Genetic Diversity, Population Structure, and Conservation Units of Castanopsis sclerophylla (Fagaceae)
Round 1
Reviewer 1 Report
I have reviewed the MS titled "Genetic diversity, population structure and conservation units of Castanopsis sclerophylla (Fagaceae)", which has been submitted to Forests. In this study, the genetic diversity and population structure of C. sclerophylla have been investigated by using chloroplast DNA sequences and nuclear microsatellite markers.
Although it is a local species, I find this study interesting, especially from a methodological point of view, and it serves as a basis for similar experiences globally.
Study strengths
Studying genetic population structure is fundamental to understanding ecological and evolutionary dynamics.
The number of samples is appropriate for this type of study, it is even good to see that a total of 320 individuals of C. sclerophylla were sampled from 15 natural populations.
The number of nuclear SSR primers (32) is enough, and previously tested, based on high polymorphism and repeatability.
Additionally, I have found some comments that I detail below.
Majors
The information presented in lines 65 to 68 is not of interest since there are many studies using these molecular marker techniques. Replace the content showing results from the literature, which relate the ecological aspect with genomic information, especially considering the species under study (or species belonging to this vegetation type; Subtropical evergreen broad-leaved forest (EBLF)).
Please explain in the text, why sixteen out of 32 loci (i.e., 50%) significantly deviated from the HWE, in the Discussion section. In this regard, please perform PCA analysis (or equivalent) in order to complement Structure results, but consider all SSRs (32) in the analysis (include Figure in Results, and discuss it in the Discussion section).
At some points in the discussion section, there are many repetitions of the results. At the beginning of the discussion, for example, results are repeated. Please start with a more general sentence linking it to recent literature, for example:
“Studying population structure has made an essential contribution to understanding evolutionary processes and demographic history in forest ecology research (Maldonado et al. 2022). In this study, significant genetic differentiation among the western, central and eastern groups of C. sclerophylla has been evidenced”.
(Eliminate “Table 5” from line 255, accordingly).
Reference:
Maldonado, C.; Mora-Poblete, F.; Echeverria, C.; Baettig, R.; Torres-Díaz, C.; Contreras-Soto, R.I.; Heidari, P.; Lobos, G.A.; do Amaral Júnior, A.T. A Neural Network-Based Spectral Approach for the Assignment of Individual Trees to Genetically Differentiated Subpopulations. Remote Sens. 2022, 14, 2898.
In this regard, please avoid repetition of the results in the Discussion section (additionally, avoid to mention Figures and Tables).
Minors
Eliminate “with STRUCTURE” from the Abstract. Specific software information is not recommended to appear in Abstract, because many software determining genetic structure are available currently.
Line 117. Replace revealed by inferred. Population genetic structure were “inferred”.
Author Response
Please see the attachment
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Manuscript Number: forests-1768021
Title: Genetic diversity, population structure and conservation units of Castanopsis sclerophylla (Fagaceae)
Authors: Shuang Chen, et al.
Remarks:
This submitted paper investigated genetic diversity and structure in cpDNA sequence and SSR markers in Castanopsis sclerophylla populations in southern mountainous area of China, and the authors found genetic structure in accordance to the phylogeographic backgrounds of the species. Although the paper seems to be weak in terms of novelty, I consider that the writing style is well and the research content is worthy for the conservation and utilizing programs of the species.
I recommend the authors on a few major points regarding with the statement of scientific novelty and the targeted populations, and also several minor points regarding with insufficient explanation and word uses. Please reconsider to improve the paper.
Major Comments:
1. I wonder that the major distribution of C. sclerophylla, as far as seeing Fig. 4c, is different from the range of sampling populations (as in Fig. 1); the species’ major distribution seems to be more southern area of China, and the sampling populations are northernmost (or outside of the distribution?) of the species. If so, the authors do not examine genetic variation of the whole species. How do the authors consider about this?
I consider that the authors should explain more clearly on what populations and why they investigate. This major problem may be related with the unclear explanation of the main theme of the study in Introduction, together with the weakness in explanation of novelty as commented above. For example, if the authors focus mainly on the northernmost (mountainous) populations of the species, they should express on the title (like “… of northernmost populations in Castanopsis sclerophylla”, and so on) and explain the backgrounds in Introduction section. Please reconsider again to make clear what the authors want to examine by using the sampled populations.
2. Concerning with the problem as in the Major comment #1, in Introduction section, the authors should reconsider and make clear what is the main theme of the study and what is the scientific novelty in the study. Unless the readers do not appreciate the value of the paper.
One of idea is to notice that the northernmost and mountainous populations of this species may have a specific phylogenetic population history patterns and that may reflect the specific genetic structure, also may be related with the species characteristics. This is an example, but I recommend strongly the authors to reconsider what they can appeal, by analyzing the sampled populations.
Minor Comments:
Abstract
3. Lines 14-, and so on. Throughout the paper, I think that “east-west differentiation” should be rewrote into “eastern-to-western differentiation” or “longitudinal differentiation”.
Materials and Methods
4. Table 1. It is more readable if the column of region groups (western, central and eastern) of populations are provided.
5. Fig. 1. Please explain the difference of the circle size.
6. Fig. 1. I wonder the reason of the SYT population having the H3 cluster, despite of its eastern distribution. Please provide an explanation.
7. Lines 116-: “SSRs deviated from HWE were excluded from further analysis”. Please provide the reason, such as the presence of null alleles or the non-neutrality, and so on.
8. Lines 124-. The “groups” are not yet defined here. Groups based on what?
Results
9. Lines 159-. Please explain the content of “16 variable sites”; such as the numbers of Insertion/Deletion sites and SNP sites.
10. Table 2. Similar to the comment #4, it is more readable if the column of region groups is provided.
11. Lines 170-. “out of 32 loci …” -> “out of 32 SSR loci …”.
12. Lines 172-. “The observed alleles …” -> “The observed number of alleles …”.
13. Table 4. Similar to the comments #4 and #10, it is more readable if the column of region groups is provided. And, in the caption, “… of Castanopsis sclerophylla” -> “… of Castanopsis sclerophylla at the 16 SSR loci.”
14. Fig. 2. Similar to comments on the Tables 1, 2 and 4, it is more readable if the separation of region groups is provided.
15. Fig. 3. In the caption, “… of Castanopsis sclerophylla” -> “… of Castanopsis sclerophylla based on scenario 6.” (Is this OK?)
16. Lines 243-. The readers do not see where are “Guangdong and Guangxi provinces” and “Hunan and Jiangxi provinces”. Please consider to make the readers find the name of provinces in the map (Fig. 1 or 4?).
17. Lines 245-. “This species contracted …” -> “Distribution of this species contracted …”
18. Fig. 4. The map c) is cut in half.
Discussion
19. Lines 293-: “Significant negative correlation between genetic diversity (AR) and latitude …”. I do not see this results anywhere in the text or Figs/Tables.
Author Response
Please see the attachment
Author Response File: Author Response.pdf
Reviewer 3 Report
General comments
This paper uses chloroplast sequencing and microsatellite genotyping to examine the phylogeography of a broad-leaf tree species in China. The manuscript fits well within the scope of the journal.
The methods applied are suitable for their data (although I don’t know much about DIYABC and ecological niche modeling) and the conclusions drawn are appropriate given their results. I mainly have minor comments on style, which are detailed below. One section that should be expanded is the STRUCTURE results. It is usual to also report on other values of K, not just the optimal value, to see if further genetic structuring within the data is present.
Specific comments
Abstract
Line 15: insert ‘has’ between divergence and arisen
Line 18 and throughout the manuscript: Last glacial maximum should be Last Glacial Maximum
Line 21: insert ‘a’ between as and key
Line 22: Change to ‘Our study supplies important information for prioritizing the conservation and sustainable utilization of C. sclerophylla, and provides insight….’
Introduction
Line 33: Change ‘supposed’ to ‘thought’
Line 34: Change ‘were’ to ‘was’
Line 39: Change ‘pattern’ to ‘patterns’
Line 43: ‘great chance’ to ‘opportunity’
Ln 44: Delete ‘The’ from the start of the sentence
Ln 47: Delete ‘population’
Ln 55: Change ‘popularly’ to ‘widely’
Ln 59: Change to ‘Only small-scale studies on the genetic diversity of C. sclerophylla, such as in the region of Qiandao Lake, have been ….’
Ln 64: Delete ‘and get insights for its scientific conservation and utilization’.
Ln 69: Change ‘to the’ to ‘in’
Ln 72: change ‘to infer’ to ‘of inferring’
Materials and Methods
Ln 76: Change ‘sample’ to ‘sampled’ and delete ‘kept’
Figure 1: A scale bar for the map would be useful.
Ln 95: Change ‘that’ to ‘those’
Ln 96: Delete ‘the’ before flourochromes.
Ln 102: Applied Bio systems should be Applied Biosystems
Ln 107: DNAsp should be DnaSP
Ln 108: Change ‘by the’ to ‘with’
Ln 120: Were the ten runs in STRUCTURE averaged e.g., with CLUMPP?
Ln 122: Change ‘were’ to ‘was’
Ln 128: Delete ‘The’ at the start of this sentence.
Ln 137: Delete ‘of’ between one million and simulations
Ln 140: Change this sentence to ‘In addition to our sampling locations, 119 sites where C. sclerophyllahas been recorded were obtained from the Chinese Virtual Herbarium.’
Ln 153: Change ‘were’ to ‘was’
Results
Ln 158: Change to ‘An alignment of 1171 bp of chloroplast DNA was obtained in which 6 haplotypes ……’
Ln 161: Change to ‘…there was a significant difference…’
Ln 163: Change to ‘The three most common haplotypes were H3, H2 and H1, which mainly occurred in the western, central and eastern regions (Figure 1), respectively.
Ln 166: Add ‘a’ between ‘had’ and ‘close relationship’
Table 2: Should the first line of the first two columns in the table be in bold and underlined or not? It doesn’t look correct as it is.
Ln 170: I think it would be worth adding ‘SSR’ or ‘microsatellite’ between ‘32’ and ‘loci’ just to make it completely clear that the chloroplast loci are no longer being discussed.
Ln 180: Change ‘index’ to the plural ‘indices’
Ln 192: I think it would be worth reporting on higher levels of K because they may also indicate further structuring. See:
Meirmans PG. Seven common mistakes in population genetics and how to avoid them. Mol. Ecol. 2015;24: 3223-3231.
Even if additional clusters are just split across all populations this is worth noting with a sentence like: At higher values of K additional clusters were split across all populations.
Ln 193: The split is not quite as clear-cut as written here – some of these populations had individuals assigned with high Q-value to each cluster e.g., populations XS, WM, DX and QDH.
Ln 203: Change to’… C. sclerophyllamainly existed within populations (Table 5)…’
Ln 210: Change ‘were’ to ‘are’ and ‘The scenario 6…’ to ‘Scenario 6…’
Ln 213: Change ‘The scenario 6…’ to ‘Scenario 6…’
Table 6: The spacing in the first column should be wider so that the heading of column 1 can be read easily.
Ln 239: Add ‘the’ before highest contributions
Ln 244: Change to ‘Castanopsis sclerophyllamainly occurred in more northern regions such as some parts of Hunan and Jiangxi provinces at this time, and the high-suitable area showed some discontinuity in the east-west direction. ‘
Ln 248: add ‘the’ between ‘occupied’ and ‘much’
Discussion
Ln 256: Change ‘ancient’ to ‘ancestral haplotype’
Ln 259: Change ‘arisen’ to ‘arose’
Ln 261: Delete ‘the’ before genetic east-west differentiation
Ln 266: insert ‘the’ before ‘Himalayan orogeny’
Ln 280: Change ‘Appropriate’ to “Suitable’
Ln 281: Change ‘chance’ to ‘an opportunity’
Ln 283: Change ‘are’ to ‘is the’
Ln 284: Change to ‘…lowlands serve as a putative dispersal corridor for many plant species, increasing the population …’
Ln 284: Add ‘and’ between ‘ranges’ and ‘even’
Ln 287: Change to ‘ …the distribution of C. sclerophylla…’
Ln 289: Change to ‘ancient haplotype H1 was found…
Ln 290: Change to ‘The southern limit of these three mountain ranges is the Nanling mountain range…’
It would be good to show the Nanling mountain range on the map of Figure 1, since it is mentioned in the text.
Ln 292: Delete ‘the’ before ‘dual roles’
Ln 295: Change to ‘…which is consistent with the suggestion that the Luoxiao…
Ln 296: Change ‘of’ to ‘for’
Ln 297: Change ‘was’ to ‘is’
Ln 299: Change ‘pollens disperse’ to ‘pollen dispersal’
Ln 301: Change ‘by the second contact’ to ‘by secondary contact’
Ln 303: Change to ‘…be considered as separate conservation units because they possessed distinct chloroplast haplotypes, and the Luoxiao Mountain range should be considered as the key region for conservation of C. sclerophylladue to it containing the highest diversity of SSR alleles.’
Conclusions
Ln 309: Delete ‘the’ from between ‘by’ and ‘isolation’
Ln 311: Change ‘become’ to ‘became’
Ln 316: insert ‘understanding’ between ‘implications for’ and ‘the dynamics’
References
Ln 337: Change ‘last’ to ‘Last’
Ln 405: The title of this paper should be in lower case letters to be consistent with the other references.
Author Response
Please see the attachment
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Manuscript Number: forests-1808654_v2
Title: Genetic diversity, population structure and conservation units of Castanopsis sclerophylla (Fagaceae)
Authors: Shuang Chen, et al.
This resubmitted paper has improved substantially. I comment one more time on a few points. Please reconsider.
1. Authors’ response on the major comment #1:
The major distribution of C. sclerophylla is consistent with the range of sampling populations. At line 52, we explained “It is mainly distributed on area in the south of the Yangtze River and the north of the Nanling mountains in China.” We have shown the Nanling mountain range on the map of Figure 1. Actually, we did not found other population in the south of WM.
At line 70, we explained our main aim “In this study, we used nuclear SSR markers and chloroplast DNA sequences to study the genetic diversity and population structure of C. sclerophylla in subtropical China with the specific aim of inferring the population evolutionary history of this canopy tree species in EBLF.”
- Comments:
I understand that the study range covers the current major distribution of the species. If so, I recommend the authors to add an additional figure of the actual, current distribution range in Fig. 4. This will make the readers catch the distribution history more clearly.
And, it is better to show what is “the Yangtze River” (the northern limit of the species) in Fig. 1.
2. Authors’ response on the minor comment #6:
At line 289 of the Discussion section, we explained “Lying in the midst of a longitudinal depression between China’s western highlands and the coastal ranges of Fujian province is the Chiang-nan hilly region, where lowlands serve as a putative dispersal corridor for many plant species, increasing the population connectivity among the Xuefeng, Luoxiao and Wuyi mountain ranges and even the Zhejiang hilly region [46, 47], thus interpret why haplotype H3 was found in SYT and YQ.”
- Comments:
The revised sentence is long, so it is better to divide into two sentences.
3. Authors’ response on the minor comment #19:
At line 302, we have explained that this result was not shown. “Significant negative correlation between genetic diversity (AR) and latitude (result no shown) was revealed in central populations.”
- Comments:
It is better to revise as “Significant negative correlation between genetic diversity (AR) and latitude was revealed in central populations (result no shown).”
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
