Population Survey Combined with Genomic-Wide Genetic Variation Unravels the Endangered Status of Quercus gilva

Round 1

Reviewer 1 Report

The manuscript describes a relevant research topic and brings important contributions to species conservation. However, I suggest that authors carefully proofread the text to make it grammatically correct and easier to understand.

 

The introduction section contextualizes the research theme, explains the importance of the study, and stated the objectives clearly.

 

In the material and methods section, the authors inform that they sampled 65 individuals for genetic analysis (up to 3 individuals per population) (l. 121). The small sample size may not represent properly the genetic diversity of each population and may lead to biased conclusions. See Marandel et al. (2020) and Nazareno et al. (2017) for a more detailed discussion. The authors should increase the sample size or explain why the small sample size is sufficient and state what limitations this choice may bring to the study.

 

Populations are coded with the province name's initial letter followed by letters present in the names of the cities. For readers who don’t know the area, it may be hard to interpret the results and follow the discussion, but with some effort, it can be understood. However, sample names in Fig 2 and Fig S1 do not follow this code. So it is impossible to understand the correlation between the genetic structure/phylogeny presented in the figures and the geographical distribution of the samples. In Fig S1, there is a legend with sample sites codes, but there are too many colors and the codes seem to be ordered randomly, which makes it even harder to understand. The authors should use the same codes throughout the manuscript. 

 

The authors highlight that there are differences in the phylogenetic structures between NJ and ML trees (l. 240). However, the differences were neither described nor discussed. The differences and the consequences of the conclusion should be discussed. 

 

The results of the cross-validation for the admixture analysis (l. 247) are not presented in Fig S1 or anywhere else in the manuscript or the supplementary material. 

 

The PCA results shown in Fig S2 also have a legend with sample sites codes ordered randomly, with too many colors. At least the most different samples should have their sample sites indicated in the figure. 

 

The authors start the discussion section with a series of contextualization sentences. In this section, it is more important to discuss the authors' findings, so it should begin in l. 297. Information in lines 288 to 297 can be presented in the introduction section. 

 

In line 335, the authors state that the analyses support strong bottleneck events due to natural selection. It’s not clear which data lead to this conclusion. There are multiple analyses that can be performed to test for bottlenecks, and for natural selection. Neither of these tests was described in this manuscript. 

 

Another important analysis to evaluate population stability and extinction risks is the effective population size. This parameter can be estimated from the sample size, coancestry, and inbreeding coefficients (Cockerham 1969, Vencovsky and Crossa 2003).

 

The last sentence in the discussion section indicates which populations should be used as seeds source for restoration projects. It is important to highlight limitations and risks when we use seeds from areas with different environmental conditions for restoration purposes. There may be outbreeding depression cases, which were not evaluated in this study. 

 

The first two sentences in the conclusion section should be in the introduction (l. 341 - 344). 

 

 

 

Cockerham, C. C. (1969). Variance of gene frequencies. Evolution, 72-84.

Marandel, F, Charrier, G, Lamy, J-B, Le Cam, S, Lorance, P, Trenkel, VM. Estimating effective population size using RADseq: Effects of SNP selection and sample size. Ecol Evol. 2020; 10: 1929– 1937. https://doi.org/10.1002/ece3.6016

Nazareno, A.G., Bemmels, J.B., Dick, C.W. and Lohmann, L.G. (2017), Minimum sample sizes for population genomics: an empirical study from an Amazonian plant species. Mol Ecol Resour, 17: 1136-1147. https://doi.org/10.1111/1755-0998.12654

Vencovsky, R., & Crossa, J. (2003). Measurements of representativeness used in genetic resources conservation and plant breeding. Crop Science, 43(6), 1912-1921.

Author Response

COMMENTS FOR AUTHOR:

Reviewer 1

Comments and Suggestions for Authors

The manuscript describes a relevant research topic and brings important contributions to species conservation. However, I suggest that authors carefully proofread the text to make it grammatically correct and easier to understand.

Answer: We have improved the whole manuscript based on the suggestions of three reviewers. Then, the text has been corrected by the English native speakers (www.editage.cn).

 

The introduction section contextualizes the research theme, explains the importance of the study, and stated the objectives clearly.

Thank you for your positive feedback on the introduction section.

 

In the material and methods section, the authors inform that they sampled 65 individuals for genetic analysis (up to 3 individuals per population) (l. 121). The small sample size may not represent properly the genetic diversity of each population and may lead to biased conclusions. See Marandel et al. (2020) and Nazareno et al. (2017) for a more detailed discussion. The authors should increase the sample size or explain why the small sample size is sufficient and state what limitations this choice may bring to the study.

Answer: Thanks for your suggestion. By resampling two to 20 individuals, Nazareno et al. (2017) have demonstrated that relatively small sample size may be sufficient for accurate estimates of genetic diversity. This result is also confirmed in the literature based on resequencing techniques, where some populations are analyzed based three or even fewer individuals (Huang et al., 2018; An et al., 2020; Lam et al., 2010; Guan et al., 2022). Meanwhile, the high mapping rate and coverage of resequencing technology also ensure the quality and accuracy of genetic loci (Hu et al., 2021). Our study showed the average mapping rate of 89.5% with the reference genome and the average coverage rate of 10x, which greatly improved the quality of SNP and the accuracy of analysis.

Reference:

An, Y., Mi, X., Zhao, S., Guo, R., Xia, X., Liu, S., & Wei, C. (2020). Revealing Distinctions in Genetic Diversity and Adaptive Evolution Between Two Varieties of Camellia sinensis by Whole-Genome Resequencing. Frontiers in plant science, 11, 603819.

Huang, L., Yang, M., Li, L., Li, H., Yang, D., Shi, T., & Yang, P. (2018). Whole genome re-sequencing reveals evolutionary patterns of sacred lotus (Nelumbo nucifera). Journal of integrative plant biology, 60(1), 2-15.

Hu, T., Chitnis, N., Monos, D., & Dinh, A. (2021). Next-generation sequencing technologies: An overview. Human Immunology, 82(11), 801–811.

Nazareno, A. G., Bemmels, J. B., Dick, C. W., & Lohmann, L. G. (2017). Minimum sample sizes for population genomics: an empirical study from an Amazonian plant species. Molecular Ecology Resources, 17(6), 1136-1147.

Guan, X., Zhao, S., Xiang, W., Jin, H., Chen, N., Lei, C., ... & Xu, L. (2022). Genetic Diversity and Selective Signature in Dabieshan Cattle Revealed by Whole-Genome Resequencing. Biology, 11(9), 1327.

Lam, H. M., Xu, X., Liu, X., Chen, W., Yang, G., Wong, F. L., ... & Zhang, G. (2010). Resequencing of 31 wild and cultivated soybean genomes identifies patterns of genetic diversity and selection. Nature genetics, 42(12), 1053-1059.

 

Populations are coded with the province name's initial letter followed by letters present in the names of the cities. For readers who don’t know the area, it may be hard to interpret the results and follow the discussion, but with some effort, it can be understood. However, sample names in Fig 2 and Fig S1 do not follow this code. So it is impossible to understand the correlation between the genetic structure/phylogeny presented in the figures and the geographical distribution of the samples. In Fig S1, there is a legend with sample sites codes, but there are too many colors and the codes seem to be ordered randomly, which makes it even harder to understand. The authors should use the same codes throughout the manuscript.

Answer: Thank you. We have improved the Figure 2 and Figure S1 to follow the population codes in Figure 1 and Table S1. Then we checked whole manuscript to make sure that the same codes are used throughout the manuscript. The new Figures are as follows.

 

Figure 2 Neighbor-joining phylogenetic tree and population structure of Quercus gilva. Fagus sylvatica was used as the outgroup for the phylogenetic analysis. The figure does not show the out-group. Population codes abbreviations are the same as in Table 2.

Figure S1 The maximum-likelihood (ML) phylogenetic tree of the 22 populations of Quercus gilva. The abbreviations of population codes are the same as in Table 2.

 

The authors highlight that there are differences in the phylogenetic structures between NJ and ML trees (l. 240). However, the differences were neither described nor discussed. The differences and the consequences of the conclusion should be discussed.

Answer: Thank you. We have added the results and discussion for this part. The details are as follows.

The NJ and ML phylogenetic trees were constructed using 4,020,695 SNPs in the single-copy genes. The NJ and ML trees consistently showed that individuals from the Zherong, Fujian (FZR), Dongkou, Hunan (HDK), and Xiangxiang, Hunan (HXX) populations did not cluster into one lineage. According to the NJ and ML trees, all the Q. gilva individuals could be divided into three major groups: West, Central, and East groups. Generally, the populations from Guizhou and western Hunan provinces comprised the western group. The populations from Eastern Hunan, Jiangxi, Fujian, and most of Zhejiang provinces formed the central group. Populations from South Korea, Japan, and ZZS (Zhoushan, Zhejiang) formed the main part of eastern group (Figure 2). There were three main differences in the phylogenetic structures between the NJ and ML trees of Q. gilva populations (Figure 2 and S1). First, the GLP population (Liping, Guizhou) was nested into the central group on the ML tree, whereas the western group was nested in the NJ tree. Second, the FMQ population (Minqing, Fu-jian) was nested into the central group on the ML tree, whereas the eastern group was nested in the NJ tree. Finally, compared to the NJ tree, the ML tree had four clear clades for the Central and East groups (Figure 2 and S1).

The results of the cross-validation for the admixture analysis (l. 247) are not presented in Fig S1 or anywhere else in the manuscript or the supplementary material.

Answer: We have added the results of the cross-validation for the admixture analysis. Following the order of description, we left the phylogeny as Figure S1 and added this result as Figure S2.

Figure S2 ADMIXTURE bar plots for the proportion of genetic membership to each ancestry assuming (K = 1 to 10) ancestral populations.

 

The PCA results shown in Fig S2 also have a legend with sample sites codes ordered randomly, with too many colors. At least the most different samples should have their sample sites indicated in the figure.

Answer: Thank you. We have improved this figure (it is now figure S3. The current version is as follows.

Figure S3 PCA (principal component analysis) of 65 individuals of Q. gilva. The cycles with different colours represent the different populations. The details of abbreviation codes for populations showed in Table 2.

 

The authors start the discussion section with a series of contextualization sentences. In this section, it is more important to discuss the authors' findings, so it should begin in l. 297. Information in lines 288 to 297 can be presented in the introduction section.

Answer:  Thank you. We have reorganized this paragraph as follows.

“Our assessment showed that Q. gilva is an endangered (EN) species as per the EN-A4acd+B2ab(ii,iv) criteria. According to our extensive field survey and more than 30 literature sources on Q. gilva, we found that this species has suffered massive population decline and will be facing accelerated declines in the future. During the last 100 years, many natural populations have been logged for industrial timber, agriculture, and economic development. Currently, natural communities dominated by Q. gilva are rare, and most of the existing Q. gilva are scattered in other forest communities with ancient trees. More than 80% of Q. gilva populations occurred in the Fengshui forests or forests surrounding shrines and temples. Most of these populations were very small, or even just individual ancient trees. These populations have no natural regeneration of young adults and seedlings and thus seem to have no future. Therefore, legislation is required to protection of this endangered species, and actively assisted in the restoration of small populations. To date, Q. gilva has been listed as vulnerable (VU) in the Korea Red Data Book [41], endangered (EN) or critically endangered (CR) in several districts of Japan [14], and has also been described as a rare and endangered tree species in China [15]. The assessment results show a large disparity between the local government and the IUCN. Based on our global study, we suggested that the IUCN elevates the threatened category of Q. gilva from LC to EN.”

 

In line 335, the authors state that the analyses support strong bottleneck events due to natural selection. It’s not clear which data lead to this conclusion. There are multiple analyses that can be performed to test for bottlenecks, and for natural selection. Neither of these tests was described in this manuscript.

Answer: Indeed, “Due to natural selection” is not very precise. We have corrected this sentence as follows.

“According to the genetic diversity and LD, the strong bottleneck was detected for the small populations of FZR, ZJN, and JWY.”

Also, please check this reference published in Nature Genetics. Based on the genetic diversity and LD, we could say that populations with low genetic diversity and the LD haven’t dropped to half after strong bottleneck events.

The reference is “Wei et al., Whole-genome resequencing of 445 Lactuca accessions reveals the domestication history of cultivated lettuce. Nature Genetics, https://doi.org/10.1038/s41588-021-00831-0”.

 

Another important analysis to evaluate population stability and extinction risks is the effective population size. This parameter can be estimated from the sample size, coancestry, and inbreeding coefficients (Cockerham 1969, Vencovsky and Crossa 2003).

Answer: Thank you for your suggestions. In this study, we mainly focused on the population survey and genetic diversity. Thus, we haven’t done further analyses of effective population size.

 

The last sentence in the discussion section indicates which populations should be used as seeds source for restoration projects. It is important to highlight limitations and risks when we use seeds from areas with different environmental conditions for restoration purposes. There may be outbreeding depression cases, which were not evaluated in this study.

Answer: Thank you. We have improved this part.

“It is important to highlight limitations and risks when we use seeds from areas with different environmental conditions for restoration purposes. Thus, we will continue to study their adaptive evolution under the climate change in the future, which will give us more detailed guidance on provenance application.”

 

The first two sentences in the conclusion section should be in the introduction (l. 341 - 344).

Answer: Thank you. Our original intention is that these two sentences helped us to lead the conclusion. This will help the readers to get the heart of this study.

 

Cockerham, C. C. (1969). Variance of gene frequencies. Evolution, 72-84.

Marandel, F, Charrier, G, Lamy, J-B, Le Cam, S, Lorance, P, Trenkel, VM. Estimating effective population size using RADseq: Effects of SNP selection and sample size. Ecol Evol. 2020; 10: 1929– 1937. https://doi.org/10.1002/ece3.6016

Nazareno, A.G., Bemmels, J.B., Dick, C.W. and Lohmann, L.G. (2017), Minimum sample sizes for population genomics: an empirical study from an Amazonian plant species. Mol Ecol Resour, 17: 1136-1147. https://doi.org/10.1111/1755-0998.12654

Vencovsky, R., & Crossa, J. (2003). Measurements of representativeness used in genetic resources conservation and plant breeding. Crop Science, 43(6), 1912-1921.

Thank you for these useful references.

Author Response File: Author Response.docx

Reviewer 2 Report

My assessment of this MS is essentially based on two critical issues that could invalidate the scientific robustness of the research.
First of all, the application of the IUCN protocol is highly questionable (most likely wrong for what I see) because several mandatory parameters have not been evaluated by the authors to arrive at this type of evaluation; specifically:
- the EOO value must not be calculated separately (per country) but should be a single value for the entire distribution area of this species (and, in this specific case, considering the distribution type the value should be corrected with the Delaunay triangulation);
- the Future-AOO it is a parameter that is not considered by the IUCN (we should considere a reduction of AOO but applying the criterion A instead of B);
- a continous decline (sensu IUCN) for the species must be demonstrated/justified;
- to support the formula (line 196) you need to calculate and enter the number of locations (sensu IUCN, based on the main threat);
- the letter c in the formula is poorly justified for a tree species as it refers to annual fluctuations in the number of individuals (mainly for annual plants).
Without these adjustments the IUCN evaluation cannot be accepted.

My second point concern the sampling effort for the molecular analysis: in my opinion the number of plants sampled per population is too low to be representative. In this case I would like the authors to support this low number (in relation to a very large global population) with a solid rationale or with adequate reference methodological references.

Author Response

COMMENTS FOR AUTHOR:

Reviewer 2

Comments and Suggestions for Authors

My assessment of this MS is essentially based on two critical issues that could invalidate the scientific robustness of the research.

Answer: We have improved the manuscript and replied your critical issues step by step in the following parts.

 

First of all, the application of the IUCN protocol is highly questionable (most likely wrong for what I see) because several mandatory parameters have not been evaluated by the authors to arrive at this type of evaluation; specifically:

- the EOO value must not be calculated separately (per country) but should be a single value for the entire distribution area of this species (and, in this specific case, considering the distribution type the value should be corrected with the Delaunay triangulation);

Answer: Please check the Table 1. We have listed the EOO for the entire distribution area of this species. The separately value is the information that helps us to understand the distribution status in each country. We used the standard method provided by the IUCN to calculate the EOO.

 

- the Future-AOO it is a parameter that is not considered by the IUCN (we should considere a reduction of AOO but applying the criterion A instead of B);

Answer: Thank you for your suggestion. We have added and improved this part as follows.

“Based on the population status, we estimated that more than 40% of the AOO after three generations (future-AOO) would be lost. Considering the populations without information, we inferred that more than 50% of the AOO would be lost in the next three generations.”

 

- a continous decline (sensu IUCN) for the species must be demonstrated/justified;

Answer: Yes, we have demonstrated that the AOO will be lost more than 50% in the next three generations. Based on the information of indigenous people, almost all the old trees (except individuals located in Fengshui forests and temples) have been deforested during the last 100 years.

 

- to support the formula (line 196) you need to calculate and enter the number of locations (sensu IUCN, based on the main threat);

Answer: We have calculated the number of locations in Table S2.

 

- the letter c in the formula is poorly justified for a tree species as it refers to annual fluctuations in the number of individuals (mainly for annual plants).

Answer: Thank you. We have corrected this part. The current version is “According to the IUCN Red List categories and criteria, the conservation status of Q. gilva is also endangered under the formula EN-B2ab(ii, iv)).” 

 

Without these adjustments the IUCN evaluation cannot be accepted.

Answer: Now, we have improved the results part of this assessment. The current version of this part is as follows.

3.1. Reassessment of Q. gilva

After collating all the distribution data of Q. gilva from different resources, there were a total of 108 known populations in East Asia (68 populations in China, 35 in Japan, and 5 in South Korea). According to the information gathered from indigenous people, almost all old trees (except individuals located in Fengshui forests and temples) have been deforested during the last 100 years. Based on this information, Q. gilva can be listed as endangered as per the EN-A4ad criteria.

Although the EOO of Q. gilva was very high, the area in South Korea was very small. The AOO of Q. gilva was less than 500 km² with the largest being in China (272 km2) and the smallest in South Korea (20 km²). More than half of the known populations have been surveyed in China. Only one large population had more than 500 individuals, and most of the surveyed populations were very small, with fewer than 30 individuals. There were even some occurrences with only one individual (Table 1 and Table S2). Based on the population status, we estimated that more than 40% of the AOO after three generations (future-AOO) would be lost. Considering the populations without information, we inferred that more than 50% of the AOO would be lost in the next three generations. Finally, we estimated that there were fewer than 10,000 individuals within the distribution area of Q. gilva. During the last 100 years, the main threat to Q. gilva in natural populations has been logging and wood harvesting, as it is used as a biological resource. To support the rapid development of society, expansion of land under agriculture, residential use, and transportation infrastructure has also led to the destruction of natural Q. gilva populations. According to our survey, most of the current populations were conserved in the Fengshui forests near the villages, and forests surrounding shrines and temples with severe fragmentation. According to the IUCN Red List categories and criteria, the conservation status of Q. gilva is also determined to be endangered as per the EN-A4c and B2ab(ii, iv) criteria.

 

My second point concern the sampling effort for the molecular analysis: in my opinion the number of plants sampled per population is too low to be representative. In this case I would like the authors to support this low number (in relation to a very large global population) with a solid rationale or with adequate reference methodological references.

Answer: Thank you, I understand your doubt. There is no problem for the sampling with 3 individuals for each population under the whole genome resequencing data. This sampling was based on the adequate methodological references.  

By resampling two to 20 individuals, Nazareno et al. (2017) have demonstrated that relatively small sample size may be sufficient for accurate estimates of genetic diversity. This result is also confirmed in the literature based on resequencing techniques, where some populations are analyzed based three or even fewer individuals (Huang et al., 2018; An et al., 2020; Lam et al., 2010; Guan et al., 2022). Meanwhile, the high mapping rate and coverage of resequencing technology also ensure the quality and accuracy of genetic loci (Hu et al., 2021). Our study showed the average mapping rate of 89.5% with the reference genome and the average coverage rate of 10x, which greatly improved the quality of SNP and the accuracy of analysis.

References:

An, Y., Mi, X., Zhao, S., Guo, R., Xia, X., Liu, S., & Wei, C. (2020). Revealing Distinctions in Genetic Diversity and Adaptive Evolution Between Two Varieties of Camellia sinensis by Whole-Genome Resequencing. Frontiers in plant science, 11, 603819.

Huang, L., Yang, M., Li, L., Li, H., Yang, D., Shi, T., & Yang, P. (2018). Whole genome re-sequencing reveals evolutionary patterns of sacred lotus (Nelumbo nucifera). Journal of integrative plant biology, 60(1), 2-15.

Hu, T., Chitnis, N., Monos, D., & Dinh, A. (2021). Next-generation sequencing technologies: An overview. Human Immunology, 82(11), 801–811.

Nazareno, A. G., Bemmels, J. B., Dick, C. W., & Lohmann, L. G. (2017). Minimum sample sizes for population genomics: an empirical study from an Amazonian plant species. Molecular Ecology Resources, 17(6), 1136-1147.

Guan, X., Zhao, S., Xiang, W., Jin, H., Chen, N., Lei, C., ... & Xu, L. (2022). Genetic Diversity and Selective Signature in Dabieshan Cattle Revealed by Whole-Genome Resequencing. Biology, 11(9), 1327.

Lam, H. M., Xu, X., Liu, X., Chen, W., Yang, G., Wong, F. L., ... & Zhang, G. (2010). Resequencing of 31 wild and cultivated soybean genomes identifies patterns of genetic diversity and selection. Nature genetics, 42(12), 1053-1059.

Reviewer 3 Report

Quercus gilva owns landscaping, ecological and commercial values. It is benefit to the genetic resources conservation and innovation that population investigation and genetic diversity analyses by SNPs is conducted. The subject of this paper is appropriate for publication in Diversity and the work represents a new, original, and interesting contribution. The structure and organization of the paper appear to be sound in general, but some aspects should be clarified and/or more thoroughly explained and justified (see below).  Materials and methods section  The sample size of individuals is small in each population. How many population do you conduct field survey? Why do you select 22 population for genetic diversity analyses?  Results section In table 1  The total number of populations and NISS are false. Do the SNPs for phylogenetic and population structure analyses distribute evenly in each chromosome? What is the mean of samples codes in Figure 2? Why is the population codes used? How was the conclusion that the evolutionary path of Q. gilva was from southwest to east of China, and then to Japan and South Korea deduced?

Author Response

COMMENTS FOR AUTHOR:

Reviewer 3

Comments and Suggestions for Authors

Quercus gilva owns landscaping, ecological and commercial values. It is benefit to the genetic resources conservation and innovation that population investigation and genetic diversity analyses by SNPs is conducted. The subject of this paper is appropriate for publication in Diversity and the work represents a new, original, and interesting contribution. The structure and organization of the paper appear to be sound in general, but some aspects should be clarified and/or more thoroughly explained and justified (see below).

Thank you for your positive evaluation.

 

Materials and methods section

The sample size of individuals is small in each population. How many populations do you conduct field survey? Why do you select 22 population for genetic diversity analyses?

Answer: First, I understand your doubt for the sampling of each population. There is no problem for the sampling with 3 individuals for each population under the whole genome resequencing data. This sampling was based on the adequate methodological references. Second, there are totally 40 populations where have conducted field survey in the mainland of China. We have added this information in the Methods. Third, the selected 22 populations used for genetic diversity analyses were based on the following rules. We tried our best to collect all the populations of Q. gilva. We totally collected 46 populations of Q. gilva. Then, if the populations were collected from the same city, we only used one population. If the number of individuals in a given population was smaller than five, we will filtered this population. Finally, we used 22 populations for our analyses.

 

Results section

In table 1  The total number of populations and NISS are false.

Answer: Thank you. We have corrected the total number of populations. The correct total number of populations is 108. There is a mistake of the NISS in China and the total number. We have corrected it to 23 populations in China of NISS and 46 populations for the total of NISS.

Do the SNPs for phylogenetic and population structure analyses distribute evenly in each chromosome?

Answer: Yes, the SNPs distribute evenly in each chromosome.

What is the mean of samples codes in Figure 2?

Answer: The mean of samples codes is the code of each individual. According to the suggestion of reviewer 1, we have changed now the samples codes to the population codes.

 

Why is the population codes used?

The population codes could improve the readability of the tables and figures.

 

How was the conclusion that the evolutionary path of Q. gilva was from southwest to east of China, and then to Japan and South Korea deduced?

Answer: Based on the phylogenomic trees, we could conclude that the populations from southwest of China are closer to the outgroup, followed by the populations from central and East of China, and the populations from South Korea and Japan are located at the very end of the phylogenetic tree.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

All comments and questions from the review were addressed by the authors. I would like to thank them for the suggested readings. 

Author Response

All comments and questions from the review were addressed by the authors. I would like to thank them for the suggested readings. 

Answer: Thank you for your positive reflect.

Reviewer 2 Report

Dear Authors,

I appreciated the work done to improve the MS; however, some points remain which need to be addressed.

In order to apply the EN category there must be a maximum of 5 locations (sensu IUCN); in Table S2 there are >100 "locations" (and this does not allow assigning the EN category), but I doubt that these have not been identified following the IUCN protocol ("The term ‘location’ defines a geographically or ecologically distinct area in which a single threatening event can rapidly affect all individuals of the taxon present", IUCN Guidelines 2022, p. 61). 

In the result section, please, present the total values of EOO and AOO, the separation on a country-level is a different thing (which concerns the distribution more than the concepts of EOO and AOO).

Author Response

Responses to Reviewers

Manuscript ID “diversity-2095823”

“Population survey combined with genomic-wide genetic variation unravel the endangered status of Quercus gilva”.

Our itemized answers and remarks are highlighted in blue.

 

COMMENTS FOR AUTHOR:

Reviewer 2

Comments and Suggestions for Authors

I appreciated the work done to improve the MS; however, some points remain which need to be addressed.

Answer: Thank you for your suggestions.

 

In order to apply the EN category there must be a maximum of 5 locations (sensu IUCN); in Table S2 there are >100 "locations" (and this does not allow assigning the EN category), but I doubt that these have not been identified following the IUCN protocol ("The term ‘location’ defines a geographically or ecologically distinct area in which a single threatening event can rapidly affect all individuals of the taxon present", IUCN Guidelines 2022, p. 61).

Answer: Thank you. We have checked here. This part related to the EN-Ba. In the condition “a”, there are two demands joined with “or” (Severely fragmented OR Number of locations). It means the assessment just accorded with one of them is enough. For the assessment Quercus gilva, we used the “severely fragmented”  as our criteria. We haven’t used the criteria that a maximum of 5 locations for the EN category.

We described this part in the results as follow. “According to our survey, most of the current populations were conserved in the Fengshui forests near the villages, and forests surrounding shrines and temples with severe fragmentation.”

 

In the result section, please, present the total values of EOO and AOO, the separation on a country-level is a different thing (which concerns the distribution more than the concepts of EOO and AOO).

Answer: The total values of EOO and AOO were calculated based on all the GPS information of Quercus gilva. The values of country-based EOO and AOO just help us to understand the distribution of this species in each country.