Comparative Survey of Morphological Variations and Plastid Genome Sequencing Reveals Phylogenetic Divergence between Four Endemic Ilex Species

Round 1

Reviewer 1 Report

Ilex Plastid Manuscript Review

 

The manuscript by Su et al. entitled: “Comparative Survey of Morphological Variations and Plastid Genome Sequencing Revealed the Phylogenetic Divergence between Four Endemic Ilex

Species” contains novel information. No further analyses are needed by a major re-write, with focus on proper syntax, grammar and usage is required.

 

In addition to a major editing and rewrite there are some additional minor comments/concerns. Specifically,

 

Lines 46-49 – An example of poor and unclear sentence structure.

 

Lines 58-60 – An example of poor punctuation and grammar.

 

Line 106 – Are these samples per species or in total across all species?

 

Line 131 -- Why only three species, where is the fourth?

 

Line 150 – Are these numbers correct, i.e., 8, 5, 4, 3, 3 and 3? If so, I do not understand what they correspond to.

 

Line 161 – Why were these species selected? Not that they are wrong, just provide some justification.

 

Line 177 -- I would recommend limiting this analysis, and data presented in Table 1, to just the three species you use in the plastid analysis; it will be simpler and more consistent for the reader.

 

Line 178 -- This is not the right term; dominant should be changed to largest [maybe?].

 

Line 180 – Same concern as noted just above.

 

Lines 183-185 -- I would recommend the authors run correlations man the various leaf traits.

 

Line 203 -- The inner gray circle in this figure in not legible, please improve and add more description in the figure title.

 

Line 236 -- The “Numbers” column is unnecessary, delete.

 

Line 240 – “Using the program MISA, ”, This is a good method for reminding the reader/reviewer of how the data was generated. Use this style more often in the manuscript.

 

Line 244 – “looked identical” is not an expectable way of describing data; “looked” is subjective, improve this with a statistic.

Line 260 -- How did we get to 8 species? Where did this data come from? If it preexisted this study, it should be mentioned in the Intro and then in Methods regarding where it is located and how it will be used. If it did not preexist this study, then how and where this data came from needs to be described in the Methods section.

 

Line 302 -- Given the conservative sequence structure this figure could be moved to the supplemental materials section.

 

Line 307 – Define/justify this choice of accessions, why 15? Does this group/number include the 8 species? Or just the 3 or 4 main species? Clarify.

 

Line 321 – Show or list the citation at the end of this sentence.

 

Lines 356-357 – I did not see this data presented in the Results section.

 

Lines 390-393 -- Ok, but how can this data be used? Could aid in field identification of the various species?

 

Lines 435-439 -- True, good point.

Author Response

The manuscript by Su et al. entitled: “Comparative Survey of Morphological Variations and Plastid Genome Sequencing Revealed the Phylogenetic Divergence between Four Endemic Ilex Species” contains novel information. No further analyses are needed by a major re-write, with focus on proper syntax, grammar and usage is required. In addition to a major editing and rewrite there are some additional minor comments/concerns.

R1: Thanks. The texts of the manuscript have been substantially revised based on the experienced English language edit by MDPI (Certificate number: 21888)

Specifically, Lines 46-49 – An example of poor and unclear sentence structure.

R2: Thank you for the comments, and it has been revised! 

Lines 58-60 – An example of poor punctuation and grammar.

R3: This has been revised.

Line 106 – Are these samples per species or in total across all species?

R4: It is per species. This has been revised in line 313.

Line 131 -- Why only three species, where is the fourth?

R5: According to the description in materials and methods, the plastid genome of I. latifolia has been previously sequenced. The genomic sequencing data are available in NCBI database (see ref 32). However, the extensive analyses of the genomic sequences have not been conducted in I. latifolia.

Line 150 – Are these numbers correct, i.e., 8, 5, 4, 3, 3 and 3? If so, I do not understand what they correspond to.

R6: The detailed information has been updated in line 421-423.

Line 161 – Why were these species selected? Not that they are wrong, just provide some justification.

R7: Good comments. Except for the ornamental and pharmaceutical use (see discussion), these four species have a more abundant distribution in the plot, suggesting the sample (e.g., seed, fruit, and flower) harvest is easier to handle than other Ilex. However, the comparative analyses will be extended to additional Ilex until the samples and sequencing data are available soon.

Line 177 -- I would recommend limiting this analysis, and data presented in Table 1, to just the three species you use in the plastid analysis; it will be simpler and more consistent for the reader.

R8: This comment might be interesting! The comparative analyses in our paper were performed based on the patterns of the morphology, DNA content, and the plastid genomes between four Ilex species, not just three.

Line 178 -- This is not the right term; dominant should be changed to largest [maybe?].

R9: This typo has been revised. 

Line 180 – Same concern as noted just above.

R10: Revised. 

Lines 183-185 -- I would recommend the authors run correlations man the various leaf traits.

R11: This comment has been argued in the discussion (line 956-1041). Association analyses are not entirely convincing and redundant in four Ilex species, perhaps. Theoretically, the statistical analyses with the increased capacity of specimens (>100 species) would provide a plausible correlation between various morphological traits, which belong to other projects.

Line 203 -- The inner gray circle in this figure in not legible, please improve and add more description in the figure title.

R12:  Thanks, and this typo has been revised.

Line 236 -- The “Numbers” column is unnecessary, delete.

R13：This comment might be interesting. If the number column is not necessary, how is the entire table necessary? The annotated plastid genes are relatively conserved within the plant kingdom. Besides, the numbers have been used for the presentation in the results and discussion, suggesting that these numbers are not redundant.

Line 240 – “Using the program MISA, ”, This is a good method for reminding the reader/reviewer of how the data was generated. Use this style more often in the manuscript.

R14: Yes, this is an exquisite style that has been used in the revised manuscript.  

Line 244 – “looked identical” is not an expectable way of describing data; “looked” is subjective, improve this with a statistic.

R15: It has been revised.

Line 260 -- How did we get to 8 species? Where did this data come from? If it preexisted this study, it should be mentioned in the Intro and then in Methods regarding where it is located and how it will be used. If it did not preexist this study, then how and where this data came from needs to be described in the Methods section.

R16: This comment is quite impressive. The accession numbers of 15 Ilex species (including 8 species for plastome comparison) together with 4 species in outgroups have been described in the legend of Figure 6. Does that mean the suggestion of moving these accession numbers into materials and methods? Besides, all of the original sequencing data (5 Ilex) used have been processed and reprogrammed for the comparative analyses in the manuscript, indicating that our work is novel rather than verification of the previous data.    

Line 302 -- Given the conservative sequence structure this figure could be moved to the supplemental materials section.

R17: This comment might be interesting. Figure 5 was used to compare the junctions in IRs/SC regions, which is particularly important in the core structure.

Line 307 – Define/justify this choice of accessions, why 15? Does this group/number include the 8 species? Or just the 3 or 4 main species? Clarify.

R18: This comment might be interesting. So far, only 15 Ilex species plastid genome was sequenced and publically available in NCBI. Undoubtedly, the 8 Ilex species were included in the phylogenetic analyses (please check the Ilex name?). Also, the accession numbers were presented in the legend of Figure 6.

Line 321 – Show or list the citation at the end of this sentence.

R19: Thanks. The ref has been added.

Lines 356-357 – I did not see this data presented in the Results section.

R20: This is not the original data in the result, but the cited reference was used for the discussion.

Lines 390-393 -- Ok, but how can this data be used? Could aid in field identification of the various species?

R21: This is general knowledge, so the answer is positive.

Lines 435-439 -- True, good point.

R22: Many thanks!

Reviewer 2 Report

The authors analyzed morphological data of four Ilex species sampled on a forest plot located on Mount Huangshan in China. Additionally, they conducted plastid genome sequencing to identify genetic variation among these and additional Ilex species, which was used for phylogenetic analysis.

The analysis is sound, albeit some additional information should be added. Further, the English language needs to be revised. Please find more specific suggestions in the following:

Line 33: Please delete “For the first time”.

Lines 40-41: The meaning of this sentence is not clear. The only living woody dioecious genus in the angiosperms (which is not true)? The only genus with 700 species?

Lines 89-90: Was there a reason to specifically select these four out of 12 available Ilex species?

Line 118: Please add the post-hoc test, which was used to detect differences among morphological traits.

Line 137 ff.: More information about the assembly and annotation should be added. Please add, which reference was used for the reference-based assembly. Further, it should be added, which data base (e.g., nr.) was used for the BLAST searches is NCBI.

Table1: “Species” should be replaced by “trait” (or the like) in the table.

Line 182: In my opinion “drastically” should be deleted, since the differences in STD were only drastically different between I. latifolia and the other species (differences among the other Ilex species were also significant but not “drastically” different).

Line 184: The correlation between SLA and STD could be tested by a correlation analysis.

Line 186: Regarding Table1 there are statistically significant differences for these traits among species. Maybe, you meant no distinct differences?

Lines 321-322: Please add a reference.

Figure 6: Please add to the figure caption that bootstrapping values are displayed (which numbers belong to which test?).

Author Response

The authors analyzed morphological data of four Ilex species sampled on a forest plot located on Mount Huangshan in China. Additionally, they conducted plastid genome sequencing to identify genetic variation among these and additional Ilex species, which was used for phylogenetic analysis. The analysis is sound, albeit some additional information should be added. Further, the English language needs to be revised.

R1: Thanks. The texts of the manuscript have been substantially revised based on the experienced English language edit by MDPI (Certificate number: 21888)

Please find more specific suggestions in the following:

Line 33: Please delete “For the first time”.

R2: Thanks, it has been deleted.

Lines 40-41: The meaning of this sentence is not clear. The only living woody dioecious genus in the angiosperms (which is not true)? The only genus with 700 species?

R3: Thanks, and this sentence has been corrected in line 39-40.

Lines 89-90: Was there a reason to specifically select these four out of 12 available Ilex species?

R4: Good comments. Except for the ornamental and pharmaceutical use, these four species have a more abundant distribution in the plot, suggesting the sample (e.g., seed, fruit, and flower) collection is easier to do than other Ilex. However, the comparative analyses will be extended to additional Ilex until the samples and sequencing data are available shortly.

Line 118: Please add the post-hoc test, which was used to detect differences among morphological traits.

R5: Absolutely, we did. The superscripted letters indicated Duncan’s multiple range test and the different letters suggested the significant difference between four species. The detailed description has been added to the legend of Table 1 and materials and methods.

Line 137 ff.: More information about the assembly and annotation should be added. Please add, which reference was used for the reference-based assembly. Further, it should be added, which data base (e.g., nr.) was used for the BLAST searches is NCBI.

R6: Thanks, and it has been corrected.

Table1: “Species” should be replaced by “trait” (or the like) in the table.

R7: This typo has been corrected.

Line 182: In my opinion “drastically” should be deleted, since the differences in STD were only drastically different between I. latifolia and the other species (differences among the other Ilex species were also significant but not “drastically” different).

R8: This typo has been corrected.

Line 184: The correlation between SLA and STD could be tested by a correlation analysis.

R9: It appears to be not convincing for running a correlation analysis within limited species. This comment has been argued in the discussion (line 956-1041). Theoretically, the statistical analyses with the increased capacity of specimens (>100 species) would provide a plausible correlation between various morphological traits, which belong to other projects.

Line 186: Regarding Table1 there are statistically significant differences for these traits among species. Maybe, you meant no distinct differences?

R10: This comment is quite confusing. We never mean this! If no distinct differences were deduced from the statistical analyses, the significant differences would be useless in any comparative study. How would it be possible or logical to recommend authors to run the post-hoc test (multiple range test)?

Lines 321-322: Please add a reference.

R11: Thanks, and the reference has been added.

Figure 6: Please add to the figure caption that bootstrapping values are displayed (which numbers belong to which test?).

R12: These descriptions have been squeezed in materials and methods in line 436-439 in order to reduce the space of the descriptions in the legend

Round 2

Reviewer 1 Report

All recommended edits and changes have been addressed.