Morphological diversity within a core collection of subterranean clover (Trifolium subterraneum L.): Lessons in pasture adaptation from the wild

Abdi I. Abdi; Phillip G. H. Nichols; Parwinder Kaur; Bradley J. Wintle; William Erskine

doi:10.1371/journal.pone.0223699

Peer Review History

Original SubmissionSeptember 25, 2019
19 Nov 2019 Decision Letter - Sergio Rossi, Editor PONE-D-19-26944 Morphological diversity within a core collection of subterranean clover (Trifolium subterraneum L.): lessons in pasture adaptation from the wild PLOS ONE Dear Dr Nichols, 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 reviewers were genrally satisfied by the work. They suggest some detailed modifications to imporve the manuscript. Please consider them in your revised version. We would appreciate receiving your revised manuscript by Jan 03 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. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols 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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. 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, Sergio Rossi 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 [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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: 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 ****** 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 ****** 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 authors present A LOT of data to show that subterranean clover is morphologically highly diverse. This claim is supported by the results. General comments: the terms 'diversity' and 'variation' seem to be used interchangeably, please be more clear, also in the hypotheses. Presentation of the results of the statistical analyses is not always clear. The manuscript is fairly easy to read, even though there are lots of technical details, in my opinion the English is good. Line 193 and further on: did you account for an edge effect? Line 238: Explain how are the parameter values determined? Figures 2-6: some kind of error measure would greatly improve these figures. Figs 3-6 are stacked bar charts, whereas fig2 is a side-by-side bar chart, consistency could be improved. Fig 5 and 6 lack a legend. Line 348: in addition to the reference please state how you calculated heritability. Line 355 and table 3: presentation of this result not very clear, all differences ere significant, differences between what? Same for line 365 and further on. flowering time is different among ALL 125 genotypes? Would be more clear if F and p-values would be mentioned. Figure 2, legend: 'values are the mean of four replicate single plants' If measurements are done on four out of 97 or 28 plants This should be specified in the methods sections. Table 5: maybe color-code significance for easier reading/interpretation of the table? Reviewer #2: The authors present a comprehensive study on a core collection of subterranean clover species consisting of 97 lines, which accounted for approximately 80% of the genetic diversity of the global subterranean clover collection along with 28 Australian cultivars. Assessments were made based on morphological marker traits, agro-morphological traits and phytoestrogen levels in subterranean sp. Trait associations were evaluated based on correlation. It is this reviewer’s opinion that the manuscript presents a comprehensive body of work that will contribute towards developing new cultivars better-suited for establishment in southern Australian agro-climatic conditions. The manuscript is organised in a logical manner, in sound English with only minor typographical errors. General comment: 1. Please note that throughout the manuscript the abbreviation ssp is misrepresented as spp, intermittently and requires correction. Specific comments: 1. Line 150: should read “DNA was extracted from each member of this subset and an 20 additional Australian cultivars. Forty eight SSR primers spread across each of the eight subterranean clover chromosomes….” 2. Line 306: It is best to be more descriptive with the method used. For example the “alcohol” used in this method is ethanol. 3. While the method for quantifying isoflavones used in this study is valid, the authors should further identify the limitations in using thin layer chromatographic techniques due to the semi quantitative nature and elude to the fact that more precise LC-MS approaches can be useful for absolute quantification of isoflavones. It is logical that the choice of method was due to the high-throughput nature and lower cost associated with TLC, nevertheless I suggest the authors acknowledge that newer technologies can be utilised. 4. The authors observed associations between several morphological markers and environmental factors and suggest that the adaptions are possibly associated with the regulation of genes or interaction between several genes. The authors should also however elude to possibility of epigenetic regulation leading to observed traits. Reviewer #3: Dear author, the manuscript is very interesting and technically sound. I think it is an important study of morphological diversity of Trifolium subterraneum compiling a large data set of several countries. It was possible to link morphological marker traits with environmental factors, being a great contribute to this research subject. However, I suggest an improvement of the graphic quality of figures 4 and 5 (legends…) and a revision of typos: see, e.g., line 109 “Chile, Iran Portugal and South” should be replaced by ““Chile, Iran, Portugal and South”. Kind regards. ****** 6. 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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.0223699.r001
Revision 1
2 Dec 2019 Author Response In this response, Line numbers refer to those in the original submitted manuscript. Review 1 General comments The authors present A LOT of data to show that subterranean clover is morphologically highly diverse. This claim is supported by the results. The manuscript is fairly easy to read, even though there are lots of technical details. In my opinion the English is good. The terms 'diversity' and 'variation' seem to be used interchangeably, please be more clear, also in the hypotheses. The term ‘variation’ has been replaced with ‘diversity’ throughout the manuscript for consistency. Presentation of the results of the statistical analyses is not always clear. Concerns with the presentation of some individual statistical analyses raised by Reviewer 1 have been addressed in the comments below. The other reviewers did not raise any concerns about the statistical analyses. Specific comments attached to manuscript Line 193 and further on: did you account for an edge effect? The issue of potential edge effects has been clarified with the additional sentence “This spacing was sufficient to allow plants to grow without competition for light and moisture and prevented edge effects”. It is also clarified in the following sentence referring to additional plants to calibrate shoot dry weight measurements by adding “using the same spacing”. Line 238: Explain how are the parameter values determined? Determination of plant dry weight has been explained better by referring specifically to the calibration plants used to estimate the regression equation. Figures 2-6: some kind of error measure would greatly improve these figures. Figures 2-6 are frequency distributions of qualitative, rather than quantitative, traits and consequently, there are no appropriate statistical error measurements that can be displayed. Titles for the vertical axes of each of these graphs have been updated for greater clarity to indicate that data in each case represents numbers of genotypes in each category. Figs 3-6 are stacked bar charts, whereas fig2 is a side-by-side bar chart, consistency could be improved. Figure 2 has been changed to a stacked bar chart for consistency. Fig 5 and 6 lack a legend. Legends for figures 5 and 6 have been removed from the caption and placed directly onto the figures for greater clarity. Line 348: in addition to the reference please state how you calculated heritability. The calculation of heritability has been clarified by the following sentence: “Broad-sense heritability (H2) was estimated for each quantitative trait as the percentage of the phenotypic variance attributable to genotypic variance, in accordance with Falconer et al. [38]”. Line 355 and table 3: presentation of this result not very clear, all differences were significant, differences between what? Same for line 365 and further on. Differences were highly significant for all traits measured among the 125 genotypes at a level of P ≤ 0.001. This is explained in the caption for Table 3. The footnote “ P ≤ 0.05, P ≤ 0.01, *P ≤ 0.001” at the bottom of Table 3 is superfluous and a potential source of confusion for this table and has been removed. Flowering time is different among ALL 125 genotypes? Would be more clear if F and p-values would be mentioned. A value of P ≤ 0.001 for flowering time is given in Table 3, indicating a significant difference between the 125 genotypes for flowering time. The P value (P ≤ 0.001) has been added to the text in Line 365, as suggested, for greater emphasis. Figure 2, legend: 'values are the mean of four replicate single plants' If measurements are done on four out of 97 or 28 plants This should be specified in the methods sections. The methodology of measuring flowering time of each genotype on four single plants is explained in detail in the Materials and Methods. The sentence “Values are the mean of four replicate single plants” has been removed from the caption for Figure 2 for simplicity. Information has also been removed from subsequent captions, where it is explained in detail in the Materials and Methods and is not required for interpretation of the figures. Table 5: maybe color-code significance for easier reading/interpretation of the table? The different levels of significance have been colour-coded for each trait correlation in Table 5, as suggested by the reviewer. The same colour-coding has also been applied to Tables 6-9. The colour scheme can be modified (or deleted) to suit the journal, according to the editor’s suggestions. Review 2 General comments The authors present a comprehensive study on a core collection of subterranean clover species consisting of 97 lines, which accounted for approximately 80% of the genetic diversity of the global subterranean clover collection along with 28 Australian cultivars. Assessments were made based on morphological marker traits, agro-morphological traits and phytoestrogen levels in subterranean sp. Trait associations were evaluated based on correlation. It is this reviewer’s opinion that the manuscript presents a comprehensive body of work that will contribute towards developing new cultivars better-suited for establishment in southern Australian agro-climatic conditions. The manuscript is organised in a logical manner, in sound English with only minor typographical errors. Throughout the manuscript the abbreviation “ssp.” is misrepresented as “spp”, intermittently and requires correction. The abbreviation for subspecies has been corrected to “ssp.” throughout the manuscript. Specific comments attached to manuscript Line 150: should read “DNA was extracted from each member of this subset and an 20 additional Australian cultivars. Forty eight SSR primers spread across each of the eight subterranean clover chromosomes….” These two sentences have been corrected as advised. Line 306: It is best to be more descriptive with the method used. For example the “alcohol” used in this method is ethanol. The term “alcohol” has been corrected to “ethanol”. While the method for quantifying isoflavones used in this study is valid, the authors should further identify the limitations in using thin layer chromatographic techniques due to the semi quantitative nature and elude to the fact that more precise LC-MS approaches can be useful for absolute quantification of isoflavones. It is logical that the choice of method was due to the high-throughput nature and lower cost associated with TLC, nevertheless I suggest the authors acknowledge that newer technologies can be utilised. This point has been addressed by adding the following sentences: “This method was used due to its high-throughput and low cost, while recognising its semi-quantitative nature imposes limitations in precision. This method has also been previously used in similar genetic diversity studies by Nichols et al. [16] and Ghamkhar et al. [31]”. The authors observed associations between several morphological markers and environmental factors and suggest that the adaptions are possibly associated with the regulation of genes or interaction between several genes. The authors should also however elude to possibility of epigenetic regulation leading to observed traits. We have incorporated the reviewer’s comment on the possible role of epigenetic inheritance by adding the sentence to the Discussion “Epigenetic inheritance may also be important and needs further investigation”. The reference “Quadrana L, Colot V. Plant Transgenerational Epigenetics. Ann. Rev. Genet. 2016; 50: 467-491” has also been added. Review 3 General comments The manuscript is very interesting and technically sound. I think it is an important study of morphological diversity of Trifolium subterraneum compiling a large data set of several countries. It was possible to link morphological marker traits with environmental factors, being a great contribute to this research subject. Specific comments attached to manuscript I suggest an improvement of the graphic quality of figures 4 and 5 (legends…) The graphic quality of Figures 3, 4 and 5 have been improved. Legends for Figures 5 and 6 have been placed directly onto the figures for greater clarity. Titles for the vertical axes of each graph in Figures 2-6 have been updated to indicate that data in each case represents numbers of genotypes in each category. I suggest a revision of typos The manuscript has been proofread again and typos have been corrected. Line 109 “Chile, Iran Portugal and South” should be replaced by ““Chile, Iran, Portugal and South”. This sentence has been corrected. Attachments Attachment Submitted filename: Response to reviewers.docx* https://doi.org/10.1371/journal.pone.0223699.r002
6 Dec 2019 Decision Letter - Sergio Rossi, Editor PONE-D-19-26944R1 Morphological diversity within a core collection of subterranean clover (Trifolium subterraneum L.): lessons in pasture adaptation from the wild PLOS ONE Dear Dr Nichols, 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. -I observe that there are 9 tables and 7 figures on the main text. I guess they are too many for a traditional research paper. Please consider to replace some figs or tables in supplementary material. -Improve the quality of the figures: add major and minor marks on the axes, remove or reduce the empty spaces (ex: the empty spaces between vertical bars). We would appreciate receiving your revised manuscript by Jan 20 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. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols 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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. 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, Sergio Rossi Academic Editor PLOS ONE [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.0223699.r003
Revision 2
23 Dec 2019 Author Response Response to Academic editor’s comments 1. I observe that there are 9 tables and 7 figures in the main text. This is too many for a traditional research paper. Please consider replacing some figures or tables in the Supplementary material. I have reduced the number of figures (four) and tables (two) from the main text and moved them to the Supplementary material, as suggested. This now means the main text has three figures and seven tables. Please advise if this is still too many. In particular, the original Table 1 is now S2 Table, original Table 2 is now S3 Table, original Fig 3 is now S2 Fig, original Fig 4 is now S3 Fig, original Fig 5 is now S4 Fig and original Fig 6 is now S5 Fig. The text has been modified to take account of these changes. 2. Improve the quality of the figures: add major and minor marks on the axes, remove or reduce the empty spaces (ex: the empty spaces between vertical bars). The graphs have been improved, as suggested. Major and minor tick marks have been added to the axes and the spaces between vertical bars in the bar graphs have been markedly reduced. Attachments Attachment Submitted filename: Response to academic editor comments.docx https://doi.org/10.1371/journal.pone.0223699.r004
27 Dec 2019 Decision Letter - Sergio Rossi, Editor Morphological diversity within a core collection of subterranean clover (Trifolium subterraneum L.): lessons in pasture adaptation from the wild PONE-D-19-26944R2 Dear Dr. Nichols, 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, Sergio Rossi Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0223699.r005
Formally Accepted
31 Dec 2019 Acceptance Letter - Sergio Rossi, Editor PONE-D-19-26944R2 Morphological diversity within a core collection of subterranean clover (Trifolium subterraneum L.): lessons in pasture adaptation from the wild Dear Dr. Nichols: 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 Prof. Sergio Rossi Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0223699.r006

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