PONE-D-20-21986

16S rRNA gene amplicon-based metagenomic analysis of bacterial communities in the rhizospheres of selected mangrove species from Mida Creek and Gazi Bay, Kenya

PLOS ONE

Dear Dr. Muwawa,

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.

In particular the reviewers have identified major flaws in the experimental design and the analysis that need to be addressed before considering this contribution for publication. I therefore encourage the authors to provide a revised version that address all the issues detected by the reviewers.

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PLOS 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: Partly

Reviewer #2: Partly

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: I Don't Know

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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: Yes

Reviewer #3: Yes

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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

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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: Overall the study is well presented and a lot of work has been done. However there is a major flaw in the experimental design, authors had only collected (multiple samples) only from one tree for each species so that the results cannot be generalized in any way.

Revised the text a lot of hyperbole example myriad of bacterial communities and repetition e.g. mangrove ecosystem ( 3 times in 5 lines 64-68)

Introduction: lines 70 -79 Authors refer to another studies but they simply report the main conclusion without any explanation on what they expect to find based on this results or how this study will help in their own one.

Methods:

Number of samples: even we the authors try to maximize the replication within the sampling campaign having only one tree for specie lead to a high uncertainty of the conclusion. Peculiar condition, maybe not notable, could been presented and the results cannot be generalized, it required to have more than one tree per specie sampled to corroborate the results.

How common are each species in the two sites? one is dominant and the remaining 3 rare or they shared the same % across the mangrove? please provide this data

Fig. 2 color should not be shared between phylum and genus graph

Results: Authors refer to data as no difference in depth is present for almost all the entire Results section with the exeption of the RDA results in which the differential depth is considered. If the authors want to include this factor also the previous section should include (as supplementary or how they prefer) this differentiation and data should be analyzed accordingly.

Fig. 5 need to be improved hardly readable

Discussion:

Authors refer across the text to their results without including reference to figure or table please amend. A big part of the discussion is devoted to a sort of list of which organism had been found but without adding any other information or discussion point, this part of the text should be cut or more discussion point should be inserted or merged with the following section about the putative pathway in microbial communities.

Reviewer #2: In this manuscript, Muwawa and collaborators investigated the bacterial community associated to the rhizosphere of four different mangrove species, in two distinct locations, along the coast of Kenya. The two locations present a different level of anthropogenic pollution and among these sites the authors investigated different factors that can affect the bacterial community composition associated to the mangrove sediments, highlighting the mangrove species and the chemical properties of the sediments as important factors that determine the bacterial community composition in the mangrove ecosystem.

I found this work interesting and well written. The authors through a combination of high-throughput sequencing data and sediment chemical analysis provided results that suggest how the root system of each mangrove species is important in determining the diversity and the composition of bacterial communities associated to mangrove sediments. However, I found some aspects that may require more information to strengthen the authors findings. I will report my comments in the next paragraphs.

Major issues

1. While I am convinced about the species-specific effect of route exudate on the bacterial community composition associated to the mangrove rhizosphere, I have found along the paper some contradictory parts, especially in the discussion paragraph, between the importance of location and nutrients concentration. The authors showed that the geographical location significantly affected the diversity of the bacterial community, also if it is not the main affecting factor. Authors also stated the importance of the chemical composition of the sediments, since Mida creek is characterized by a higher concentration of nutrients that decrease the bacterial diversity, due to more intense anthropogenic activities.

If it is possible, to clarify and better investigated which factors are more affecting the bacterial community composition, I think that the authors should consider to include in the analysis bulk soil samples as control, collected far from the plant roots in the two sites, to have a comparison of the bacterial community and the chemical composition associated to the sediment not affected by the mangrove root system. In this way it should be possible to consider if there are variations in the bacterial community composition that are due to differences in the sediment characteristics, that are not influenced by the presence of the different mangroves species and which parameters could be affected by anthropogenic activities.

2a. In general, I think that the authors should give more information regarding some aspects of the experiments, some also linked to my previous comment. The authors initially explained that the two sites along the Kenyan coast have been chosen due to the different level of pollution in the two areas due to anthropogenic activities, but they never mentioned which are the main parameters that could be affected in the mangrove ecosystems. Moreover, when the authors compared the two sites, they described them using different parameters. For instance, for Mida creek they provided information on the temperature range and the range of rain precipitation, while for Gazi Bay they provided the average temperature and humidity. If it is possible, I suggest that the parameters adopted to described the areas would be the same (line 110-117).

2b. In line 117-121 Authors gave a general overview on how the four mangrove species are distributed in Kenyan mangrove forest, but they don’t provide details on the distribution that each species covers in the two studied areas. Is the distribution and the proportion of the four species different in the two locations? Where the different three were collected? in the seaward side or in the middle zone? Was the tide affecting differently the mangroves sampled in the two location or at the different depths considered?

Maybe the authors could provide more information on the experimental design to better describe the mangroves in the two sampling sites and exclude that other environmental factors could have affected the bacterial community composition in the rhizosphere beside the species selection effect of the plants.

2c. Were the temperature and tide level homogeneous in the sampling sites for all the samples or there were significant variations in these parameters? These variables are usually very important and can deeply affect the bacterial community composition in mangrove sediments. May the authors comment if these parameters have been taken in consideration?

3. The functional predictions of the mangrove sediment bacterial community from 16S rRNA amplicon data has to be consider as a first overview of the bacterial community metabolic potential. The data obtained are good for the purpose of this manuscript but further studies need to investigate more in details these results, in future works. Due to the low resolution at low taxonomic level of amplicon data, the prediction could be affected by substantial bias that metagenomic or metatranscriptomic analysis can resolve.

Minor issues

1. In Figure S2 to highlight the importance of the plants on the bacterial community composition in the rhizosphere, could be more useful to see how many OTUs are in common between the same mangrove species in the 2 different locations. May the author add a Venn diagram panel also with this comparison?

I also found strange that Gazy bay does not have any OTU not shared with Mida creek. The addition of this panel may immediately give an idea on how many OTUs are shared between the rhizosphere of the same mangrove species in the two different locations, strengthen the importance of the species-specific plant effect in the selection of bacterial communities associated to the rhizosphere independently by the geographical location.

2. Probably in line 241 the authors are referring to Figure S3 panel A instead C.

3. In Figure 2 panel B I would suggest to align in the graph the relative abundance of the genus less abundant than 5%. In this way would be easier for the reader to appreciate the differences between the genus that the authors took in consideration in the analysis.

4. I suggest to add the taxonomic bacterial composition at family level in the supplementary material since authors described these results between line 279 and 293.

5. In Figure 6 change the color code for the sites, black and purple are difficult to be distinguished.

6. I found the paragraph from line 510 in the discussion redundant with the paragraph in the results section starting at line 265. I suggest that the authors rephrase the discussion paragraph avoiding the detail of the percentage already reported in the result section and focus more they comment more on their findings.

Reviewer #3: General

The work is a very interesting contribute. I recommend authors to play more attention to the clarity of sentences and writing errors. Data supports authors’ discussion and conclusions. Discussion could be more rich especially in exploring diversities in microbial communities composition in the two sites (playing attention to role of pollution that is the main difference between the two sites described by authors since the very beginning) or among different species.

Minor review

Note 1: check if measure unit and negative signs are written beside numerical data with appropriate space (especially lines 113-134).

Lines 70-78 : check for some errors in the language.

Lines 70: I’d suggest to summarize a little bit more in details the ref (10) experimental design. The sense of sentence at line 74-75 is not clear.

Lines 82-83: the phrase “due to inadequate efforts spent in exploring the mangrove habitats for microbial diversity” is maybe too strong (especially without reference).

Lines 94: better describe what kind of pollution resources are present in Mida Creek site.

Lines 109-116: data of temperature and humidity are not described homogeneously for the two sites.

Lines 138-141: method should be described more in details or a reference should be present.

Lines 452-54 are the exact copy of lines 57-58.

Lines 500-524: check for typos and errors in language.

Major review

For the description of a proper vertical distribution I’d suggest to the authors to sample at 3 different depths instead of two (done in Wu et al. 2016 – ref 10).

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Reviewer #1: No

Reviewer #2: Yes: Alan Barozzi

Reviewer #3: No

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PONE-D-20-21986R1

16S rRNA gene amplicon-based metagenomic analysis  of bacterial communities in the rhizospheres of selected mangrove species from Mida Creek and Gazi Bay, Kenya

PLOS ONE

Dear Dr. Muwawa,

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.

Although the manuscript was significantly improved, the author still need to provide a clear explanation of the term rhizosphere. The revised version provides an explanation of the methods use to retrieve the rhizopsheric soil that does not match with a rigorous definition of rhizosphere. Rhizosphere is that portion of sediment attached to the root. Specific procedures are needed to separate the rhizosphere from the root from the sediment surrounding the root. See the comment of the reviewer 4 for more detail. If unable to provide such methods the authors can think to remodel the title and the main message of the paper, that is still valuable especially in ecosystems such as mangroves where microbial studies are poor.

Please submit your revised manuscript by Jan 22 2021 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Marco Fusi

Academic Editor

PLOS ONE

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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 #2: (No Response)

Reviewer #3: All comments have been addressed

Reviewer #4: (No Response)

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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 #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: 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 #2: Yes

Reviewer #3: Yes

Reviewer #4: 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 #2: Yes

Reviewer #3: Yes

Reviewer #4: No

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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 #2: I found the revised version of the manuscript of Muwawa and collaborators clearly improved. The authors put a good effort to comply with the reviewers’ comments and I do not have further clarification to ask to the authors. However, I reported a very few minor comments, that I think need to be addressed before the publication.

Minor comments

- In the abstract line 27, Mida Creek and Gazi Bay are both in capital letters, while in the manuscript after this point, the capital letter was removed in the second part of the name. In line 112 happened the same. Give a check and be consistent.

- In lines 50-52, in the brackets there is a space between the numbers and the percentage symbol. I suggest to remove it and in general to keep the same style along the all text (i.e. in line 52 there is no space).

- In lines 125-126 “Gazy bay and Mida creek had low tides and temperature of 28°C and 27°C.” Are the authors referring at the conditions found at the time of the sampling?

Remove this sentence here, since it is out of context and it is repeated already in the collection of samples paragraph, where gives important information on the conditions in the sampling sites.

- In Figure 1 panel B, (old Figure 2) my comment was misunderstood. If it is possible, I suggested to align in the graph, in panel B, the relative abundance of the genus less abundant than 4%. In this way it would be easier for the reader to appreciate the differences between the genus that the authors took in consideration in the analysis and there will not be some taxa before and after the “others” bar, like the author did in panel A.

- Maybe it is my mistake but I didn’t fine the Supplementary Figure S4 mentioned in line 313.

- In line 351 probably the authors are referring to figure 2 and not 3.

- For Figure 4, there is only the caption title. A small description of the figure could help the reader to understand it.

- Lines 545-548, the two sentences are quite repetitive. Try to rephrase them better.

Reviewer #3: In my opinion the corrections applied are adequate.

The paper is now more clear and precise in form and contents.

Line 67-69: check the sentence grammar.

Reviewer #4: General:

In the manuscript titled “16S rRNA gene amplicon-based metagenomic analysis of bacterial communities in the rhizospheres of selected mangrove species from Mida Creek and Gazi Bay, Kenya” by Muwawa et al., the rhizosphere of four different mangrove species at two sites along the coast of Kenya is analysed for sedimentary conditions and the microbial community composition. The study shows how different areas within the forest dominated by different tree species contain varying bacterial communities. Furthermore, the authors identify potential drivers of bacterial community composition. This work provides novel insights into the bacterial communities of mangrove soils in eastern Africa, and adds to a growing datatbase of mangrove microbiomes across the globe. I commend the authors for their work and the neat presentation of the manuscript. I recommend however, to complete a thorough grammar-, spell-, and punctuation-check on the entire document as there were too many mistakes for me to point out.

I do not agree with the definition of rhizospheric sediment as it is presented in the manuscript, as it seems that the samples were taken from below the trees and with roots present in the sample, but there is no evidence that this area is actively mediated by root exudates (I would have sampled the soil directly off the roots). As there may be wider definitions of what the rhizosphere is (I am aware the methodology followed Wu et al. 2016), I would like to see it defined for this study, and the sampling method justified.

Throughout the document, there is an inconsistency using R. mucronate instead of R. mucronata

Abstract:

There is no need for specification of methods (such as which alpha diversity metrics used) and p-values in the abstract

Introduction:

Lines 125-126: This sentence gets repeated in the “collection of samples” section. Can be removed here.

Lines 126-133: The contribution of each species is unclear: Do all four species together contribute to 80% of the mangrove forest? If yes, what is the contribution of each species? This doesn’t need to be exact, but if the microbiome varies between these stands, it would be good to know their approximate extent.

Line 142: Four trees of each species means 4x4 =16 individual trees were sampled. If this is the case, I apologise, please ignore the comment. However, I believe “one tree each of the four species was sampled”, is what was actually done.

Lines 143-145: I find the description of the sampling procedure confusing. It may help to break down the sentences as much as possible to keep each action simple and clear. A diagram of the sampling design would help immensely.

Line 146: When calculating the number of samples a 4x4x2x2 structure is used, while it is described in the text as 4x8x2. Keep it consistent for clarity.

Line 198: I wonder, why OTUs were used for community metrics but ASVs for the functional alignment? ASVs and OTUs are two methods, that rely on very different ideas. In this way, I do not think the community data and the functional data can be compared.

Results:

Line 351: Figure mention should be Fig. 2 instead of Fig. 3

Lines 394-398: The wording in these sentences doesn’t quite fit the analysis. The environmental factors are able to separate the sites/samples, ie pH separated the samples along the y axis (Gazi vs Mida). pH has an effect on the bacterial community in all sites but is here able to explain some of the differences between them.

Lines 416-420: The interpretation of the variance partitioning results is not quite accurate. The “interaction zones” (overlap of the diagram) are additive to the influence of a single factor. This means that the variation explained by sediment chemical properties alone was 6% and when an interaction with species was inferred it was 11%. Hence, the interaction doesn’t reduce the influence of the factors but adds another layer of information.

Discussion:

I would like to see a note in the discussion in which the authors discuss the potential effect of intertidal zonation on the microbiome. They show nicely how the tree species have an effect on the bacterial community, but fail to mention in the discussion, that the trees follow a strict zonation from sea to land. While the study is not designed to disentangle the question whether the trees or their intertidal position determine the microbiome more, this is an important point to mention. Especially as the sedimentary conditions have such a large influence too.

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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 #2: Yes: Alan Barozzi

Reviewer #3: No

Reviewer #4: Yes: Timothy Thomson

[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.]

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Attachments

Attachment

Submitted filename: Review II.docx

PONE-D-20-21986R2

16S rRNA gene amplicon-based metagenomic analysis  of bacterial communities in the rhizospheres of selected mangrove species from Mida Creek and Gazi Bay, Kenya

PLOS ONE

Dear Dr. Muwawa,

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.

The authors were able to address all the reviewer and editor points but there is still two minor changes that need to be solved as indicated by the reviewer, before to consider the manuscript for publication. 

Please submit your revised manuscript by Mar 28 2021 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Marco Fusi

Academic Editor

PLOS ONE

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Reviewers' comments:

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Comments to the Author

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Reviewer #4: (No Response)

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Reviewer #4: Yes

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

Reviewer #4: Yes

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Reviewer #4: Yes

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Reviewer #4: Yes

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6. Review Comments to the Author

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Reviewer #4: I found my comments on the manuscript by Muwawa et al. addressed satisfyingly.

Two minor issues with the wording may be resolved as suggested to ensure clarity of the study site and design. If these two sentences can be fixed, I am happy to recommend this mauscript for publication.

Methods:

Line 124-127: A suggestion: The mangrove forests in both sites display a similar zonation and mangrove species contribution among the four dominant species A. marina (30%), R. mucronata (25%), S. alba (15%), and C. tagal (10%), which contribute about 80% of the total mangrove formation in both forests.

Lines 140-142: The sentences seem repetitive. You also use the word species in a ambivalent way, which makes it very hard to understand. I would suggest a very simple sentence such as: “From pure stands of S. alba, R. mucronata, C. tagal, and A. marina, one specimen (or tree) was selected per stand. The selected trees were spaced at least 10 m apart.”

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Reviewer #4: Yes: Timothy Thomson

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16S rRNA gene amplicon-based metagenomic analysis  of bacterial communities in the rhizospheres of selected mangrove species from Mida Creek and Gazi Bay, Kenya

PONE-D-20-21986R3

Dear Dr. Muwawa,

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.

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Marco Fusi

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PLOS ONE

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