Reproductive compatibility in Capsicum is not necessarily reflected in genetic or phenotypic similarity between species complexes

Catherine Parry; Yen-Wei Wang; Shih-wen Lin; Derek W. Barchenger

doi:10.1371/journal.pone.0243689

Peer Review History

Original SubmissionNovember 27, 2020
14 Jan 2021 Decision Letter - Dengcai Liu, Editor PONE-D-20-37323 Reproductive Compatibility in Capsicum is not Reflected in Genetic or Phenotypic Similarity Between Species Complexes PLOS ONE Dear Dr. Barchenger, 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 Feb 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, Dengcai Liu, PhD Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. 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 https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://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: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A 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: No 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: In this study, thirty-eight accessions of 15 species of Capsicum were chosen for investigating genetic diversity and hybridization compatibility as well as the relationship between species relatedness and their ability to form hybrids based on multiple methods including phylogentic reconstruction, phenotypic data, and artificial hybrid. As the authors correctly pointed out that “interspecies compatibility is not necessarily reflected in relatedness according to established Capsicum genepool complexes”. The evolutionary factors involved in the establishment of polyploids in nature may depend, at least, on the parental origin of particular genomic features (e.g. high level of genetic heterogeneity) and genetic character (e.g. the ph gene that control chromosome pairing). Tracing the successful factors in the establishment of hybrids firstly require a robust and clear phylogenetic framework and then integrate multiple disciplines to give a power evidence for clarifying the relationship between interspecies compatibility and relatedness. In fact, this is lacking in this study. As outlined in the comments below, moreover, I feel there are some substantial issues (including rewriting) to address. 1. For the first time in the paper, the appearance of species name including C. annuum (L.), C. baccatum (L.), C. chinense (Jacq.), C. frutescens (L.), and C. pubescens, should be given a full taxonomic nomenclature. 2. Many basically biological features of the genus Capsicum involving ploidy level, geographical distribution, the level of reproductive isolation should be described briefly in introduction. This would help the reader to understand the process of speciation, potential species relatedness, possible pre- and post-zygotic barriers to hybridization, and introgression in the genus 3. One of the objectives of this study was to elucidate the relationship between interspecies compatibility and relatedness through extensive interspecific hybridization and the construction of a phylogeny. How can you get it? 4. The same species without different accession number should be listed closely rather than being scattered in different volume in the table (For example, Capsicum annuum). The table should also present the species in ploidy level, origin ect. 5. A table should be given to show which sample(s) are included in reciprocal hybridizations. 6. Why the author uses the SSR for studying the relationship among sampled Capsicum species. In other word, what’s the advantage of SSR in genetic diversity study? Similarly, why the author selected the wax gene as a marker for phylogenetic reconstruction. Generally, single- or low-copy genes are less likely subject to concerted evolution, thus making themselves ideal tools for studying the origin and evolution of taxa, especially in hybrid speciation. In addition, I don’t know how the authors get the wax sequences, by direct sequencing or clone sequencing? Moreover, many information in phylogenetic reconstruction is lacking. But, the author present a phylogenetic tree inferred from the wax sequences. do you use a species as outgroup? 7. Since the experiments of artificial hybridizations were performed, results of meiotic pairing behavior should be shortly described to give the readers a clear figure. 8. I can’t pick up the species and their the groupings in the Figure 1 because the species information is lacking in figure. This actually lead to some inconvenience to readers. 9. Some species (e.g. C. eximium) seems to be non-monophyletic origin, but there is no explanation in discussion. Reviewer #2: Generally, the research demonstrated that the inter-specific compatibility may not necessarily reflect in relatedness from established Capsicum gene pool, and correction of the previous mis-identification of a number of Capsicum species This information will be valuable for future breeding programs. The following comments and suggestions may help improve the quality of the manuscript: Lane 37: The single sequence repeat (SSR) must be changed as simple sequence repeat (SSR). Lane 41-42: The description of “The World … wild relatives” can be deleted. Lane71-72: The description of “This suggestion between complex is not total” is not clear, it should be rephrased. Lane 91: The ability to hybridize might be crossability. Table 1. The Source might have a table note to refer the abbreviations for the organizations. The μl might be changed as μL through the main text. Lane 287-289: The sentence is not clear and needs to be edited. The Discussion part, the cluster analysis between Fig.1 and Fig. 5 should be compared in detail, and the contribution of genome sequencing of Capsicum for the phylogenetic study may also be mentioned. ****** 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: 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.0243689.r001
Revision 1
8 Feb 2021 Author Response Reviewer #1: In this study, thirty-eight accessions of 15 species of Capsicum were chosen for investigating genetic diversity and hybridization compatibility as well as the relationship between species relatedness and their ability to form hybrids based on multiple methods including phylogentic reconstruction, phenotypic data, and artificial hybrid. As the authors correctly pointed out that “interspecies compatibility is not necessarily reflected in relatedness according to established Capsicum genepool complexes”. The evolutionary factors involved in the establishment of polyploids in nature may depend, at least, on the parental origin of particular genomic features (e.g. high level of genetic heterogeneity) and genetic character (e.g. the ph gene that control chromosome pairing). Tracing the successful factors in the establishment of hybrids firstly require a robust and clear phylogenetic framework and then integrate multiple disciplines to give a power evidence for clarifying the relationship between interspecies compatibility and relatedness. In fact, this is lacking in this study. As outlined in the comments below, moreover, I feel there are some substantial issues (including rewriting) to address. 1. For the first time in the paper, the appearance of species name including C. annuum (L.), C. baccatum (L.), C. chinense (Jacq.), C. frutescens (L.), and C. pubescens, should be given a full taxonomic nomenclature. The authority has been added to the first mention of each species as well as in Table 1. We did not expand the abbreviated genus after first mention, as it is standard to use the abbreviated genus name after first mention (per PLOS one style). 2. Many basically biological features of the genus Capsicum involving ploidy level, geographical distribution, the level of reproductive isolation should be described briefly in introduction. This would help the reader to understand the process of speciation, potential species relatedness, possible pre- and post-zygotic barriers to hybridization, and introgression in the genus The introduction has been improved to include a description of the barriers to hybridization that are currently understood in Capsicum. The ploidy level of Capsicum species has been included, as well as a brief description of the geographical origin. 3. One of the objectives of this study was to elucidate the relationship between interspecies compatibility and relatedness through extensive interspecific hybridization and the construction of a phylogeny. How can you get it? This reviewer comment is not clear. The authors request further clarification on this point so we can appropriate respond and make necessary changes for the improvement of our manuscript. One possible response is that we constructed phylogenies based on SSR markers, targeted sequencing of the waxy gene and based on various phenotypic traits. The relatedness using these techniques was compared and contrasted with cross compatibility. 4. The same species without different accession number should be listed closely rather than being scattered in different volume in the table (For example, Capsicum annuum). The table should also present the species in ploidy level, origin ect. Table 1 was heavily revised- we listed members of the same species together and add the full species name, including authority, for each species in the table. 5. A table should be given to show which sample(s) are included in reciprocal hybridizations. All were used for crossing; therefore, such a table would not differ from Table 1. Thus, it is not necessary to make a new table for this. Furthermore, it is already clearly stated in the body of the materials and methods that “Reciprocal hybridizations were attempted among all combinations of accessions throughout the experimental period.” and therefore, no change was made here. 6. Why the author uses the SSR for studying the relationship among sampled Capsicum species. In other word, what’s the advantage of SSR in genetic diversity study? Similarly, why the author selected the wax gene as a marker for phylogenetic reconstruction. Generally, single- or low-copy genes are less likely subject to concerted evolution, thus making themselves ideal tools for studying the origin and evolution of taxa, especially in hybrid speciation. In addition, I don’t know how the authors get the wax sequences, by direct sequencing or clone sequencing? Moreover, many information in phylogenetic reconstruction is lacking. But, the author present a phylogenetic tree inferred from the wax sequences. do you use a species as outgroup? The use of SSRs, especially those evenly distributed across the genome as in our case, are widely used to support relatedness among wild and domesticated species, especially in situations where very little information is known about some of the wild species, like in Capsicum. The waxy gene has been previously used to help clarify the species of Capsicum in the past and we used the published makers of Carrizo García et al., 2016 as already cited in materials and methods and discussion points have been added. Nowhere in the manuscript did we state that understanding origin or evolution was an objective of our study. We want to understand relatedness and how that impacts cross combability. Therefore, the use of SSRs supplemented by targeted sequencing of the waxy gene and comparisons made with morphology is appropriate to answer these questions. We have added the tomato waxy gene as an outgroup. Furthermore, we have added several waxy gene sequences for different Capsicum species available on NCBI to our analysis, providing confidence in our results. 7. Since the experiments of artificial hybridizations were performed, results of meiotic pairing behavior should be shortly described to give the readers a clear figure. The role of meiotic paring was described in the results and discussion section. 8. I can’t pick up the species and their the groupings in the Figure 1 because the species information is lacking in figure. This actually lead to some inconvenience to readers. This change was made so that species and accession are listed in each figure. 9. Some species (e.g. C. eximium) seems to be non-monophyletic origin, but there is no explanation in discussion. The reviewers perceived non-monophyletic origin is likely misidentification based on the waxy gene sequence, the SSR markers, and the phenotypic characters. This is extensively discussed in the manuscript; therefore, no change was made. Reviewer #2: Generally, the research demonstrated that the inter-specific compatibility may not necessarily reflect in relatedness from established Capsicum gene pool, and correction of the previous mis-identification of a number of Capsicum species This information will be valuable for future breeding programs. The following comments and suggestions may help improve the quality of the manuscript: Lane 37: The single sequence repeat (SSR) must be changed as simple sequence repeat (SSR). This change was made throughout the manuscript. Lane 41-42: The description of “The World … wild relatives” can be deleted. This sentence provides justification for the need of our study. The crossability of Capsicum has conflicting reports, therefore, in addition to understanding the hybridization success rate we also need to ensure that the species listed in genebank repositories are accurate, which can help resolve the contradictions in research findings. Therefore, no change was made. Lane71-72: The description of “This suggestion between complex is not total” is not clear, it should be rephrased. Total was changed to absolute here. Lane 91: The ability to hybridize might be crossability. The term “ability to hybridize” is very similar to “crossability”, but the author understand that hybridization refers to the ability of the plants to produce successful offspring, while crossability is the study of hybridization. To say two species are crossable does not have the level of accuracy as to say they hybridize. No change was made here. Table 1. The Source might have a table note to refer the abbreviations for the organizations. A footnote was added to Table 1 expanding the abbreviations of the sources. The μl might be changed as μL through the main text. PLOS one requires the use of the universal system of units as prescribed by the Bureau International des Poids et Mesures, which states that liter is abbreviated with a lowercase “l”. Linked below is the brochure confirming this. Therefore, no change was made here. https://www.bipm.org/utils/common/pdf/si-brochure/SI-Brochure-9-EN.pdf Lane 287-289: The sentence is not clear and needs to be edited. This sentence has been revised to indicate that the wild species were dispersed further from the origin of the plot. The Discussion part, the cluster analysis between Fig.1 and Fig. 5 should be compared in detail, and the contribution of genome sequencing of Capsicum for the phylogenetic study may also be mentioned. We cannot conduct any analysis to compare the data presented in these two figures. However, further discussion has been added of the contribution of waxy sequencing in both our study and its context in wider literature. https://doi.org/10.1371/journal.pone.0243689.r002
16 Feb 2021 Decision Letter - Dengcai Liu, Editor PONE-D-20-37323R1 Reproductive Compatibility in Capsicum is not Reflected in Genetic or Phenotypic Similarity Between Species Complexes PLOS ONE Dear Dr. Barchenger, Thank you for substantial revisions and submitting your manuscript to PLOS ONE again. After careful consideration, we feel that it can be accepted after minor revison. Therefore, we invite you to submit a revised version of the manuscript again. Please submit your revised manuscript by Apr 02 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, Dengcai Liu, PhD Academic Editor PLOS ONE Journal Requirements: Additional Editor Comments (if provided): Dear Dr. Barchenger, Thank your great efforts to improvethe manuscript. I have two minor suggestions: (1) In the title, I think "....is not reflected..." is so strong. I suggest to revise the title as such "Reproductive compatibility in Capsicum is not necessarily reflected in genetic or phenotypic similarity between species complexes", or someone else. (2) please check the taxonomic nomenclature. For instances in page 52-53, C. annuum (L.), C. baccatum (L.), C. chinense (Jacq.), C. frutescens (L.), and C. pubescens (Ruiz & Pav.) should be C. annuum L., C. baccatum L., C. chinense Jacq., C. frutescens L., and C. pubescens Ruiz & Pav. [Note: HTML markup is below. Please do not edit.] [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.0243689.r003
Revision 2
16 Feb 2021 Author Response (1) In the title, I think "....is not reflected..." is so strong. I suggest to revise the title as such "Reproductive compatibility in Capsicum is not necessarily reflected in genetic or phenotypic similarity between species complexes", or someone else. This change was made and the title is now Reproductive Compatibility in Capsicum is not Necessarily Reflected in Genetic or Phenotypic Similarity Between Species Complexes (2) please check the taxonomic nomenclature. For instances in page 52-53, C. annuum (L.), C. baccatum (L.), C. chinense (Jacq.), C. frutescens (L.), and C. pubescens (Ruiz & Pav.) should be C. annuum L., C. baccatum L., C. chinense Jacq., C. frutescens L., and C. pubescens Ruiz & Pav. This change was made throughout the manuscript. https://doi.org/10.1371/journal.pone.0243689.r004
18 Feb 2021 Decision Letter - Dengcai Liu, Editor Reproductive Compatibility in Capsicum is not Necessarily Reflected in Genetic or Phenotypic Similarity Between Species Complexes PONE-D-20-37323R2 Dear Dr. Barchenger, 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, Dengcai Liu, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0243689.r005
Formally Accepted
24 Feb 2021 Acceptance Letter - Dengcai Liu, Editor PONE-D-20-37323R2 Reproductive Compatibility in Capsicum is not Necessarily Reflected in Genetic or Phenotypic Similarity Between Species Complexes Dear Dr. Barchenger: 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. Dengcai Liu Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0243689.r006

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