SNP-Based Analysis of Genetic Diversity and Genetic Structure in Bursaphelenchus xylophilus Populations from Guizhou Province, China
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe study contributes to genetic diversity of Bursaphelenchus xylophilus in China, by investigating the situation in Guizhou province. There is a large body literature on this topic in China. This is properly reflected in the text.
The results show very low genetic diversity and no regional pattern. A direct connection between data from other provinces would be very interesting but probably very hard due to different methods in the different studies. Under these circumstances, the results might change when more samples are considered. A direct connection between data from other provinces would be very interesting but probably very hard due to different methods in the different studies. However, the authors state clearly that they found not much genetic diversity, so the reader is not misled.
The analysis use up-to-date technology and interesting methods for the data analysis.
Line 186: Please, clarify, was STRUCTURE or ADMIXTURE used?
Figure 4: A 3D plot is not useful in this way. It would be better to have PC 1 and PC 2 in one plot, and PC 1 and PC 3 in another plot.
line 360: There is a formatting error in my PDF. Please, check!
line 102: Typo "Each"
line 102: Typo "was" -> "were", because samples were collected
line 133: Typo "the PLINK". No article is better here.
Line 164: "The Nei’s gene diversity" - no article is better here
line 209: Typo "no" -> "not"
Author Response
POINT-BY-POINT RESPONSE
Dear Referee,
Please find attached the revised version of our manuscript, “SNP-Based Analysis of Genetic Diversity and Genetic Structure in Bursaphelenchus xylophilus Populations from Guizhou Province, China” (MS number: forests-4353983). We have carefully considered all comments, and we now submit the new version. In general, we address each specific comment but for those particular cases where not, we explain our reason for doing so.
Comments to the Author
The study contributes to genetic diversity of Bursaphelenchus xylophilus in China, by investigating the situation in Guizhou province. There is a large body literature on this topic in China. This is properly reflected in the text. The results show very low genetic diversity and no regional pattern. A direct connection between data from other provinces would be very interesting but probably very hard due to different methods in the different studies. Under these circumstances, the results might change when more samples are considered. A direct connection between data from other provinces would be very interesting but probably very hard due to different methods in the different studies. However, the authors state clearly that they found not much genetic diversity, so the reader is not misled.
Reply: Many thanks for your suggestions. We try to revise the ms. following your suggestions (Line 245-352).
The analysis use up-to-date technology and interesting methods for the data analysis.
Line 186: Please, clarify, was STRUCTURE or ADMIXTURE used?
Reply: Many thanks for your suggestions. We address this issue in the new version of the ms. (Lines 200).
Figure 4: A 3D plot is not useful in this way. It would be better to have PC 1 and PC 2 in one plot, and PC 1 and PC 3 in another plot.
Reply: Many thanks for your suggestions. We revised this issue in the new version of the ms. (See Figure 4).
line 360: There is a formatting error in my PDF. Please, check!
Reply: Many thanks for your suggestions. We revised this issue in the new version of the ms. (Line 435-437).
line 102: Typo "Each"
Reply: Many thanks for your suggestions. We revised this issue in the new version of the ms. (Line 103).
line 102: Typo "was" -> "were", because samples were collected
Reply: Many thanks for your suggestions. We revised this issue in the new version of the ms. (Line 103).
line 133: Typo "the PLINK". No article is better here.
Reply: Many thanks for your suggestions. We revised this issue in the new version of the ms. (Line 136).
Line 164: "The Nei’s gene diversity" - no article is better here
Reply: Many thanks for your suggestions. We revised this issue in the new version of the ms. (Line 167).
line 209: Typo "no" -> "not"
Reply: Many thanks for your suggestions. We revised this issue in the new version of the ms. (Line 229).
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe methods are correct; the appropriate methods and statistical software were used to achieve the stated objective. I would only question the sample size (5 to 10 individuals) in each population; it would be excellent if the implications of the sample size on the results were discussed in the discussion section.
Most of the results are concrete and clear; however, the results corresponding to the clusters obtained, described in lines 180 to 192, are not consistent with the information presented in Figures 2 and 3.
The discussion is adequate; however, it needs improvement in the following areas: 1) Expand on the discussion of the results in Table 1; 2) Compare your results with those obtained in other studies using different markers; 3) Discuss the similarities or differences (and their implications) of the results from the three clustering techniques used (phylogenetic tree, AMIXTURE clustering, and principal components).
No conclusions are presented; it is necessary to include them. Formulate conclusions based on the objectives, research hypotheses, and research results. Avoid making the conclusions merely another summary of the results.
*There are other comments regarding other details in the manuscript; addressing them could improve the manuscript**
Comments for author File: Comments.pdf
Author Response
POINT-BY-POINT RESPONSE
Dear Referee,
Please find attached the revised version of our manuscript, “SNP-Based Analysis of Genetic Diversity and Genetic Structure in Bursaphelenchus xylophilus Populations from Guizhou Province, China” (MS number: forests-4353983). We have carefully considered all comments, and we now submit the new version. In general, we address each specific comment but for those particular cases where not, we explain our reason for doing so.
Comments to the Author
The methods are correct; the appropriate methods and statistical software were used to achieve the stated objective. I would only question the sample size (5 to 10 individuals) in each population; it would be excellent if the implications of the sample size on the results were discussed in the discussion section.
Reply: Many thanks for your suggestions. We address this issue in the new version of the ms. (Lines 325-331).
Most of the results are concrete and clear; however, the results corresponding to the clusters obtained, described in lines 180 to 192, are not consistent with the information presented in Figures 2 and 3.
Reply: Many thanks for your suggestions. We revised this issue in the new version of the ms. (Lines 183-211).
The discussion is adequate; however, it needs improvement in the following areas: 1) Expand on the discussion of the results in Table 1; 2) Compare your results with those obtained in other studies using different markers; 3) Discuss the similarities or differences (and their implications) of the results from the three clustering techniques used (phylogenetic tree, AMIXTURE clustering, and principal components).
Reply: Many thanks for your suggestions. We revised this issue in the new version of the ms. (Lines 244-331).
No conclusions are presented; it is necessary to include them. Formulate conclusions based on the objectives, research hypotheses, and research results. Avoid making the conclusions merely another summary of the results.
Reply: Many thanks for your suggestions. We revised this issue in the new version of the ms. (Lines 332-348).
*There are other comments regarding other details in the manuscript; addressing them could improve the manuscript**
1.These words already appear in the title; use this section to include new keywords that will help search engines find your manuscript.
Reply: Many thanks for your suggestions. We revised this issue in the new version of the ms. (Lines 24).
2.Consider whether this word is a key term for your research.For example: SNPs, He, Ho, gene flow.
Reply: Many thanks for your suggestions. We revised this issue in the new version of the ms. (Lines 24).
3.There's a period missing here.
Reply: Many thanks for your suggestions. We revised this issue in the new version of the ms. (Lines 24).
4.Support this with at least two references.
Reply: Many thanks for your suggestions. We revised this issue in the new version of the ms. (Lines 60).
5.At least one research hypothesis must be formulated.
Reply: Many thanks for your suggestions. We revised this issue in the new version of the ms. (Lines 87-88).
6.Enter the name of the country here. Insert the name of the province here.
Reply: Many thanks for your suggestions. We revised this issue in the new version of the ms. (See Figure 1).
7...., China.
Reply: Many thanks for your suggestions. We revised this issue in the new version of the ms. (Lines 108).
8.Please specify the quality, that is, demonstrate the quality using specific parameters.
Reply: Many thanks for your suggestions. We revised this issue in the new version of the ms. (Lines 115-116).
9.Please make sure this link is correct.
Reply: Many thanks for your suggestions. We revised this issue in the new version of the ms. (Lines 124).
10.I recommend following the order in which they appear in the table.
Reply: Many thanks for your suggestions. We revised this issue in the new version of the ms. (Lines 180-182).
11.In Figure 2, I did not find a group that includes individuals from both of these cities or counties. Therefore, it would be helpful to indicate here how many individuals from each city or county are included in each group. How many individuals are there in each location? Please note: indicate how many individuals are in each location.
Reply: Many thanks for your suggestions. We revised this issue in the new version of the ms. (Lines 183-200).
12.In addition, the groups must correspond to the color scheme and legend in Figure 3; according to Figure 3:
- The second group (red) includes individuals BZ6, BZ5, JS2, and RH7;
- The third group (green) includes BZ2, BZ3, FG3, and ST1;
- The fourth group (orange) includes RH4 and ST2;
- The fifth group (purple) includes CJ1, CJ4, and RJ2;
- The sixth group (light blue) includes FG1, RH2, SD1, and WS1;
- The FIRST group (blue) includes the remaining individuals.
There is a complete discrepancy; none of the groups mentioned in the text match Figure 3 (k=6).
Reply: Many thanks for your suggestions. We revised this issue in the new version of the ms. (Lines 200-210).
13.Check whether it was actually similar; the legend for Figure 4 indicates EIGHT groups—unless the legend refers to the individuals' origins rather than the groups!
Reply: Many thanks for your suggestions. We revised this issue in the new version of the ms. (See Figure 4).
13.TR_group or TR_individual? Please label each group in some way to make them easier to identify.
Reply: Many thanks for your suggestions. We revised this issue in the new version of the ms. (See Figure 4).
14.This section is for discussing results. Please do not list results here; instead, discuss them.
Reply: Many thanks for your suggestions. We revised this issue in the new version of the ms. (Lines 245-280).
15.Further discussion is needed:
- Regarding the results in Table 1: What indicated that for several populations, Ho was slightly higher than He? What did the other parameters—Fis, A, PI, and PIC—indicate? How do you explain the low number of SNPs in Xishui and its relatively higher Nei diversity index compared to the lower diversity in Renhuai and Congjiang (both of which have a high number of SNPs)?
- The introduction mentions that there are studies using other markers conducted in China. How do your results compare to those previous studies?
- Regarding the clustering, three techniques were used (phylogenetic tree, AMIXTURE clustering, and principal components); however, there is no discussion of whether these techniques yielded similar results. Please discuss this point.
Reply: Many thanks for your suggestions. We revised this issue in the new version of the ms. (Lines 244-331).
16.The research should include conclusions; please include them.
Reply: Many thanks for your suggestions. We revised this issue in the new version of the ms. (Lines 332-348).
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsGeneral remarks
- The MS deals with the destructive nematode of pines (PWN). It employs many algorithms to find the origin of the PWN through the analysis of SNP data of 60 samples derived from six counties and the respective populations from Guizhou Province. Then the MS proceeds to the estimation of the genetic structure of these populations.
- The authors used a lot of software packages to perform these analyses, and many times they use only a specific feature of a software package to perform an analysis already existing in another. For instance, the PCA analysis can be performed in the ADMIXTURE package [Ref 37] and through the EIGENSOFT v6.0 [in line 138 it is written] “Principal component analysis (PCA) was conducted using EIGENSOFT v6.0 [38] to examine the genetic structure of PWN”.
- In addition, in the MS is written [lines 150-155] that “We used TreeMix v.1.12 (parameter: -m from 0 to 6) to assess gene flow among PWN populations [42]. The optimal number of migration events was determined using the R package “OptM” [43]. Visualization of gene flow directions was performed with the R package “plotting_funcs” [I was not able to find this function]. To further estimate gene flow between populations, we employed MIGRATE v.4.4.3 [44] under the following settings: 10,000 recorded steps per chain, a burn-in of 100,000 steps per chain, and three replicate runs. 155”.
- With all these features, the MS seems to be an account of existing literature directed to students to inform them about the existing methods. The output figures and tables must be properly discussed. For example, the fact that some entries in Table 2 are not even touched, such as the XS-TR = 0.120, XS-SD = 0.155, FG-XS = 0.183, and FG-RH = 0.131 causes many questions.
Specific remarks
- L44: Pl. change to [14, 15].
- L41-42: In what way the SNP analysis will help the detection of “the transmission routes of the PWN are important for conservation and management of the forestry ecosystem”? The authors used a plenty of methods that estimate the number of migration events, or edges (m), on population trees from genome-wide allele frequency data. I hope the authors will explain that in the discussion section.
- L192: PCoA (usually used to abbreviate the Principal Coordinate Analysis) or PCA (Principal Component Analysis)?
- 192-193: The authors have to specify what they mean by writing ‘a similar pattern’.
- 224: The important Table 2 is not properly discussed, and I think it includes the most important results of the work.
Several positions in th etext need further attention.
Author Response
POINT-BY-POINT RESPONSE
Dear Referee,
Please find attached the revised version of our manuscript, “SNP-Based Analysis of Genetic Diversity and Genetic Structure in Bursaphelenchus xylophilus Populations from Guizhou Province, China” (MS number: forests-4353983). We have carefully considered all comments, and we now submit the new version. In general, we address each specific comment but for those particular cases where not, we explain our reason for doing so.
Comments to the Author
The MS deals with the destructive nematode of pines (PWN). It employs many algorithms to find the origin of the PWN through the analysis of SNP data of 60 samples derived from six counties and the respective populations from Guizhou Province. Then the MS proceeds to the estimation of the genetic structure of these populations.
The authors used a lot of software packages to perform these analyses, and many times they use only a specific feature of a software package to perform an analysis already existing in another. For instance, the PCA analysis can be performed in the ADMIXTURE package [Ref 37] and through the EIGENSOFT v6.0 [in line 138 it is written] “Principal component analysis (PCA) was conducted using EIGENSOFT v6.0 [38] to examine the genetic structure of PWN”.
Reply: Many thanks for your suggestions. We address this issue in the new version of the ms. (Lines 138-142). In addition, it raises a valid point about redundancy in the use of multiple software packages. While it's true that PCA can be performed in ADMIXTURE (e.g., via its -pca option), the choice of EIGENSOFT (specifically smartpca) is not necessarily redundant. EIGENSOFT is widely regarded as a standard tool for population genetic PCA, offering features like outlier detection, Tracy–Widom tests for significant components, and proper handling of missing data. ADMIXTURE's PCA is more of a convenience feature for quick visualization. Using EIGENSOFT does not imply ignorance of ADMIXTURE's capabilities; rather, it reflects a preference for a tool specifically optimized and validated for PCA in population genetics. That said, the broader issue—authors employing many packages for overlapping analyses—can sometimes indicate a lack of streamlining or awareness of alternative features. However, it may also be justified by differences in algorithm robustness, output formats, or integration with downstream analyses. In rigorous research, reproducibility and accuracy take precedence over minimizing the number of software packages used.
In addition, in the MS is written [lines 150-155] that “We used TreeMix v.1.12 (parameter: -m from 0 to 6) to assess gene flow among PWN populations [42]. The optimal number of migration events was determined using the R package “OptM” [43]. Visualization of gene flow directions was performed with the R package “plotting_funcs” [I was not able to find this function]. To further estimate gene flow between populations, we employed MIGRATE v.4.4.3 [44] under the following settings: 10,000 recorded steps per chain, a burn-in of 100,000 steps per chain, and three replicate runs. 155”.
Reply: Many thanks for your suggestions. We address this issue in the new version of the ms. (Lines 153-158).
With all these features, the MS seems to be an account of existing literature directed to students to inform them about the existing methods. The output figures and tables must be properly discussed. For example, the fact that some entries in Table 2 are not even touched, such as the XS-TR = 0.120, XS-SD = 0.155, FG-XS = 0.183, and FG-RH = 0.131 causes many questions.
Reply: Many thanks for your suggestions. We address this issue in the new version of the ms. (Lines 288-296).
L44: Pl. change to [14, 15].
Reply: Many thanks for your suggestions. We address this issue in the new version of the ms. (Lines 44).
L41-42: In what way the SNP analysis will help the detection of “the transmission routes of the PWN are important for conservation and management of the forestry ecosystem”? The authors used a plenty of methods that estimate the number of migration events, or edges (m), on population trees from genome-wide allele frequency data. I hope the authors will explain that in the discussion section.
Reply: Many thanks for your suggestions. We address this issue in the new version of the ms. (Lines 297-309).
L192: PCoA (usually used to abbreviate the Principal Coordinate Analysis) or PCA (Principal Component Analysis)?
Reply: Many thanks for your suggestions. We address this issue in the new version of the ms. (Lines 211).
192-193: The authors have to specify what they mean by writing ‘a similar pattern’.
Reply: Many thanks for your suggestions. We address this issue in the new version of the ms. (Lines 210-211).
224: The important Table 2 is not properly discussed, and I think it includes the most important results of the work.
Reply: Many thanks for your suggestions. We address this issue in the new version of the ms. (Lines 288-296).
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsDear authors,
Thank you for addressing my concerns. There is only a minor concern I just found.
line 21: Replace "STRUCTURE" with "ADMIXTURE". You clarified that you used ADMIXTURE, not STRUCTURE at line 200 of the current manuscript.
Author Response
POINT-BY-POINT RESPONSE
Dear Referee,
Please find attached the revised version of our manuscript, “SNP-Based Analysis of Genetic Diversity and Genetic Structure in Bursaphelenchus xylophilus Populations from Guizhou Province, China” (MS number: forests-4353983). We have carefully considered all comments, and we now submit the new version. In general, we address each specific comment but for those particular cases where not, we explain our reason for doing so.
Comments to the Author
Thank you for addressing my concerns. There is only a minor concern I just found.
line 21: Replace "STRUCTURE" with "ADMIXTURE". You clarified that you used ADMIXTURE, not STRUCTURE at line 200 of the current manuscript.
Reply: Many thanks for your suggestions. We address this issue in the new version of the ms. (Lines 21).
Author Response File: 
Author Response.docx
