Genetic Evaluation in Natural Populations of the Threatened Conifer Amentotaxus argotaenia (Hance) Pilg. (Taxaceae) Using Microsatellites
, Thuy Thi La 1, Xuyen Thi Do 1, Hai Van Do 2, Duc Minh Nguyen 3,4, Hong Phan Lan Nguyen 2, Hosakatte Niranjana Murthy 5, Long Ke Phan 6 and Tam Minh Nguyen 6,*Round 1
Reviewer 1 Report
This paper presents genetic diversity and genetic structure of a threatened and relict conifer, Amentotaxus argotaenia, using nuclear microsatellite markers in Vietnam. I know that a couple of primer notes for the species of Amentotaxus in China was published previously. However, to the best of my knowledge, this paper would be the first study revealing the nuclear genetic structure of the species of Amentotaxus in Vietnam as well as in Asia. Basic analyses were employed; this paper is well organized and concisely written (except for several parts of Introduction). I believe that this knowledge obtained in this study will facilitate the conservation management of this threatened species. Therefore, this paper is competent for the publication in Forests. But, I would provide several major and minor revisions for improving the manuscript.
Major points:
-L65-77: I feel that these sentences, involving allozyme, RAPD, and AFLP, would be unnecessary in modern paper. I would suggest removing these sentences and starting from the topic of nuclear microsatellite.
-L86-94: I think that these sentences would also be shortened or removed after moving several references to the above sentences (L78-85).
-L94-95: I would suggest that the authors mention previous papers, which revealed the genetic structure of Amentotaxus species for some populations in Asia using ISSR (Ge et al. 2005, J Plant Res 118: 415-422), mtDNA(Ge et al. 2015, Plant Mol Biol Rep 33: 264-280), and EST-SSR (Li et al. 2016, Molecules 61: 67).
-L103-104: I know that Vietnam has four species of Amentotaxus, namely, A. argotaenia, A. poilanei, A. yunnanensis, and A. hekouensis, which is a new species described in 2019 (PhytoKeys 130: 25-32). For confirmation, I would like to know whether the samples from Pu Hoat and Pu Luong populations, which are clearly differentiated from the other populations in STRUCTUEE analysis, are really A. argotaenia.
-L104: Figure 1 is missing. Please provide a geographical map with population locations. In addition, “area (Central, Northwest, and Northeast)” that we can meet in Results should be defined on the geographical map and mentioned in “Study sites” for readers unfamiliar with this region.
-L149 and L224-225: If population differentiation is strong between the areas, I would suggest adding one more hierarchy, that is “Among areas” above “Among populations” in AMOVA.
-L364-271: In Figure 3A, I did not know where the individuals exceptionally positioned (e.g. one from Nam Chang and one from Liem Phu) are at coordinates. Please show these points of the individuals clearly.
-L313: I think that genetic diversity measures obtained from cpSSR and nuclear SSR would not be comparable each other because of the difference of ploidy (haploid vs. diploid) and of the mode of inheritance (uniparental (maternal in Taxaceae?) vs. biparental).
-L337: How have the population size changed? Have population growth occurred along with the heterozygote deficiency?
-L363-364: Please show that the large geographic distance and high mountain range exists using geographical map (e.g. on Figure 1).
Minor points:
-L42: I would suggest inserting family name (Taxaceae) and ‘conifer’ in this sentence, although we can see these words in the title. But, I think that these words should also be provided in the main text.
-L121: “30 ng” would be correct if the authors used 3 ul of the diluted DNA of 10 ng/ul.
-L194-196: I think that it is unnecessary to show the total number of alleles observed over the loci.
-L194: Please unitalicized “var.”
-L426: Figure S1 entitled “Studying location of A. argotaenia” is missing.
-L564: Please italicize “Taxus wallichiana” and “mairei”
Author Response
Dear the Referee,
I thank you very much for reviewing our manuscript of ID forests-1891502. We consider carefully comments from the reviewers and have made appropriate changes in our manuscript. Our detailed explanations for the specific comments are given as below. We do hope your consideration of our revised manuscript for publishing in your journal.
We should send you many thanks and the best wishes.
Sincerely,
Dr. Nguyen Minh Tam
Dr. Nguyen Trung Thanh
The comments from Reviewer 1:
Major points:
Comment 1: L65-77: I feel that these sentences, involving allozyme, RAPD, and AFLP, would be unnecessary in modern paper. I would suggest removing these sentences and starting from the topic of nuclear microsatellite.
Our answers: We agree with the reviewer and removed these sentences.
Comment 2: L86-94: I think that these sentences would also be shortened or removed after moving several references to the above sentences (L78-85).
Our answers: We agree with the reviewer. These sentences were written shorter. We removed some references.
Comment 3: L94-95: I would suggest that the authors mention previous papers, which revealed the genetic structure of Amentotaxus species for some populations in Asia using ISSR (Ge et al. 2005, J Plant Res 118: 415-422), mtDNA(Ge et al. 2015, Plant Mol Biol Rep 33: 264-280), and EST-SSR (Li et al. 2016, Molecules 61: 67).
Our answers: We agree with the reviewer and added these references.
Comment 4: L103-104: I know that Vietnam has four species of Amentotaxus, namely, A. argotaenia, A. poilanei, A. yunnanensis, and A. hekouensis, which is a new species described in 2019 (PhytoKeys 130: 25-32). For confirmation, I would like to know whether the samples from Pu Hoat and Pu Luong populations, which are clearly differentiated from the other populations in STRUCTUEE analysis, are really A. argotaenia.
Our answers: Gao et al. (2019) found a new species A. hekouensis in two locations, one in Muong Khuong (Lao Cai) and one in Mai Chau (Hoa Binh). We have not informed this species in Pu Hoat and Pu Luong in the Central area of Vietnam.
Comment 5: L104: Figure 1 is missing. Please provide a geographical map with population locations. In addition, “area (Central, Northwest, and Northeast)” that we can meet in Results should be defined on the geographical map and mentioned in “Study sites” for readers unfamiliar with this region.
Our answers: We apologize for missing Fig.1. We agree with the reviewer and the areas added in Fig. 1.
Comment 6: L149 and L224-225: If population differentiation is strong between the areas, I would suggest adding one more hierarchy, that is “Among areas” above “Among populations” in AMOVA.
Our answers: We agree with the reviewer and added “among areas”, “among populations within areas” in Table 3.
Comment 7: L364-271: In Figure 3A, I did not know where the individuals exceptionally positioned (e.g. one from Nam Chang and one from Liem Phu) are at coordinates. Please show these points of the individuals clearly.
Our answers: In Fig. 3A, three genetic clusters were showed. Individuals in each population were assigned in one of three clusters and showed in Table S5.
Table S5. Number of individuals for each population assigned to a cluster1
|
Population |
Cluster 1 |
Cluster 2 |
Cluster 3
|
|
Xuan Truong |
0 |
25 |
1 |
|
Nam Chang |
1 |
25 |
1 |
|
Xuan Son |
0 |
20 |
1 |
|
Liem Phu |
1 |
4 |
24 |
|
Co Ma |
0 |
3 |
20 |
|
Hang Kia |
0 |
0 |
30 |
|
Pu Luong |
18 |
1 |
0 |
|
Pu Hoat |
25 |
0 |
0 |
1 Jombart, T.; Devillard, S.; Balloux, F. Discriminant analysis of principal components: a new method for the analysis of genetically structured populations. BMC Genet. 2010, 11, 94.
Comment 8: L313: I think that genetic diversity measures obtained from cpSSR and nuclear SSR would not be comparable each other because of the difference of ploidy (haploid vs. diploid) and of the mode of inheritance (uniparental (maternal in Taxaceae?) vs. biparental).
Our answers: We agree with the reviewer. We corrected it again and did not compare genetic diversity measures between use of cp SSRs and nSSRs.
Comment 9: L337: How have the population size changed? Have population growth occurred along with the heterozygote deficiency?.
Our answers: After the Botttleneck analysis, due to the reduction in genetic diversity, we suggest that there was the heterozygote deficiency over the past several decades in relation to the change in population size.
Comment 10: L363-364: Please show that the large geographic distance and high mountain range exists using geographical map (e.g. on Figure 1).
Our answers: After analysis of genetic differentiation among populations between different areas, we suggest that there are related between genetic differentiation and geographic distance and high mountain range.
Minor points:
Comment 1: L42: I would suggest inserting family name (Taxaceae) and ‘conifer’ in this sentence, although we can see these words in the title. But, I think that these words should also be provided in the main text.
Our answers: We agree with the reviewer and added “Taxaceae” and “conifer” in this sentence.
Comment 2: L121: “30 ng” would be correct if the authors used 3 ul of the diluted DNA of 10 ng/ul.
Our answers: we are sorry. We corrected it again.
Comment 3: L194-196: I think that it is unnecessary to show the total number of alleles observed over the loci.
Our answers: We agree with the reviewer and removed the total number of alleles observed over the loci.
Comment 4: L194: Please unitalicized “var.”
Our answers: We agree with the reviewer and used ‘ranged” change “varied”.
Comment 5: L426: Figure S1 entitled “Studying location of A. argotaenia” is missing.
Our answers: We agree with the reviewer. We corrected Figure 1 entitled “Studying location of A. argotaenia”. We corrected Supplementary materials.
Comment 6: L564: Please italicize “Taxus wallichiana” and “mairei”.
Our answers: We corrected it as follows: “Taxus walliciana var. mairei”.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Dear authors,
I have reviewed the article titled "Genetic evaluation in natural populations of the threatened conifer Amentotaxus argotaenia (Hance) Pilg. (Taxaceae) using microsatellites". The topic is interesting and under the scope of the journal. The article is well written but can't accept in its present form. The abstract is lengthy and needs to be re-writing to meet the standard requirement of the journal. The introduction and results also need improvements; however, the discussion is good. Moreover, Figure 1 is missing, and Figures 2 and 3 are not clear so need to redraw. Briefly, the manuscript needs minor revisions before accepting for publication in the journal ‘Forests’. Find my comments enclosed in the manuscript.
Good luck!
Comments for author File: 
Comments.pdf
Author Response
Dear the Referee,
We thank you very much for reviewing our manuscript of ID forests-1891502. We consider carefully comments from the reviewers and have made appropriate changes in our manuscript. Our detailed explanations for the specific comments are given as below. We do hope your consideration of our revised manuscript for publishing in your journal.
We should send you many thanks and the best wishes.
Sincerely,
Dr. Nguyen Minh Tam
Dr. Nguyen Trung Thanh
The comments from Reviewer 2:
Comment 1: Line 2-4: Please change title as Genetic Evaluation in Natural Populations of the Threatened Conifer Amentotaxus argotaenia (Hance) Pilg. (Taxaceae) using Microsatellites
Our answers: We thank you. We corrected them.
Comment 2: Abstract: According to journal (forests) policy, abstract should not be more 200 words and your abstract is more than 250 words. It is lengthy, so reduce it as per journal requirement.
Our answers: We agree with the reviewer and rewritten “Abstract” with 200 words. We also improved “Introduction” and “results” in our manuscript.
Comment 3: Line 39: Please delete 'conifer' because already used in the title, so replace it with some other word to enhance the visibility of your article.
Our answers: We agree with the reviewer and deleted the “conifer” and changed it with “Gene flow”.
Comment 4: Line 56: Please check the font and make sure to use a same font and format in the whole manuscript.
Our answers: We checked the font and formatted it again.
Comment 5: Line 94-96: Please replace so far, there have been no studies investigating the genetic variability and population genetic structure of A. argotaenia in Vietnam, which is one of the prerequisites for establishing effective conservation activities with so far, there is a need to investigate the genetic variability and population genetic structure of A. argotaenia in Vietnam, which is one of the prerequisites for establishing effective conservation activities.
Our answers: We agree with the reviewer and changed it according to the reviewer.
Comment 6: Line 97: Please avoid to use personal nouns like I, we, etc.
Line 107: Please avoid to use personal nouns like I, we, etc.
Line 131: Please avoid to use personal nouns like I, we, etc. and check this in the whole manuscript.
Our answers: We agree with the reviewer and checked and removed “we” and rewritten them in the text.
Comment 7: Line 104 Where is the Figure 1 ???
Our answers: We are sorry. We added Figure 1.
Comment 8: Line 259: Figure 2 is not clear, so please redraw it clearly.
Our answers: We improved quality Figure 2.
Comment 9: Line 284: Figure 3: A and B part of figure should be of same size so please redraw it.
Our answers: We agree with the reviewer and improved the quality of Figure 3 and redrawn A and B part of the figure.
Author Response File: Author Response.docx
