Distinct temporal diversity profiles for nitrogen cycling genes in a hyporheic microbiome

William C. Nelson; Emily B. Graham; Alex R. Crump; Sarah J. Fansler; Evan V. Arntzen; David W. Kennedy; James C. Stegen

doi:10.1371/journal.pone.0228165

Peer Review History

Original SubmissionOctober 21, 2019
3 Dec 2019 Decision Letter - Wenzhi Liu, Editor PONE-D-19-29302 Distinct temporal diversity profiles for nitrogen cycling genes in a hyporheic microbiome PLOS ONE Dear Dr. Nelson, 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. We would appreciate receiving your revised manuscript by Jan 17 2020 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. 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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, Wenzhi Liu 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. Please include a copy of Table 2 which you refer to in your text on page 18 2. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. [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: Yes 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: The manuscript clearly illustrated the seasonal dynamic profiles of N-cycling gene diversity in a hyporheic microbiome. It is well written and the results and discussion are presented well, except for the methods which should be described in more detail. Especially for figure 10, the analogy of an electrical circuit is very imagery and easy for readers to understand the importance of diversity in biogeochemical processes. Thus, I would suggest minor revision. Some of specific comments: 1． Line 76, please illustrate the reason(s) of designing the three locations (T2, T3 and T4) and the differences between them. 2． Line 105-118, in this paragraph, too many details on the determination of diversity are given in results, which is suggested to put into the methods. 3． Line 347, to better understand the sediment and aqueous sample collection, the sampling device schematic is suggested to be attached. 4． How many samples were assigned for DNA extraction? Which DNA was assigned for sequencing (including the fraction of 16S rRNA gene and Metagenomic analysis)? And which area of 16S rRNA gene was sequenced? It is necessary to be elaborated clearly in the methods. 5． In addition, gene names should be written in the formal way, such as, change 16S to 16S rRNA gene in figure 2, and write the gene names, such as narG in italic. The time ruler in the figures is recommended to be consistent. Reviewer #2: Overall a well written paper with sufficient data to support claims. I recommend the paper be published in PLOS ONE, but have some minor revision suggestions for increasing clarity to future readers. Each suggestion is listed below: 1. For all of the heat map figures (3-6), the current image quality is low and it is impossible to read the individual gene phylotype names. Also, in these figures some of the boxes are shaded gray - it is unclear what the gray shade means/represents, it would be great if this could be clarified in the figure legend. In my opinion, I do not think the heat map figures do a good job of clearly showing changes in gene phylotype changes over time. Is it possible to graph this data another way? I was thinking an alluvial diagram would be a better way to visualize the phylotype changes. I think better graphics would increase reader accessibility. 2. As currently written, it is hard for non-experts to follow the results descriptions of nitrification and denitrification genes and keep straight which genes belong to which metabolic pathway. Adding short clarifying sentences would help. For example, in line 120 it would be helpful to add a sentence about denitrification genes being discussed - similar to what is said about nitrification around line 128-129. If there is space, it would be useful to create a diagram that lists the steps of nitrification and denitrification in sequential order and bold/highlight each of the genes in these metabolisms you searched for during your analyses. It would be great if this diagram was in the introduction so the reader could always refer to it. This sort of graphic would help guide readers as to why it is unexpected nxrA showed little correlation with amoA (line 175-176). In the discussion (line 209-211, 223-225) you mention breaking the nitrification and denitrification process into component steps, yet these steps are unclear in the results, as it seems each gene is discussed individually. Adding a sentence in the results identifying which genes make up component steps and highlighting these components steps in the proposed nitrification/denitrification figure listed above would be useful for readers to follow your work. 3. Line 146-148 is confusing and unclear yet seemingly a critical conclusion from this work. You state that Figure 8 suggests that establishment of novel organisms, with the genes you looked at, is rare. How do you reach this conclusion? Would new unique microbes with the genes you are interested in cause an increase in diversity and a steeper curve in on the inverse Simpson plot? A brief justification would be useful. 4. If space, please spell out what RPKM stands for in Figure legend 9 (line 179). 5. A small typo exists at the end of line 215-216. 6. Line 405 - the HMM table needs to be table 2, and this number needs to be corrected in line 396 ****** 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: Yes: Anne E. Booker [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. https://doi.org/10.1371/journal.pone.0228165.r001
Revision 1
6 Jan 2020 Author Response 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 manuscript has been revised and file names have been changed to meet PLOS ONE's style requirements. 1. Please include a copy of Table 2 which you refer to in your text on page 18 Table 2 was mislabeled in the initial submission. The error has been corrected. 2. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. The requested text has been added. Review Comments to the Author Reviewer #1: The manuscript clearly illustrated the seasonal dynamic profiles of N-cycling gene diversity in a hyporheic microbiome. It is well written and the results and discussion are presented well, except for the methods which should be described in more detail. Especially for figure 10, the analogy of an electrical circuit is very imagery and easy for readers to understand the importance of diversity in biogeochemical processes. Thus, I would suggest minor revision. Some of specific comments: 1. Line 76, please illustrate the reason(s) of designing the three locations (T2, T3 and T4) and the differences between them. The three locations were spatially-separated replications sampling the hyporheic zone. This has been noted in the text. 2. Line 105-118, in this paragraph, too many details on the determination of diversity are given in results, which is suggested to put into the methods. We felt that it is important for the reader to understand the data types used in the work to properly assess our analysis, thus we decided to emphasize the methods used to generate the sequences and how we define ‘phylotype’ in this work. 3. Line 347, to better understand the sediment and aqueous sample collection, the sampling device schematic is suggested to be attached. A supplemental figure portraying the sampling setup has been added to the manuscript. 4. How many samples were assigned for DNA extraction? Which DNA was assigned for sequencing (including the fraction of 16S rRNA gene and Metagenomic analysis)? And which area of 16S rRNA gene was sequenced? It is necessary to be elaborated clearly in the methods. T4 is the only site for which we are presenting sequence data. For each timepoint, two sandpacks were collected, combined and homogenized prior to DNA extraction. Genomic DNA preparations were divided and subjected to amplicon and shotgun sequencing. The V4 region of the 16S rRNA gene was used for amplicon analysis, as proscribed by the Earth Microbiome Project. The Methods section has been edited to include this information. 5. In addition, gene names should be written in the formal way, such as, change 16S to 16S rRNA gene in figure 2, and write the gene names, such as narG in italic. The time ruler in the figures is recommended to be consistent. We feel it is fair to use abbreviations in the key for Figure 2 (now Figure 3), however, we have altered the legend text to directly associate the abbreviations used with the formal gene names. Gene names in the text have been italicized, and figures have been updated to have uniform time scale axes. Reviewer #2: Overall a well written paper with sufficient data to support claims. I recommend the paper be published in PLOS ONE, but have some minor revision suggestions for increasing clarity to future readers. Each suggestion is listed below: 1. For all of the heat map figures (3-6), the current image quality is low and it is impossible to read the individual gene phylotype names. Also, in these figures some of the boxes are shaded gray - it is unclear what the gray shade means/represents, it would be great if this could be clarified in the figure legend. Presentation of large data sets is always challenging. We have supplied figures in the highest resolution specified by the journal. Perhaps a solution is to put larger versions of the figures in Supplemental Information. We have requested guidance from the editor on this matter. We have stipulated in the Figure 3 (now Fig 4) legend that gray shading indicates no observation of the phylotype at that timepoint. 2. In my opinion, I do not think the heat map figures do a good job of clearly showing changes in gene phylotype changes over time. Is it possible to graph this data another way? I was thinking an alluvial diagram would be a better way to visualize the phylotype changes. I think better graphics would increase reader accessibility. An alluvial diagram is an interesting idea, but in practice, our data is more numerical than categorical, and we feel that the number of phylotypes being tracked in the figures would strain the interpretability of such a figure. 3. As currently written, it is hard for non-experts to follow the results descriptions of nitrification and denitrification genes and keep straight which genes belong to which metabolic pathway. Adding short clarifying sentences would help. For example, in line 120 it would be helpful to add a sentence about denitrification genes being discussed - similar to what is said about nitrification around line 128-129. If there is space, it would be useful to create a diagram that lists the steps of nitrification and denitrification in sequential order and bold/highlight each of the genes in these metabolisms you searched for during your analyses. It would be great if this diagram was in the introduction so the reader could always refer to it. This sort of graphic would help guide readers as to why it is unexpected nxrA showed little correlation with amoA (line 175-176). We have edited the text to give more context to the functions of the genes in question. In addition, we have added a summary figure (Figure 1) delineating the denitrification and nitrification processes. 4. In the discussion (line 209-211, 223-225) you mention breaking the nitrification and denitrification process into component steps, yet these steps are unclear in the results, as it seems each gene is discussed individually. Adding a sentence in the results identifying which genes make up component steps and highlighting these components steps in the proposed nitrification/denitrification figure listed above would be useful for readers to follow your work. The component steps are simply the individual enzymatic reactions that compose the pathway, which is why each gene is discussed individually. 5. Line 146-148 is confusing and unclear yet seemingly a critical conclusion from this work. You state that Figure 8 suggests that establishment of novel organisms, with the genes you looked at, is rare. How do you reach this conclusion? Would new unique microbes with the genes you are interested in cause an increase in diversity and a steeper curve in on the inverse Simpson plot? A brief justification would be useful. This figure has been expanded and the text has been edited to better relate the changes in phylotype composition for each gene from both a richness and diversity (inverse Simpson) perspective. 6. If space, please spell out what RPKM stands for in Figure legend 9 (line 179). This edit has been made. 7. A small typo exists at the end of line 215-216. The text has been edited. 8. Line 405 - the HMM table needs to be table 2, and this number needs to be corrected in line 396 The text has been edited accordingly. Attachments Attachment Submitted filename: Response Letter.docx https://doi.org/10.1371/journal.pone.0228165.r002
9 Jan 2020 Decision Letter - Wenzhi Liu, Editor Distinct temporal diversity profiles for nitrogen cycling genes in a hyporheic microbiome PONE-D-19-29302R1 Dear Dr. Nelson, 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, Wenzhi Liu Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0228165.r003
Formally Accepted
10 Jan 2020 Acceptance Letter - Wenzhi Liu, Editor PONE-D-19-29302R1 Distinct temporal diversity profiles for nitrogen cycling genes in a hyporheic microbiome Dear Dr. Nelson: I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Wenzhi Liu Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0228165.r004

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