Comparative Analysis of Chloroplast Genomes Reveals Phylogenetic Relationships and Variation in Chlorophyll Fluorescence In Vitis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Editor,

Here is a review with comments for the manuscript entitled "Comparative Analysis of Chloroplast Genomes Reveals Phylogenetic Relationships and Contributions to Photosynthesis in Vitis" by Teng et al.

General comments

The manuscript presented by Teng et al. deals with a comparative and phylogenetic analysis of nine chloroplast genomes of Vitis species. The results reveal that these complete genomes present conservation in overall structure and gene content, punctuated by seven high-variation regions (including rbcL and ndhF). Photosynthetic performance and related traits measured for four cultivars demonstrate significant differences attributable to genotype. In addition, maximum likelihood phylogenetic analysis, including 32 complete cpDNAs, resolved four main clades matching historical breeding and taxonomic divisions: Muscadinia, American grape species, Eurasian cultivated varieties, and the wild V. amurensis.

Although the exhaustive integration of assembly, annotation, comparative genomics, phenotypic analysis, and phylogenetic reconstruction provides robust frameworks for grape molecular systematics and breeding studies, the manuscript contains noticeable grammatical issues throughout several sections, as well as lapses in formal structure and clarity. For example, frequent sentences reduce clarity, especially where complex ideas or multiple results are mentioned without appropriate punctuation or conjunctions.  Ambiguous constructions are common, such as “At the same, seven natural variation sites were identified...” (missing “time” and clarity of context). Also, unnecessary repetition of terms (e.g., listing cultivar names repeatedly) and missing articles, for example “The grape belongs to Vitaceae family” (should be: “The grape belongs to the Vitaceae family”), are frequent in the introduction and discussion sections. The use of unclear phrases, e.g., “the classification of different populations provides a theoretical basis…” would benefit from rewording and clearer linking between ideas. The manuscript also lapses into informal language or redundancy (such as listing method steps as complete sentences with incomplete subjects or verbs). In some analytic sections, inconsistent tense usage occurs, switching between present and past when describing results, for example “The CI- RAS-3 infrared gas analyzer ... is used to perform the measurements” vs. “Parameters recorded included...”, which should be standardized for scientific reporting. The attribution of findings to previous literature is sometimes unclear; citation integration could be improved for fluency, for example “This evidence aligns with previous studies that have identified distinct clades within the genus…” instead of “As shown in Figure 10, the constructed…”. Transitions between paragraphs and sections are often abrupt, lacking connective or overview sentences to guide the reader from context to results and implications. Reporting of results uses unexplained abbreviations and excessive numerical data without proper introduction or summary, making interpretation difficult for broader audiences.

 

Other examples to correct are:

1) Line 27-29 - Original  “Nevertheless, further sequence comparisons revealed seven regions of high variation, including rbcL, ndhF, rps16-trnQ, ndhC- trnV, accD-psaI, ndhF-rpl32, and trnL-ccsA.”

Improve suggested: “However, additional sequence comparisons revealed seven regions with high variation, including the genes rbcL and ndhF, and the intergenic regions rps16-trnQ, ndhC-trnV, accD-psaI, ndhF-rpl32, and trnL-ccsA.”

2) Line 31-34 - Original: “Besides, maximum likelihood ML phylogenetic trees and photosynthetic index measurements, it was revealed that the developmental characteristics of grape photosynthesis, might be related to the evolutionary origins of different populations.”

Improve suggested: “Additionally, the study’s maximum likelihood phylogenetic trees and photosynthetic index measurements suggest that developmental characteristics of grape photosynthesis may be related to the evolutionary origins of different populations.”

3) line 50-54. The taxonomic level of genus must be italicized, but the word genus should not be italicized.

4) line 92. There is a “m” at the end of the line. What does it mean?

5) Every time "9 species" is mentioned, it should be "nine species." In scientific numeration, numbers from one to nine are written in words, and then you can use the numbers 10, 11, and so on.

6) Line 140-147. Very difficult to understand this paragraph. Please rewrite.

7) line 153-154. I think that "Experimental design" is unnecessary.

8) line 166-177. As in other parts of the document, several references to programs and methods are missing.

9) Line 202-214. Do you use the complete genomes for these analyses?. In Figure 6, it is indicated that "The relevant colored boxes reveal locally collinear blocks, which present homologous gene clusters." However, it is not clear which regions are colored and with what color. If there are non-homologous or transposed regions, it is suggested to use only the PCGs. MAGA11 is MEGA11? (line 212). Again, references for MAFFT and MEGA 11 are missing.

In short, while the article is scientifically interesting and could certainly be published, it would benefit from careful and much more rigorous editing to correct fragmented sentences, missing articles, awkward phrasing, inconsistencies in verb tenses, and improve overall cohesion, making its arguments more transparent and more accessible to international scientific readers.

Comments on the Quality of English Language

Although English is fine, you should thoroughly review your grammar.

Author Response

Comments 1

General comments

The manuscript presented by Teng et al. deals with a comparative and phylogenetic analysis of nine chloroplast genomes of Vitis species. The results reveal that these complete genomes present conservation in overall structure and gene content, punctuated by seven high-variation regions (including rbcL and ndhF). Photosynthetic performance and related traits measured for four cultivars demonstrate significant differences attributable to genotype. In addition, maximum likelihood phylogenetic analysis, including 32 complete cpDNAs, resolved four main clades matching historical breeding and taxonomic divisions: Muscadinia, American grape species, Eurasian cultivated varieties, and the wild V. amurensis.

Although the exhaustive integration of assembly, annotation, comparative genomics, phenotypic analysis, and phylogenetic reconstruction provides robust frameworks for grape molecular systematics and breeding studies, the manuscript contains noticeable grammatical issues throughout several sections, as well as lapses in formal structure and clarity. For example, frequent sentences reduce clarity, especially where complex ideas or multiple results are mentioned without appropriate punctuation or conjunctions.  Ambiguous constructions are common, such as “At the same, seven natural variation sites were identified...” (missing “time” and clarity of context). Also, unnecessary repetition of terms (e.g., listing cultivar names repeatedly) and missing articles, for example “The grape belongs to Vitaceae family” (should be: “The grape belongs to the Vitaceae family”), are frequent in the introduction and discussion sections. The use of unclear phrases, e.g., “the classification of different populations provides a theoretical basis…” would benefit from rewording and clearer linking between ideas.

Response

Thank you for your reviewing. Your affirmation and recognition of “Comparative Analysis of Chloroplast Genomes Reveals Phylogenetic Relationships and Variation in Chlorophyll Fluorescence in Vitis” research paper will be of great help to our follow-up scientific research. We sincerely appreciate your positive assessment of our work and your valuable suggestions. We have carefully proofread the entire manuscript to address the grammatical issues and improve clarity. Specifically, we have corrected missing articles, rephrased ambiguous constructions, and strengthened logical connections between ideas. Unnecessary repetitions have been removed, and sentence structures have been optimized throughout the text. As follows,

At the same time, seven natural variation sites were identified in the amino acid sequences of rbcL and ndhF.

The results showed that '5BB' exhibited higher values in the parameters φE₀, ψE₀, Eto/CSm, and the Performance Index (PIabs), indicating that it possesses the strongest photosynthetic capacity.

Specifically, the rootstock varieties clustered within the American grape clade. Cultivars including wine-making or table grape varieties were classified within the V. vinifera clade, while 'Shuanghong' was grouped within the V. amurensis clade.

Taxonomically, the grape belongs to the Vitaceae family and the Vitis genus.

 

Comments 2

The manuscript also lapses into informal language or redundancy (such as listing method steps as complete sentences with incomplete subjects or verbs). In some analytic sections, inconsistent tense usage occurs, switching between present and past when describing results, for example “The CI- RAS-3 infrared gas analyzer ... is used to perform the measurements” vs. “Parameters recorded included...”, which should be standardized for scientific reporting.

Response

Thanks for your comment. We have thoroughly revised the manuscript to eliminate informal language and redundancy. Method steps have been rewritten with complete sentence structures, and tense usage has been standardized to the past tense throughout the analytical sections for consistent scientific reporting. we have made the corresponding modification (page 3 and 4, line 141 to 154). The full is as follows,

In May 2025, key physiological traits were measured on functional leaves. The net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs), and intercellular CO₂ concentration (Ci) were recorded using a CI-RAS-3 infrared gas analyzer (PP Systems, MA, USA) [24] . Chlorophyll a fluorescence was assessed with a Handy Plant Efficiency Analyzer (PEA, Hansatech Instruments Ltd., UK), which captured OJIP transient curves. The derived parameters are listed in Table S2 [25]. Total chlorophyll was extracted and quantified following the method of Lichtenthaler [26]. Briefly, functional leaves were homogenized in 95% (v/v) ethanol, and chlorophyll content was determined spectrophotometrically. All measurements were conducted with five biological replicates per cultivar.

 

Comments 3

The attribution of findings to previous literature is sometimes unclear; citation integration could be improved for fluency, for example “This evidence aligns with previous studies that have identified distinct clades within the genus…” instead of “As shown in Figure 10, the constructed…”. Transitions between paragraphs and sections are often abrupt, lacking connective or overview sentences to guide the reader from context to results and implications. Reporting of results uses unexplained abbreviations and excessive numerical data without proper introduction or summary, making interpretation difficult for broader audiences.

Response

Thank you for these insightful suggestions. We have thoroughly revised the manuscript to improve academic fluency. Citations are now integrated more naturally into the narrative, transitions between paragraphs and sections have been strengthened with connective phrases, and all abbreviations are defined upon first use. Numerical data are now preceded by explanatory summaries to enhance readability for a broad audience (page 14, line 390 to 393). As follows,

This evidence aligns with previous studies that have identified distinct clades within the genus: our phylogenetic analysis divided the tree into four clades, consisting of Vitis rotundifolia, American grape species, Vitis amurensis, and Vitis vinifera (Eurasian grape) varieties.

 

Comments 4

Other examples to correct are:

1) Line 27-29 - Original  “Nevertheless, further sequence comparisons revealed seven regions of high variation, including rbcL, ndhF, rps16-trnQ, ndhC- trnV, accD-psaI, ndhF-rpl32, and trnL-ccsA.”

Improve suggested: “However, additional sequence comparisons revealed seven regions with high variation, including the genes rbcL and ndhF, and the intergenic regions rps16-trnQ, ndhC-trnV, accD-psaI, ndhF-rpl32, and trnL-ccsA.”

Response

According to your suggestion, we have made the corresponding modification (page 1, line 27-30). The full is as follows,

However, additional sequence comparisons revealed seven regions with high variation, including the genes rbcL and ndhF, and the intergenic regions rps16-trnQ, ndhC-trnV, accD-psaI, ndhF-rpl32, and trnL-ccsA.

 

Comments 5

2) Line 31-34 - Original: “Besides, maximum likelihood ML phylogenetic trees and photosynthetic index measurements, it was revealed that the developmental characteristics of grape photosynthesis, might be related to the evolutionary origins of different populations.”

Improve suggested: “Additionally, the study’s maximum likelihood phylogenetic trees and photosynthetic index measurements suggest that developmental characteristics of grape photosynthesis may be related to the evolutionary origins of different populations.”

According to your suggestion, we have made the corresponding modification (page 1, line 31-35).As follows,

Additionally, the study’s maximum likelihood (ML) phylogenetic trees and photosynthetic index measurements suggest that developmental characteristics of grape photosynthesis may be related to the evolutionary origins of different populations.

 

Comments 5

3) line 50-54. The taxonomic level of genus must be italicized, but the word genus should not be italicized.

Response

Thanks for your comment, we have made the corresponding modification.

 

Comments 6

4) line 92. There is a “m” at the end of the line. What does it mean?

Response

We thank the reviewer for spotting this error. The stray character "m" on line 92 has been removed from the manuscript.

 

Comments 7

5) Every time "9 species" is mentioned, it should be "nine species." In scientific numeration, numbers from one to nine are written in words, and then you can use the numbers 10, 11, and so on.

Response

Thanks for your comment, we have made the corresponding modification.

 

Comments 7

6) Line 140-147. Very difficult to understand this paragraph. Please rewrite.

Response

We thank the reviewer for highlighting the lack of clarity in this paragraph. We have completely rewritten the section (Lines 139-154) to improve logical flow and simplify sentence structures, making the scientific content more accessible to readers. The full is as follows,

They received standard pest control, disease control and horticultural practices. The fifth to seventh functional leaves, from bottom to top, were selected as the test material. In May 2025, key physiological traits were measured on functional leaves. The net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs), and intercellular CO₂ concentration (Ci) were recorded using a CI-RAS-3 infrared gas analyzer (PP Systems, MA, USA) [23] . Chlorophyll a fluorescence was assessed with a Handy Plant Efficiency Analyzer (PEA, Hansatech Instruments Ltd., UK), which captured OJIP transient curves. The derived parameters are listed in Table S2 [24]. Total chlorophyll was extracted and quantified following the method of Lichtenthaler [25]. Briefly, functional leaves were homogenized in 95% (v/v) ethanol, and chlorophyll content was determined spectrophotometrically. All measurements were conducted with five biological replicates per cultivar.

 

Comments 8

7) line 153-154. I think that "Experimental design" is unnecessary.

Response

According to your suggestion, we have made the corresponding modification. Delete “Experimental design”

 

Comments 9

8) line 166-177. As in other parts of the document, several references to programs and methods are missing.

Response

We thank the reviewer for this suggestion. We have carefully checked the Materials and Methods section, revised unclear descriptions, and added all missing references to software and methods. The added references are as follows,

Bolger, A.M.; Lohse, M.; Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 2014, 30, 2114-2120.

Jin, J.-J.; Yu, W.-B.; Yang, J.-B.; Song, Y.; DePamphilis, C.W.; Yi, T.-S.; Li, D.-Z. GetOrganelle: a fast and versatile toolkit for accurate de novo assembly of organelle genomes. Genome biology 2020, 21, 241.

Wick, R.R.; Schultz, M.B.; Zobel, J.; Holt, K.E. Bandage: interactive visualization of de novo genome assemblies. Bioinformatics 2015, 31, 3350-3352.

Zheng, S.; Poczai, P.; Hyvönen, J.; Tang, J.; Amiryousefi, A. Chloroplot: an online program for the versatile plotting of organelle genomes. Frontiers in Genetics 2020, 11, 576124.

 

Comments 10

9) Line 202-214. Do you use the complete genomes for these analyses?. In Figure 6, it is indicated that "The relevant colored boxes reveal locally collinear blocks, which present homologous gene clusters." However, it is not clear which regions are colored and with what color. If there are non-homologous or transposed regions, it is suggested to use only the PCGs. MAGA11 is MEGA11? (line 212). Again, references for MAFFT and MEGA 11 are missing.

Response

We thank the reviewer for their insightful comments. The analysis was performed using complete chloroplast genomes, and this has been clarified in the Materials and Methods section. The description of Figure 6 has been revised for improved clarity. The figure illustrates gene collinearity analysis, where colored blocks represent locally collinear blocks (LCBs) and connecting lines indicate homologous gene relationships. Each box corresponds to a gene, representing its approximate size and genomic position. The conserved arrangement of LCBs and genes across genomes indicates the absence of major inversions or rearrangements in the chloroplast genome structure. The typographical error "MAGA11" has been corrected to "MEGA11" throughout the manuscript, and appropriate citations for both MAFFT and MEGA11 have been added (page 5, line 219). The added references are as follows,

Katoh, K.; Standley, D.M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular biology and evolution 2013, 30, 772-780.

Tamura, K.; Stecher, G.; Kumar, S. MEGA11: molecular evolutionary genetics analysis version 11. Molecular biology and evolution 2021, 38, 3022-3027.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This paper describes the chloroplast genomes of nine representative grape cultivars and analyzed differences in photosynthetic capacity in four representative cultivars. Overall, the paper was clearly written, easy to follow, and the analyses were thorough. I only have a few minor comments:

Abstract: L29 (“At the same”) and the sentence in L31-32 in the abstract were awkward or incomplete

L51: the word “genus” should be in normal case.

L54: Italicize the subgenus name Vitis.

L85: “Sequence comparisons” instead of “sequence comparative”

L146: The sentence starting with “Assessed” is incomplete

L164: I don’t see the accession numbers?

L206: Please provide an appropriate reference for the “previous study”

L212: MEGA 11 instead of MAGA11?

L224: “Chloroplast genomes” is a more appropriate term to use here instead of “accessions”

Figure 1: Is this based on a representative genome or the pan-genome generated from the 9 chloroplast genomes? How were the gene positions labelled? 

 

Author Response

Comments 1

This paper describes the chloroplast genomes of nine representative grape cultivars and analyzed differences in photosynthetic capacity in four representative cultivars. Overall, the paper was clearly written, easy to follow, and the analyses were thorough. I only have a few minor comments:

Abstract: L29 (“At the same”) and the sentence in L31-32 in the abstract were awkward or incomplete

Response

Thank you for your reviewing. Your affirmation and recognition of “Comparative Analysis of Chloroplast Genomes Reveals Phylogenetic Relationships and Variation in Chlorophyll Fluorescence in Vitis” research paper will be of great help to our follow-up scientific research. We sincerely appreciate your positive assessment of our work and your valuable suggestions .The incomplete phrase "At the same" in line 29 has been corrected to "At the same time," and the awkward sentence in lines 31-32 has been rewritten for clarity and grammatical completeness. The full is as follows, 

Additionally, the study’s maximum likelihood (ML) phylogenetic trees and photosynthetic index measurements suggest that developmental characteristics of grape photosynthesis may be related to the evolutionary origins of different populations.

 

Comments 2

L51: the word “genus” should be in normal case.

Response

Thanks for your comment, we have made the corresponding modification.

 

Comments 3

L54: Italicize the subgenus name Vitis.

Response

Thanks for your comment, we have made the corresponding modification.

 

Comments 4 

L85: “Sequence comparisons” instead of “sequence comparative”

Response

Thanks for your comment, we have made the corresponding modification.

 

Comments 5

L146: The sentence starting with “Assessed” is incomplete

Response

Thanks for your comment, we have made the corresponding modification (page 3 ,line 146 to 149 ). The full is as follows,

Chlorophyll a fluorescence was assessed with a Handy Plant Effi-ciency Analyzer (PEA, Hansatech Instruments Ltd., UK), which captured OJIP transient curves. The derived parameters are listed in Table S2 [25].

 

Comments 6

L164: I don’t see the accession numbers?

Response

We thank the reviewer for this comment. The GenBank accession numbers for all chloroplast genome sequences analyzed in this study have now been provided in Supplementary Table S1 below.

Table S1. Eight grape germplasm resources.
Number	species	accession numbers
1	V.berlandieri×V.riparia ‘ 5BB ’	SRR26559662
2	V. berlandieri×V.rupestris ‘ 1103P ’	SRR26391998
3	V.vinifera ×V.labrusca ‘ Shine Muscat ’	SRR15219641
4	V.vinifera ‘ Thompson Seedless’	SRR21423876
5	V.labrusca × V.riparia ‘ Beta ’	SRR26559467
6	V.vinifera ‘ Muscat Hamburg ’	SRR10439055
7	V.vinifera ‘Merlot ’	SRR26163229
8	Vitis amurensis ‘ Shuanghong ’	SRR16038735

 

Comments 7

L206: Please provide an appropriate reference for the “previous study”

Response

We thank the reviewer for this suggestion. An appropriate citation to the "previous study" has been added at line 206, and the corresponding reference is now included in the reference list.

 

Comments 8

L212: MEGA 11 instead of MAGA11?

Response

Thanks for your comment, we have made the corresponding modification (page 5, line 219).

 

Comments 9

L224: “Chloroplast genomes” is a more appropriate term to use here instead of “accessions”

Response

Thanks for your comment, we have made the corresponding modification (page 6, line 233).

 

Comments 10

Figure 1: Is this based on a representative genome or the pan-genome generated from the 9 chloroplast genomes? How were the gene positions labelled?

Response

We thank the reviewer for this question. Figure 1 is a schematic representation of the chloroplast genome structure based on the reference genome NC_007957. The diagram was generated using the online tool Chloroplot, which annotated the gene positions directly from the GenBank file . A detailed description of this methodology has been included in the 'Materials and Methods' section.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript titled “Comparative Analysis of Chloroplast Genomes Reveals Phylogenetic Relationships and Contributions to Photosynthesis in Vitis” presents a phylogenetic analysis using complete sequences from nine chloroplast genomes. The analysis allowed the identification of the expected differences in the junctions between genes within the chloroplast genome, but it also revealed variations in two coding genes. The authors not only reported the presence of these variations in certain cultivars but also measured the photosynthetic rate and successfully established a relationship between the two variables. The findings are significant and particularly important for clarifying the phylogenetic relationships within the genus Vitis.

Major Revisions:

-The authors should modify the title so that it more clearly reflects the results obtained, specifically the part “Contributions to Photosynthesis in Vitis.” This phrase is very general. I think it should be more specific.

-Several aspects of the methodology require further detail, particularly in the section “Data Sources and Analysis”. In this section, the authors should describe DNA isolation, DNA quality assessment, the sequencing technology used, etc. If there is previous methodology followed by the authors, the corresponding reference must be cited. The library preparation, the specific kit used and the equipment involved are not mentioned. These details are crucial and should be prioritized over general descriptions of sequencing procedures.

-The results obtained from the sequencing should be described in more detail. What was the number of reads obtained? What quality criteria were applied for sequence filtering, and how was the assembly performed? Although it is understood that complete and circular chloroplast genomes were obtained for all species, this is not clearly detailed in the Results section of the manuscript.

-The reported phylogeny should be constructed using another robust approach such as the Bayesian posterior probability. This method is recommended to support the results obtained by ML, especially for the inferences proposed by the authors.

Minor Revisions:

Line 70: The entire sentence requires the addition of a reference.

Line 85: The word sequence should not be capitalized; change it to lowercase.

Line 87: This is the first time cpDNA is mentioned, so the term should be written in full, followed by the abbreviation in parentheses.

Line 92: Remove the letter m at the end of the sentence.

Line 104: Rephrase the group of sentences referring to “gaps remain within the genus Vitis” to improve clarity and readability.

Line 168: Clarify what is meant by “identify potential anomalies.” FastQC is used solely for analyzing sequences and establishing the criteria to be applied in the subsequent trimming program.

Line 170: Trimmomatic is software. You should explicitly indicate this and include the corresponding reference.

Line 175: Is Bandage a software? If so, specify this and provide the appropriate reference.

Line 206: You mention a “previous study.” You need to specify which study this refers to and cite it properly.

Line 216: What kind of statistical analyses were performed? Excel is a package of different statistical tools; you need to specify which were used.

Line 226: Which individuals have 91 protein-coding genes? According to Table 1, only one species has a maximum of 89 PCGs.

Line 255: Add a reference for this paragraph.

Line 261: Is this figure from each chloroplast genome or from all? Since there are several types, you should specify which of the nine cp genomes it corresponds to.

Line 272: I believe part B of the figure is not relevant. Therefore, I recommend removing it.

Line 285: What do you mean by “significant differences”? Is there any statistical support for this statement?

Line 357: Why was the Beta cultivar not chosen for the analysis?

Line 402: Correct the word Disscussion to Discussion.

Line 418: Add a reference to support that statement.

Line 428: Rephrase the sentence to improve clarity.

Line 451: Add a reference to support the authors’ claims.

Line 461: I recommend comparing the obtained results with a species more closely related to Vitis, since rice was used in the comparison.

Line 466: The word Advacements should be corrected; I suggest using Advancements or Progress.

Line 489: What kind of information did the authors use to infer this statement? The presented phylogeny does not appear to support this conclusion.

Comments on the Quality of English Language

The manuscript contains valuable information, but the writing style in certain sections hinders smooth readability. This could be improved if the text were reviewed by a native English speaker. I recommend doing so or using the professional editing services offered by some scientific journals.

Author Response

Comments 1

The manuscript titled “Comparative Analysis of Chloroplast Genomes Reveals Phylogenetic Relationships and Contributions to Photosynthesis in Vitis” presents a phylogenetic analysis using complete sequences from nine chloroplast genomes. The analysis allowed the identification of the expected differences in the junctions between genes within the chloroplast genome, but it also revealed variations in two coding genes. The authors not only reported the presence of these variations in certain cultivars but also measured the photosynthetic rate and successfully established a relationship between the two variables. The findings are significant and particularly important for clarifying the phylogenetic relationships within the genus Vitis.

Major Revisions:

 

-The authors should modify the title so that it more clearly reflects the results obtained, specifically the part “Contributions to Photosynthesis in Vitis.” This phrase is very general. I think it should be more specific.

Response

Thank you for your reviewing. Your affirmation and recognition of “Comparative Analysis of Chloroplast Genomes Reveals Phylogenetic Relationships and Variation in Chlorophyll Fluorescence in Vitis” research paper will be of great help to our follow-up scientific research. The title has been revised to more specifically reflect our experimental findings, and now reads: "Comparative Analysis of Chloroplast Genomes Reveals Phylogenetic Relationships and Variation in Chlorophyll Fluorescence in Vitis".

 

Comments 2

Several aspects of the methodology require further detail, particularly in the section “Data Sources and Analysis”. In this section, the authors should describe DNA isolation, DNA quality assessment, the sequencing technology used, etc. If there is previous methodology followed by the authors, the corresponding reference must be cited. The library preparation, the specific kit used and the equipment involved are not mentioned. These details are crucial and should be prioritized over general descriptions of sequencing procedures.

The results obtained from the sequencing should be described in more detail. What was the number of reads obtained? What quality criteria were applied for sequence filtering, and how was the assembly performed? Although it is understood that complete and circular chloroplast genomes were obtained for all species, this is not clearly detailed in the Results section of the manuscript.

Response

We thank the reviewer for these insightful comments regarding methodological details. We have revised the "Data Sources and Analysis" section to provide greater clarity. Our study utilized existing whole genome sequences downloaded from public databases (NCBI), including both published data and sequences previously generated and deposited by our research group. All accession numbers are provided in Supplementary Table S1. Since no new wet-lab experiments (such as DNA extraction, library preparation, or sequencing) were conducted for this specific bioinformatic analysis, these technical details are not applicable. We have ensured that all references to previously established methodologies are now properly cited in the revised manuscript.

 

We have revised the "Data Sources and Analysis" section to provide greater clarity. Specifically, we downloaded the whole genome sequences of eight target varieties from public databases. Within the research scope, we processed a dataset of 8samples through the following bioinformatic pipeline: Raw sequencing data first underwent quality assessment using FastQC to evaluate sequence quality metrics , followed by adapter trimming and quality filtering using Trimmomatic with default parameters. De novo assembly was then performed using GetOrganelle (v1.7.1) under default configurations , with resultant assembly graphs validated using Bandage . The assembled plastomes were annotated using GeSeq with Vitis vinifera (NC_007957) as the reference , and finally visualized using Chloroplot to generate the genome maps . All accession numbers are provided in Supplementary Table S1. Since no new wet-lab experiments (such as DNA extraction, library preparation, or sequencing) were conducted for this specific bioinformatic analysis, these technical details are not applicable. We have ensured that all references to previously established methodologies are now properly cited in the revised manuscript.

 

Comments 3

The reported phylogeny should be constructed using another robust approach such as the Bayesian posterior probability. This method is recommended to support the results obtained by ML, especially for the inferences proposed by the authors.

Response

We thank the reviewer for this valuable suggestion. ML seeks the tree topology that maximizes the probability of observing the aligned sequence data under a given evolutionary model, with nodal support typically assessed through non-parametric bootstrap resampling. In contrast, Bayesian inference incorporates prior knowledge about parameters alongside the sequence data to estimate the posterior probability distribution of trees, directly quantifying phylogenetic uncertainty through Markov chain Monte Carlo sampling. Following the reviewer's suggestion, we constructed a phylogeny of the genus using Bayesian inference based on 32 chloroplast genomes(Figure 1). The resulting tree topology is largely congruent with that obtained from the maximum likelihood (ML) analysis.This additional analysis substantially strengthens confidence in our phylogenetic conclusions.

 

Figure 1. Phylogenetic tree of bayesian inference (BI) based on complete chloroplast ge-nome sequence.

 

Comments 4

Minor Revisions:

Line 70: The entire sentence requires the addition of a reference.

Response

Thanks for your comment, we have made the corresponding modification. The added references are as follows,

Liu, M.; Xu, Y.; Song, Y.; Fan, D.; Li, J.; Zhang, Z.; Wang, L.; He, J.; Chen, C.; Ma, C. Hierarchical Regulatory Networks Reveal Conserved Drivers of Plant Drought Response at the Cell‐Type Level. Advanced Science 2025, 12, 2415106.

 

Comments 5

Line 85: The word sequence should not be capitalized; change it to lowercase.

Response

Thanks for your comment, we have made the corresponding modification.

 

Comments 6

Line 87: This is the first time cpDNA is mentioned, so the term should be written in full, followed by the abbreviation in parentheses.

Response

Thanks for your comment, we have made the corresponding modification.

 

Comments 7

Line 92: Remove the letter m at the end of the sentence.

Response

Thanks for your comment, we have made the corresponding modification.

 

Comments 8

Line 104: Rephrase the group of sentences referring to “gaps remain within the genus Vitis” to improve clarity and readability.

Response

According to your suggestion, we have made the corresponding modification(page 3 ,line 103 to 106 ). As follows, 

CpDNA has been extensively applied in studies of various plants. However, significant research gaps persist regarding its use within the genus Vitis, where critical questions remain unanswered.

 

Comments 9

Line 168: Clarify what is meant by “identify potential anomalies.” FastQC is used solely for analyzing sequences and establishing the criteria to be applied in the subsequent trimming program.

Line 170: Trimmomatic is software. You should explicitly indicate this and include the corresponding reference.

Line 175: Is Bandage a software? If so, specify this and provide the appropriate reference.

Response

We thank the reviewer for these constructive suggestions. We have revised the manuscript accordingly. The phrase "identify potential anomalies" has been clarified to specify the standard quality metrics assessed by FastQC. Furthermore, both Trimmomatic and Bandage are now explicitly identified as software tools, and their corresponding citations have been added to the reference list. The added references are as follows,

Bolger, A.M.; Lohse, M.; Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 2014, 30, 2114-2120.

Wick, R.R.; Schultz, M.B.; Zobel, J.; Holt, K.E. Bandage: interactive visualization of de novo genome assemblies. Bioinformatics 2015, 31, 3350-3352.

 

Comments 10

Line 206: You mention a “previous study.” You need to specify which study this refers to and cite it properly.

Response

We thank the reviewer for this suggestion. The "previous study" cited at line 213 has now been clearly identified as [23], and the full citation has been added to the reference list. The added references are as follows,

Song, Y.; Wang, L.; Zhang, L.; Li, J.; Teng, Y.; Zhang, Z.; Xu, Y.; Fan, D.; He, J.; Ma, C. Unified Assembly of Chloroplast Genomes: A Comparative Study of Grapes Representing Global Geographic Diversity. Horticulturae 2024, 10, 1218.

 

Comments 11

Line 216: What kind of statistical analyses were performed? Excel is a package of different statistical tools; you need to specify which were used.

Response

According to your suggestion, we have made the corresponding modification (page 5, line 223 to 230). The full is as follows, 

Phenotypic traits and physiological indices of plants were recorded and organized using Microsoft Excel 2016. Statistical analysis was performed using SPSS 19.0 (IBM Corp., USA) through one-way ANOVA to assess differences among treat-ments or varieties.

 

Comments 11

Line 226: Which individuals have 91 protein-coding genes? According to Table 1, only one species has a maximum of 89 PCGs.

Response

We thank the reviewer for spotting this error, we have made the corresponding modification (page 5, line 233 to 237). The full is as follows,

In the analysis of nine Vitis Chloroplast genomes (Table 1; Figure 1,Table S4), chloroplast genome annotation identified a total of 133 to 134 genes across the specimens, comprising 88 to 89 protein-coding genes (PCGs), 37 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes.

 

Comments 12

Line 255: Add a reference for this paragraph.

Response

According to your suggestion, we have made the corresponding modification. The added references are as follows,

Parvathy, S.T.; Udayasuriyan, V.; Bhadana, V. Codon usage bias. Molecular biology reports 2022, 49, 539-565.

 

Comments 13

Line 261: Is this figure from each chloroplast genome or from all? Since there are several types, you should specify which of the nine cp genomes it corresponds to.

Response

We thank the reviewer for this question. Figure 1 is a schematic representation of the chloroplast genome structure based on the reference genome NC_007957. The diagram was generated using the online tool Chloroplot, which annotated the gene positions directly from the GenBank file . A detailed description of this methodology has been included in the 'Materials and Methods' section.

 

Comments 13

Line 272: I believe part B of the figure is not relevant. Therefore, I recommend removing it.

Response

According to your suggestion, we have made the corresponding modification. The full is as follows.

 

Figure 2. Relative synonymous codon usage (RSCU) of protein-coding genes (PCGs) in the chloroplast genomes of nine grape varieties.

 

 

 

Comments 13

Line 285: What do you mean by “significant differences”? Is there any statistical support for this statement?

Response

We thank the reviewer for pointing out this issue. The phrase "significant differences" has been removed from line 285, as it was not supported by statistical evidence. The sentence has been revised accordingly to maintain accuracy.

 

Comments 14

Line 357: Why was the Beta cultivar not chosen for the analysis?

Response

We thank the reviewer for this relevant question. The selection of plant materials for this study was based on their agronomic importance, specifically focusing on commercially adopted rootstocks and scion cultivars. This strategy was designed to facilitate a comparative analysis of photosynthetic capacity across genotypes with distinct practical applications in viticulture.

 

Comments 15

Line 402: Correct the word Disscussion to Discussion.

Response

According to your suggestion, we have made the corresponding modification.

 

Comments 16

Line 418: Add a reference to support that statement.

Response

According to your suggestion, we have made the corresponding modification.

 

Comments 17

Line 428: Rephrase the sentence to improve clarity.

Response

According to your suggestion, we have made the corresponding modification (page 15, line 419 to 423). As follows,

The identified simple sequence repeats (SSRs) serve as valuable molecular markers. These resources will support future studies on genetic diversity and phylogenetic relationships within the genus Vitis.

 

Comments 18

Line 451: Add a reference to support the authors’ claims.

Response

According to your suggestion, we have made the corresponding modification. The added references are as follows,

Miglani, G.S.; Kaur, R.; Sharma, P.; Gupta, N. Leveraging photosynthetic efficiency toward improving crop yields. Journal of Crop Improvement 2021, 35, 361-402.

 

Comments 19

Line 461: I recommend comparing the obtained results with a species more closely related to Vitis, since rice was used in the comparison.

Response

According to your suggestion, we have made the corresponding modification (page 16, line 462-464). The full is as follows,

In studies of grape, it has been demonstrated that PIabs exhibits a strong correlation with grain yield, suggesting its utility as a reliable proxy for assessing yield potential .

 

Comments 20

Line 466: The word Advacements should be corrected; I suggest using Advancements or Progress.

Response

According to your suggestion, we have made the corresponding modification. The full is as follows. 

 

Comments 21

Line 489: What kind of information did the authors use to infer this statement? The presented phylogeny does not appear to support this conclusion.

Response

We thank the reviewer for raising this point, which allows us to clarify the basis for our statement. Our inference is primarily derived from the fundamental topological structure of the phylogenetic tree presented in Figure10. The phylogeny clearly segregates all genotypes into four major, well-supported clades. Crucially, Clade I is exclusively composed of the two Muscadinia genotypes, forming a distinct lineage that is phylogenetically separate from the remaining three clades, which collectively encompass all genotypes belonging to Subg. Euvitis. This clear separation at the subgeneric level—where all Muscadinia samples cluster together in one clade, entirely distinct from the clades of Euvitis—is the primary evidence supporting our statement regarding the phylogenetic distinction between the two subgenera.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors, a brief summary of your reviewed manuscript.

The revised manuscript has improved significantly. The focus of the study is easier to recognize, and the reduction of trivial information improves the reading flow. All comments were addressed and recommendations have been realized. There are still some parts that should be revised again as the content is unclear or the writing is scattered. See comments below:

Minor comments:

• line 60: Include a space between “species identification [13].In recent years”
• variable sites, especially the parsimony informative sites.
• line 871: This section is not clear. “However, significant research gaps persist regarding its use within the genus Vitis, where critical questions remain unanswered. Such as, the number of publicly available complete cpDNA sequences remains limited, particularly for wild relatives and indigenous cultivars, constraining a comprehensive assessment of the genus’s chloroplast diversity [9,19,23]”. I think that should be a comma, not a period, in “remain unanswered. Such….”
• Line 93-95. I think this objective represents an activity rather than a goal per se. I think it could be rephrased as "Infer a robust phylogeny to establish a phylogenetically informed classification system for grape cultivars."
• Line 98: “Plant Material Collection and Preparation” should be deleted
• Line 101: “Experimental planting:” should be deleted
• 113-114. This section is not clear. What means “..Chlorophyll a fluorescence was assessed with a Handy Plant Efficiency…”
• 132-133. Include a period after reference 30. “for subsequent analysis[30]Subsequently, de novo assembly of chloroplast genomes was conducted…”
• Lines 133-135. If the conditions of runs were by default setting in “using GetOrganelle (version 1.7.1) with default settings [31]”, why mention and apply the parameters? “… the chloroplast type was “embplant_pt”, the 134 assembly process was iterated 10 times, and we specified a k-mer size of 121 for the 135 SPAdes assembly algorithm.”?
• Line 138 include a space before Annotation
• Line 142: Italicize Vitis vinifera.
• Line 145: One could think why the same reference sequence (Vitis vinifera) had different reference (34 in line 142 and 35 here)?. But In think that you could put the number of the reference after the program version, and resolve this confusion.
• Line 147. Please delete “meticulously”. All our analyses must be meticulous.
• Line 167-168. I think that this paragraph should be performed.
• Line 242-243. The caption of the figure is not clear respect to the colours. LSC is light blue?. Please contrast with the colours of (and explain) JLB, JSB, JSA and JLA in the leyend.
• Line 266-267. Just a suggestion: explore the output file formats of Mauve, perhaps you can obtain a better representation of this figure.
• Line 291. Delete one period after (Table 2)
• Line 330. Delete the period.
• Line 340. Include a space between genes(Table 1).
• Line 346. Include a space in …bility.In this study
• Line 362 Include a space Vitis [19].However
• Line Italicize the scientific names.
• Line 407 Include a space in (Figure 10).As…
• Line 353 I saw that you not refer to figures 5 and 6 in this section. My impression is that such figures seem to point to something similar, therefore I suggest that if possible, they be placed in supplementary material.

 

Author Response

Comments 1

Reviewer #1: The revised manuscript has improved significantly. The focus of the study is easier to recognize, and the reduction of trivial information improves the reading flow. All comments were addressed and recommendations have been realized. There are still some parts that should be revised again as the content is unclear or the writing is scattered. See comments below:

Minor comments:

line 60: Include a space between “species identification [13].In recent years”

variable sites, especially the parsimony informative sites.

Response

Thank you for your reviewing. Your affirmation and recognition of “Comparative Analysis of Chloroplast Genomes Reveals Phylogenetic Relationships and Variation in Chlorophyll Fluorescence in Vitis” research paper will be of great help to our follow-up scientific research.

About formatting problem，I have carefully revised the full manuscript.

 

Comments 2

line 79-81: This section is not clear. “However, significant research gaps persist regarding its use within the genus Vitis, where critical questions remain unanswered. Such as, the number of publicly available complete cpDNA sequences remains limited, particularly for wild relatives and indigenous cultivars, constraining a comprehensive assessment of the genus’s chloroplast diversity [9,19,23]”. I think that should be a comma, not a period, in “remain unanswered. Such….”

Response

We appreciate the reviewer's keen observation regarding the sentence structure. The use of a period indeed fragmented the thought. Following the suggestion, we have replaced it with a comma to properly connect the general statement with the specific example introduced by 'such as'(page 2, line 79-82). The corrected text is:

However, significant research gaps persist regarding its use within the genus Vitis, such as the limited number of publicly available complete cpDNA sequences, particu-larly for wild relatives and indigenous cultivars, which constrains a comprehensive assessment of the genus's chloroplast diversity [9,19,22].

 

Comments 3

Line 93-95. I think this objective represents an activity rather than a goal per se. I think it could be rephrased as "Infer a robust phylogeny to establish a phylogenetically informed classification system for grape cultivars."

Response

Thanks for your comment. Based on the reviewer's comments, we have revised the article (page 2, line 93-95). As follow,

Infer a robust phylogeny to establish a phylogenetically informed classification system for grape cultivars.

 

Comments 4

Line 98: “Plant Material Collection and Preparation” should be deleted

Line 101: “Experimental planting:” should be deleted

Response

According to your suggestion, we have made the corresponding modification.

 

Comments 5

113-114. This section is not clear. What means “..Chlorophyll a fluorescence was assessed with a Handy Plant Efficiency…”

Response

We thank the reviewer for pointing this out. The original sentence was intended to describe the methodology for measuring chlorophyll a fluorescence transients and the OJIP test. We agree that the phrasing could be improved for clarity. Following the reviewer's comment, we have revised the sentence to:

Chlorophyll a fluorescence transients were measured, and the OJIP test was conducted, using a Hansatech Handy PEA photosynthesis efficiency analyzer (Hansatech Instru-ments Ltd., Norfolk, England).

 

Comments 6

132-133. Include a period after reference 30. “for subsequent analysis[30]Subsequently, de novo assembly of chloroplast genomes was conducted…”

Response

We thank the reviewer for pointing out this oversight. The missing period has been added after reference [30] in the revised manuscript (page 3, line 129).

 

Comments 7

Lines 133-135. If the conditions of runs were by default setting in “using GetOrganelle (version 1.7.1) with default settings [31]”, why mention and apply the parameters? “… the chloroplast type was “embplant_pt”, the 134 assembly process was iterated 10 times, and we specified a k-mer size of 121 for the 135 SPAdes assembly algorithm.”?

Response

We thank the reviewer for pointing out this inconsistency. We agree that mentioning specific parameters after stating the use of "default settings" was confusing. These parameters were indeed part of the default configuration in GetOrganelle version 1.7.1. To avoid any ambiguity, we have revised the sentence to simply state the use of the software with its default settings, as shown below:

Subsequently, the chloroplast genome was assembled using GetOrganelle (version 1.7.1) with default settings [30].

 

Comments 8

Line 138 include a space before Annotation

Line 142: Italicize Vitis vinifera.

Response

According to your suggestion, we have made the corresponding modification.

 

Comments 9

Line 145: One could think why the same reference sequence (Vitis vinifera) had different reference (34 in line 142 and 35 here)?. But In think that you could put the number of the reference after the program version, and resolve this confusion.

Response

We thank the reviewer for this suggestion. We have revised the text to place the reference number after the program version, which clarifies that the same reference sequence is used throughout, thus resolving the confusion.

 

Comments 10

Line 147. Please delete “meticulously”. All our analyses must be meticulous.

Response

We agree with the reviewer and have deleted the word "meticulously" as suggested.

 

Comments 11

Line 167-168. I think that this paragraph should be performed.

Response

In response to your comment, we have performed a revision of the indicated paragraph. The mention of TBtools has been deleted from the text. This is because its use was exclusively for generating the previous Figure 2B. Since this figure has been removed to streamline the presentation, retaining the corresponding methodological description could cause confusion (page 4, line 167-171). The specific modifications are as follows,

The graphical representations in this study, such as bar plots, line charts, and scatter plots, were plotted using Origin 2022 (OriginLab, USA). Subsequently, the final figures were composed and arranged by assembling the individual plots in Microsoft Power-Point 2019 to create publication-ready multi-panel figures.

 

Comments 12

Line 242-243. The caption of the figure is not clear respect to the colours. LSC is light blue?. Please contrast with the colours of (and explain) JLB, JSB, JSA and JLA in the leyend.

Response

We have clarified the figure caption as suggested. The definitions for the junction sites JLB, JSB, JSA, and JLA have been explicitly added to the legend. Furthermore, the color representation has been confirmed: the LSC region is indeed depicted in light blue (page 7 ,line 238-242). The specific modifications are as follows,

Figure 4. Comparison of junctions between LSC (light blue), SSC (light green), and IR(orange) re-gions among nine Vitis complete chloroplast genomes. The distance in the figure is not to scale. JLB represents the junction between the LSC and IRb, JSB indicates the junction between the SSC and IRb, JSA reverse the junction between the SSC and IRa, JLA refers to the junction between the LSC and IRa.

 

Comments 13

Line 266-267. Just a suggestion: explore the output file formats of Mauve, perhaps you can obtain a better representation of this figure.

Response

We thank the reviewer for this helpful suggestion. We have explored the output formats of Mauve and have replaced the original figure with a new version generated from a higher-quality to ensure a clearer and more precise representation. As follow,

 

 

Figure 6. Chloroplast genome mauve alignment of nine grape species. With V. vinifera ‘Pinot Noir’ set as a reference genome. The relevant colored boxes reveal locally-collinear blocks, which present homologous gene clusters.

 

Comments 14

Line 291. Delete one period after (Table 2)

Response

According to your suggestion, we have made the corresponding modification.

 

Comments 15

Line 330. Delete the period.

Response

According to your suggestion, we have made the corresponding modification.

 

Comments 16

Line 340. Include a space between genes(Table 1).

Response

According to your suggestion, we have made the corresponding modification.

Comments 17

Line 346. Include a space in …bility.In this study

Response

We thank the reviewer for pointing out this typographical error. The missing space after the period has been added in Line 343 of the revised manuscript.

 

Comments 18

Line 362 Include a space Vitis [19].However

Response

We thank the reviewer for pointing out this typographical error. The missing space after the period has been added in Line 362 of the revised manuscript.

 

Comments 19

Line Italicize the scientific names.

Response

We thank the reviewer for reminding us of this important convention. We have now carefully reviewed the entire manuscript and italicized all scientific names throughout the text.

 

Comments 20

Line 407 Include a space in (Figure 10).As…

Response

We thank the reviewer for pointing out this typographical error. The missing space after the period has been added in Line 408 of the revised manuscript.

 

Comments 21

Line 353 I saw that you not refer to figures 5 and 6 in this section. My impression is that such figures seem to point to something similar, therefore I suggest that if possible, they be placed in supplementary material.

Response

We thank the reviewer for their careful observation regarding the placement of Figures 5 and 6. The reviewer is correct that these figures are related. In response to the comment, we have now explicitly referenced Figure 5 and 6 in the designated section (Lines 355-358) to ensure its relevance is clear to the reader. As follow,

Based on the sequence alignment and collinearity analysis (Figures 5, 6), the chloro-plast genomes of the nine Vitis species exhibited high conservation in both UTR and CDS regions. No inversions or genomic rearrangements were detected, further con-firming their evolutionary stability.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have followed my recommendations and corrected the errors I pointed out in the previous version. Therefore, I agree with the revised manuscript. There are only a few minor revisions:

Line 125: The phrase “with specific accession numbers detailed” is repeated.

Line 128: The authors mentioned that they analyzed a dataset consisting of eight samples. However, on line 138 they mentioned that there are nine genomes for annotation. I understand that they are not including the reference genome since this genome has already been annotated. I recommend to check the numbers.

Line 142: Write the scientific name correctly in italics: Vitis vinifera

Line 167: To using? Check the grammar

Author Response

Comments 1

The authors have followed my recommendations and corrected the errors I pointed out in the previous version. Therefore, I agree with the revised manuscript. There are only a few minor revisions:

Line 125: The phrase “with specific accession numbers detailed” is repeated.

Response

Thank you for your reviewing. Your affirmation and recognition of “Comparative Analysis of Chloroplast Genomes Reveals Phylogenetic Relationships and Variation in Chlorophyll Fluorescence in Vitis” research paper will be of great help to our follow-up scientific research.

We thank the reviewer for pointing out this duplication. The repeated phrase "with specific accession numbers detailed" has been deleted in the revised manuscript (page 3, line 122).

 

Comments 2

Line 128: The authors mentioned that they analyzed a dataset consisting of eight samples. However, on line 138 they mentioned that there are nine genomes for annotation. I understand that they are not including the reference genome since this genome has already been annotated. I recommend to check the numbers.

Response

We thank the reviewer for this astute observation and the opportunity to clarify. The reviewer is correct in their understanding. We have corrected the error on line 133.

 

Comments 3

Line 142: Write the scientific name correctly in italics: Vitis vinifera

Response

We thank the reviewer for pointing this out. The scientific name has now been correctly italicized as Vitis vinifera.

 

Comments 4

Line 167: To using? Check the grammar

Response

We have corrected the grammatical error on line 162. As follow,

Using MEGA11 with 1000 bootstrap replicates under the GTRGAMMA model, a maximum likelihood (ML) phylogenetic tree was constructed[40]

Author Response File: Author Response.pdf