Genetic Patterns and Diversity of Postintroduction of Metasequoia glyptostroboides (Hu and W. C. Cheng) in Ningbo Forest Farm, China
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsIn this study, Genetic diversity analysis of six groups including mother trees and seedlings of Metasequoia glyptostroboides was evaluated using 6 SSR markers. The researchers found a relatively high genetic diversity and notable genetic differentiation among the introduced M. glyptostroboides.
There are important flaws in the manuscript listed below:
- One of the objectives of this research, lines 81-82, is to "confirm the validity and repeatability of the previously developed primers." This cannot be considered an objective, as SSR markers are species-specific and we anticipate that the used SSRs can be amplified in PCR. Therefore, I recommend deleting this objective.
- Regarding materials and methods, lines 90-91, the samples taken from three areas are from 17 to 50. As the diversity parameters calculated in the manuscript are affected by the number of samplings, these significant differences may cause some bias in the calculated parameters. Why were the number of samples not the same? How do you explain the effect of sample differences on the calculated parameters? The same problems can be observed in the number of samples for each group, ranging between 9 and 41.
- To determine the genetic similarity between samples, the Jaccard genetic similarity coefficient was used (as stated in lines 148-149). However, since SSR markers are co-dominant markers, using the Dice similarity coefficient was preferred.
- In figure 3, the numbers mentioned in each tie of the dendrogram seem to be bootstrap values. Explanations about bootstrapping in the figure legend need to be mentioned.
- In Table 6, I recommend adding another source of variation between the three forest areas. A discussion of genetic diversity analysis within and between these areas could be added.
Comments on the Quality of English LanguageThe quality of English language needs minor revision.
Author Response
Reviewer1
In this study, Genetic diversity analysis of six groups including mother trees and seedlings of Metasequoia glyptostroboides was evaluated using 6 SSR markers. The researchers found a relatively high genetic diversity and notable genetic differentiation among the introduced M. glyptostroboides.
There are important flaws in the manuscript listed below:
One of the objectives of this research, lines 81-82, is to "confirm the validity and repeatability of the previously developed primers." This cannot be considered an objective, as SSR markers are species-specific and we anticipate that the used SSRs can be amplified in PCR. Therefore, I recommend deleting this objective.
Response: Based on your suggestion, we have removed this objective content.
Regarding materials and methods, lines 90-91, the samples taken from three areas are from 17 to 50. As the diversity parameters calculated in the manuscript are affected by the number of samplings, these significant differences may cause some bias in the calculated parameters. Why were the number of samples not the same? How do you explain the effect of sample differences on the calculated parameters? The same problems can be observed in the number of samples for each group, ranging between 9 and 41.
Response: Thank you for bringing up this important point regarding the number of samples in our study. We would like to clarify that we conducted comprehensive sampling of all available individuals in the forest areas. The variability in the number of samples taken from different areas is primarily due to differences in the number of parent trees and the resulting uneven production of seedlings. We acknowledge that the unequal sampling may introduce bias in the calculated diversity parameters. We had discussed the potential impact of sample differences on the statistical analysis of diversity indices. Thank you for highlighting this concern.
To determine the genetic similarity between samples, the Jaccard genetic similarity coefficient was used (as stated in lines 148-149). However, since SSR markers are co-dominant markers, using the Dice similarity coefficient was preferred.
Response: According to your suggestion, we have redrawn Figure 3 using Dice similarity coefficient.
In figure 3, the numbers mentioned in each tie of the dendrogram seem to be bootstrap values. Explanations about bootstrapping in the figure legend need to be mentioned.
Response: We have added an explanation of bootstrapping in the caption of Figure 3 and redraw the figure.
In Table 6, I recommend adding another source of variation between the three forest areas. A discussion of genetic diversity analysis within and between these areas could be added.
Response: We appreciate your insightful feedback. However, after careful consideration, we believe that the current results already encompass the necessary information on genetic diversity analysis within and between the Groups. We feel that additional analysis or discussion in this regard may not add significant value to the interpretation of the study results. Thank you for understanding our perspective.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsDear authors,
Here are my comments and recommendations.
Line 53 - 61. Remove this text (starting from "Initially"). I don't think there is much value in this information to the reader.
L. 78 - remove the comma after "study".
L. 82 - change "validate" to "validity"
Materials and methods. Please add a short paragraph explaining the reason for low sample number
L. 95 - change "polymorphic" to "polymorphism"
L. 100 - replace "screened out" with "selected for this study"
Header of Table 2. - use "primer pairs", not "primers"
Please use full name for Ta in Table 2.
L. 109 - please provide the catalogue number for the DNA extraction kit.
L. 111 - change "system" to "reaction", in this paragraph also provide the manufacturer, kit name and catalogue number for the PCR reagents used.
L. 116 - here you mention 55 C as the annealing temperature. Was this actually the temperature for all the primer pairs despite the information provided in Table 2? If not, please state that annealing temperatures were set as indicated in Table 2.
L. 119 - delete "After completion of the fluorescent PCR amplification,".
L. 121 - manufacturer and cat. no. for the size standard missing.
L. 121 - 122 - remove information about peak sizes of the size standard.
L. 124 - "F" missing in the name of fluorescent dye
L. 126 - manufacturer and cat. no. for the formamide missing.
L. 164 - provided link not working. Probably a typo in the link.
L. 174 - remove "polymorphic"
L. 177 - you indicate the peak sizes using decimal points. That's a bit uncommon in my experience. As there is no thing as a partial nucleotide, the results are typically rounded to the closest number without decimal points. I leave this to your consideration whether to change this.
Header Table 3. - Please change to "Polymorphism information for six SSR loci in M. glyptostroboides."
Header of Table 4 - please insert "consisting of" before "mother". No need for capital letter in the word "mother" in this sentence.
Header and top row of Table 5 appears twice! Please fix this.
Header of Table 6 - please change to "Analysis of intragroup and intergroup molecular variance"
Line 357 - you probably mean "uniformity of the germplasms"? Please express the sentence containing this fragment more clearly.
Your conclusions about results of PCA analysis - I don't agree to the statements that, 1.) the uniformity of the germplasms indicates a high level of genetic diversity and, 2.) PCoA analysis categorized the 106 germplasms into six major groups.
What I see in Figure 4 is that AS and SLGS form separate clusters but the rest of groups overlap with at least one other group so you can't tell that they are categorized into separate groups.
What I think about your statement about uniformity of the germplasms indicates a high level of genetic diversity - at first I would wish yo better understand what exactly you mean by uniformity, secondly, if genetic diversity would be high, would you see any clustering or grouping at all. Also, as you mention in the discussion, the genetic diversity of M. glyptostroboides is lower than in gymnosperms. So is the genetic diversity shown in Fig. 4 high? Compared to what?
I my opinion you have to rewrite chapter 3.7 of your manuscript to express yourself more clearly and not to cause contradictions with how the data in Figure 4 can be interpreted.
L. 388 - please use "polymorphism" instead of "polymorphic"
L. 390 - what do you mean by "traditional molecular markers"? Isozymes? Please specify. Some researchers already call SSR markers traditional markers.
L. 391 - change "all six primers" to "the selected six primer pairs"
L. 392 - delete "marker"
L. 392-394 - delete text starting with the word "used" until the word "technology" (including these words)
L. 403 - there is a space before the dot
L. 407 - "related to habitat loss" sounds wrong, please try to rephrase
L. 419 - is it possible to make a conclusion about the ranking of the six groups taking into account the small sample number of some of them?
You should discuss the potential effects of the sample size on your results and conclusions. At present moment I see just a little bit of discussion into this topic in lines 450-452. In my opinion this is insufficient.
Does Figure 6 really support your statement in L. 475-476?
L. 485 - I suggest using "restoration of M. glyptostroboides populations" instead of "rejuvenation..."
The Supplementary file is not very useful and is not mentioned in text. I recommend removing it.
Author Response
Reviewer2
Dear authors,
Here are my comments and recommendations.
Line 53 - 61. Remove this text (starting from "Initially"). I don't think there is much value in this information to the reader.
Response: Agreed, we have removed the content.
- 78 - remove the comma after "study".
Response: We have removed the comma.
- 82 - change "validate" to "validity"
Response: We replaced the words.
Materials and methods. Please add a short paragraph explaining the reason for low sample number
Response: We added an explanation for the low number of LX individuals in Section 2.1.
- 95 - change "polymorphic" to "polymorphism"
Response: We replaced the words.
L.100 - replace "screened out" with "selected for this study"
Response: We replaced the phrase.
Header of Table 2. - use "primer pairs", not "primers"
Response: We made the substitution.
Please use full name for Ta in Table 2.
Response: We replaced the abbreviation with the full name.
L.109 - please provide the catalogue number for the DNA extraction kit.
Response: We added the category number for the DNA extraction kit.
- 111 - change "system" to "reaction", in this paragraph also provide the manufacturer, kit name and catalogue number for the PCR reagents used.
Response: We made the substitution and provided the requested information.
- 116 - here you mention 55 C as the annealing temperature. Was this actually the temperature for all the primer pairs despite the information provided in Table 2? If not, please state that annealing temperatures were set as indicated in Table 2.
Response: According to your suggestion, we have rewritten the expression of annealing temperature.
L.119 - delete "After completion of the fluorescent PCR amplification,".
Response: We deleted the sentence.
L.121 - manufacturer and cat. no. for the size standard missing.
Response: We supplemented the information.
- 121 - 122 - remove information about peak sizes of the size standard.
Response: We removed peak sizes.
O.124 - "F" missing in the name of fluorescent dye
Response: We added an "F".
L.126 - manufacturer and cat. no. for the formamide missing.
Response: We supplemented the information.
- 164 - provided link not working. Probably a typo in the link.
Response: We checked and corrected the link.
- 174 - remove "polymorphic"
Response: We deleted the word.
177 - you indicate the peak sizes using decimal points. That's a bit uncommon in my experience. As there is no thing as a partial nucleotide, the results are typically rounded to the closest number without decimal points. I leave this to your consideration whether to change this.
Response: We rounded the numbers.
Header Table 3. - Please change to "Polymorphism information for six SSR loci in M. glyptostroboides."
Response: According to your suggestion, we have rewritten the title of Table 3.
Header of Table 4 - please insert "consisting of" before "mother". No need for capital letter in the word "mother" in this sentence.
Response: According to your suggestion, we inserted the phrase and corrected the word.
Header and top row of Table 5 appears twice! Please fix this.
Response: This should be due to the automatic addition of headings across pages of the table. We have checked the manuscript.
Header of Table 6 - please change to "Analysis of intragroup and intergroup molecular variance"
Response: According to your suggestion, we have adjusted the title.
Line 357 - you probably mean "uniformity of the germplasms"? Please express the sentence containing this fragment more clearly.
Your conclusions about results of PCA analysis - I don't agree to the statements that, 1.) the uniformity of the germplasms indicates a high level of genetic diversity and, 2.) PCoA analysis categorized the 106 germplasms into six major groups.
What I see in Figure 4 is that AS and SLGS form separate clusters but the rest of groups overlap with at least one other group so you can't tell that they are categorized into separate groups.
What I think about your statement about uniformity of the germplasms indicates a high level of genetic diversity - at first I would wish yo better understand what exactly you mean by uniformity, secondly, if genetic diversity would be high, would you see any clustering or grouping at all. Also, as you mention in the discussion, the genetic diversity of M. glyptostroboides is lower than in gymnosperms. So is the genetic diversity shown in Fig. 4 high? Compared to what?
I my opinion you have to rewrite chapter 3.7 of your manuscript to express yourself more clearly and not to cause contradictions with how the data in Figure 4 can be interpreted.
Response: Thank you for your valuable feedback and suggestions on our manuscript. We have carefully reviewed your points and taken them into consideration for the revision of Chapter 3.7. Regarding the clarification of the sentence containing the fragment "uniformity of the germplasms" in line 357, we will ensure to rephrase the statement for better clarity and accuracy. We appreciate your insights on the conclusions drawn from the PCA analysis results. We understand your concerns about the interpretation of Figure 4 and the categorization of germplasms into separate groups based on PCoA analysis. In the revised version, we will provide a more objective description of the results and avoid making statements that could potentially lead to contradictions with the data presented in Figure 4.
L.388 - please use "polymorphism" instead of "polymorphic"
Response: We made the substitution.
M.390 - what do you mean by "traditional molecular markers"? Isozymes? Please specify. Some researchers already call SSR markers traditional markers.
Response: We supplemented the contrast with traditional markers.
- 391 - change "all six primers" to "the selected six primer pairs"
Response: We made the replacement according to your suggestion.
- 392 - delete "marker"
Response: We deleted the word.
- 392-394 - delete text starting with the word "used" until the word "technology" (including these words)
Response: According to your suggestion, I have deleted this paragraph.
L.403 - there is a space before the dot
Response: We deleted the Spaces.
L.407 - "related to habitat loss" sounds wrong, please try to rephrase
Response: We've rewritten that sentence to make sure it's clear.
- 419 - is it possible to make a conclusion about the ranking of the six groups taking into account the small sample number of some of them?
Response: In Section 4.2 of the discussion, based on the indicators of genetic diversity, we ranked the six groups and three forest areas separately.
You should discuss the potential effects of the sample size on your results and conclusions. At present moment I see just a little bit of discussion into this topic in lines 450-452. In my opinion this is insufficient.
Response: We appreciate your feedback regarding the potential effects of sample size on our results and conclusions. We have taken your suggestion into consideration and have supplemented our discussion on this topic in various sections, including the abstract and materials and methods.
Does Figure 6 really support your statement in L. 475-476?
Response: We have rewritten chapter 4.3.
L.485 - I suggest using "restoration of M. glyptostroboides populations" instead of "rejuvenation..."
Response: According to your suggestion, we made a replacement.
The Supplementary file is not very useful and is not mentioned in text. I recommend removing it.
Response: We deleted the Supplementary file.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThis manuscript provides an in-depth analysis of the genetic diversity and structure of Metasequoia glyptostroboides populations in the Ningbo Forest Farm using SSR markers and capillary electrophoresis. The study is well-motivated, addressing conservation concerns for this endangered species and offering practical recommendations for germplasm management.
The methodological framework is robust, and the results are presented with clarity. The authors effectively analyze genetic variation, differentiation, and population structure, highlighting the absence of a founder effect and the implications of inbreeding within certain subpopulations. The findings contribute valuable insights into the conservation of M. glyptostroboides, particularly the importance of tailored management strategies for populations exhibiting high genetic diversity.
Nevertheless, there are areas that require improvement. The discussion could benefit from a more comprehensive contextualization of the results, particularly in relation to similar studies on other endangered or relict species. The conclusion could also outline more specific directions for future research, such as exploring additional molecular markers or assessing long-term genetic trends.
Overall, the manuscript offers a significant contribution to conservation genetics, though revisions are necessary to enhance its clarity and depth.
Author Response
Reviewer3
This manuscript provides an in-depth analysis of the genetic diversity and structure of Metasequoia glyptostroboides populations in the Ningbo Forest Farm using SSR markers and capillary electrophoresis. The study is well-motivated, addressing conservation concerns for this endangered species and offering practical recommendations for germplasm management.
The methodological framework is robust, and the results are presented with clarity. The authors effectively analyze genetic variation, differentiation, and population structure, highlighting the absence of a founder effect and the implications of inbreeding within certain subpopulations. The findings contribute valuable insights into the conservation of M. glyptostroboides, particularly the importance of tailored management strategies for populations exhibiting high genetic diversity.
Nevertheless, there are areas that require improvement. The discussion could benefit from a more comprehensive contextualization of the results, particularly in relation to similar studies on other endangered or relict species. The conclusion could also outline more specific directions for future research, such as exploring additional molecular markers or assessing long-term genetic trends.
Response: We appreciate for providing a positive review of our manuscript and acknowledging the significance of our study on the genetic diversity and structure of M. glyptostroboides populations. In response to your suggestions and the feedback from other reviewers, we have revised the discussion section to enhance the contextualization of our results by incorporating citations from similar studies on other endangered or relict species. Furthermore, we have included perspectives on future molecular studies.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have adequately addressed all concerns and have made the necessary revisions to the manuscript.
Comments on the Quality of English LanguageThe quality of the English language is fine,
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors have addressed and responded to all the points raised.
