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Dear Authors,
I am pleased to inform you that the manuscript has improved after the last revision and can be accepted for publication.
Congratulations on accepting your manuscript, and thank you for your interest in submitting your work to PeerJ.
With Thanks
[# PeerJ Staff Note - this decision was reviewed and approved by Paula Soares, a PeerJ Section Editor covering this Section #]
Dear Authors
The manuscript still needs a minor revision before publication. The authors are invited to revise the paper in light of all the reviewers' suggestions. Please note that the requested changes are required for publication.
With Thanks
**PeerJ Staff Note:** Please ensure that all review, editorial, and staff comments are addressed in a response letter and that any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
The authors have satisfactorily addressed my previous concerns and modified the manuscript accordingly. Additional background information has been incorporated, enriching the Introduction. The language and style have been revised, improving readability, and clarifications have been added for several specific terms. Figures and legends have also been improved. However, I have a few additional minor comments:
Figure 1: The legend states, “The gray arrows show that genes inside the cycle are transcribed clockwise, and genes outside the circle are transcribed counterclockwise.” However, I cannot find the gray arrows in the figure.
Figure 4: Please check the image quality. In the version I reviewed (PDF), details such as arrows and gene names are not visible.
Figure 6: The authors stated, “As clarified in the legend of Figure 6, branch lengths represent the expected number of substitutions per site, and a scale bar is provided.” However, this information still does not appear in the legend.
Raw data: The accession numbers for the assembled chloroplast genomes work, but the records have not yet been released. In addition, the raw sequence data have not been deposited. I am not certain whether this is mandatory, so I leave this decision to the editor.
As requested, the authors have added more details regarding sampling, DNA extraction, sequencing, and data analysis, improving the reproducibility of the study.
Overall, the authors have addressed my concerns and incorporated most of my suggestions. They have also done a good job of contextualizing their work within current knowledge of Ficus phylogenetic relationships and plastome genomics, and of clarifying the potential applications of their findings. I believe this manuscript is now suitable for publication.
In this revision, the authors took the feedback from three independent reviewers and strengthened their manuscript following the guidance of the peer reviews. The manuscript reads well, and represents a meaningful contribution. This reviewer was focused on lacking literature citations and context for the work, and that has been rectified in the revision.
The approach has had some edits made that make it better justified.
Findings are appropriately described and interesting.
The edits have been made in accordance with the suggestions of the three reviewers.
Dear Authors
The manuscript cannot be accepted for publication in its current form. The manuscript requires substantial revision to meet the journal's standards. The authors are invited to revise the paper in consideration of all suggestions made by both reviewers, including the reviewer who initially rejected the manuscript. Please note that the requested changes are required for publication.
With Thanks
**PeerJ Staff Note:** Please ensure that all review, editorial, and staff comments are addressed in a response letter and that any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
The introduction is clear and informative. As someone who isn't an expert in chloroplast genome structure, I found it helpful for understanding the results. Here are some specific comments:
lines 54-55: “are important for traditional medicine and food production, and often used as a species.” What did you mean with this?
lines 55-57: Could you maybe expand on what is currently known about the evolutionary relationships within the genus? The citation to Devi et al. (2022) does not provide information on this topic. Also, consider changing that citation.
lines 64-65: have any barcode markers been identified and used specifically for Ficus before this study?
Raw data: A data deposition statement is included and the information is also mentioned in the main manuscript. The accession numbers for assembled cp genomes work but the records have not been released yet. I would suggest depositing the raw reads as well, as encouraged by the PeerJ policy.
Figure 1. The image quality is good, as well as labeling. Could use some extra description. What do the gray bars in the inner circle represent? The legend says "physical maps" but the picture shows a single one. Which genome are you showing? Or is it just a general representation?
Figure S1. Check legend. For panel A it says “(F: positive; R: reverse; C: Complement; P: Palindromic; T: tandem)” but those letters are not in the figure. For panel B it says “Number of various repeat types”, should it be “various tandem repeat types”. Lastly, for panel C it says “The proportion of repeat types in each species” but there is only one pie chart and it is not separated by species.
Figure 2: legend is missing for panel A. Also, when looking at the heatmap, codons ATG and TGG have a value of 0, not 1 as expected for codons with no bias. Have you changed the scale of the heatmap to make 0 the no-bias value?
Figure 3: Needs some extra description of the components of the figure in the legend. Also, there is an arrow towards the right side of the sequences that I am not sure what is indicating.
Figure 5: Graph axes units are not defined neither in the figure nor in the legend.
Figure 6: What does the branch length represent? Please add to legend and report likelihood of the topology.
This is a nice and complete work. The research question is well defined and its contribution to knowledge is clearly stated. Methods are explained in a straightforward way but at some points important details necessary for replicability are lacking. Here are some specific comments:
lines 79-81: Check sentence structure. Also, did you sample and then deposit the material at the Guangxi Institute of Botany? If so, maybe change "source" for "voucher storage" or something of the sort in Supplementary Table 1.
line 82: extra t in “complied”
line 85: For DNA extraction, what was the starting material? What tissue was it? Was it fresh or dry?
line 87: “High-quality DNA samples were fragmented to construct paired-end libraries” How was the DNA fragmented and what kind of libraries did you use? This information is not provided in Duan et al. (2022) either.
line 92: what was the quality threshold?
lines 94-95: What species was used as the reference? Is that cp genome sequence publicly available? Please provide the accession number.
line 116: Was F. hirta chosen as the reference for any particular reason? If so, could you clarify?
line 125: Could you mention the genus/species of the outgroup and the reason for choosing it?
line 129: Please explain how the parameters of the runs in MrBayes were set.
lines 138-140: check sentence structure. The legend of Supplementary Table S3 says "Bold type indicates species sequenced in this study; data for others were downloaded from GenBank." but I think it does not correspond to that table.
line 142: “The total number of annotated genes in Ficus plastomes was 130, comprising 85 (79+6)” What does 79+6 stand for? Maybe I am asking something obvious, but I think it is worth clarifying.
lines 181-183: Isn’t TGG the only codon for tryptophan? If it is, there could be no way of finding codon bias, so I think it is misleading to report it this way.
line 189: what do JL and JS stand for?
line 257: Duplications are not mentioned in the results section, could you expand a bit on this? I have never worked with chloroplasts so I don't know if the clarification I am asking for is too obvious. You can ignore my comment if it is.
lines 277-279: a reference here would be useful.
no comment
This paper reports the assembly of chloroplast genomes from seven Ficus species. The following are some questions and suggestions regarding the article.
1. Abstract. Some descriptions in the Conclusion section are inappropriate.
(1) The molecular markers analyzed in this study should be described as having potential applications rather than simply being referred to as "useful".
(2) The statement “contributing to a clearer understanding of Ficus evolution” is not well – justified. Since only a relatively small number of sequences was used in the article and the result of the phylogenetic analysis of Ficus is rather rough.
2. Background. As stated in the article, the genus Ficus (Moraceae) is a large and ecologically important group. Therefore, a great deal of research has been conducted on it. For example, numerous chloroplast genomes of Ficus have been published in the NCBI database, and one can search for 96 reference sequences related to Ficus. Moreover, five of the seven sequences in this study have also been previously published in NCBI. However, this information and related research are rarely mentioned or cited in this manuscript. It is recommended to enrich the introduction section and provide more detailed information about past studies.
3. Results and Discussion.
(1) The text repeatedly mentions the “conservation in content and numbers of genes in Ficus plastomes”. However, according to Table S3 and the description in the text (L143 - 144), the number of genes in F. esquiroloana is three fewer than those in other species. The missing genes should be identified and the implications of them should be discussed, too, such as biological significance of these missing genes hold? whether it is unique evolutionary adaptations or functional losses in F. esquiroloana? Adding this information will provide a more comprehensive understanding of gene conservation and variation within the Ficus genus.
(2) This article presents the chloroplast genomes of seven Ficus species, five of which have already been published in the NCBI database. Could you elaborate on the differences between the previously published chloroplast genomes and the newly assembled ones in this study? These differences may include variations in sequence quality, gene annotations, or structural features. Clarifying these distinctions will help assess the novelty and value of the current research.
(3) In the sentence “Phylogenetic reconstruction based on 79 chloroplast PCGs robustly resolved the relationships among the seven Ficus species, supporting their monophyly and clarifying intrageneric clades with strong statistical support.” the reference of “Their” is ambiguous. Is it refers to the seven Ficus species？Given that monophyly typically refers to a group of organisms that share a common ancestor and include all of its descendants, and a single sequence cannot be considered monophyletic. or refers to Ficus？However, considering the context, referring to the Ficus genus as monophyletic is also inaccurate for only ficus, one genus in the text.
4. Tables and figures. Inadequate Resolution, Inappropriate Descriptions, and Labeling.
Figure 1. (1) Incomplete or Overlapped Gene Names. Some gene names in the figure are incomplete or overlapping, which significantly impairs the readability and interpretability of the data presented. (2) Incorrect Annotation of the rpl2 Gene: Given that rpl2 is located within the inverted repeat region, the sequences spanning from the start of the IRA region to the IRA/LSC boundary should all be identified as the rpl2 gene. However, the figure only shows a small portion of it, leading to a misrepresentation of the gene's actual location and extent.
Figure 2. what is the A? what is the B? The title of Figure 2 does not accurately reflect the content of the image. Moreover, Figure 2B does not align with the description provided in the text, creating confusion and making it difficult for readers to connect the visual data with the written explanations.
Figure 3. There is a noticeable difference in the size of the rpl2 gene depicted in Figure 3, while the sizes are the same in the provided GB file. Why?
Figure 6. The terms "Subgen. Ficus" and "Subgen. Urostigma" are used in Figure 6 without any prior description or explanation in the text. To enhance the comprehensibility of the figure, it is crucial to define these subgenera and provide relevant background information.
Figure S1. (1) The description in the figure does not correspond to the content in the text. For instance, the text on L150 - 153 refers to Figure S1, not S1A. (2) The title of Figure S1C states that it shows the proportion of repeat types in each species, yet there is only one set of data presented. It is unclear whether these data represent all seven species equally or if they belong to a single species. If it is for one species, the specific species should be identified.
Table S2. Optimization of Table Content. The table would benefit from a more focused presentation of data. Columns with identical content could be either omitted or merged to streamline the table, and present more different or key information, such as length, subgenus, etc.
Table S3. (1) The note at the back of Table S3 mentions that "Bold type indicates species sequenced in this study; data for others were downloaded from GenBank." However, the bold type is not visible in the table, and it is unclear which species are referred to as "others." Clarification is needed to ensure transparency and accuracy in data representation. (2) What does the last line of “Protein coding%” mean? Please explain and modify.
GB file. Some annotations of the rpl2 genes in the GB file are incomplete. A thorough review and modification of these annotations are necessary to ensure the accuracy and completeness of the genomic data.
5. Other Issues
(1) Lack of Accessible Original Data. The article does not provide original data. Furthermore, the login number provided in the article has not been released in GenBank.
(2) Absence of Assembly Coverage Information. The article did not mention the coverage of assembly process, which will enable readers to better evaluate the reliability and accuracy of the genomic assemblies presented in the study.
(3) The first project listed under the funding section of the article does not have a project number.
(4) Some problems in writing. Please conduct a comprehensive check of the full text to ensure its quality.
L53-55：Economically, several species, such as F. carica (the common fig), are important for traditional medicine and food production, and often used as a species. In the end of the sentence, “as a species” means what.
L79-81: All seven Ficus species were included in this study (F. esquiroliana, F. pandurata, F. formosana, F. erecta, F. carica, F. hirta, and F. stenophylla) the voucher specimen storage information is shown in Supplementary Table S1.
These should be two sentences.
L81-82: The sampling of seven species was approved by the Guangxi Zhuang Autonomous Region of China and compliedt local policy requirements. “compliedt” should be ““complied”.
A detailed description of the experimental procedures, data processing steps, and analytical algorithms used would greatly enhance the reproducibility and comprehensibility of the study.
(1) Please supplement the method used in generating Figure 2B!
(2) In the process of assembling the chloroplast genomes, what sequence is used as the reference seed? Was it a single reference sequence or multiple ones? If a single reference was used, please specify which one. If multiple references were employed, the sentence should be revised to “with related Ficus cp genomes as reference seeds” to accurately reflect the assembly approach.
(3) The outgroup species used in the phylogenetic analysis should be explicitly pointed out.
(4) Lack of Accessible Original Data. The article does not provide original data. Furthermore, the login number provided in the article has not been released in GenBank.
(5) Absence of Assembly Coverage Information. The article did not mention the coverage of assembly process, which will enable readers to better evaluate the reliability and accuracy of the genomic assemblies presented in the study.
The conclusions should be connected to the original question investigated, and should be limited to those supported by the results. So, the description “Further expansion of plastome sampling across the genus, integration with nuclear and mitochondrial genomic data, and population-level sampling will be instrumental for a comprehensive understanding of the evolutionary history, biogeography, and adaptive diversification of Ficus.” (L297-300) is inappropriate.
The manuscript is written very clearly and is an easy and compelling read. The presentation of the work is very professional. The most important critique is that there are quite a few highly relevant literature citations that were not included in the manuscript, so the introduction and discussion sections are not as fully developed or presented in full context of the current available literature as they need to be. I have attached a PDF with comments, and I am very specific on which articles may be good to review, interpret, and cite. I also point out areas where the text needs to be expanded with this added context from the literature. The figures and tables are appropriate, and I think Figure S1 should be included in the main manuscript.
The sampling, data collection, and analysis design is appropriate and repeatable. The methods address the research questions, and are clear.
The findings are valid based on the data and analyses. The authors do a good job effectively interpreting and presenting the outcomes without over interpreting the data. As commented above, a better contextualization with relevant published literature that was missed needs to be done in the revision.
The attached PDF has many minor comments to fix a few grammatical and typographical errors. The PDF also details further the need to review and cite highly related, relevant, recent literature using plastid genomes in the Moraceae for similar work.
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