Karyotypes of 10 Anuran Species from the Qinghai–Tibetan Plateau

Round 1

Reviewer 1 Report

Dear Editor: I carefully read the manuscript entitled "Karyotypes of 10 amphibians from the Qinghai-Tibetan Plateau."

 

Despite its potential interest, the manuscript has serious deficiencies in theoretical content and interpretation of the obtained results. I believe that the work deserves greater dedication and the use of other cytogenetic techniques, particularly classical techniques if the authors intend to provide more information about the karyotypic constitution and aid in result interpretation.

 

I consider the mere comparison of chromosome measurements obtained from previous reports to be precarious and lacking validity.

 

Line 64: An inversion on chromosome No. 6 (ADD "PAIR") was found between two subspecies of O. xiangchengensis.

Line 91-94: These paragraphs should be in the Materials and Methods section.

Table 1: Table 1 could be formatted in a way that is less tedious to read. Species could be listed on the Y-axis and the chromosome pairs on the X-axis, with AR, CI, and other data in each corresponding cell. Refer to examples in Ferro et al. 2020 and Cardozo et al. 2011.

Results: What do you mean by "large and small chromosomes"?

Line 96: 2n = 24??? The karyotype indicates 2n = 22.

Line 99: Could you please provide a description of the secondary constrictions?

Line 101: The secondary constriction over 2p interstitial in S. boulengeri is not evident in the plate.

Line 108: 6q inter??? DNA satellites are located in the distal region.

Line 113-117: Were the Bufotes species analyzed 2n or 4n? How can you be sure?

Line 163: With the obtained data, assuming this is too risky. Why not consider that the position of the secondary constrictions, possibly coinciding with the nucleolar organizing regions, is polymorphic between populations, as observed in numerous amphibian species?

Line 226: Or could the observed difference be simply due to the measurement of chromosomes and the criteria used to establish their homology? Additionally, it seems there may be a comparison problem influenced by the condensation of the obtained metaphase chromosomes.

Line 241: If Bufotes zandaensis is tetraploid (as questioned earlier), why was the karyotype arranged by pairs?

 

Author Response

Revewer1

• Despite its potential interest, the manuscript has serious deficiencies in theoretical content and interpretation of the obtained results. I believe that the work deserves greater dedication and the use of other cytogenetic techniques, particularly classical techniques if the authors intend to provide more information about the karyotypic constitution and aid in result interpretation.

Answer: Thank you for your comments. This study primarily focused on the identification of 10 Anuran species and their karyotypes collected from the Qinghai-Tibet Plateau (QTP). I agree that incorporating more cytogenetic techniques, such as C-banding or FISH, would be beneficial for detecting karyotypic variations. These techniques are often more effective in karyotype comparisons among different geographic populations of the same species. However, we did not conduct karyotype comparisons among multiple geographic populations within a single species in this study. Our specific focus was on the karyotype analysis of frogs and toads in the QTP, rather than comparing different populations within a specific species. Therefore, we primarily focused on variations of chromosome numbers, centromere positions, and secondary constriction locations. By comparing karyotypes, we discussed the role of karyotype variations in phylogenetic evolution, highlighting the potential value of karyotype variations in species diversity. Certainly, in the future, if we conduct in-depth karyotype analysis on an interesting species, we would be very willing to utilize additional techniques for further research.

 

• I consider the mere comparison of chromosome measurements obtained from previous reports to be precarious and lacking validity.

Answer: Thank you for your comments. We agree that comparing different measurements from various studies can introduce certain uncertainties, not only in chromosome measurements but also in morphological measurements and other aspects. This can be attributed to differences in the people who conducting the measurements, as well as differences in methods and standards used. Therefore, in this study, we adopted unified standards for measurement and analysis (revised manuscript: line 74-83) to minimize such errors, allowing for the comparison of measurement data from previous studies we referenced. In fact, the measurement methods and description ways we employed align with the majority of previous studies. Therefore, we think that comparing our chromosome measurements to previous studies is valid.

 

• Line 64: An inversion on chromosome No. 6 (ADD "PAIR") was found between two subspecies of O. xiangchengensis.

Answer: Thanks for your comments. We have corrected this grammatical error.

 

• Line 91-94: These paragraphs should be in the Materials and Methods section.

Answer: Thanks for your comments. Due to the repetition of information with the materials and methods section (revised manuscript: Line 65-66, 76-83), we have decided to remove this paragraph from the results.

 

• Table 1: Table 1 could be formatted in a way that is less tedious to read. Species could be listed on the Y-axis and the chromosome pairs on the X-axis, with AR, CI, and other data in each corresponding cell. Refer to examples in Ferro et al. 2020 and Cardozo et al. 2011.

Answer: Thanks for your comments. In the initial manuscript, Table 1 displays the location information of the samples. Based on the content you mentioned, I assume you are referring to Table 2, which presents chromosome measurement data. We agree with your point. We adjusted the format of this Table. It is presented from page 7 to page 11 in the revised version.

 

• Results: What do you mean by "large and small chromosomes"?

Answer: In a species’ karyotype, “large chromosomes” and “small chromosomes” refer to the relatively longer and shorter chromosomes, respectively, in a specific karyotype. This expression is commonly used in karyotype studies of the Anura (frogs and toads). For example, “The karyotype shows a distinct demarcation in size between the larger elements (triplets 1 to 6) … in the smaller chromosomes of triplets 6 to 11….” (Stöck et al., 1999). “Chromosome numbers 1 and no. 6 consisted of two large homomorphic metacentric (MM) chromosomes and two small homomorphic metacentric (mm) chromosomes….” (Qing et al., 2012).

Qing, L., Xia, Y., Zheng, Y., and Zeng, X. (2012). A de novo case of floating chromosomal polymorphisms by translocation in Quasipaa boulengeri (Anura, Dicroglossidae). PLoS One 7, e46163.

• Line 96: 2n = 24??? The karyotype indicates 2n = 22.

Answer: Thanks for your comments. We have corrected this error, and the correct result should be 2n=26. In the revised manuscript Page 4 Line 88-89 “T The karyotype of Oreolalax xiangchengensis consists 26 chromosomes, including 6 pairs of large chromosomes and 7 pairs of small chromosomes (Figure 1A; Figure 2A).”

 

• Line 99: Could you please provide a description of the secondary constrictions?

Answer: Thanks for your comments. The description of the secondary constriction location of Oreolalax xiangchengensis is in the revised manuscript Page 4 Line 89-90 “The SC is located on the 6q^inter and the AR ranges from 1.03~1.71.”

 

• Line 101: The secondary constriction over 2p interstitial in S. boulengeri is not evident in the plate.

Answer: Thanks for your comments. The secondary constriction in S. boulengeri indeed appear less pronounced in Figure 2, whereas they are clearly visible in the karyotype photograph provided in Figure 1B. We have inspected the original photograph (The following picture) of S. boulengeri in Figure 2 and have confirmed that the secondary constriction over 2p interstitial is unmistakable. Therefore, we confirm the accuracy of the result, and we will reprocess the image of Figure 2B to enhance its clarity and avoid any misunderstandings (In the revised manuscript Page 6).

• Line 108: 6q inter??? DNA satellites are located in the distal region.

Answer: Answer: Thanks for your comments. The secondary constriction of B. gargarizans is indeed located at the distal end of No. 6 chromosome (6q^ter). The corrected result is in the revised manuscript Page 4 Line 100 “All chromosomes are metacentric, and the AR ranges from 1.06 to 1.63. The SC is located on 6q^ter”.

 

• Line 113-117: Were the Bufotes species analyzed 2n or 4n? How can you be sure?

Answer: Thanks for your comments. According to previous research, the genus Bufotes includes diploid (2n = 22) (Stöck et al., 1999), triploid (3n = 33) (Stöck et al., 1999), and tetraploid (4n = 44) species (Stöck et al., 2001). Furthermore, the distribution, origin, and phylogenetic relationships of these polyploid species have been preliminarily elucidated (Dufresnes et al., 2019). In this study, the chromosome numbers of Bufotes taxkorensi and Bufotes zamdaensis were found to be 44, which is the same as the tetraploid number reported in previous research. Therefore, we confirm that they are tetraploid.

Stöck, M., Schmid, M., Steinlein, C., and Grosse, W.R. (1999). Mosaicism in somatic triploid specimens of the Bufo viridis complex in the Karakoram with examination of calls, morphology and taxonomic conclusions. Italian Journal of Zoology 66, 215-232.

Stöck, M., Günther, R., and Böhme, W. (2001). Progress towards a taxonomic revision of the Asian Bufo viridis group: current status of nominal taxa and unsolved problems (Amphibia: Anura: Bufonidae). Zool Abh Staatl Mus Tierkunde Dresden 51, 253–319.

Dufresnes, C., Mazepa, G., Jablonski, D., Oliveira, R.C., Wenseleers, T., Shabanov, D.A., Auer, M., Ernst, R., Koch, C., Ramirez-Chaves, H.E., et al. (2019). Fifteen shades of green: The evolution of Bufotes toads revisited. Molecular Phylogenetics and Evolution 141.

 

• Line 163: With the obtained data, assuming this is too risky. Why not consider that the position of the secondary constrictions, possibly coinciding with the nucleolar organizing regions, is polymorphic between populations, as observed in numerous amphibian species?

Answer: Thanks for your comments. We agree with your point. In this study, we observed variations in the secondary constriction positions among different geographic populations of Oreolalax xiangchengensis. This could be attributed to chromosomal inversions or simply changes in the position of NORs (nucleolar organizing regions). Therefore, we have made revisions to our previous discussion. In revised manuscript Page 12 Line 144-146 “This suggests that there may be a chromosomal inversion or changes in the NORs (nucle-olar organizing regions) locations occurring between different geographic populations of the O. xiangchengensis.”.

 

• Line 226: Or could the observed difference be simply due to the measurement of chromosomes and the criteria used to establish their homology? Additionally, it seems there may be a comparison problem influenced by the condensation of the obtained metaphase chromosomes.

Answer: (1) This study performed measurements on 10 repeated cells for each individual chromosome, ensuring the validity of our statistical analysis. The establishment of homology was based on the relative length and arm ratio as reference characteristics. This approach is commonly employed in most studies for statistical analysis, homology establishment, comparison, and discussion. Therefore, we believe that the differences obtained through measurement and comparison are valid and can provide valuable information and insights. (2) Chromosome morphology can be influenced by its condensation level. Even within the same batch of experimental cells, the degree of condensation may not be identical. Therefore, both the previous and current studies have relied on statistical analysis of metaphase chromosomes from multiple cells as results. Additionally, the indices we used for analysis, such as arm ratio (AR), relative length (RL), and centromeric index (CI), are all relative measures rather than absolute values. Hence, even if differences in condensation states arise due to variations in experimental procedures, the statistically analyzed data still retain comparability.

 

• Line 241: If Bufotes zandaensis is tetraploid (as questioned earlier), why was the karyotype arranged by pairs?

Answer: Thanks for your comments. For a tetraploid species, it is ideal to arrange the chromosomes in tetrads, and indicate which chromosome set each pair of chromosomes in the tetrad comes from. While, this species is recorded as an allotetraploid, its two chromosome sets have different origins. The corresponding chromosome pairs in these two sets may have different morphologies. Therefore, we cannot determine which pairs of chromosomes correspond to each other, as well as which chromosome set they belong to. So we arranged the chromosomes in pairs instead of in tetrads.

Reviewer 2 Report

Dear authors, after a thorough review of your manuscript, I would like to provide some suggestions that can enhance the overall quality of your work. To maintain the originality of the text, it would be best to avoid adding new information, sentences, or paragraphs and refrain from removing any crucial details. By implementing these suggestions, your paper can achieve its intended purpose and be further improved. The title of your paper should be a more specific and accurate title like "Chromosomes  in the Summit: the Karyotypes  of ten Anuran Species from Quing-Tibetan Plateau"   instead of the ambiguous term "Amphibians."

When writing the abstract, aim for brevity and avoid mentioning any results. 

Introduction:When writing the introduction section, it is crucial to clearly state the problem to be addressed, its significance, and its objective. Including hypotheses may also be helpful, but presenting results should be reserved for later in the paper. It's important to note that any text between lines N°23 and N°28 should not be included in the introduction.

Material and methos: Regarding the study's materials and methods, it is deemed satisfactory, although it would be more helpful to include the number of plates examined for each species. Alternatively, incorporating this information into the karyotype description would be advantageous.

Results: Below are the indicated changes for your reference.

 

1. Can you provide information on the chromosomal count of X. xiangchengensis? I'm curious to know if Line N°96 has a chromosomal count of 2n=24 or 2n=22, as indicated in Figure 2A. Also, have there been any inconsistencies observed among the studied subspecies?

2. It is necessary to include a bar in Fig. 2, and this must be done without any exceptions.

3. Were there any instances of sexual chromosomes found among the populations under examination?

4. Are there any differences in the karyotypes of S. boulengeri among the five analyzed localities in terms of chromosomal variations?

Discussion:

Let's discuss the information presented, which can be quite diverse and overwhelming. I suggest splitting it into two sections to make it easier to understand. The first section can compare the data collected, and presenting it in a table would be ideal. The second section can focus on the most important aspects, such as polyploidy, mechanisms of chromosomal evolution, and the differences in chromosomes among the areas and species studied.

Summary:

1. Clarify the purpose and objective of the manuscript in the introduction section to help the reader understand the significance of the research.

2. Use a more specific and accurate title to reflect the paper's content and attract the intended audience.

3. Organize the discussion section into two parts to make it more manageable and easier to understand for the reader.

 

Author Response

Revewer2

Dear authors, after a thorough review of your manuscript, I would like to provide some suggestions that can enhance the overall quality of your work. To maintain the originality of the text, it would be best to avoid adding new information, sentences, or paragraphs and refrain from removing any crucial details. By implementing these suggestions, your paper can achieve its intended purpose and be further improved.

• The title of your paper should be a more specific and accurate title like "Chromosomes in the Summit: the Karyotypes  of ten Anuran Species from Quing-Tibetan Plateau"   instead of the ambiguous term "Amphibians."

Answer: Thanks for your comments. We are very willing to accept your suggests. We have modified the title a more accurate version “Karyotypes of 10 Anuran species from Qinghai-Tibetan Plateau” in revised manuscript.

 

• When writing the abstract, aim for brevity and avoid mentioning any results.

Answer: Thanks for your comments. We have rewritten the Abstract section and removed the content about results in the revised manuscript (Line 17-24).

 

• Introduction: When writing the introduction section, it is crucial to clearly state the problem to be addressed, its significance, and its objective. Including hypotheses may also be helpful, but presenting results should be reserved for later in the paper. It's important to note that any text between lines N°23 and N°28 should not be included in the introduction.

Answer: Thanks for your comments. We have made modifications to the Introduction section, especially in removing the six lines that contain the results of this study. The modified version is in revised manuscript Page 2 Line 58-62: “In this study, we investigated the karyotypes of 10 anuran species in the QTP and explored variations in chromosome number and structure. These data will contribute to revealing the genetic diversity of amphibians in the QTP. Furthermore, our karyotypic data will provide support for research on environmental adaptation and conservation of amphibians.”.

 

• Material and methods: Regarding the study's materials and methods, it is deemed satisfactory, although it would be more helpful to include the number of plates examined for each species. Alternatively, incorporating this information into the karyotype description would be advantageous.

Answer: Thanks for your comments., We supplemented statements regarding the number of plates and repetitions in the Methods section. in the revised manuscript Page 2 Line 72, 75-76: “Mitotic metaphase cells were obtained from bone marrow using the procedures de-scribed by Schmid [23], and each individual produced four slices.” And “Ten mitotic metaphase cells were randomly selected for measurement from each species.”

 

Results: Below are the indicated changes for your reference.

• Can you provide information on the chromosomal count of X. xiangchengensis? I'm curious to know if Line N°96 has a chromosomal count of 2n=24 or 2n=22, as indicated in Figure 2A. Also, have there been any inconsistencies observed among the studied subspecies?

Answer: Thanks for your comments. (1) We have corrected this error, and the correct result should be 2n=26. In the revised manuscript Page 4 Line 88-89 “The karyotype of Oreolalax xiangchengensis consists 26 chromosomes, including 6 pairs of large chromosomes and 7 pairs of small chromosomes (Figure 1A; Figure 2A).”. (2) The number of chromosomes is consistent among different subspecies of O. xiangchengensis, and there are differences in the location of secondary constriction and the chromosome measurements. In the revised manuscript Page 12 Line 140-144 “The karyotype of O. xiangchengensis population from Zhongdian County (ZD), Yunna Province has been reported by Li [25]. They have 26 chromosomes, including 3 pairs of SM (No. 3, 4, 5) and 10 pairs of M, with one pair SC on the 6q^per. On the other hand, the karyotype of population from Weixi County (WX), Yunnan Province only has one pair of SM (No. 3), and the SC is on the 6q^inter.”

 

• It is necessary to include a bar in Fig. 2, and this must be done without any exceptions.

Answer: Thanks for your comments. In the modified version of Figure 2, a bar has been added after the karyotype of each species. In the revised manuscript Page 6.

 

• Were there any instances of sexual chromosomes found among the populations under examination?

In this study, all the samples used were discovered to have sex chromosomes.

Answer: Thanks for your comments. No sexual chromosomes were detected in all individuals in this study.

 

• Are there any differences in the karyotypes of S. boulengeri among the five analyzed localities in terms of chromosomal variations?

Answer: Thanks for your comments. In this study, five individuals of Scutiger boulengeri were collected from three different locations (Ding jie County, Dingqing County, Zhongba County) in Xizang Province, China. After measurement and comparison, it was found that their chromosome morphology was similar, and the locations of secondary constriction and chromosome composition were identical (2p^inter, 20M + 6SM). Therefore, the karyotypes of S. boulengeri in these three counties within Xizang province, China are identical.

 

Discussion:

• Let's discuss the information presented, which can be quite diverse and overwhelming. I suggest splitting it into two sections to make it easier to understand. The first section can compare the data collected, and presenting it in a table would be ideal. The second section can focus on the most important aspects, such as polyploidy, mechanisms of chromosomal evolution, and the differences in chromosomes among the areas and species studied.

Answer: Thanks for your comments. We are glad to make modifications to the Discussion section according to your suggestions. We divided the Discussion into two sections: “Karyotype Comparison” and “Karyotypic Differences and Phylogenetic Differentiation”. Additionally, we presented the mentioned karyotype data in the new table (Table 3). The new Discussion section is in the revised manuscript Page 12-14, while the Table 3 is on page 15.

Summary:

1. Clarify the purpose and objective of the manuscript in the introduction section to help the reader understand the significance of the research.

Answer: Thanks for your comments. We have accepted your suggestions and completed the revision of the introduction section.

2. Use a more specific and accurate title to reflect the paper's content and attract the intended audience.

Answer: Thanks for your comments. We have revised the title according to your suggestion.

3. Organize the discussion section into two parts to make it more manageable and easier to understand for the reader.

Answer: Thanks for your comments. As you suggested, we Organized the discussion section into two parts and added the Table 3 to show the karyotype data.

Author Response File: Author Response.pdf

Reviewer 3 Report

This study presents karyological analyzes for ten amphibian species providing new data to those available from the literature and the first karyotype description for Duttaphrynus himalayanus and Bufotes zamdaensis.

   The Ms is worthy of publication after an English revision (English language, especially in the discussion, needs a thorough revision as several phrases are unclear).

Below, some minor comments:

 

Page 2 Lines 63 - 66: Please, give entire scientific names in this section

In legend of Table 2 specify values with SE?

IN the discussion I suggest to remove the acronim for the locality. It could help the reader

Page 10 Lines 160-162: Some language mistakes, I suggest rewriting: Furthermore, despite the species' high diversity and significant morphological differences in the genus Oreolalax, genetic distances based on mitochondrial DNA are relatively closer [28]. 

Page 10 Line 168: incorrect form of verb “suggests”

Page 10 Line 170: Ranges

   The Ms is worthy of publication after an English revision (English language, especially in the discussion, needs a thorough revision as several phrases are unclear).

Author Response

Revewer3

This study presents karyological analyzes for ten amphibian species providing new data to those available from the literature and the first karyotype description for Duttaphrynus himalayanus and Bufotes zamdaensis.

 

• The Ms is worthy of publication after an English revision (English language, especially in the discussion, needs a thorough revision as several phrases are unclear).

Answer: Thanks for your comments. We have made language revisions to the manuscript, particularly in the Discussion section, where numerous sentences have been modified or rewritten. The modified discussion in the revised manuscript is from Page 12 Line 138 to Page 14 Line 243.

 

Below, some minor comments:

• Page 2 Lines 63 - 66: Please, give entire scientific names in this section

Answer: Thanks for your comments. We are glad to accept your suggestions. Additionally, based on suggests from other reviewers, we have made modifications to this paragraph. The Introduction section no longer includes any species names. Therefore, we have made modifications in the “Results” section by using the entire scientific names of each species the first time they were mentioned in the text.

 

• In legend of Table 2 specify values with SE?

Answer: Thanks for your comments. In Table 2, the values represent the mean and SD. We have provided illumination in Table 2 of revised manuscript (Page 7) to avoid misunderstandings.

 

• IN the discussion I suggest to remove the acronym for the locality. It could help the reader

Answer: Thanks for your comments. We have removed all the acronym of the locality in the discussion section and replaced them with their full names.

 

• Page 10 Lines 160-162: Some language mistakes, I suggest rewriting: Furthermore, despite the species' high diversity and significant morphological differences in the genus Oreolalax, genetic distances based on mitochondrial DNA are relatively closer [28].

Answer: Thanks for your comments. Thank you for your suggestions and assistance. We are glad to accept the version you have provided. The modified sentence is in the revised manuscript Page 13 Line 227-229: “Furthermore, despite the species' high diversity and significant morphological differences in the genus Oreolalax, genetic distances based on mitochondrial DNA are relatively closer [38].”

 

• Page 10 Line 168: incorrect form of verb “suggests”

Answer: Thanks for your comments. We have corrected this grammatical error. In the revised manuscript Line 204-206 “These results suggest that the main characteristics of karyotypes among different populations of R. chaochiaoensis were constant, while the AR and SC positions show rich variations.”

 

• Page 10 Line 170: Ranges

Answer: Thanks for your comments. We have corrected this grammatical error. In the revised manuscript Line 208 “The chromosome number of Megophryidae species ranges from 22 to 30 [14].”

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Dear Authors

Despite the fact that in the previous review, I made my comments on the problem of drawing conclusions based on the measurements of the chromosomes and the assignment of their position in the karyotype based on it. I consider that the manuscript has been improved in a certain respect. As a comment on this review, I suggest that statements about possible implications of the findings made be less risky, since the homology between chromosome pairs cannot be specified in the vast majority of cases. therefore, there should be fewer statements and more perspectives to continue investigating about the karyotypic evolution of the taxa analyzed and their related ones. 

A number of minor revisions are presented. However, the entire manuscript should be reviewed based on these considerations, which will allow for a somewhat more elaborate discussion section and less based on ad-hoc hypotheses

Line 184: The problem of working and drawing conclusions with the number of chromosome pairs from different studies can lead us to consider, as in this case, that two chromosome pairs (18 and 20), both of very similar size and morphology, are considered a variation. When in reality it is very likely that it is only due to a problem of establishing homologies and how the karyotype is ordered.

Line 194: or it is simply due to a problem in establishing chromosomal homologies. It may be a possibility and should be mentioned in the manuscript.

Line 254: I suggest you be less risky with affirmations. The presence of variation will not necessarily indicate subspecies or species differentiation.

 

 

Author Response

Revewer1

Dear Authors

 

• Despite the fact that in the previous review, I made my comments on the problem of drawing conclusions based on the measurements of the chromosomes and the assignment of their position in the karyotype based on it. I consider that the manuscript has been improved in a certain respect. As a comment on this review, I suggest that statements about possible implications of the findings made be less risky, since the homology between chromosome pairs cannot be specified in the vast majority of cases. therefore, there should be fewer statements and more perspectives to continue investigating about the karyotypic evolution of the taxa analyzed and their related ones.

Answer: Thank you for your comments. We agree with your viewpoint. Due to the potential problems that may exist in establishing chromosomal homologies and the order, there is a risk of comparing the measurements from different researches. Therefore, when discussing chromosome differences, we will mention the possible effects of establishing chromosomal homologies and the order. These modifications will be specifically reflected in the revisions to your following suggestions.

 

A number of minor revisions are presented. However, the entire manuscript should be reviewed based on these considerations, which will allow for a somewhat more elaborate discussion section and less based on ad-hoc hypotheses

• Line 184: The problem of working and drawing conclusions with the number of chromosome pairs from different studies can lead us to consider, as in this case, that two chromosome pairs (18 and 20), both of very similar size and morphology, are considered a variation. When in reality it is very likely that it is only due to a problem of establishing homologies and how the karyotype is ordered.

Answer: Thank you for your comments. We agree with your point. We have made modifications to the discussion section about G. sichuanensis. In the revised manuscript Page 12-13, Line 186-190 “Due to the similar chromosome morphology in G. sichuanensis, the differences in SC positions could be attributed to SC location polymorphism, or potential differences in measurement and ordering processes. Therefore, more cytogenetic techniques are needed to be conducted in different G. sichuanensis populations.”

 

• Line 194: or it is simply due to a problem in establishing chromosomal homologies. It may be a possibility and should be mentioned in the manuscript.

Answer: Thank you for your comments. We agree with your point. The measurement differences in Nanorana parkeri may probably be due to a problem in establishing chromosomal homologies. We mentioned it in the manuscript. In the revised manuscript Page 3, Line 196-199 “It is difficult to determine whether these chromosomal differences arise from karyotypic variations or simply are caused by a problem in establishing chromosomal homologies. Therefore, further research is needed using more samples and additional techniques.”

 

Line 254: I suggest you be less risky with affirmations. The presence of variation will not necessarily indicate subspecies or species differentiation.

Answer: Thank you for your agreement. We agree with your point. We have made modifications to Abstract, Discussion and Conclusion sections. We have removed the sentence “This chromosomal variation may be an important driving forces behind the differentiation of the O. x. xiangchengensis and O. x. deqinicus.” from the Discussion section. Additionally, we have rewritten the relevant sentences in the Abstract and Conclusion sections. In the revised manuscript Page 1, Line 22-25 “Furthermore, the different locations of the secondary constriction between the Weixi and Zhongdian population of O. xiangchengensis support that there are karyotypic variation between the two subspecies (O. xiangchengensis xiangchengensis vs. O. xiangchengensis deqinicus).” And Page 17, Line 257-258 “Furthermore, there are differences in the secondary constriction locations between the two subspecies of O. xiangchengensis (O. x. xiangchengensis and O. x. deqinicus).”

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear authors:

                     I have reviewed the new manuscript and agree with the changes introduced. And I think it should be published.  

 

 

Author Response

Revewer2

Dear authors:

 

I have reviewed the new manuscript and agree with the changes introduced. And I think it should be published.

 

Answer: Thank you very much for your suggestions and assistance in the previous review.

Author Response File: Author Response.pdf