivity) and Maule (high conductivity), and based on ambient conductivities, they tested three experimental conductivity levels: low (180 μS/cm), medium (500 μS/cm) and high (1400 μS/cm) different salinity levels were prepared with aquarium sea salt dissolved in distilled water. Response variables measured were time to death, and behavior (swimming, sheltering, walking, pushing up). This assessment of behavioral responses (i.e., non-lethal) was an interesting addition to the standard survival study. The experiments found significant differences between basins – big differences. This is most unusual and suggests measurable local adaptation or acclimation.

One factor that is only lightly addressed is this species in this region exhibits significant genetic differentiation among basins (Sabando et al. 2011) – suggesting either local adaptation with little or no gene exchange, or possibly some closely related species or subspecies that are unrecognized and responding to a strong habitat template.

Specific comments;

I assume the experiments were conducted at 20 C, same as the acclimation temperature.

Survival time – Table 1 indicates that basin matters, and that the effect of conductivity was not significant (p=0.08), but the results present a bunch of antidotal assessements of conductivity effects. Table 1 results and the Survival results need to be in agreement – at least justify the details by calling the factorial result “nearly significant” and not “marginal”.

In the behavioral tests, why do you think the BC x Basin was significant? Why would behavioral responses differ between basins within a species?

In the behavioral tests, do you believe behavioral responses are in some what adaptive with regard to salinity?

The results presented here make one question standard bioassay results – where was the original test population from? Have laboratory conditions changed the test organisms?

**********

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

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

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

Reviewer #1: No

Reviewer #2: No

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

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

Geographical origin determines responses to salinity of Mediterranean caddisflies

PONE-D-19-19484R1

Dear Dr. Ramos-Jiliberto,

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

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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

With kind regards,

Rachel A Hovel

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

This revision has sufficiently addressed reviewer comments and outstanding concerns on this manuscript, and I am pleased to accept it. Please make sure to thoroughly proofread the manuscript before submission. For example, Line 71 now reads: "We conducted this study usin as a model speciesgthe...". It will be your responsibility to identify and remedy typographical or other errors.

Reviewers' comments: