Complete Chloroplast Genome of Clethra fargesii Franch., an Original Sympetalous Plant from Central China: Comparative Analysis, Adaptive Evolution, and Phylogenetic Relationships

Round 1

Reviewer 1 Report

The publication "Complete Chloroplast Genome of Clethra fargesii, an Original Sympetalous Plant from Central China: Comparative analysis, Adaptive Evolution, and Phylogenetic Relationship" was written in good language, in accordance with the accepted rules. Work methodology described correctly, techniques adequate to the work performed. I only noticed some minor inaccuracies, which I present below:

- line 119 - should be rather 34 than 55,

- line 385 - should be comma rather than dash,

- line 427 - "1990; Volume 56, pp. 120–156" - should be corrected,

- line 440 - " 2005; Volume 14, pp. 238–241" - should be corrected,

 - line 537 - the year should be bold,

- line 539 - Solanum tuberosum must be Italic.

Author Response

Note: the following numbers of pages and lines are under using the "Track Changes" function in Microsoft Word.

 

- line 119 - should be rather 34 than 55,

The reference number has been changed and “55” has been deleted.

Line 135

 

- line 385 - should be comma rather than dash,

Revised.

Line 390

 

- line 427 - "1990; Volume 56, pp. 120–156" - should be corrected,

The reference format of book section may be no problem.

Line 435

 

- line 440 - " 2005; Volume 14, pp. 238–241" - should be corrected,

The reference format of book section may be no problem.

Line 448

 

 - line 537 - the year should be bold,

This reference has been replaced.

Line 554-547

 

- line 539 - Solanum tuberosum must be Italic.

Revised.

Line 549

 

Reviewer 2 Report

Following are my comments on the manuscript (1166046) titled “Complete chloroplast genome of Clethra fargesii, an original sympetalous plant from central China: Comparative analysis, adaptive evolution, and phylogenetic relationship” by Ding and coworkers.

Note that special formatting (e.g., italics) may not be preserved in this online review.

 

In the title and a few other places in the manuscript the authors use the phrase “chloroplast genome,” which is a common practice in the literature. However, the isolation of chloroplasts from other types of plastids prior to DNA extraction was not reported and would be unusual. Since the genomes of chloroplasts, leucoplasts, amyloplasts, and other types of plastids were apparently extracted as a pool from which the sequence was assembled, the phrase: “chloroplast genome” is technically more accurate as either “plastid genome” or “plastome” here and throughout the manuscript.

 

Title     Here and elsewhere (pp. 2, 14, and 15) in the manuscript the authors repeatedly use the confusing phrase “original sympetalous plants.” If the intended meaning is that Clethra represents an early diverging lineage with sympetalous corollas, then this phrase should be reworded, and an appropriate citation is also needed.

 

Title     The phrase “...phylogenetic relationship...” should be “...phylogenetic relationships...”

 

Line 18             The word “features” should be plural in the phrase “...analyzed the chloroplast genome features and compared them to...” (and change “it” to “them”).

 

Lines 26 and 29 “...Clethra...” is misspelled in two places in the abstract.

 

Line 30             The phrase “...reflecting from few positive selection genes...” should be “...reflected by few positively selected genes...” (four changes needed).

 

Lines 42-43      The phrase “...ranging from subtropics to tropics areas...” should be “...ranging from subtropical to tropical areas...”

 

Lines 53-54      The phrase “...based on the revise of morphological taxonomy...” should be “...based on the revision of morphological taxonomy...”

 

Lines 69-70      The phrase “...as DNA barcoding to identify species...In recent several years, there has been...” should be “...as DNA barcodes to identify species...In several recent years, there have been...” (three changes).

 

Line 74             The GenBank accession number of Clethra delavayi is given here, but GenBank numbers for other Ericales used in the manuscript are not specified. Perhaps the accession numbers for the 40 species of Ericales could be added as a supplemental table.

 

Page 2, Methods           According to standard practice an herbarium specimen voucher of the plant, from which DNA was extracted for research purposes, is usually collected and deposited in an herbarium.

 

Line 130           The phrase “...and then concatenated them using...” should be “...and then concatenated using...” (remove “them”).

 

Lines 150-151   The authors state that the “...ML tree and Bayesian tree were recombined based on the consistent topological structures...” This is an interesting approach but needs further explanation.

 

Line 171, Fig. 1 legend. The phrase “...Drawn genes inside the circle...” should probably be “...Genes drawn inside the circle...”

 

Line 175 Table 1           Here the authors compare plastome structure of Clethra fargesii with “seven related species.” The same species are selected elsewhere (Figs. 2, 3). In Figs. 4 and 6 a subset of these species is used. However, I am unable to find the selection criteria for choosing particular species for these comparisons. All of the 40 species in their study are “...related species in the Ericales order...” and could be used in all these comparisons. The authors need to clearly explain why different subsets of the Ericales were selected for various comparisons.

 

Line 175 Table Heading             The specific epithet “fargesii” is misspelled.

 

Lines 183-185   The phrase “...one tRNA genes...18 genes has introns...” should be “...one tRNA gene...18 genes have introns...”

 

Lines 189-190   Here the authors use the word “...lain...” twice. The same word is found in line 339 (“...value is lain in the LSC region...”). A clearer word choice in all cases would be “found.”

 

Line 194           The phrase “...chloroplast genome of C. fargesii is almost similar to C. delavayi...” should be “...chloroplast genome of C. fargesii is most similar to C. delavayi...”

 

Lines 197-198   The authors state that “...two Ericaceae species and two Actinidiaceae species (Saurauia tristyla and Actinidia eriantha) are closer to two Clethra species...than the other four species...” This is unclear. Relationships in phylogenetic trees are ambiguously defined when using terms like “closer” or “more closely related” here and elsewhere in the manuscript (e.g., see lines 206-207; 223-224; 263-264). Moreover, the reasons for detailed comparisons of these few species are not stated (see comment above). These issues need to be corrected.

 

Lines 202-203   The phrase “...expansion of IR regions leads to only thousands of base pairs remained in the SSC regions...” would be clearer as “...expansion of IR regions reduces the length of the SSC regions to about 2,000 – 3,000 bp...”

 

Line 204           The phrase “...Besides, under the long selective evolutionary, the vast rearrangement...” is not grammatically correct, likely because something is missing. A correction is needed.

 

Lines 213 – 230            The mVISTA analysis fails to demonstrate any novel plastome features and should either be deleted or moved to the supplemental material.

 

Lines 236 – 243             The authors oversimplify the interesting and complex structural differences among plastomes of Ericales stating only that “...Some inversion and rearrangement regions were observed...” (line 239). The results of the MAUVE analysis should be more thoroughly discussed.

 

Page 8, Fig. 4    The specific epithet of Actinidia eriantha (fourth row in figure) is misspelled.

 

Line 315           The phrase “...the other complex SSRs are lower frequencies...” should be “...the other complex SSRs are at lower frequencies...”

 

Lines 317-318   The phrase “...the number of dinucleotide SSRs is significant more than mononucleotide and A/T  (no C/G) is the only mononucleotide SSRs type in two species...” should be

“...the number of dinucleotide SSRs is significantly greater than mononucleotide SSRs and A/T  (not C/G) is the only mononucleotide SSR type in the two species...”

 

Line 325           The phrase “...The phenomenon reflected that the SSRs preferred to distribute in the...” would be less anthropomorphic as “...The SSRs were disproportionately found in the...”

 

Pages 12-13, Figs. 8 and 9         The quantities/units are not labelled on the vertical axes of these graphs and should be added.

 

Line 359         The phrase “...The ratio of non-synonymous (dN) and synonymous (dS), dN/dS, has been...” should be “...The ratio of non-synonymous (dN) to synonymous (dS) substitutions, dN/dS, has been...” (add “to” and “substitutions”).

 

Lines 366-367   The phrase “...positive selection sites were detected...which distributes in...” should be “...positively selected sites were detected...which were distributed in...” (four changes needed).

 

Line 379           The phrase “...topological structure is highly conforms to the phylogenetic relationship...” should be “...topological structure that is highly consistent with the phylogenetic relationships...”

 

Lines 403-404   The phrase “...Clethra species have a high conserved chloroplast genome...” should be “...Clethra species have a highly conserved chloroplast genome...”

----- ----- ----- ----- ----- ----- ----- ----- ----- -----

The manuscript by Ding and coworkers is a well-organized and well researched manuscript that reports a detailed characterization of the newly sequenced plastome of Clethra fargesii in the context of other complete plastomes from Ericales. This is a generally valuable study, like many other complete plastome studies that are currently published.

 

However, there are a number of minor weaknesses of this manuscript in its present form.

• The authors should collect and deposit an herbarium voucher specimen of the plant that was the source of the DNA for the newly sequenced plastome.
• For their phylogenetic analysis the authors need to explain how they combined ML and Bayesian inference trees.
• The selection criteria for choosing different subsets of species for various comparisons between the newly sequenced plastomes and existing plastomes need to be given.
• When the authors describe relationships in phylogenetic trees, they use ambiguous language throughout the manuscript. Rather than stating relationships as being “closer” they should indicate which specific sister clades in their phylogenetic tree are being discussed.
• The mVISTA analysis can be deleted from this paper since it fails to inform the reader of new information in the newly sequenced plastome.
• Structural rearrangements shown in MAUVE analysis are inadequately discussed. There are several potentially interesting features that are indicative of molecular evolutionary events in the plastomes of Ericales that should be described.
• Finally, there are problems with the English language/grammar and choice of certain inappropriate words. I have highlighted some, but not all of these in my review (please see above). The language in the paper needs to be improved.

Author Response

Note: the following numbers of pages and lines are under using the "Track Changes" function in Microsoft Word.

 

In the title and a few other places in the manuscript the authors use the phrase “chloroplast genome,” which is a common practice in the literature. However, the isolation of chloroplasts from other types of plastids prior to DNA extraction was not reported and would be unusual. Since the genomes of chloroplasts, leucoplasts, amyloplasts, and other types of plastids were apparently extracted as a pool from which the sequence was assembled, the phrase: “chloroplast genome” is technically more accurate as either “plastid genome” or “plastome” here and throughout the manuscript.

The words of “plastid genome” or “plastome” have been replaced with chloroplast genome.

Line 57

Line 81

Line 103

Line 390

 

Title     Here and elsewhere (pp. 2, 14, and 15) in the manuscript the authors repeatedly use the confusing phrase “original sympetalous plants.” If the intended meaning is that Clethra represents an early diverging lineage with sympetalous corollas, then this phrase should be reworded, and an appropriate citation is also needed.

On second thought, the phrase of “original sympetalous plants” should not be emphasized, I have deleted them and it is only existed in pp.2 and title as a introduction of Clethra.

Line 33

Line 392

Line 410

 

Title     The phrase “...phylogenetic relationship...” should be “...phylogenetic relationships...”

Changed.

Line 4

 

Line 18             The word “features” should be plural in the phrase “...analyzed the chloroplast genome features and compared them to...” (and change “it” to “them”).

Changed.

Line 17

 

Lines 26 and 29 “...Clethra...” is misspelled in two places in the abstract.

Changed.

Line 25

Line 28-29

 

Line 30             The phrase “...reflecting from few positive selection genes...” should be “...reflected by few positively selected genes...” (four changes needed).

Changed.

Line 29-30

 

Lines 42-43      The phrase “...ranging from subtropics to tropics areas...” should be “...ranging from subtropical to tropical areas...”

Changed.

Line 43

 

 

Lines 53-54      The phrase “...based on the revise of morphological taxonomy...” should be “...based on the revision of morphological taxonomy...”

Changed.

Line 53

 

Lines 69-70      The phrase “...as DNA barcoding to identify species...In recent several years, there has been...” should be “...as DNA barcodes to identify species...In several recent years, there have been...” (three changes).

Changed.

Line 69-70

 

 

Line 74             The GenBank accession number of Clethra delavayi is given here, but GenBank numbers for other Ericales used in the manuscript are not specified. Perhaps the accession numbers for the 40 species of Ericales could be added as a supplemental table.

I have added a supplemental table. See Supplementary Materials: Table S4: NCBI accession number of 40 chloroplast genomes.

Line 418

 

Page 2, Methods           According to standard practice an herbarium specimen voucher of the plant, from which DNA was extracted for research purposes, is usually collected and deposited in an herbarium.

Changed sentences “The materials of C. fargesii were collected from Hubei Nanhe National Nature Reserve, China (31°53′45″N, 111°30′30″E, Alt. 1130 m ). The fresh leaves were kept in silica gels for preservation [22] and the voucher specimens (Voucher Number: HGW-2041) were later deposited at the Herbarium of Wuhan Botanical Garden, CAS (HIB) (China).”

Line 88-96

 

Line 130           The phrase “...and then concatenated them using...” should be “...and then concatenated using...” (remove “them”).

Changed.

Line 146

 

 

Lines 150-151   The authors state that the “...ML tree and Bayesian tree were recombined based on the consistent topological structures...” This is an interesting approach but needs further explanation.

Changed sentences “The ML tree and Bayesian tree were manually recombined using AI software based on the consistent topological structures.”

Line 170

 

Line 171, Fig. 1 legend. The phrase “...Drawn genes inside the circle...” should probably be “...Genes drawn inside the circle...”

Changed.

Line 209

 

Line 175 Table 1           Here the authors compare plastome structure of Clethra fargesii with “seven related species.” The same species are selected elsewhere (Figs. 2, 3). In Figs. 4 and 6 a subset of these species is used. However, I am unable to find the selection criteria for choosing particular species for these comparisons. All of the 40 species in their study are “...related species in the Ericales order...” and could be used in all these comparisons. The authors need to clearly explain why different subsets of the Ericales were selected for various comparisons.

The seven Ericale species were selected to verify the conservation of C. fargesii chloroplast genome based on the related gradient close to C. fargesii in phylogeny. Further, multiple genome alignment analysis was done using the Mauve program [31]. Excluding two Ericaceae species, six Ericales species chloroplast genomes were used to analysis junction characteristics and codon usage bias considering the cases of great gene losses and extreme expansion or contraction of IR region in the chloroplast genomes of Ericales species.

Line 117-126

 

Line 175 Table Heading             The specific epithet “fargesii” is misspelled.

Changed.

Line 213

 

 

Lines 183-185   The phrase “...one tRNA genes...18 genes has introns...” should be “...one tRNA gene...18 genes have introns...”

Changed.

Line 199

 

Lines 189-190   Here the authors use the word “...lain...” twice. The same word is found in line 339 (“...value is lain in the LSC region...”). A clearer word choice in all cases would be “found.”

Changed.

Line 204

Line 343

 

Line 194           The phrase “...chloroplast genome of C. fargesii is almost similar to C. delavayi...” should be “...chloroplast genome of C. fargesii is most similar to C. delavayi...”

Changed.

Line 216

 

Lines 197-198   The authors state that “...two Ericaceae species and two Actinidiaceae species (Saurauia tristyla and Actinidia eriantha) are closer to two Clethra species...than the other four species...” This is unclear. Relationships in phylogenetic trees are ambiguously defined when using terms like “closer” or “more closely related” here and elsewhere in the manuscript (e.g., see lines 206-207; 223-224; 263-264). Moreover, the reasons for detailed comparisons of these few species are not stated (see comment above). These issues need to be corrected.

Corrected.

Line 219-222

Line 228-229

Line 231-248

Line266-268

 

Lines 202-203   The phrase “...expansion of IR regions leads to only thousands of base pairs remained in the SSC regions...” would be clearer as “...expansion of IR regions reduces the length of the SSC regions to about 2,000 – 3,000 bp...”

Changed.

Line 226

 

Line 204           The phrase “...Besides, under the long selective evolutionary, the vast rearrangement...” is not grammatically correct, likely because something is missing. A correction is needed.

Deleted.

Line 226

 

Lines 213 – 230            The mVISTA analysis fails to demonstrate any novel plastome features and should either be deleted or moved to the supplemental material.

Deleted.

Page 7

 

Lines 236 – 243             The authors oversimplify the interesting and complex structural differences among plastomes of Ericales stating only that “...Some inversion and rearrangement regions were observed...” (line 239). The results of the MAUVE analysis should be more thoroughly discussed.

Added “The two Ericaceae species may be prone to the structural mutations in DNA strands and accumulate constantly due to the low GC content in their chloroplast genomes under the long evolutionary history. Whereas the two Clethra species and other four Ericales species present more conserved, which leads to a growing divergence in the genome structure of them from that of the two Ericaceae species.”

Line 236-241

 

Page 8, Fig. 4    The specific epithet of Actinidia eriantha (fourth row in figure) is misspelled.

Changed.

Page 8

 

Line 315           The phrase “...the other complex SSRs are lower frequencies...” should be “...the other complex SSRs are at lower frequencies...”

Changed.

Line 319

 

Lines 317-318   The phrase “...the number of dinucleotide SSRs is significant more than mononucleotide and A/T  (no C/G) is the only mononucleotide SSRs type in two species...” should be      “...the number of dinucleotide SSRs is significantly greater than mononucleotide SSRs and A/T  (not C/G) is the only mononucleotide SSR type in the two species...”

Changed.

Line 321-322

 

Line 325           The phrase “...The phenomenon reflected that the SSRs preferred to distribute in the...” would be less anthropomorphic as “...The SSRs were disproportionately found in the...”

Changed.

Line 329-330

 

Pages 12-13, Figs. 8 and 9         The quantities/units are not labelled on the vertical axes of these graphs and should be added.

Changed.

Page 11

 

Line 359         The phrase “...The ratio of non-synonymous (dN) and synonymous (dS), dN/dS, has been...” should be “...The ratio of non-synonymous (dN) to synonymous (dS) substitutions, dN/dS, has been...” (add “to” and “substitutions”).

Changed.

Line 363

 

Lines 366-367   The phrase “...positive selection sites were detected...which distributes in...” should be “...positively selected sites were detected...which were distributed in...” (four changes needed).

Changed.

Line 368-373

 

 

Line 379           The phrase “...topological structure is highly conforms to the phylogenetic relationship...” should be “...topological structure that is highly consistent with the phylogenetic relationships...”

Changed.

Line 384-385

 

 

Lines 403-404   The phrase “...Clethra species have a high conserved chloroplast genome...” should be “...Clethra species have a highly conserved chloroplast genome...”

Changed.

Line 409-410