Seven Complete Chloroplast Genomes from Symplocos: Genome Organization and Comparative Analysis
Round 1
Reviewer 1 Report
The review of manuscript entitled: "Seven complete chloroplast genomes from Symplocos: genome, organization and comparative analysis" (forests - 1152203) can be found in attached .pdf file.
Comments for author File: 
Comments.pdf
Author Response
Response to Reviewer 1
Reviewer 1 comments
The review of manuscript: forests - 1152203 Title: Seven complete chloroplast genomes from Symplocos: genome, organization and comparative analysis
The reviewed paper reports the results of chloroplast genome sequencing of four species representing genus Symplocos. Complete chloroplast genome sequences of S. chinensis for. pilosa, S. prunifolia, S. coreana and S. tanakana were characterized and used for comparative and evolutionary studies. The reviewed paper is an example of an interesting study with correct and complementary methodological side. The described results are complementary to previous studies on the evolution and diversity of genus Symplocos. The applied methodology, high-throughput sequencing, provide a high-quality data with a number of applications. Here, authors not only report complete chloroplast genomes for four Symplocos species, but also based on the molecular data verified the systematic relationships of the species. Furthermore, they identified plastome regions characterized with the highest variation and propose them as a potential DNA barcodes for Symplocos species. The observations included in the reviewed manuscript may become a valuable element of the discussion not only in case of studies on evolution and diversity of genus Symplocos, but also for other closely related taxa.
Objective of the paper is clear and unambiguous. The article is well structured, the methodology is correct and suitable for realization of the paper objectives. However, many improvements are needed especially in section devoted to description of results. Furthermore, the Introduction is scant and does not provide sufficient background for the topic (it consist of 34 lines, out of which 17 are devoted to general cp genome structure, 10 lines include information about genus Symplocos and characterization of studied species, and finally 7 lines describes the aim of the study. Discussion is also unsatisfactory (consist of 4 paragraphs squeezed into 34 lines) and in some places confusing. English language and style of the whole text is correct with rather minor drawbacks. More exhaustive comments are listed below in the detailed review report.
L.15
replace "better conserved" with "more conserved"
Response: This has now been revised as suggested.
L.27
replace "protein-expressing" with "protein-coding"
Response: This has now been revised as suggested.
L.28
replace "transgenic RNA genes" with "transfer RNA genes"
Response: This has now been revised as suggested.
L.28-29
replace "...independent replicating genomes..." with "...independently replicated genomes..."
Response: This has now been revised as suggested.
L.33
I suggest to replace "...has been deleted." with "... has been lost" or "...is not observed."
Response: This has now been revised as suggested.
L.36
replication???
Response: We have changed this to “gene replication.”
L.37
there should be rather "...intron gain/loos have occurred..."
Response: This has now been revised as suggested.
L.39
Chloroplast are UNIPARENTALLY inherited but it means that depend on the systematic group they could be transmitted maternally or paternally!
Response: This has now been revised as suggested.
- 43
"area boundary" should be deleted
Response: This has now been revised as suggested.
L.60
replace "..whole CP genome..." with "...complete CP genome sequences..."
Response: This has now been revised as suggested.
- 61
apply the italic font for S. coreana
Response: This has now been revised as suggested.
- 64-65
The sentence about the approaches applied for cp genome assembly should be a part of Materials and Methods section. Moreover, authors should specify the reference genomes used during that process - I can only guess that these were the same genomes that were used for cp genome annotation.
Response: This has now been revised as suggested.
L.81
replace "...87 coding proteins..." with "...87 protein-coding genes..."
Response: This has now been revised as suggested.
L.80-87
I suggest to rephrase this section by giving the specific data on:
- the number of unique genes
- the number of duplicated genes
- number of conserved chloroplast ORFs (ycf)
This improvement will make the observed number of genes to be concordant with generally expected number of genes in chloroplast genomes referred in introduction section (L.26-28).
Response: This has now been revised as suggested.
L.87-89
I suggest to rephrase this sentence: "The rps12 was confirmed to be trans-splicing gene which consist of three exons: exon1 was found in the LSC region, while exon2 and exon3 were located in the IR regions."
Response: We have accepted the comments and made some corrections in the revised manuscript, for accuracy. Please check.
L.97-100
sentences about the character of JSB junctions are not clear
I suggest also to explain here why one copy of the ycf1 is called pseudogene, you can apply here the abbreviation/symbol for pseudogene (e.g. Greek letter 'psi' ψ) and used it also on Fig.3 to distinguish between ycf1 and ψycf1
Response: This has now been revised as suggested.
- 114
I strongly encourage the authors to share with the readers the detailed data on SSRs (not only motif but also length, start and end position, location) and long-repeat analyses (length of the repeat, starting site of both (A, B) repeats, location of A and B repeats, region in which repeat A and B can be found, repeat type). It become a standard to provide with the manuscript (as a supplementary files) more precise information about SSRs and long-repeats identified in reported chloroplast genomes of four Symplocos species.
Response: This has now been revised as suggested.
L.115
I suggest to rephrase that sentence: "...and refers to tandem repeats of 1-6 nucleotide motifs."
Response: Thank you for the remark. In line with your suggestion, we revised this sentence, for accuracy.
L.126
preliminary repeats???
Response: We have changed this to “reverse repeats.”
L.142
You may strengthen the value of the paper by adding the information on classification of SNPs detected within coding sequences i.e. how many of them are synonymous or non-synonymous substitutions.
Furthermore, as you performed the sliding window analysis you may present the plot of pi value (sequence divergence) obtained for studied Symplocos species.
Response: The information has now been included, in the manuscript text or in a supplementary data file (Data File S3). As we made a comprehensive comparison of the seven species, we could not compare the number of synonyms or nonsynonymous substitutions for each species.
L.147
Provide the explanation what p-distance stands for, whether it is pi (π) value which express the sequence divergence (estimated in DnaSP) or something else.
Response: This has now been revised as suggested.
- 180
not clear, what structures?, LSC, SSC and both IRs? Please be more specific.
Response: This has now been revised as suggested.
L.191
Quite ambiguous. Please explain that. Do you think that the cited literature is the correct reference here (Parks et al. 2009)?
Response: This has now been revised as suggested.
L.195-196
This sentence like the others in this paragraph are very general statements with no details. Finally, your discussion on structure of junctions between LSC, SSC and IR region in was squeezed in 6 lines.
Response: This has now been revised as suggested.
- 198-202
It is hard to follow whether you are writing about Symplocos or generally about cp genomes. Only analysis of references allowed me (more or less) to follow your concept. However check, whether all cited literature is correct - reference nr 38 refers to the DOGMA which is a tool for used for genome annotation.
Response: This has now been revised as suggested.
- 202-203
Can you give any examples here on both markers and species?
Response: →In this study, seven Symplocos species are described and 17 genes were identical.
- 203
"...all 17 genes...", why all? What group it is? Why did you distinguish that group of genes?
Response: This has now been revised as suggested.
- 204
"well-preserved" - not clear expression
Response: This has now been revised as suggested.
L.205
wrong reference: reference 14 is not Wang et al. (additionally no year was given here).
I guess your intention here was Wang et al. 2004 (reference 37).
Response: This has now been revised as suggested.
L.205-207
It is a true but you ignored here a fact that Wang et al. 2004 analyzed only 4 loci: nuclear ITS and 3 chloroplast loci. As a consequence the discriminatory power of such data appeared as not sufficient - BUT they analyzed 111 species whereas you include only 11, therefore it is very hard to make any comparison between these two studies.
Response: This has now been revised as suggested.
L.240
preliminary sequence?
Response: This has now been revised as suggested.
L.248-249
Please provide the accession numbers for all cp genomes used in phylogenetic analyses.
Response: This has now been revised as suggested.
L.251
Please provide the information how did you choose the substitution model for the analysis.
Response: This has now been revised as suggested.
- 258-259
replace "...could be obtained..." with "...were provided..."
Response: This has now been revised as suggested.
L.261
replace "transformation studies" with "genetic transformation"
Response: This has now been revised as suggested.
L.262
How genetic transformation fits/correspond to ongoing conservation of these plants?
Response: This has now been revised as suggested.
L.278
Please check carefully reference citation throughout the entire manuscript as some mistakes was found.
Response: This has now been revised as suggested.
Table 2
rps12 is a trans-spliced gene which consist of three exons, so I wonder why you marked it as a gene containing one intron.
Response: This has now been revised as suggested.
Figure 7
Provide the capital letter in the name of the genus for Pterostyrax hispidus
Response: This has now been revised as suggested.
Author Response File: 
Author Response.docx
Reviewer 2 Report
This manuscript describes the comparison of seven chloroplast genome for Symplocos. I found the manuscript to be interesting and well written. I just have a few comments for the authors.
1) I would like to see a little more information in the introduction/results indicating why Chloroplast genomes were analyzed and not the full genome.
2) What is the importance of Symplocos? I think 1-2 sentences indicating the importance of this species is needed in the introduction.
3) Line 61, s.coreana needs to be italicized.
4) In Table 4 and Figure 5, since no Complement Repeats were found I think this should be removed from the table and the legend in figure 5.
5) I am confused by supplemental table 1. Is site the position in the gene or in the chloroplast genome? Thats needs to be indicated. I also think it should be labeled as position and not site. I am also confused what Column "SNP" is. What does the number indicate?
6) The Raw SNP data should be provided as a supplemental file or available to download from a data repository.
Author Response
Response to reviewer 2
Reviewer 2 comments
This manuscript describes the comparison of seven chloroplast genome for Symplocos. I found the manuscript to be interesting and well written. I just have a few comments for the authors.
1) I would like to see a little more information in the introduction/results indicating why Chloroplast genomes were analyzed and not the full genome.
Response: This has now been revised as suggested.
.
2) What is the importance of Symplocos? I think 1-2 sentences indicating the importance of this species is needed in the introduction.
Response: This has now been revised as suggested.
3) Line 61, s.coreana needs to be italicized.
Response: This has now been revised as suggested.
4) In Table 4 and Figure 5, since no Complement Repeats were found I think this should be removed from the table and the legend in figure 5.
Response: This has now been revised as suggested.
5) I am confused by supplemental table 1. Is site the position in the gene or in the chloroplast genome? Thats needs to be indicated. I also think it should be labeled as position and not site. I am also confused what Column "SNP" is. What does the number indicate?
Response: Your concerns have been addressed. This table is now in Supplementary Data File S3.
6) The Raw SNP data should be provided as a supplemental file or available to download from a data repository.
Response: → Since the complete chloroplast genome sequences have been deposited in the NCBI (as explained in the manuscript file), the relevant data can be extracted from the database should anyone wish to do the analysis themselves. We therefore did not provide the raw SNP data with the manuscript.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Dear Editor and Authors,
below I included my comments to the new version of the manuscript.
L.47-48
"...only some fragmentary sequences and partial intergenic spacers were used in the analyses..."
It's not precise and a little bit ambiguous statement, isn't it. It is hard to understand your intention. Please be more specific. Please specify whether it is also protein-coding genes or only non-coding sequences.
L.83-84
replace "The tRNA sequences were conformed using tRNAscan-SE" with "The tRNA sequences were confirmed using tRNAscan-SE"
L.109
replace "ML analysis was performed using RAxML as a GTR+I+G model" with " ML analysis was performed using RAxML and a GTR+I+G model"
L.149 and 150
apply italic font for the ψycf1
L. 167-168
replace " We identified 44–59 repeats were across the genomes" with "We identified 44–59 repeats across the genomes"
L.170 and 171
replace "harbor" with "harbored" - as you use past tense for describing the results, heck throughout the entire test the consistency in the used form of narration
L.242
Please specify that this sentence concern four Symplocos cp genomes by adding appropriate information e.g. "The reported here CP genomes of Symplacos species are well conserved..."
L. 243
rephrase "including four CP molecular structures." with "including its traditional quadripartite molecular structures."
L.251-252
I suggest to replace " ...used as molecular markers for genetic diversity analysis and also as indicators to understand the evolution history" with " used as molecular markers for genetic diversity analysis and genetic evolution studies"
L.257-258
"However, some Ericales CP genome families differ significantly from others, confirming rearrangements and changes in the IR region length during evolution"
I think that after such introduction some details are needed here.
L.264-265
"However, the use of a few markers when classifying closely related species is inadequate because each marker shows little variability [60]."
What was your intention here? Are you willing to say that there are still some plant species, especially those which are closely related, for which there is no sufficient DNA barcodes? If yes please be more specific - express your thoughts in more clear way, and add in the sentence above that you are writing about chloroplast CDS and intergenic regions which could be used as a DNA barcodes.
L.267
instead??? I do not understand your intention here as it means "alternatively" or "rather". In previous sentence you were talking about genes which has no differences in their sequences among studied species, and in this sentence you are talking about " generally well-preserved CDS". What did you mean here?
L.271
there should be rather ITS not "nr-ITS"
L.272-273
"Furthermore, Fritsch et al. sampled 74 species and their results were consistent with Wang's findings [18]"
Ok, but what did Wang found and described in the cited paper?
L.273
You should add here that it was 30 Samplocos species.
L.297
replace "demonstrate" with "demonstrated"
Figure 5
The quality (resolution) of the figure should be improved (the species names are blur)
Figure 6
Not sufficient quality (resolution) of the figure.
Figure 8
Not sufficient quality (resolution) of the figure make the species names are hard to read.
Author Response
Please see the attachment
Author Response File: Author Response.docx
