Genetic Variation of Schizothorax wangchiachii Populations Between the Jinsha and Yalong Rivers Using Simplified Genome Sequencing
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors1. The sample sizes for all populations are too small, with the largest group containing only ten individuals. Consequently, the conclusions of this study may not accurately reflect the genetic information among populations.
2. The formatting of three-line tables (e.g., Table 1, Table 2) does not conform to standard conventions.
3. While the authors employed genotyping-by-sequencing (GBS) for SNP detection, they did not justify why this method was chosen over whole-genome resequencing. Furthermore, the suitability of GBS for Schizothorax species and the potential genotyping biases due to missing data were not discussed.
4. The specific thresholds used for SNP filtering—such as minor allele frequency, missing rate, and sequencing depth—were not detailed. It is recommended to supplement the Methods section to ensure the reproducibility of the results.
5. Principal component analysis (PCA) revealed no clear population structure (PC1 and PC2 together explained only 6.41% of the variance), whereas ADMIXTURE and neighbor-joining (NJ) tree analyses indicated some degree of clustering among populations. The authors should further explore this inconsistency.
6. In the TreeMix analysis, the selection of the number of migration events lacks sufficient justification. Likelihood values or statistical criteria such as AIC were not provided; it is recommended to include the model selection process.
7. Figure 2 provides incomplete information on SNP types and distribution. The exact transition/transversion ratio is not presented, and the chromosomal density plots (C and D) lack axis labels. More detailed captions are needed.
8. The gene flow heatmap in Figure 5 is difficult to interpret due to the absence of a color scale legend and unclear indications of migration direction and intensity. Adding a legend and annotations is advised.
9. The analysis of the causes underlying the decline in genetic diversity is insufficient. Although human activities such as hydropower development and overfishing are suggested as potential factors, direct evidence—such as historical fishing pressure or habitat destruction in the study area—is lacking.
10. The inferred causal relationship between the uplift of the Qinghai–Tibet Plateau and population historical dynamics is weakly supported. The temporal correlation between changes in effective population size (Nâ‚‘) and plateau uplift events lacks robust geochronological evidence.
11. The formatting of some references does not comply with journal guidelines (e.g., page numbers, author abbreviations). It is recommended to unify the citation style according to the journal’s requirements.
12. The Conclusion section is overly general and does not clearly outline the specific implications of this study for conservation strategies. It is suggested to integrate findings such as low genetic diversity and restricted gene flow to propose concrete conservation recommendations.
Author Response
Response to Reviewer 1
Comment 1. The sample sizes for all populations are too small, with the largest group containing only ten individuals. Consequently, the conclusions of this study may not accurately reflect the genetic information among populations.
Response 1: Thanks for your constructive suggestions. We agree with your point of view that the small sizes of populations may not completely reflect the genetic information. In this study, the individuals were collected from wild resources. The Chinese Ministry of Agriculture and Rural Affairs has issued a ten-year fishing ban plan for the Yangtze River from 2020 to 2030 years. Thereby within the limited time frame allowed (from 2021 to 2023 years), we were unable to collect a wider variety of samples. This is our responsibility. Thus, the limitation of this study has been presented in “5. Conclusion” part (Lines 701-704).
Comment 2. The formatting of three-line tables (e.g., Table 1, Table 2) does not conform to standard conventions.
Response 2: The formatting of Tables has been modified in the revised manuscript.
Comment 3. While the authors employed genotyping-by-sequencing (GBS) for SNP detection, they did not justify why this method was chosen over whole-genome resequencing. Furthermore, the suitability of GBS for Schizothorax species and the potential genotyping biases due to missing data were not discussed.
Response 3: The background of genotyping-by-sequencing (GBS) has been added in “1. Introduction” part in this study (Lines 106-125).
Comment 4. The specific thresholds used for SNP filtering—such as minor allele frequency, missing rate, and sequencing depth—were not detailed. It is recommended to supplement the Methods section to ensure the reproducibility of the results.
Response 4: The method used for SNP filtering has been added in the revised manuscript (Lines 180-187).
Comment 5. Principal component analysis (PCA) revealed no clear population structure (PC1 and PC2 together explained only 6.41% of the variance), whereas ADMIXTURE and neighbor-joining (NJ) tree analyses indicated some degree of clustering among populations. The authors should further explore this inconsistency.
Response 5: In our study, 75 individuals from 10 populations was not distinct differentiation structure in PCA analysis. Then, based on admixture K value, 75 individuals were grouped into a single genetic cluster only when K=1 as the optimal value. In addition, NJ tree analyses indicated some degree of clustering among populations. We have discussed the reason in Discussion part (Lines 540-571).
Comment 6. In the TreeMix analysis, the selection of the number of migration events lacks sufficient justification. Likelihood values or statistical criteria such as AIC were not provided; it is recommended to include the model selection process.
Response 6: The detailed description has been added to the text (Lines 221-226, 231-232).
Comment 7. Figure 2 provides incomplete information on SNP types and distribution. The exact transition/transversion ratio is not presented, and the chromosomal density plots (C and D) lack axis labels. More detailed captions are needed.
Response 7: The exact counts and transition/transversion ratio have been added to“3.2 Clustering analysis of differential SNPs”part (Lines 253-254). The new Figure 2 has been redrawn.
Comment 8. The gene flow heatmap in Figure 5 is difficult to interpret due to the absence of a color scale legend and unclear indications of migration direction and intensity. Adding a legend and annotations is advised.
Response 8: The detailed legend and annotations have been added to the revised manuscript (Lines 382-384).
Comment 9. The analysis of the causes underlying the decline in genetic diversity is insufficient. Although human activities such as hydropower development and overfishing are suggested as potential factors, direct evidence—such as historical fishing pressure or habitat destruction in the study area—is lacking.
Response 9: The short-scaled Schizothorax wangchiachii is an endemic fish species in the upper reaches of the Yangtze River, primarily distributed in the Jinsha River and Yalong River basins [1, 2]. As a food source, this fish is fresh and palatable, making it a major target for capture fisheries [1-3]. A resource survey conducted from 2004 to 2011 revealed that the development and construction of cascade hydropower projects in the Jinsha and Yalong River basins have led to rapid changes in the aquatic ecological environment of the region. Furthermore, regarding the observed low level of genetic diversity among geographical populations of S. wangchiachii compared to other schizothoracin species, historical population dynamics under the influence of glacial cycles and geological events—such as recent population expansion—likely constitute key factors contributing to its reduced genetic diversity. Particularly for schizothoracin fishes distributed in the Hengduan Mountain region of the Qinghai-Tibet Plateau, which are cold-water adapted, glacial-interglacial cycles and geological events have significantly shaped genetic variation and population history [4].
The above comments have added to the revised text. Please check lines 474-487, 523-529.
References:
- Yan, W.B.; Zhu, T.B.; Wu, X.B.; Yang, D.G.; Chen, L. An observation of spawning behavior of Schizothorax wangchiachii. Freshw.Fish. 2017, 47, 7.
- Zhang, S.L.; Zhang, J.B.; Qiao, Y.; Wang, L.T.; Hou, Y.Q. Experimental study on aerobic swimming performance and behavior ofSchizothorax wangchiachii J. Hydrol. 2016, 37, 56–62.
- Gao, S.B.; Tang, H.Y.; Qiao, Y.; Yang, Z.; Chen, J.S. Status of fishery resources in the mainstream of the lower reaches of Jinsha J. Hydrol. 2013, 34, 44–49.
- Chen W, Du K, He S. Genetic structure and historical demography of Schizothorax nukiangensis (Cyprinidae) in continuous habitat.[J]. Ecology & Evolution, 2015, 4, 984-995.
Comment 10. The inferred causal relationship between the uplift of the Qinghai–Tibet Plateau and population historical dynamics is weakly supported. The temporal correlation between changes in effective population size (Nâ‚‘) and plateau uplift events lacks robust geochronological evidence.
Response 10: In our study, the population evolutionary history of S. wangchiachii is highly consistent with the late stage of the Extensive Glacial Period (EGP, 0.5-0.17 Ma) during the Quaternary. The Qinghai-Tibet Plateau experienced four to five distinct glacial cycles throughout the Quaternary, including not only the EGP but also the Last Glacial Period (LGP, 0.08-0.01 Ma) and the Last Glacial Maximum (LGM, 0.021-0.017 Ma) [1-3]. Population expansion events of this species were primarily concentrated in the later phases of the EGP, LGP, and LGM, or during the subsequent interglacial periods. This temporal congruence suggests that Quaternary glacial-interglacial cycles were likely a key driver of population dynamics in S. wangchiachii. Above comments have been added to the revised manuscript (Lines 596-607).
References:
- Jing, M.C.; Yang, G.L.; Sun, N.D. Study on the climatic changes between the last interglacial age and the last glacial age recorded by ostracoda in eastern Qaidam Basin. Journal of Earth Sciences and Environment, 2024, 26, 83-87.
- Yi, C.L.; Cui, Z.J.; Xiong, H.G. Numerical periods of Quaternary glaciations in China. Quaternary Sciences, 2005, 25, 609-619.
- Li, X.L.; Yang, K.; Tong, L.T.; Hou, F.X.; Liu, Q.; Li, Q.; Li, J.X.; Lu, Y.; Song, Z.B. Zhaobin Song . Phylogeography of Schizopygopsis malacanthus Herzenstein (Cypriniformes, Cyprinidae) in relation to the tectonic events and Quaternary climatic oscillations in the Shaluli Mountains Region[J]. Zoology (Jena, Germany), 2020, 143,
Comment 11. The formatting of some references does not comply with journal guidelines (e.g., page numbers, author abbreviations). It is recommended to unify the citation style according to the journal’s requirements.
Response 11: The styles of references in the text have been revised.
- The Conclusion section is overly general and does not clearly outline the specific implications of this study for conservation strategies. It is suggested to integrate findings such as low genetic diversity and restricted gene flow to propose concrete conservation recommendations.
Response 12: The conservation recommendations of S. wangchiachii have been added to the text (Lines 683-700).
Reviewer 2 Report
Comments and Suggestions for AuthorsI recommend acceptance pending modest revision. The paper is a clear and well structured study of the population structure of Schizothorax wangchiachii, an economically valuable species in an interesting system draining the eastern Tibetan Plateau. The results demonstrate lower genetic diversity and differentiation than generally would be expected, but some populational differentiation and evidence of some local genetic adaptation. I look forward to the publication of this manuscript.
I have one more general comment requiring more substantial addition.
The abstract and conclusion discuss the value of the research to current conservation and management of the species. The discussion does not directly address this as a coherent area. The results show things that might suggest different interpretations in a conservation or management context: lower genetic difersity and differentiation, but detectable differentiation in three populations. Although considered a panmictic population, the higher Fst value for one upstream population and phylogenetic population-structure analysis suggest the upstream BD, GT, and SWL populations are genetically more distinct. Should this distinctness merit more conservation attention, or treatment of these populations as separate management units? You mention the hydropower barriers in this region too. Does the lower genetic variation among populations elsewhere suggest the need for management as a single large population? Does the distribution of likely high altitude-associated genes suggest more consideration be given to local adaptation or suggest that human movement of individuals between some areas could be problematic? Do you consider the panmixis enough that the more distinct populations don't need separate conservation or management consideration? Does their lower genetic diversity require more or less conservation or management consideration? Overall, what do your results taken together suggest for future conservation and management?
Smaller-scale revisions:
Simple Summary
Lines 10-12, 14-17. The first and last two sentences of the Simple Summary are unclear in what they are saying. They need to be substantially revised for clarity.
Abstract
Line 20. Change to "with large population sizes."
Lines 21-23. Change to "production are. We used genotype sequencing (GBS) technology to analyze the genetic diversity and population structure of 10 wild populations in the Jinsha River and Yalong River basins of the upper Yangtze River." -I think the authors used the prior structure (that was difficult to understand) to try to maintain passive voice. Active voice is fine to use in scientific publications in the biological sciences and is often required in the most high-profile journals. When it increases clarity, it definitely can and should be used.
Line 27. Change to "SNPs. The Panzhihua" (deleted "then,")
Line 28. Delete "a shared" (Gene flow by definition describes shared genes across populations.)
Introduction
Line 45. Delete "From the perspective of the development process," (This is obvious from the rest of the sentence.)
Line 86. Delete "wild resources"
Materials and Methods
Lines 89-94. Mention which fin was (or which fins were) clipped for the samples.
Results
Figure 2. Either the figure needs to be increased in size or the text on the figures need to be increased in size to be visible. Currently the text is almost unreadable.
Figure 4. Either the figure needs to be increased in size or the text on the figures need to be increased in size to be visible. Currently the text is almost unreadable.
Line 256. Change to "Historic Population Dynamics Analysis" or "Historical Population Dynamics Analysis"
Figure 7A. Either the figure part A needs to be increased in size or the text on the figures needs to be increased in size to be visible. Currently the text is almost unreadable.
Discussion
Line 322. Change to "may be the result of the release of captive bred individuals into the wild populations."
Line 367 and 368. I'm not sure what "dangerous beaches" entail.
Line 379. Change to "Uplift of the Qinghai Tibet Plateau and Historical Population Dynamics"
Line 399. Change to "Adaptive Evolution and Molecular Mechanisms on the Qinghai Tibet Plateau."
Line 405. Change to "Relative to a lowland fish such as..."
Conclusion
Lines 439-440. Change the first sentence to something like "We analyzed the structure and genetic variation of Schizothorax wangchiachii using GBS technology."
Author Response
Response to Reviewer 2
Comment 1: The abstract and conclusion discuss the value of the research to current conservation and management of the species. The discussion does not directly address this as a coherent area. The results show things that might suggest different interpretations in a conservation or management context: lower genetic difersity and differentiation, but detectable differentiation in three populations. Although considered a panmictic population, the higher Fst value for one upstream population and phylogenetic population-structure analysis suggest the upstream BD, GT, and SWL populations are genetically more distinct. Should this distinctness merit more conservation attention, or treatment of these populations as separate management units? You mention the hydropower barriers in this region too. Does the lower genetic variation among populations elsewhere suggest the need for management as a single large population? Does the distribution of likely high altitude-associated genes suggest more consideration be given to local adaptation or suggest that human movement of individuals between some areas could be problematic? Do you consider the panmixis enough that the more distinct populations don't need separate conservation or management consideration? Does their lower genetic diversity require more or less conservation or management consideration? Overall, what do your results taken together suggest for future conservation and management?
Response 1: Thanks for your constructive suggestions. In the present study, the lower genetic diversity in wild populations of Schizothorax wangchiachii. Fortunately, based on the higher Fst value and phylogenetic population-structure analysis, the upstream BD, GT, and SWL populations are genetically more distinct, which suggests this distinctness merit need more conservation attention and could be as a separate management units. The Chinese Ministry of Agriculture and Rural Affairs has issued a ten-year fishing ban plan for the Yangtze River from 2020 to 2030 years. The Jinsha River, Yalong River, and their tributary, the Xianshui River, where S. wangchiachii live, all belong to the upper reaches of the Yangtze River. In this context, S. wangchiachii have been conserved in those tributaries. In our study, the upstream of Jinsha River (including BD, GT, and SWL populations) are likely as an important refuge for S. wangchiachii, and should be established as a nature reserve. Thus, protecting and maintaining the high genetic diversity in those wild populations may be critical for the future.
Otherwise, the enrichment of selected sites and energy metabolism and cellular processes in S. wangchiachii contributes to adaptation to high-altitude and low-temperature environments in our study. Therefore, in order to protect suitable living conditions, more efforts should be made to preserve its existing beneficial environment, such as reducing the construction of dams.
Those suggestions have been added to the Conclusion part (Lines 683-700).
Comment 2: Lines 10-12, 14-17. The first and last two sentences of the Simple Summary are unclear in what they are saying. They need to be substantially revised for clarity.
Response 2: The Simple Summary part has been revised (Lines 10-17).
Comment 3: Abstract.
Line 20. Change to "with large population sizes."
Lines 21-23. Change to "production are. We used genotype sequencing (GBS) technology to analyze the genetic diversity and population structure of 10 wild populations in the Jinsha River and Yalong River basins of the upper Yangtze River." -I think the authors used the prior structure (that was difficult to understand) to try to maintain passive voice. Active voice is fine to use in scientific publications in the biological sciences and is often required in the most high-profile journals. When it increases clarity, it definitely can and should be used.
Line 27. Change to "SNPs. The Panzhihua" (deleted "then,")
Line 28. Delete "a shared" (Gene flow by definition describes shared genes across populations.)
Response 3: The words and sentences in Abstract have been modified in the revised manuscript (Lines 23, 25-27, 33-34).
Comment 4: Introduction. Line 45. Delete "From the perspective of the development process," (This is obvious from the rest of the sentence.). Line 86. Delete "wild resources".
Response 4: Those words have been deleted in the text (Lines 54, 131).
Comment 5: Materials and Methods. Lines 89-94. Mention which fin was (or which fins were) clipped for the samples.
Response 5: Thanks for your professional advice. In our study, the dorsal fin of a fish was clipped for the sample (Lines 140).
Comment 6: Results.
Figure 2. Either the figure needs to be increased in size or the text on the figures need to be increased in size to be visible. Currently the text is almost unreadable.
Figure 4. Either the figure needs to be increased in size or the text on the figures need to be increased in size to be visible. Currently the text is almost unreadable.
Figure 7A. Either the figure part A needs to be increased in size or the text on the figures needs to be increased in size to be visible. Currently the text is almost unreadable.
Response 6: Figure 1, 4, and 7A have been modified in the revised manuscript.
Comment 7: Line 256. Change to "Historic Population Dynamics Analysis" or "Historical Population Dynamics Analysis".
Response 7: Those words have been changed in the text (Line 389).
Comment 8: Discussion.
Line 322. Change to "may be the result of the release of captive bred individuals into the wild populations."
Line 367 and 368. I'm not sure what "dangerous beaches" entail.
Line 379. Change to "Uplift of the Qinghai Tibet Plateau and Historical Population Dynamics"
Line 399. Change to "Adaptive Evolution and Molecular Mechanisms on the Qinghai Tibet Plateau."
Line 405. Change to "Relative to a lowland fish such as..."
Response 8: The sentence "may be the result of the release of captive bred individuals into the wild populations." have been added in the text (Lines 485-487).
In our study, "dangerous beaches" is best rendered as a hazardous river reach—commonly described as“rapids” or “dangerous shoals”-characterized by a relatively steep bed gradient, shallow flow and/or bed obstructions (boulders, ledges, reefs) that induce high velocity, intense turbulence (whitewater), hydraulic jumps and localized vortices; such reaches are important agents of sediment sorting and channel change and constitute significant navigation and safety hazards. In order to express more scientifically, the words "dangerous beaches" have been changed to“dangerous shoals”(Line 556).
The title “4.3 Uplift of the Qinghai Tibet Plateau and historical population dynamics” has been added to the text (Lines 572-573).
The title “4.4 Adaptive Evolution and Molecular Mechanisms on the Qinghai Tibet Plateau” has been added to the text (Lines 611-612).
The sentence "Relative to a lowland fish such as..." has been added to the text (Line 620).
Comment 9: Conclusion.
Lines 439-440. Change the first sentence to something like "We analyzed the structure and genetic variation of Schizothorax wangchiachii using GBS technology."
Response 9: This sentence has been modified (Line 666-667).
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsIt can be accepted
