Genetic Diversity of Five Pelodiscus sinensis Sub-Populations in the Dongting Lake Basin Based on Cytb and 12S rRNA Markers
, Hewei Xiao 1,†, Qifan Wu 1, Lu Tian 1, Feng Gao 1, Cheng Li 1, Jiajia Ni 2, Zhitao Peng 1,* and Jin Xiang 1,*
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors Dear Editor, Here is a review with general and specific comments for the manuscript entitled “Genetic Diversity of Five Pelodiscus sinensis Populations in the Dongting Lake Basin Based on Cytb and 12S rRNA Genes”, by Zuo et. al. General commentsThe manuscript is based on analyses of two mitochondrial markers, Cytb (659 bp) and 12S rRNA (406 bp), to investigate the genetic diversity and structure of five geographically distinct wild populations of the Chinese soft-shell turtle (Pelodiscus sinensis) in the Dongting Lake basin. The work aims to provide fundamental insights into the conservation and sustainable utilization of these turtle populations. The study involved alignment, genetic diversity indices, phylogenetic trees, haplotype network diagrams, AMOVA, and neutrality tests (Tajima's D and Fu's Fs) using sequences from 71 turtles sampled from five sites across the Dongting Lake Basin: Junshan (JS), Hanshou (HS), Yuanjiang (YJ), Shaoyang (SY), and Hengyang (HY). The study suggests that all populations, except SY, showed high genetic diversity. The SY population, likely impacted by habitat fragmentation and human influence, showed notably lower diversity. Most population pairs showed weak or no differentiation, with high gene flow indicating strong genetic connectivity.
Although the use of two mitochondrial markers may seem old-fashioned today, given the prevalence of whole mitochondrial genome sequencing, multilocus analysis, and NGS technologies, I still believe these analyses are valid and that the manuscript could be worth publishing. It is noteworthy, however, that several studies on the same species and with the same markers are available, which the authors could have used to strengthen their sample size, both in terms of N and geographic range. Some population samples are relatively small (n = 10), which may affect the resolution of rare alleles and, in particular, the interpretation of mismatch distribution analysis. Wider sampling (with data obtained from NCBI) across China and East Asia could provide a broader context. Because of this, conclusions about high genetic diversity, population homogeneity, and risk of diversity decline should be considered with caution. Furthermore, if the findings are used as a genetic baseline for resource protection and sustainable aquaculture development in this economically important species, it is essential to consider them as preliminary. Specific Comments:
Line 1-2: In the title, you could change the word genes to markers, so that “genetic” and “genes” are similar words.
Lines 10 to 13: Please correct email address
Line 17. Please change haplatypes to haplotypes. On the same line, please change 6 by six.
Lines 21-23. This paragraph is confusing: what does “was significantly different from the other populations, despite belonging to the same population” mean? which means “Genetic variation within the P. sinensis population was much higher than that between populations. Was intrapopulation variation greater than interpopulation variation?” Please clarify this confusing paragraph. Line 32: Please change “…in the Trinychidae family” to “…belonging to the Trinychidae family.”
Line 48. Please indicate the current status of the Chinese soft-shell turtle (Vulnerable).
Lines 68-73. All the information presented here can be used to strengthen the analyses, as noted in the general comments.
Line 138. The comparison with teleost fish seems misguided. There are many turtle species with which you can compare your results.
Lines 140-162. I have concerns about the use of the concept of loci, as you are using only mitochondrial markers; therefore, strictly speaking, you are using a single locus. When using S, please refer to segregating sites, which is the correct meaning. Line 222. Haplotype network analysis is not used to observe phylogenetic patterns, but rather genetic and geographic associations.
Line 234. Just a suggestion: population dynamics analyses can be greatly enriched with Bayesian skyline plots. Please consider all these comments as constructive, and they could greatly help you improve the manuscript for publication. Comments on the Quality of English Language
The quality of english language must be improved
Author Response
Comment
Dear Editor, Here is a review with general and specific comments for the manuscript entitled “Genetic Diversity of Five Pelodiscus sinensis Populations in the Dongting Lake Basin Based on Cytb and 12S rRNA Genes”, by Zuo et. al.
The manuscript is based on analyses of two mitochondrial markers, Cytb (659 bp) and 12S rRNA (406 bp), to investigate the genetic diversity and structure of five geographically distinct wild populations of the Chinese soft-shell turtle (Pelodiscus sinensis) in the Dongting Lake basin. The work aims to provide fundamental insights into the conservation and sustainable utilization of these turtle populations. The study involved alignment, genetic diversity indices, phylogenetic trees, haplotype network diagrams, AMOVA, and neutrality tests (Tajima's D and Fu's Fs) using sequences from 71 turtles sampled from five sites across the Dongting Lake Basin: Junshan (JS), Hanshou (HS), Yuanjiang (YJ), Shaoyang (SY), and Hengyang (HY). The study suggests that all populations, except SY, showed high genetic diversity. The SY population, likely impacted by habitat fragmentation and human influence, showed notably lower diversity. Most population pairs showed weak or no differentiation, with high gene flow indicating strong genetic connectivity.
Response
Thank you very much for reviewing our manuscript and providing very insightful comments. These comments are very important for us to revise the manuscript and improve its readability. We have revised our manuscript according to these comments.
Comment
Although the use of two mitochondrial markers may seem old-fashioned today, given the prevalence of whole mitochondrial genome sequencing, multilocus analysis, and NGS technologies, I still believe these analyses are valid and that the manuscript could be worth publishing. It is noteworthy, however, that several studies on the same species and with the same markers are available, which the authors could have used to strengthen their sample size, both in terms of N and geographic range. Some population samples are relatively small (n = 10), which may affect the resolution of rare alleles and, in particular, the interpretation of mismatch distribution analysis. Wider sampling (with data obtained from NCBI) across China and East Asia could provide a broader context. Because of this, conclusions about high genetic diversity, population homogeneity, and risk of diversity decline should be considered with caution. Furthermore, if the findings are used as a genetic baseline for resource protection and sustainable aquaculture development in this economically important species, it is essential to consider them as preliminary.
Response
Thank you very much for your comments. Although whole mitochondrial genome sequencing, multilocus analysis, and genome NGS technologies have been used to analyze genetic polymorphisms in Chinese soft-shelled turtles, the data available at present cannot be called population genetic diversity studies, and only a small number of individuals are analyzed. We performed supplementary analysis with some sequences of the cytb gene supplemented according to your comment (Figure S1), but since most studies reported that cytb and 12S rDNA sequences were not available, we did not perform further supplementary analysis of the 12S rDNA sequences.
Comment
Line 1-2: In the title, you could change the word genes to markers, so that “genetic” and “genes” are similar words.
Response
We have revised the word genes to markers according to your comment.
Comment
Lines 10 to 13: Please correct email address
Response
We have corrected the email address according to your comment.
Comment
Line 17. Please change haplatypes to haplotypes. On the same line, please change 6 by six.
Response
We have revised our manuscript according to your comment.
Comment
Lines 21-23. This paragraph is confusing: what does “was significantly different from the other populations, despite belonging to the same population” mean? which means “Genetic variation within the P. sinensis population was much higher than that between populations. Was intrapopulation variation greater than interpopulation variation?” Please clarify this confusing paragraph.
Response
Thank you for your comment. It should be “The Junshan sub-population was significantly different from the other sub-populations (P<0.05), and gene exchange was weak, despite belonging to the same population. Genetic variation within the P. sinensis sub-population was much higher than that between sub-populations.” We have revised the description.
Comment
Line 32: Please change “…in the Trinychidae family” to “…belonging to the Trinychidae family.”
Response
We have revised the sentence according to your comment.
Comment
Line 48. Please indicate the current status of the Chinese soft-shell turtle (Vulnerable).
Response
Thank you very much for your comment. We have revised the status according to your comment.
Comment
Lines 68-73. All the information presented here can be used to strengthen the analyses, as noted in the general comments.
Response
Thank you for your comment. We have used the Cytb data reported by Xiong et al. to supplementary analysis. However, we were unable to obtain data reported by Lu et al.
Comment
Line 138. The comparison with teleost fish seems misguided. There are many turtle species with which you can compare your results.
Response
Thank you for your comment. We have revised our manuscript according to your comment.
Comment
Lines 140-162. I have concerns about the use of the concept of loci, as you are using only mitochondrial markers; therefore, strictly speaking, you are using a single locus. When using S, please refer to segregating sites, which is the correct meaning.
Response
Thank you very much for your comments. We fully agree with your comments. We have revised our manuscript according to your comment.
Comment
Line 222. Haplotype network analysis is not used to observe phylogenetic patterns, but rather genetic and geographic associations.
Response
Thank you very much for your comment. We have revised the error according to your comment.
Comment
Line 234. Just a suggestion: population dynamics analyses can be greatly enriched with Bayesian skyline plots. Please consider all these comments as constructive, and they could greatly help you improve the manuscript for publication.
Response
Thank you very much for your comment. Although Bayesian skyline plots are widely used in population dynamics analyses, we believe that our current analyses are enough. In order not to complicate the problem, we have not provided Bayesian skyline plots. But we still appreciate your comment.
Comments on the Quality of English Language
The quality of english language must be improved
Response
Thank you very much for your comment. We have improved the quality of English language according to your comment.
Reviewer 2 Report
Comments and Suggestions for AuthorsZuo et al.'s paper, "Genetic Diversity of Five Pelodiscus sinensis Populations in the Dongting Lake Basin Based on Cytb and 12S rRNA Genes," analyzes the mitochondrial DNA diversity of the P. sinensis turtle from a Chinese region. I recommend against its publication in Animals because its scope is too limited and the presentation is of insufficient quality.
The paper examines only five populations of P. sinensis from a relatively small geographic region. This sample size is limited compared to studies such as Fritz et al. (2010). While this limitation could potentially be offset by methodological depth, a study relying solely on two mitochondrial genes represents outdated practice in current phylogenetic research. The analysis might yield interesting insights if the populations showed substantial divergence, but they appear to be both recently diverged and connected within the same water system, resulting in minimal detectable differences.
Regarding the analysis, portions of the paper suggest the authors may be unfamiliar with standard terminology or experiencing translation difficulties. For example: "A total of 25 nucleotide variation loci (S) were detected in the Cytb gene of the five soft-shell turtle populations, accounting for 3.79% of the total loci. This included five single information loci and 20 abbreviated information loci. All variation loci were either transformation or transversion, with a ratio of 6:1." The term "transformations" likely refers to transitions. Similarly, "single information loci" and "abbreviated information loci" appear to be non-standard terminology—while the intended meaning can be inferred, these phrases are not established terms in the scientific literature.
The paper's conclusions are also questionable. The authors state: "In this study, the average values of Hd and Pi for the Cytb and 12S rRNA genes in the five populations were 0.750 and 0.609, and 0.016 and 0.010, respectively. These values indicate that the genetic diversity of Chinese soft-shell turtle populations was relatively high (Hd >0.5, Pi >0.005) [30], and that they have strong breeding potential." From my perspective, Hd values of 0.75 and Pi values of 0.01 actually indicate relatively low genetic diversity. While these indices are admittedly taxon-specific, the authors' interpretation appears overly optimistic.
Overall, I recommend rejecting this paper. Even if all linguistic and terminological issues were addressed, the fundamental problems of limited data and weak conclusions would remain.
Author Response
Comment 1: Zuo et al.'s paper, "Genetic Diversity of Five Pelodiscus sinensis Populations in the Dongting Lake Basin Based on Cytb and 12S rRNA Genes," analyzes the mitochondrial DNA diversity of the P. sinensis turtle from a Chinese region. I recommend against its publication in Animals because its scope is too limited and the presentation is of insufficient quality.
Response: Thank you for your comment. We did not submitted our manuscript to Animals. We submitted our manuscript to Diversity for considering publishing. We believe that our study is in line with the scope of Diversity, and we believe that it is of great significance to follow up and investigate the population genetic diversity of P. sinensis in time to protect the wild populations of P. sinensis.
Comment 2: The paper examines only five populations of P. sinensis from a relatively small geographic region. This sample size is limited compared to studies such as Fritz et al. (2010). While this limitation could potentially be offset by methodological depth, a study relying solely on two mitochondrial genes represents outdated practice in current phylogenetic research. The analysis might yield interesting insights if the populations showed substantial divergence, but they appear to be both recently diverged and connected within the same water system, resulting in minimal detectable differences.
Response: Thank you for your comment. We admit that the geographic region of our study is smaller than that of Fritz et al. (2010, DOI: 10.1007/s13127-010-0007-1), but our sample size is not smaller than that of Fritz et al. (2010). Moreover, although the geographic region studied by Fritz et al. (2010) is larger than ours, they only collected a small number of samples at multiple sampling sites, which did not reflect the diversity of the population, and they did not study the genetic diversity of the Pelodiscus sinensis population, but the diversity at the level of the genus Pelodiscus. Furthermore, although whole mitochondrial genome sequencing, multilocus analysis, and genome NGS technologies have been used to analyze genetic polymorphisms in Chinese soft-shelled turtles, the data available at present cannot be called population genetic diversity studies, and only a small number of individuals are analyzed. Most genetic diversity studies of the Pelodiscus sinensis populations are still used traditional single marker analysis.
Comment 3: Regarding the analysis, portions of the paper suggest the authors may be unfamiliar with standard terminology or experiencing translation difficulties. For example: "A total of 25 nucleotide variation loci (S) were detected in the Cytb gene of the five soft-shell turtle populations, accounting for 3.79% of the total loci. This included five single information loci and 20 abbreviated information loci. All variation loci were either transformation or transversion, with a ratio of 6:1." The term "transformations" likely refers to transitions. Similarly, "single information loci" and "abbreviated information loci" appear to be non-standard terminology—while the intended meaning can be inferred, these phrases are not established terms in the scientific literature.
Response: Thank you very much for your comment. We have revised the academic terminologies according to your comment.
Comment 4: The paper's conclusions are also questionable. The authors state: "In this study, the average values of Hd and Pi for the Cytb and 12S rRNA genes in the five populations were 0.750 and 0.609, and 0.016 and 0.010, respectively. These values indicate that the genetic diversity of Chinese soft-shell turtle populations was relatively high (Hd >0.5, Pi >0.005) [30], and that they have strong breeding potential." From my perspective, Hd values of 0.75 and Pi values of 0.01 actually indicate relatively low genetic diversity. While these indices are admittedly taxon-specific, the authors' interpretation appears overly optimistic.
Response: Thank you for your comment. As description by Grant et al. (1998), Hd >0.5, Pi >0.005 indicated a relatively high genetic diversity. If you have more reference to show the standard is unreasonable, we very much welcome you to provide it to us.
Comment 5: Overall, I recommend rejecting this paper. Even if all linguistic and terminological issues were addressed, the fundamental problems of limited data and weak conclusions would remain.
Response: Thank you very much for your comment. We have performed supplementary analysis with some sequences of the cytb gene from Xiong et al. (2019). Moreover, although the genetic diversity of Chinese soft-shelled turtle populations has been reported multiple times, these studies rarely provide accurate population-level Cytb and 12S rDNA sequences, although a small number of mitochondrial whole genomes have been reported in Chinese soft-shelled turtle samples.
Reviewer 3 Report
Comments and Suggestions for AuthorsGenetic Diversity of Five Pelodiscus sinensis Populations in the Dongting Lake Basin Based on Cytb and 12S rRNA Genes by Zou et al.
The study explained the genetic diversity of soft-shelled turtle in five populations by using markers of Cytb and 12S rRNA genes.
They obtained the DNAs and processed with PCR and get the sequences of the 71 individuals. The results were analysed via ANOVA and relevant tests. The results presented in meaningful way and heatmap diagrams and network diagrames were also produced.
I anly have a comment on the mismatch graphs wheter they should be presented in the manuscript or not. I would suggest them to provide as suplement.
The rest of the results were presented well and discussed with relevant literature.
Author Response
Comment
Genetic Diversity of Five Pelodiscus sinensis Populations in the Dongting Lake Basin Based on Cytb and 12S rRNA Genes by Zou et al.
The study explained the genetic diversity of soft-shelled turtle in five populations by using markers of Cytb and 12S rRNA genes.
They obtained the DNAs and processed with PCR and get the sequences of the 71 individuals. The results were analysed via ANOVA and relevant tests. The results presented in meaningful way and heatmap diagrams and network diagrames were also produced.
I only have a comment on the mismatch graphs whether they should be presented in the manuscript or not. I would suggest them to provide as supplement.
The rest of the results were presented well and discussed with relevant literature.
Response
Thank you very much for reviewing our manuscript. We have removed the mismatch graphs to Supplemental Materials (Fig. S1). Thank you for your comment.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsDear Editor, I have revised the second version of the manuscript by Zhiliang Zuo et al. I appreciate you considering the comments made on the first version. This second version is much improved. The authors respond clearly and with arguments to each of the comments. There is only minor details that remain, as detailed below.
1) Lines 120 and 141: Notice that it must be just "segregating sites (S)", not "nucleotide segregating sites"
2) lines 113-124. I noticed that there are no citations for ContigExpress 9.1.0, ClustalW, MEGA11, DNAsp, and Arlequin. However, I am unsure whether the Diversity journal requires or considers it necessary to cite the authors of each software.
Author Response
Comment
1) Lines 120 and 141: Notice that it must be just "segregating sites (S)", not "nucleotide segregating sites"
Comment
Thank you for your comment. We have revised our manuscript according to your comment.
Comment
2) lines 113-124. I noticed that there are no citations for ContigExpress 9.1.0, ClustalW, MEGA11, DNAsp, and Arlequin. However, I am unsure whether the Diversity journal requires or considers it necessary to cite the authors of each software.
Response
Thank you for your comment. We have added the citations for ContigExpress 9.1.0, ClustalW, MEGA11, DNAsp, and Arlequin according to your comment.
Reviewer 2 Report
Comments and Suggestions for AuthorsI have reviewed the revised manuscript and the authors' responses. While I still believe the paper has limitations, the authors have adequately addressed all comments. Therefore, if the Editor deems this paper acceptable, I have no objections to its acceptance.
Author Response
Comment
I have reviewed the revised manuscript and the authors' responses. While I still believe the paper has limitations, the authors have adequately addressed all comments. Therefore, if the Editor deems this paper acceptable, I have no objections to its acceptance.
Response
Thank you very much for your comment. We admit that our manuscript still has limitations. Especially, we did not use genome sequencing / mitochondrial genome sequencing technologies for genetic diversity analysis. We appreciate your determination to apply the most advanced technology to scientific research, which will undoubtedly get more accurate information. However, due to the limitation of funds and experimental conditions, omics sequencing technologies has not been widely used in large-scale population genetic diversity analysis. But we do not deny that this is an important trend in the future. Therefore, we are still grateful for your comments and will consider using them for reference in future study.
