PONE-D-24-55509Genetic variability in microbial eukaryotes reshapes marine biodiversity assessment in the age of amplicon sequencingPLOS ONE

Dear Dr. Trubovitz,Thank you for submitting your manuscript to PLOS ONE. I have received evaluations by four experts in the field  - all evaluations were enthusiastic and in support of your work, while providing constructive suggestions to revise the work. I recommend the authors pay attention to the following points raised by the reviewers: 

• Analysis of the variable regions of the long-read amplicons to confirm whether the long-read level corresponds to the same variability of the short-read level (e.g., is the v4 region the same across many of these long-read ASVs);
• Analysis of degree of similarity/differences between ASVs of the same or between different morphospecies;
• Methodological clarifications of the analytical pipeline (which identification guides used; specific DADA2 parameters; changes to accommodate long read sequencing);
• Reformatting of Table 1 based on taxonomy rather than alphabetically;
• Clarification of the cause for low passing rates of CCS reads in the Quality and Length filtering step;
• Analysis of the proportion of ASVs shared between juvenile and mature radiolarians to support statements of overestimations of species richness in radiolarian metabarcoding surveys; 
• Several of the reviewers made specific suggestions to include specific literature to support and/or discuss your findings.

I am very much looking forward to a revised version of this manuscript.

Please submit your revised manuscript by Mar 07 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,

Claudia Isabella Pogoreutz

Academic Editor

PLOS ONE

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. 2. Thank you for stating the following financial disclosure:  [This project was supported by the National Science Foundation Postdoctoral Research Fellowship in Biology Award #2109767 (to ST) and the Simons Collaboration on Ocean Processes and Ecology p49802 (to DAC). MMS was supported by a postdoctoral fellowship from the Beatriu de Pinós programme of the Government of Catalonia's Secretariat for Universities and Research of the Ministry of Economy and Knowledge (grant #2021BP00068).].  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. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

[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

Reviewer #3: Yes

Reviewer #4: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: 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: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: 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

Reviewer #3: Yes

Reviewer #4: 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: General Comments:

Trubovitz et al present a fascinating exploration of the intraspecific and intragenomic variability within polycystine Radiolarians. This work is vital as it allows researchers to connect the vast amount of morphological data available for the clade with the equally as vast genetic data recently generated. The methods are thorough, and the researchers went to great lengths to ensure their sequences were biologically representative (great stringent workflow for filtering reads) and data was sound. One key finding is that intragenomic variability within radiolaria is likely a significant source of the total genetic richness being measured in metabarcoding surveys, therefore these studies should account for this variability within their study groups. In line with this, the ASV diversity within single species was also very interesting to see. A suggestion I have that would further support/confirm the results would be to analyze the variable regions of the long-read amplicons so that you can see if the variability you see at the long-read level corresponds to the same variability at the short-read level. For instance, is the V4 region (the most commonly used in metabarcoding surveys) the same across many of these long-read ASVs? Overall, I feel that this study is suitable for publication after some minor clarifications and this quick V4 analysis. Given my background is predominately in metabarcoding and marine microbial diversity, and not in Radiolaria specifically, I would also suggest that an expert in Radiolaria be consulted before publication.

Introduction:

Line 48- “morphologically-referenced sequence databases”. I am unsure about this definition. PR2, which is the example used, is based on phylogenetics. The sequences are curated based on where they fall in maximum-likelihood trees. Yes, some sequences which originate from cultures have an original morphologically linked ID, but if a phylogenetic tree shows it branching elsewhere, the taxonomic assignment of the sequence will be changed.

Lines 53-54- I’m not so sure that morphology-based taxonomy is the gold standard in protistology. Most work in the last 10-20 years has focused on phylogenomics to denote new lineages of the eukaryotic tree of life and reclassify those that were taxonomically assigned morphologically in the past.

Line 224- This sentence is a little clunky. Please re-phrase.

Methods:

Line 800- Did a single researcher perform the taxonomic classification? Were any other researchers consulted for the morphological classifications? What was the identification guide used?

Line 819- What parameters were used for quality and length filtering in DADA2? Which unique changes were made to the workflow to account for the long-reads?

I would suggest looking into the V4 region (most common region for metabarcoding studies) of these long read ASVs to see if they correspond to the same amount of variability, as this is what would really reveal if the different ASVs you see at the long-read level correspond to the variability at the short read level.

Results & Discussion:

Figure 1- a short title within or above each chart would be useful for data interpretation.

Heatmaps- the light shading for high abundance and dark shading for low abundance seems reversed for me. I would suggest switching these around or changing the color scheme all together to be more distinct (might help to also remove the gray background?)

Beautiful images and vizualization of the data in Fig 7, great job!

Line 605- “not inconsistent” to “consistent”

Reviewer #2: This manuscript entitled " Genetic variability in microbial eukaryotes reshapes marine biodiversity assessment in the age of amplicon sequencin" provided much information and discussion on the intragenomic and intraspecies genetic variation in one group of marine protists, the polycystine Radiolaria. The authors analyzed the combined microscopy and long-read 18S rRNA gene amplicon data sequencing data and made interesting and important findings that will have great implications for the interpretation of metabarcoding data. The manuscript is well-written, the text is well structured, clear and easy to understand. The results are well illustrated and the manuscript is accompanied by a large amount of supplementery material (photos of all 173 isolated specimens used in analyses, heatmaps illustrating ASV presence and abundance in all analyzed specimens, expanded phylogenetic tree based on the dominant ASVs and ect.). The authors have done extensive and important research. This manuscript is helpful to readers and researchers who are working on or beginning the metabarcoding study of marine protists.

My minor comments are addressed below:

Line 193. “…relative abundance (scaled from 0 to 1)…” If I understand correctly, relative abundance is calculated as n%/100%. Please clarify this in the caption to the figure. You use “%” in the text (lines 177-190). The discrepancy between the figure and the description is a bit confusing.

Lines 207-209. … I would suggest to be more specific. I would suggest adding exact copy numbers of the 18S rRNA gene to the phrases... “astoundingly high…”, “…similar …” “…fewer copies…”

Line 252. Table 1. The list is presented in alphabetical order by morphospecies name. I would suggest reformatting this list based on taxonomy (orders Nassellaria, Collodaria, Spumellaria). In my opinion, this way the data will look more structured and will correspond to its description.

It should also be noted that the main objective of this study is to investigate the intragenomic and intraspecies genetic variation in the polycystine Radiolaria and integrating morphological and molecular information. However, in my opinion it would be interesting to estimate the degree of similarity (or the degree of differences) between sequences of different ASVs of one sample/morphospecies and/or between different morphospecies (for example based on p-distans). I recommend this aspect needs to be addressed in the future. It will add useful information and clarity to the study. And it will probably allow us to determine the boundaries of intragenomic and intraspecies genetic variation among polycystine Radiolaria.

Reviewer #3: The article is well written and structured, interpreting complex data with remarkable clarity. The organization of the content ensures a seamless flow, making the information accessible. It demonstrates the high intragenomic variance in polycystine Radiolaria and the importance of incorporating this variability in future metabarcoding survey.

My main concern would be regarding the Table 3: More than half of input CCS reads was filtered out at the Quality & Length filtering step. However, about 89% reads passed after removing primers and filtering in the literature cited (Line 823). Is there any possible cause to the low passing rate? Was the Q score of raw data tested in other quality control tools? It'd be better if an explanation of the low passing rate is included.

Besides it, I only have a few minor comments and suggestions:

Line 29 – 33: It is a very nice summary of results. But it would be nice to expand a little if possible.

Line 66 – 70: I believe there are also other research using metabarcoding as the Tara Oceans expedition and generated similar data. I think you can compare or add a few more references to back it up.

Line 377-383: It’d be more persuasive with some references. Are there previous research discuss the relation between genetic variation and morphological differences in Radiolaria or protists?

Line 512: Is the question mark inside brackets a typo?

Line 557 – 567: In addition to ASV richness, do these juvenile radiolarians share some of ASVs with their mature specimens? If no ASV is shared, it could support the overestimation of species richness in metabarcoding surveys.

Reviewer #4: The paper “Genetic variability in microbial eukaryotes reshapes marine biodiversity assessment in the age of amplicon sequencing” by Turbovitz and co-authors have used a combined microscopy and long-read sequencing approach to address the relationship between molecular data and morphologically defined species of Radiolaria. The authors show that more specimens should be examined via combined microscopy and molecular analyses to understand more about radiolarian life cycles and phylogenetic relationships.

Their results demonstrates that intraspecies and intragenomic variation in radiolarians is a likely reason for the discrepancy between morphological richness and genetic richness, as the average specimen in their dataset yielded 12 unique ASVs. These findings have major implications for diversity interpretations in metabarcoding studies and should be followed up in future studies by protistologists in general, since intragenomic variation might also be high in other protist groups.

The manuscript is very well written, and it was a pleasure to read it. The findings by Turbovitz and co-authors can be interesting for many researchers.

I only have a few minor comments for the authors:

• L.88: Although it is natural to make a selection of papers to cite, I recommend that the authors also include Krabberød et al. 2011 in the list of papers cited here (https://doi.org/10.1371/journal.pone.0023526)

• I believe the correctly spelled version of i.e./e.g. is with a trailing comma (i.e.,/e.g.,)?

• More a question than a comment: I think it would have been interesting to see what happened to the ASVs if these were clustered into OTUs at different levels (99%, 98%, 97% etc.) – maybe something to consider.

**********

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

Reviewer #3: No

Reviewer #4: Yes:  Marit F. M. Bjorbækmo

**********

[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.]

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 PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Genetic variability in microbial eukaryotes reshapes marine biodiversity assessment in the age of amplicon sequencing

PONE-D-24-55509R1

Dear Dr. Trubovitz,

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.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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 help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- 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.

Kind regards,

Claudia Isabella Pogoreutz

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

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: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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: Yes

Reviewer #2: Yes

**********

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: Yes

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 #1: (No Response)

Reviewer #2: The authors have satisfactorily addressed all my comments, revised the manuscript accordingly and added important clarifications.

Also, the authors conducted an additional analysis in the part of comparing the variability of the short V4 region (the most common region for metabarcoding studies) and the long read ASVs of 18S rRNA. This allowed them to draw important conclusions for interpreting metabarcoding data within marine protists.

The manuscript is much improved and can be accepted for publication.

**********

7. 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

**********