Analysis of the Chloroplast Genome of Ficus simplicissima Lour Collected in Vietnam and Proposed Barcodes for Identifying Ficus Plants
Round 1
Reviewer 1 Report
Need to corrected grammatical error, missing full stop and references style. Add new literature from Ficus sarmentosa and phylogenetic analysis. Add plant sample photo in the plant materials section as well as the person/organization that identify the plant species.
Comments for author File: 
Comments.pdf
Author Response
Dear Reviewer
Thank you very much for your comments. We have carefully reviewed your comments and have thoroughly revised our manuscript. We also provide a point-by-point response to your concerns. Revised portions are marked in blue in the attached manuscript.
We appreciate your warm work earnestly and hope that the revised version is now suitable for publication and look forward to your approval.
Sincerely,
Mau Hoang Chu
Corresponding author:
TNU- University of Education, Viet Nam
Response to the reviewers
1) Comment 1: There was one literature related to cp genome study and phylogeny. Zhang et al (2022); Comparison of chloroplast genomes and phylogenomics in the Ficus sarmentosa complex (Moraceae). PLoS ONE 17(12): e0279849.
Our response: We have added to the reference list. “[8] Zhang Z, Zhang D-S, Zou L, Yao C-Y (2022) Comparison of chloroplast genomes and phylogenomics in the Ficus sarmentosa complex (Moraceae). PLoS ONE 17(12): e0279849. https://doi.org/10.1371/journal.pone.0279849
2) Comment 2: “as potential DNA...”
Our response: - Added the word "potential" on line 64"
3) Comment 3: Suggested to include the plant sample photo
Our response: Sample image of F. simplicissima Lour m3 plant has been added to the manuscript
4) Comment 4: state the unit; w/v?
Our response: Added unit %- 0.8%
5) Comment 5: grammatical error; should past tense
Our response: Corrected "is" to "was".
6) Comment 6: grammatical error; was
Our response: Corrected "is" to "was"
7) Comment 7: the scientific name should be italic
Our response: Corrected “F. simplicissima” on line 144.
8) Comment 8: missing full stop at the end of sentences
Our response: Added punctuation on line 143
9) Comment 9: missing full stop at the end of sentences
Our response: Added punctuation on line 157
10) Comment 10: 100%?
Our response: Added unit “%”.
11) Comment 11: should be italic
Our response: Corrected.
12) Comment 12: Suggest to write the accession number in the text for easier referring
Our response: Added the accession number into the text.
13) Comment 13: Is LSC regions have benefited in identification species?
Our response: Analysis of previous studies showed that the large single-copy (LSC) region of chloroplast DNA is highly efficient in species identification.
Ludan et al. (2020) suggested that the large single-copy (LSC) region functions as a highly effective and efficient molecular marker for accurate authentication of medicinal Dendrobium species
References: Ludan Li , Yu Jiang , Yuanyuan Liu , Zhitao Niu , Qingyun Xue , Wei Liu và Xiaoyu Ding, (2020), The large single-copy (LSC) region functions as a highly effective and efficient molecular marker for accurate authentication of medicinal Dendrobium species. Acta Pharm Sin B. 10(10), pp. 1989–2001.doi: 10.1016/j.apsb.2020.01.012.
14) Comment 14: The scientific name of species should be italic. Some references are not follow the journal requirement.
Our response: We have checked the list of references and edited the scientific name of species to italicized.
We have added nine references to the list of references, which are [8], [26], [32], [33], [34[, [35], [36], [56], [57].
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Author Response File: 
Author Response.docx
Reviewer 2 Report
In this study, the authors newly sequenced the chloroplast genome of Ficus simplicissima and performed its features, codon usage bias, repeats, comparative, and phylogenetic analyses. Our understanding of the characteristics and variability of the chloroplast genome still requires considerable work. The concept of this study is generally correct. However, the newly sequenced and assembled genome does not appear to provide any new insights. I encountered aspects in the manuscript that diminish its scientific value and require improvement. Here I provide several comments that may be of utility to the authors.
1. I'd like to point out that there have previously been three published studies comparing the chloroplast genomes of Ficus species. (seen below). Notably, in these studies, the total number of newly sequenced Ficus species was 10, 8 and 10, respectively. All the analyses mentioned in this paper have already been done in the three studies. Compared to the earlier studies, what distinguishes this study as novel?
Huang Y, Li J, Yang Z, et al. Comprehensive analysis of complete chloroplast genome and phylogenetic aspects of ten Ficus species[J]. BMC plant biology, 2022, 22(1): 1-15.
Xia X, Peng J, Yang L, et al. Comparative Analysis of the Complete Chloroplast Genomes of Eight Ficus Species and Insights into the Phylogenetic Relationships of Ficus[J]. Life, 2022, 12(6): 848.
Zhang ZR, Yang X, Li WY, et al. Comparative chloroplast genome analysis of Ficus (Moraceae): Insight into adaptive evolution and mutational hotspot regions[J]. Frontiers in plant science, 2022, 13.
2. Sequencing of only one species and its comparison with other public species without any specific reason does not guarantee publication in 2022, especially when similar studies have already been published. Authors should concentrate on why they chose to research Ficus and give a thorough justification for the study's purpose and significance.
3. It doesn't appear that the annotated results of the newly assembled Ficus simplicissima's chloroplast genome has been published to the NCBI database. Have the authors provided other public access to verify the accuracy of the data?
4. Materials and Methods. Some programs include a version; others don't.
5. Besides the maximum likelihood (ML) phylogeny, bayes tree was also suggested added.
6. The best substitution model in phylogenetic analysis should be stated.
7. Please check the manuscript throughout, the name of the gene should be in italics.
8. Check the manuscript carefully; "Ficus" is not italicized in some places.
9. The results displayed in Figure 1 suggest that the authors did not manually correct the GeSeq annotation results. Duplicated genes and indeterminate genes such as trnH-GUG, trnM-CAU/trnI-CAU etc. appear in the figure.
10. It is advised to consult some literature for discussion since codon use analysis is not currently being discussed. The following articles can be cited:
Song W, Chen Z, Shi W, et al. Comparative Analysis of Complete Chloroplast Genomes of Nine Species of Litsea (Lauraceae): Hypervariable Regions, Positive Selection, and Phylogenetic Relationships[J]. Genes, 2022, 13(9): 1550.
Song W, Ji C, Chen Z, et al. Comparative analysis the complete chloroplast genomes of nine Musa Species: Genomic features, comparative analysis, and phylogenetic implications[J]. Frontiers in Plant Science, 2022, 13.
Trofimov D, Cadar D, Schmidt-Chanasit J, et al. A comparative analysis of complete chloroplast genomes of seven Ocotea species (Lauraceae) confirms low sequence divergence within the Ocotea complex[J]. Scientific reports, 2022, 12(1): 1-13.
11. What criteria did the authors use to decide which of the other five Ficus species to compare? based on what? The writers ought to have made a comment about this clearly.
12. Please clarify why only several species were chosen for phylogenetic study since there are more than 30 complete chloroplast genome sequences of Ficus species in the NCBI database.
13. Why did the psbA-trnH intergenic spacer region, which did not exhibit a high degree of diversity in comparative analyses, get chosen for phylogenetic analysis of Ficus species?
14. Please manully checked the IRScope results, for instance, the length of ycf1 was 1106bp, not a multiple of 3.
Author Response
Dear Reviewer
Thank you very much for your comments. We have carefully reviewed your comments and have thoroughly revised our manuscript. We also provide a point-by-point response to your concerns. Revised portions are marked in blue in the attached manuscript.
We appreciate your warm work earnestly and hope that the revised version is now suitable for publication and look forward to your approval.
Sincerely,
Mau Hoang Chu
Corresponding author:
TNU- University of Education, Viet Nam
Response to the reviewer
1) Comment 1: I'd like to point out that there have previously been three published studies comparing the chloroplast genomes of Ficus species. (seen below). Notably, in these studies, the total number of newly sequenced Ficus species was 10, 8 and 10, respectively. All the analyses mentioned in this paper have already been done in the three studies. Compared to the earlier studies, what distinguishes this study as novel?
Our response: The study on sequencing the F. simplicissima chloroplast genome collected in Thai Nguyen (Vietnam) has been carried out since 2021, but we have not yet published it in time. The study's objective was to analyze the chloroplast genome of F. simplicissima and compare it with some other species in the genus Ficus to find potential molecular markers for identification and phylogenetic analysis in the genus Ficus.
This study analyzes the chloroplast genome of the F.simplicissima plant collected from the northern mountainous region of Vietnam and looks for differences from the chloroplast genomes of F.simplicissima on GenBank. However, we have not found any F. simplicissima chloroplast genome sequence on GenBank.
When we performed chloroplast genome sequencing of the specimen collected from Thai Nguyen (Vietnam), there were not yet publications by Huang et al. (2022). Of the three references, only Huang et al. (2022) reported on the chloroplast genome of F.simplicissima, while the study of Xia et al. (2022) and Zhang et al. (2022) did not find the chloroplast genome data of F.simplicissima. We discussed and cited the study of Huang et al. (2022) in the reference list.
2) Comment 2: Sequencing of only one species and its comparison with other public species without any specific reason does not guarantee publication in 2022, especially when similar studies have already been published. Authors should concentrate on why they chose to research Ficus and give a thorough justification for the study's purpose and significance.
Our response: Ficus is a large genus containing about 850 species (Shanahan et al., 2001). Species in the Ficus genus are widely distributed in subtropical and tropical regions (Ber & Corner, 2005). The presence of many species in the Ficus genus is both food for animals and medicine for human diseases. They also play a vital role in the tropical forest ecosystem (Dev et al., 2011).
Comparative analysis of the whole chloroplast genome can provide an accurate and rapid method for species and subspecies differentiation (Li et al., 2015). When the sample of medicinal plants is deformed or in powder form, using DNA barcodes to identify medicinal plants will bring high efficiency.
References
Shanahan, M., So, S., Compton, S., and Corlett, R. (2001). Fig-eating by vertebrate frugivores: A global review. Biol. Rev. Camb. Philos. Soc. 76, 529–572. doi: 10.1017/S1464793101005760
Berg, C. C. and, Corner, E. J. H. (2005). Flora Malesiana Series I - Seed Plants. Leiden: National Herbarium
Dev, S. A., Kjellberg, F., Hossaert-Mckey, M., and Borges, R. M. (2011). Fine-scale population genetic structure of two dioecious Indian keystone species. Ficus hispida and Ficus exasperata (Moraceae). Biotropica 43, 309–316. doi: 10.1111/j.1744-7429.2010.00704.x
Li, X., Yang, Y., Henry, R. J., Rossetto, M., Wang, Y., and Chen, S. (2015). Plant DNA barcoding: From gene to genome. Biol. Rev. Camb. Philos. Soc. 90, 157–166. doi: 10.1111/brv.12104
3) Comment 3: It doesn't appear that the annotated results of the newly assembled Ficus simplicissima's chloroplast genome has been published to the NCBI database. Have the authors provided other public access to verify the accuracy of the data?
Our response: We completed the sequencing of the F. simplicissima chloroplast genome in Thai Nguyen and submitted it to GenBank in November 2022, and was granted the code BankIt2647431 Ficus_simplicissima_m3 OP928145 on December 5, 2022.
We have added the cp genome sequence of F. simplicissima, submitted to GenBank as Supplementary 2 with the revised manuscript.
4) Comment 4: Materials and Methods. Some programs include a version; others don't.
Our response: We only listed in the Materials and Methods section those versions used in F. simplicissima chloroplast genome sequencing and analysis.
5) Comment 5: Besides the maximum likelihood (ML) phylogeny, bayes tree was also suggested added.
Our response: In this paper, we only work on mega software with maximum likelihood (ML) phylogeny, and we will use the Bayes tree in future studies
6) Comment 6: The best substitution model in phylogenetic analysis should be stated.
Our response: The best alternative model in phylogenetic analysis is maximum likelihood (ML). In evolutionary analysis by ML, the evolutionary history was inferred by using the Maximum Likelihood method and Tamura-Nei model [1]. The bootstrap consensus tree inferred from 1000 replicates [3] is taken to represent the evolutionary history of the taxa analyzed [3]. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [3]. Initial tree(s) for the heuristic search were obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 1.9641)).
References
- Tamura K. and Nei M. (1993). Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution10:512-526.
- Kumar S., Stecher G., Li M., Knyaz C., and Tamura K. (2018). MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Molecular Biology and Evolution35:1547-1549.
- Felsenstein J. (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution39:783-791.
7) Comment 7: Please check the manuscript throughout, the name of the gene should be in italics.
Our response: We have checked and edited the gene names in the manuscript, and the gene names were italicized.
8) Comment 8: Check the manuscript carefully; "Ficus" is not italicized in some places.
Our response: We have checked and edited the genus name "Ficus" in the manuscript, and "Ficus" was italicized.
9) Comment 9: The results displayed in Figure 1 suggest that the authors did not manually correct the GeSeq annotation results. Duplicated genes and indeterminate genes such as trnH-GUG, trnM-CAU/trnI-CAU etc. appear in the figure.
Our response: The circular map of the chloroplast genome of F. simplicissima m3 in Figure 1 was exported from the software and we ask for permission to remain as it is and without further editing, because the visibility of duplicated genes and indeterminate genes does not change chloroplast genome sequence of F. simplicissima m3.
10) Comment 10: It is advised to consult some literature for discussion since codon use analysis is not currently being discussed. The following articles can be cited:
Our response: We have added a discussion of the use of codons and added to the reference list articles as the reviewer suggested.
11) Comment 11: What criteria did the authors use to decide which of the other five Ficus species to compare? based on what? The writers ought to have made a comment about this clearly.
Our response: All six complet chloroplast genomes including F. simplicissima Lour m3 were compared using the mVISTA online tool (https://genome.lbl.gov/vista/mvista/submit.shtml), with the global multiple alignment model (LAGAN). The F. hista chloroplast genome was used as the reference and the RankVISTA probability threshold was set to 0.5.
12) Comment 12: Please clarify why only several species were chosen for phylogenetic study since there are more than 30 complete chloroplast genome sequences of Ficus species in the NCBI database.
Our response: At the time of our analysis, we did not find data on the chloroplast genome of F. simplicissima on GenBank. Based on BLAST analysis on NCBI, the criteria to select chloroplast genomes of some species for phylogenetic analysis in this study are based on the total score and query cover.
13) Comment 13: Why did the psbA-trnH intergenic spacer region, which did not exhibit a high degree of diversity in comparative analyses, get chosen for phylogenetic analysis of Ficus species?
Our response: According to Burgess et al. (2011), up to 97% accurate identification of Canadian temperate plant samples was possible based on five gene regions rbcL, matK, rpoC1, psbA-trnH, and atpF-atpH. Research by Newmaster et al. (2008) showed that using matK and psbA-trnH data could identify more than 94% of species in the Myristicaceae family. The study's results using the psbA-trnH marker to identify the above plant objects are the basis for us to choose psbA-trnH for phylogenetic analysis of Ficus species.
We have added this content to the discussion section.
References
Burgess, K. S.; Fazekas, A. J.; Kesanakurti, P. R.; Graham, S. W.; Husband, B. C.; Newmaster, S. G.; Percy D.M.; Hajibabaei, M.; Barrett, S.C.H. Discriminating plant species in a local temperate flora using the rbcL+matK DNA barcode. Methods Ecol. Evol. 2011, 2, 333–340. https://doi.org/10.1111/j.2041-210X.2011.00092.x
Newmaster, S. G.; Fazekas, A. J.; Steeves, R. A.; and Janovec, J. Testing candidate plant barcode regions in the Myristicaceae. Mol. Ecol. Resour. 2008, 8, 480–490. https://doi.org/10.1111/j.1471-8286.2007.02002.x
14) Comment 14: Please manully checked the IRScope results, for instance, the length of ycf1 was 1106bp, not a multiple of 3.
Our response: We have manually checked the IRScope results in detail; however, we have not yet to find any changes, including the ycf1 gene. So we ask for permission to keep the status quo.
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Author Response File: Author Response.docx
