Cryptic functional diversity within a grass mycobiome

Cedric Ndinga-Muniania; Nicholas Wornson; Michael R. Fulcher; Elizabeth T. Borer; Eric W. Seabloom; Linda Kinkel; Georgiana May

doi:10.1371/journal.pone.0287990

Peer Review History

Original SubmissionApril 6, 2023
5 May 2023 Decision Letter - Tzen-Yuh Chiang, Editor PONE-D-23-10504Cryptic functional diversity within a grass mycobiomePLOS ONE Dear Dr. Ndinga-Muniania, 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. Please submit your revised manuscript by Jun 19 2023 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. 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Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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: Andropogon gerardii leaf associated fungal communities were examined using two different methods of isolating - either sectioning or macerating the leaves. Surprisingly or not, the two different isolation methods yielded taxa representing different lineages. The acquired isolates were evaluated for their C use on the BIOLOG system. A collection of isolates was divided into fast- and slow-growing fungi by a cut off OD in the BIOLOG plates and the similarity of observed functional traits were used to estimate their distribution over evolutionary history. Further, the fast- and slow-growing isolates’ niche width is estimated by number of C-substrates among the 95 that were available on the BIOLOG plates. These analyses highlight the difference between the observed slow- and fast-growing isolates with lower mean but higher variance among the former isolates. The contribution arrives to three conclusions: 1) that A. gerardii endophytes include fast and slow growing members; 2) that the slow and fast-growing isolates differ in their niche breadth and variability; 3) that these differences may be conserved or deep-rooted in phylogenies as the assemblages could be distinguished on high levels of taxonomic hierarchy. While #1 may be rather expected, #3 is intriguing and may stimulate some further research to confirm. Whether supported or not is irrelevant as the conclusion is quite thought-provoking and supported by the limited data available from the reported studies. The authors conclude that the communities include undescribed, functionally and phylogenetically distinct fungi providing insight into the stochastic/deterministic processes underlying the assembly of foliar fungal communities. In more general terms, the reported research all falls in line with widespread functional diversity within fungal guilds and that - although the traits (C-use) are variable - they are conserved within lineages. I have a few general concerns and some minor suggestions. First, although perhaps based on a small sample size (12 leaves and 240 random isolates for the analyses), the analyses are meticulous and lead to well-justified conclusions. However, I ask authors to acknowledge the caveat of small sample size in generalizing their conclusions. Second, I am uncertain if metabarcoding is appropriate to use here. Perhaps this is more in line with ITS barcode identification of acquired isolates. Thirds, does the melanization of the fungal hyphae affect the OD, therefore potentially resulting in grouping of light and dark cultures erroneously? MINOR COMMENTS: Line 60: “important but diverse” - Maybe important and diverse would be better? Line 67: need Line 102: often dominate. Remove “often” Line 122: Additional background information on the age and management of the grass is necessary. The leaf samples were collected in August after flowering time - would this effect fungal endophytes? Line 122: Some treatment plots were treated with the nitrogen fertilizer. Although perhaps beyond the scope of the current study, where there any effect of this treatment of the fungal communities? Lines 137-138: It is unclear how the fungal colonies were collected from 1.5mL Eppendorfs. It seems that fast growing endophytes would take over the limited space and it would be hard to isolate slow-growing isolates? Lines 143–144: More details are needed on the isolation process. How were single taxon pure cultures confirmed? Line 145: Can we make conclusions about the growth of the fungi on artificial media such as 2% PDA for 6 months? As stated earlier in the text endophytes are plant symbionts, how can we ensure the same functional behavior of the fungi on the media without the plant symbiont? Line 173: This assumes that all cultures sporulated. Were the isolates screened for conidia before selecting them for further assays. ****** 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 ******** [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. https://doi.org/10.1371/journal.pone.0287990.r001
Revision 1
12 Jun 2023 Author Response Responses to the Academic Editor 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. Respose: We made changes to our funding information and wish our Financial Disclosure to read as follows: This study was supported by a National Science Foundation (NSF) Macrosystems Biology grant (NSF-DEB 00037623) to co-PIs EB, ES, LK, GM). Support was also provided from the NSF Long Term Ecological Research (NSF-DEB-1234162 and NSF-DEB-1831944 to Cedar Creek LTER) and Research Coordination Network (NSF-DEB-1042132) programs. Support to CNM was provided by the NSF-DEB, a Dissertation Fellowship from the Graduate School at University of Minnesota, and from the Graduate Program in Plant and Microbial Biology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Responses to Reviewers' comments Review Comments to the Author: Reviewer #1: Andropogon gerardii leaf associated fungal communities were examined using two different methods of isolating - either sectioning or macerating the leaves. Surprisingly or not, the two different isolation methods yielded taxa representing different lineages. The acquired isolates were evaluated for their C use on the BIOLOG system. A collection of isolates was divided into fast- and slow-growing fungi by a cut off OD in the BIOLOG plates and the similarity of observed functional traits were used to estimate their distribution over evolutionary history. Further, the fast- and slow-growing isolates’ niche width is estimated by number of C-substrates among the 95 that were available on the BIOLOG plates. These analyses highlight the difference between the observed slow- and fast-growing isolates with lower mean but higher variance among the former isolates. The contribution arrives to three conclusions: 1) that A. gerardii endophytes include fast and slow growing members; 2) that the slow and fast-growing isolates differ in their niche breadth and variability; 3) that these differences may be conserved or deep-rooted in phylogenies as the assemblages could be distinguished on high levels of taxonomic hierarchy. While #1 may be rather expected, #3 is intriguing and may stimulate some further research to confirm. Whether supported or not is irrelevant as the conclusion is quite thought-provoking and supported by the limited data available from the reported studies. The authors conclude that the communities include undescribed, functionally and phylogenetically distinct fungi providing insight into the stochastic/deterministic processes underlying the assembly of foliar fungal communities. In more general terms, the reported research all falls in line with widespread functional diversity within fungal guilds and that - although the traits (C-use) are variable - they are conserved within lineages. Response: Thank you! We also hope that our results will stimulate further research. As we acknowledge below, while the sampling is based on relatively few leaves (12), the observations are consistent across these leaves, and reporting this new observation of functionally distinct endophyte assemblages will spur further research. Reviewer #1: I have a few general concerns and some minor suggestions. First, although perhaps based on a small sample size (12 leaves and 240 random isolates for the analyses), the analyses are meticulous and lead to well-justified conclusions. Response: Thank you, we did take great care in the analyses and distinguishing conclusions justified from the results, and inferences such as those regarding assembly processes. Reviewer #1: However, I ask authors to acknowledge the caveat of small sample size in generalizing their conclusions. Response: We agree. While we did sample a fairly large number of isolates for functional analyses (240), at lines 417-419, we now acknowledge that sampling only 12 leaves (3 plants from each of 4 plots) limits our abilities to generalize to endophytic communities generally. Reviewer #1: Second, I am uncertain if metabarcoding is appropriate to use here. Perhaps this is more in line with ITS barcode identification of acquired isolates. Response: We understand that “metabarcoding” is a term used in many ways and thus potentially confusing. However, there may be a misunderstanding here because we do not use metabarcoding approaches (community sequencing) or terminology. We used Sanger sequencing of the fungal ITS barcode. We made edits in lines 152-160 that we hope will clarify our methods. Reviewer #1: Thirds, does the melanization of the fungal hyphae affect the OD, therefore potentially resulting in grouping of light and dark cultures erroneously? Response: We had not considered this and appreciate the question. The more melanized fungi such as those in the order Xylariales were most often also slow-growing, which is contrary to what we would expect if melanization upwardly biased the OD readings. We have now included that information at lines 303-305. MINOR COMMENTS: Line 60: “important but diverse” - Maybe important and diverse would be better? Response: Done. Line 67: need Response: Done. Line 102: often dominate. Remove “often” Response: As A. gerardii is not dominant across the whole region of the Great Plains, we edited the text at lines 101-102 for more clarity. Line 122: Additional background information on the age and management of the grass is necessary. Response: We provided edits for clarity on the management of the experimental NutNet site (lines 118-129). In short, it was established in 2007 and nutrient treatments have been applied annually since then. https://doi.org/10.1371/journal.pone.0287990.r002
19 Jun 2023 Decision Letter - Tzen-Yuh Chiang, Editor Cryptic functional diversity within a grass mycobiome PONE-D-23-10504R1 Dear Dr. Ndinga-Muniania, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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 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, Tzen-Yuh Chiang Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0287990.r003
Formally Accepted
12 Jul 2023 Acceptance Letter - Tzen-Yuh Chiang, Editor PONE-D-23-10504R1 Cryptic functional diversity within a grass mycobiome Dear Dr. Ndinga-Muniania: I'm 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 let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Tzen-Yuh Chiang Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0287990.r004

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