Phylogenetics Study to Compare Chloroplast Genomes in Four Magnoliaceae Species

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for your submission and your great work.

I have some questions and concerns that I would like you to address.

1- Please change the title of the manuscript to something " Phylogenetics study to compare...".

2- In Introduction- Add why phylogenetic studies are important for conservation and how it has been used earlier in other species, especially using cp genome.

3- My most important concern is the language and editing of the manuscript. Please remove dash from different places. Sometimes there are dash inserted within a word. Also, send the manuscript to an English language expert before resubmission for review.

4- Section 2.2- Rephrase line 101. 

5- Did you submit the genome sequence in NCBI database and have a submission ID. Please include the submission ID in section 2.2 so that it can be used by researchers in future.

6- Image 2- Difficult to interpret and understand. Is it comparison of the 4 species? If yes, then which region belongs to which species? 

7- The author said they used 3 methods for cp genome sequencing, which method yielded the results presented in figure 3, please specify?

8- Line 234- rephrase.

9- Figure 8- Are you saying you used 100 bootstrap values for tree construction? It's too low. I would suggest using at least 1000 bootstraps. Also present the results for all three methods used for tree construction to have a comparative picture. Please explain how many clusters were formed and did all the four species showed same distribution among three different trees.

 

 

 

 

 

 

Comments on the Quality of English Language

My most important concern is the language and editing of the manuscript. Please remove dash from different places. Sometimes there are dash inserted within a word. Also, send the manuscript to an English language expert before resubmission for review.

Author Response

Dear Reviewer,

We sincerely thank you for your valuable feedback that we need to improve the quality of our manuscript. The reviewer comments laid out below in italicized font and were all replied from point to point. Revised places were highlighted in our manuscript to be easily reviewed.

Here are our reply:

1. Please change the title of the manuscript to something "Phylogenetics study to compare...".

Reply: We sincerely thanks for your suggestion. As suggested, we revised the title from “Comparative study of chloroplast genomes in four Magnoliaceae species” into “Phylogenetics study to compare chloroplast genomes in four Magnoliaceae species”.

 

2. In Introduction- Add why phylogenetic studies are important for conservation and how it has been used earlier in other species, especially using cp genome.

Reply: Thank you for your guidance. We are also aware of the lack of this part. We supplement this part in the introduction section of the paper and add a literature citation. We added supplementary content as “These differences can be more directly demonstrated through various analyses. In order to more clearly reflect the evolutionary relationship of these four Magnoliaceae plants and distinguish the differences between them, general scholars adopted the method of building phylogenetic trees.”

 

3. My most important concern is the language and editing of the manuscript. Please remove dash from different places. Sometimes there are dash inserted within a word. Also, send the manuscript to an English language expert before resubmission for review.

Reply: We tried our best to improve language and editing of the manuscript and made some changes to the manuscript. These changes did not influence the content and framework of the paper. We invited a friend who is a native English speaker from the UK to help polish our article. Revised detail places were highlighted in our manuscript. We hope the revised manuscript could meet your requirements.

 

4. Section 2.2- Rephrase line 101.

Reply: We would like to thank you for your suggestions on adjusting the order of the article chapters. We have combined 2.2 and 2.1 into one part according to your suggestion, hoping to meet your requirements.

 

5. Did you submit the genome sequence in NCBI database and have a submission ID. Please include the submission ID in section 2.2 so that it can be used by researchers in future.

Reply: Thank you for your reminding. We are very sorry for the inconvenience caused to you due to our carelessness. We have added the submission ID of these four species in section 3.1. We added supplementary content as "NCBI: Woonyoungia septentrionalis (Dandy) YW Law: ON456177.1, Michelia champaca L.: ON456178.1, Michelia figo (Lour.) Spreng: ON456179.1, Michelia macclurei Dandy: ON456180.1.”

 

6. Image 2- Difficult to interpret and understand. Is it comparison of the 4 species? If yes, then which region belongs to which species?

Reply: Thank you for your reminding us. Our original comment on Figure 2 is indeed prone to ambiguity. We have written the note in Figure 2 in more detail, and we sincerely hope to answer your doubts.

 

7. The author said they used 3 methods for cp genome sequencing, which method yielded the results presented in figure 3, please specify?

Reply: Thank you very much for your careful questions. The result in Figure 3 is the combination of BI, ML and NJ methods, mainly on the NJ diagram. Because the clustering results of the three methods overlap, we present them in this way, and the separate results show them in the supplementary materials.

 

8. Line 234- rephrase.

Reply: We rephrased line 234 as “The results showed that after classifying the known functional genes, it was found that there are introns in 19 genes (W. septentrionalis), 21 genes (M. champaca), 19 genes (M. figo), and 20 genes (M. maclurei). In terms of intron distribution, there are 10 genes in the LSC region, 4 genes in the IR region, and 1 gene in the SSC region for the chloroplast genomes of W. septentrionalis, M. figo, and M. macclurei. There are 11 genes in the LSC region, 4 genes in the IR region, and 2 genes in the SSC region for chloroplast genome of M. macclurei.”

 

9. Figure 8- Are you saying you used 100 bootstrap values for tree construction? It's too low. I would suggest using at least 1000 bootstraps. Also, present the results for all three methods used for tree construction to have a comparative picture. Please explain how many clusters were formed and did all the four species showed same distribution among three different trees.

Reply: Thanks for you careful checks. We are sorry for our carelessness. Based on your suggestion, we added more detail in section 2.7, including the use of 1000 boot values. Moreover, the BV values in Figure 8 are only showing results, not parameters. We have also added how many clusters are in section 3.6.

 

In addition, we checked all through our manuscript and revised some grammar and language mistakes including those in section of reference. We hope our revision work will satisfy you although there might be something that still need to be improved. Still thank you for your help for perfecting our manuscript.

 

Kind regards,

Ying Hu

Reviewer 2 Report

Comments and Suggestions for Authors

 

• The introduction provides a good overview of chloroplast genomes and their utility in phylogenetic studies of plants. However, the rationale for studying these specific Magnoliaceae species could be made stronger - why were these particular 4 species chosen? What gap in knowledge is this study aiming to fill?
• The methods are generally sound, utilizing standard bioinformatic tools for chloroplast genome assembly, annotation, comparison, and phylogenetic analysis. A couple suggestions:

1. Provide more details on the specific parameters/settings used for the various software tools. For example, what substitution model was used for phylogenetic reconstruction in RAxML?
2. Use Circos to show homology.
3. Justify why coding sequences (CDS) were used for phylogenetic analysis rather than whole chloroplast genomes or translated ORFs. Using CDS may not be the best option, but further rationale would strengthen this choice.

• The results are clearly presented, with ample figures and tables. The identified mutational hotspots and areas of variation between the species provide useful molecular markers for future studies.
• The discussion could be expanded. The authors note some uncertainties and discrepancies compared to traditional taxonomy of Magnoliaceae, but don't delve deeply into potential reasons or implications. More comparison to prior phylogenetic studies of these species would also be informative.
• Overall the study design, methods, and presentation of results are reasonably strong. My main recommendations would be to expand the introduction and discussion, and provide more methodological details. The findings advance knowledge of chloroplast variation across these Magnoliaceae species.

 

Comments on the Quality of English Language

English is OK, but should provide line numbers on draft submitted to journal

Author Response

Dear Reviewer,

We sincerely thank you for your valuable feedback that we need to improve the quality of our manuscript. The reviewer comments laid out below in italicized font and were all replied from point to point. Revised places were highlighted in our manuscript to be easily reviewed.

Here are our reply:

1. Provide more details on the specific parameters/settings used for the various software tools. For example, what substitution model was used for phylogenetic reconstruction in RAxML?

 

Reply: Thank you for your guidance. We are also aware of the shortcomings of this part. We supplemented this part in Part 2.7 of the paper and added the literature citations. We added supplementary content as" The BI tree was reconstructed with GTR+I+G [25]. The ML tree was reconstructed with IQ-TREE [26] and bootstrap probability values were calculated from 1000 replicates.”

 

2. Use Circos to show homology.

 

Reply: Thank you for supplementing the method for our manuscript. According to your requirements, we used Circos software to perform sequence comparison on the collinear analysis results, and we found that this result made our display effect more intuitive and could highlight the sequence differences among the four species more directly.

 

3. Justify why coding sequences (CDS) were used for phylogenetic analysis rather than whole chloroplast genomes or translated ORFs. Using CDS may not be the best option, but further rationale would strengthen this choice.

 

Reply: Thank you for your careful reading of the manuscript. We are sorry for missing this part. We have added the reasons why we choose CDS in the introduction. We added supplementary content as" The experimental material of this study is cp genome, whose sequence length is shorter than that of nuclear gene fragments, while matk gene sequence is a research method for short fragment sequences. Therefore, CDS method will be adopted in this study to explore the phylogenetic relationship of Magnolia species.”

 

 

We tried our best to improve our manuscript and these changes were marked in highlight in revised paper. As you suggested, we revised sections of introduction and discussion, and add more comparison to prior phylogenetic studies of these species. In addition, we checked all through our manuscript and revised some grammar and language mistakes including those in section of reference. We hope our revision work will satisfy you although there might be something that still need to be improved. Still thank you for your help for perfecting our manuscript.

 

 

Kind regards,

Ying Hu

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for including the suggestions. The quality of the manuscript has improved tremendously.

Comments on the Quality of English Language

Minor changes needed.