Concepts towards Functional Eukaryotic Microbial Biogeography in the Ocean

Round 1

Reviewer 1 Report

The submitted manuscript aims to provide a review of the application of high throughput sequencing approaches of marine protists to reveal the functional protist biogeography.

However, in my opinion, the paper does not provide new information and discussion on the topic.  In addition the paper will not be very clear for non-experts or new researchers. For example, while the authors explain the principles of metabarcoding; there is no clear explanation for metagenomics and metatranscriptomics. In the end, I don't think this review MS provide novel discussion to disentangle problems associated with protist functional biogeography.

Therefore, I don't find this paper suitable for publication in the Journal of Marine Science and Engineering.

 

Author Response

We thank the reviewer for their comment.

Following the recommendations of another reviewer and the editor, we introduced each of the methods shortly in the beginning and re-structured the sections on challenges and opportunities. Additionally, we created conceptual Figures (Figures 1 and 2) that we hope will help understand the individual workflows better.

We appreciate the reviewer's concerns regarding the novelty of this review. We are aware of redundancies among reviews on omics methods, as we stated in the introduction of our review (L. 86). However, we would like to highlight that we specifically aimed to discuss the opportunities of omic research in functional protist biogeography. We hope that the conceptual figures we created will help to make this more clear.

Reviewer 2 Report

The article written by the authors is a form of review of the existing literature and research related to the study of marine microorganisms. such review articles are needed.

 However, P. J. Keeling, J. del Campo, Marine Protists Are Not Just Big Bacteria. Curr. Biol. 27, (2017). https://doi.org/10.1016/j.cub.2017.03.075 previously wrote a very similar article.

In the reviewed article, the authors seem to follow Keeling et al. [2017] and completed the literature from 2017 to 2022.

Line 503 - the authors write in press, and the article was published on June 22, 2022, i.e. before sending the manuscript to the editorial office.

 I have the impression that the authors, after rejecting the manuscript in another journal, sent it to Marine Science and Engineering, as evidenced by its preparation completely contrary to the requirements of the journal

1. Literature citation is:

 Microbial single-cell eukaryotic (protist) diversity in the ocean is rich, with a total estimate of 200,000 - 250,000 eukaryotic species (1). Protists make up a significant fraction of the total microbial community serving fundamental roles in the marine ecosystem and biogeochemical cycles. In marine environments, these small, planktonic organisms get continuously distributed through ocean currents and hydrodynamic processes and thus exposed to changing environmental conditions. Therefore, protist diversity can be understood as similar to a mosaic with different patterns at different temporal and spatial scales, breaking down into 'tiles' at the microscale (2).

and it should be

In the text, reference numbers should be placed in square brackets [], and placed before the punctuation; for example [1], [1-3] or [1,3]. For embedded citations in the text with pagination, use both parentheses and brackets to indicate the reference number and page numbers; for example [5] (p. 10). or [6] (pp. 101-105).

Moreover, the references were also not prepared in accordance with the journal's requirements.

 is: R. Kodzius, T. Gojobori, Marine metagenomics as a source for bioprospecting. Mar. Genomics. 24, 21–30 (2015).

References should be described as follows, depending on the type of work:

Journal Articles:

1. Author 1, A.B .; Author 2, C.D. Title of the article. Abbreviated Journal Name Year, Volume, page range.

Books and Book Chapters:

2. Author 1, A .; Author 2, B. Book Title, 3rd ed .; Publisher: Publisher Location, Country, Year; pp. 154–196.

3. Author 1, A .; Author 2, B. Title of the chapter. In Book Title, 2nd ed .; Editor 1, A., Editor 2, B., Eds .; Publisher: Publisher Location, Country, Year; Volume 3, pp. 154–196.

Unpublished materials intended for publication:

4. Author 1, A.B .; Author 2, C. Title of Unpublished Work (optional). Correspondence Affiliation, City, State, Country. year, status (manuscript in preparation; to be submitted).

5. Author 1, A.B .; Author 2, C. Title of Unpublished Work. Abbreviated Journal Name year, indicating stage of publication (submitted; accepted; in press).

To sum up,

the manuscript requires general improvement and systematization.

Author Response

We thank the reviewer for their comments.

First, we would like to highlight that this review is an invited contribution to the special topic "Marine Phytoplankton and Their Evolution". Therefore, this is a dedicated contribution to the topic and has not been previously submitted elsewhere.

We thank the reviewer for pointing us to the reference style and we corrected the style in our revised version accordingly.

We agree with the reviewer that Keeling & del Campo had another important contribution on methodologies to study protist diversity. Therefore, we cited this reference in our review. However, we believe that our contribution is not only "filling the literature between 2017 to 2022", but focussing more on the three different omics tools (i.e., metabarcoding, metagenomics, and metatranscriptomics) and their application in functional biogeography. In their review, Keeling & del Campo discussed the need for more functional exploration but did not outline the available resources. We tried to compile relevant information on this topic for other researchers in our text and the compiled trait tables in Table 1. We also created two conceptual figures that we hope will help clarify the link between omics' methodological workflows and their functional context (Fig. 1 and 2).

Following the advice of another reviewer's comments, we restructured our review and specifically all sections on "Challenges & opportunities for functional diversity analyses". We 1) introduced each method in their dedicated section and 2) restricted the challenges by inherent challenges of the methods, methodological challenges, and challenges of data intercomparability (e.g, reference databases). We hope these sections are more clear now.

Reviewer 3 Report

In this short review the authors explain for three HTS approaches (Metabarcoding, Metagenomics, and Metratranscriptomics) the methodology and then discuss the opportunities and challenges of each of these three methods for functional diversity analyses of the phytoplankton (in the marine realm). This is done thoroughly. However, at times they are too brisk and jumpy. I suggest (see comments lines 104-107) to be clearer and discussing the various issues in clearly distinguishable and delineated points. May I suggest categorising the various challenges into 1) inherent to the shortcomings in the data themselves, 2) incompatible methods, formats and their consequences, 3) external ones (e.g. incomplete reference databases). I suggest after the explanation of the methods also a small overview of the different analytical methods and databases that are now in vogue, if any in each of these 3 categories.

I find the “What do we need for a clear(er) picture?” paragraphs clear and well-structured

Literature list fairly complete.

 

Minor issues

49 Ubstantially

50 nano- and picoplankton size fractionS

71-72 Sentence not clear

93 Primarily used? Principal!

96 These relatively short gene sequences (as compared to whole genomes),

104-107 first comparison is between metabarcoding and classical approaches. Then that provides explanation why metabarcoding is more common than metatranscriptomics and metagenomics? Please, explain. Then, lines 107-.. straightforward follow on that line of thought but the logical transition to FAIR is not clear. It’s a different issue. Please provide clearer distinction of issues.

172 For example, results of metagenomics analyses (56) showed that....

Author Response

We thank the reviewer for their insightful and inspiring comments.

We changed our manuscript as follows:

1) We added conceptual figures (Fig. 1 and 2) on the different methods and workflows to visually support the main points we want to highlight in this review.

2) We introduced each method at the beginning of each section and hope that this makes it more clear now.

3) We restructured the challenges and opportunities sections following the reviewer's suggestion by clearly separating inherent issues, methodological biases, and external issues. We hope this reads better now.

4) We added Figures from other publications (Fig. 3 + 4) to underline potential visualization and data interpretation methods. We hope this underlines the content of this review and can act as an inspiring resource for other researchers.

 

We would like to thank the reviewer for pointing out all the minor mistakes in the text, and we want to note that we corrected them in the revised version of our manuscript accordingly.

Round 2

Reviewer 1 Report

Authors significantly revised the manuscript and current version is better than the first version. Although I still think discussed information is presented in many reviews; there is some value in this manuscript for bringing atttention to protists.

Author Response

We thank the reviewer for their remark. We agree that there are overlaps, but we are still convinced that this short review adds a more functional perspective on protist biogeography than previous reviews.

Reviewer 2 Report

The manuscript was thoroughly revised, and the authors responded to the comments and supplemented the references. In the current version, the manuscript is accepted

Author Response

We thank the reviewer for their time and valuable comments that helped improving our manuscript.