Historical Landscape Evolution Shaped the Phylogeography and Population History of the Cyprinid Fishes of Acrossocheilus (Cypriniformes: Cyprinidae) According to Mitochondrial DNA in Zhejiang Province, China

Round 1

Reviewer 1 Report

Dear authors,

according to my review, I cannot find any serious flaws regarding the introduction, the statistical analysis and the general presentation and discussion of your findings. However, my major concern is the population sample size used for the genetic analysis. We all agree that freshwater species and their corresponding populations might possess unique genetic pools due to their geographic isolation, founder effects, genetic drift, limited gene flow, adaptation to local enviroments etc..The extended genetic differentiation between populations is somewhat expected due to all the aforementioned factors. However, it is also critical to capture correctly the within population diversity for each one of them. For example, herein, you analyzed 2 specimens from Jinqing River, 6 specimens from Feiyun River,  6 specimens from Jiaoxi River, 4 specimens from Feiyun River etc. These are very low population sample sizes and cannot be considered as appropriate for a thorough and reliable (from a statistical point of view) estimation of the genetic variability for each population (intra-population variability). What is your response (comments) on that??

Author Response

Thank you for providing very important suggestions. In recent years, due to improper release by humans and environmental pollution, the population of freshwater fish has sharply declined, especially in small independent streams. The Acrossocheilus species, which is a popular wild fish locally, has also suffered from overfishing, exacerbating the decline in its population. In this study, the sampled populations were from areas that were severely damaged locally, making it extremely difficult to obtain sufficient samples. However, based on the expression of genetic diversity, we believe that the impact on understanding the genetic structure of these freshwater fish is relatively small.

Author Response File: Author Response.docx

Reviewer 2 Report

On line 27 and later in the text of manuscript, you use the term “cytochrome b (cyt-b) and control (D-loop) GENES”. However, the control region of the mtDNA is not a gene, it is a regulatory region of the molecule; it does not correspond to any protein product. Please, correct this throughout this article. In addition, when you discuss concatenated sequences cyt-b+D-loop, you can use the terms “fragment, region”, etc., but not gene. Please, check it!

Lines 57-58: May be more successful not “According to the fundamental geohistory events and the ichthyofauna…” but “According to the fundamental geohistory events and the ichthyofauna’s structure…”

Lines 70–71: I think that here and further it would be better to arrange the names of species that you give in parentheses, according to the order of cited works about them in the References. That is, the species Huigobio chenhsienensis should come first, because the article about which is the fourth in the list, and then all the others species.

Line 80: change “between Acrossocheilus species” to “of Acrossocheilus species”

Lines 81–85: Here you put a reference to the same work, namely [10] in the References, after each sentence. You can only put this reference only once – after the sentence in line 85.]

Line 86: Is the Yangtze-Pearl River one river or two? If they are two different rivers, then the Yangtze-Pearl Rivers.

Lines 88–90: I suggest you combine and rephrase two sentences in these lines because in lines 90–91 you start two sentences with the same word, namely “However”. This is not very good. And why do you use the past tense when describing species distribution? As I understand it, these species are still present in the discussed area. Wouldn't it be better to use the present tense, like this: “A. fasciatus is mainly distributed in the northern Zhejiang region, including the Qiantang River, Yongjiang River and Jiaojiang River, and A. wenchowensis – only in the southern Zhejiang region, mainly in the Ou River and its adjacent water systems.”?

Lines 104: The phrase "...which is not native to the wild area where it is introduced." must be deleted, as the term "the introduced population" implies just that.

Lines 97–114: The paragraph is too long. It could be divided into two paragraphs: the sentence "In this study, mitochondrial....." could be the beginning of a new paragraph.

Line 109: Remove “and” and replace “,” by “?”

Lines 111–114: I suggest that the authors rephrase this part of the text like this: (3) What competing scenarios of demographic and phylogenetic population history, tested using the approximate Bayesian computation (ABC) procedure, have potentially contributed to the modern distribution of A. fasciatus and A. wenchowensis.

Why doesn't a third species, Acrossocheilus kreyenbergii, appear in your goals? For what purpose was it included in your study?

In the Introduction, you touch on the level of genetic differentiation of the three species of genus Acrossocheilus. Is there any data on their differences at the level of morphology, diagnostic traits for these species? How much do they differ in morphological parameters? I think this information would also be interesting to present in the Introduction or in the Discussion when analyzing your results.

Line 121: Replace “All specimens  “ by “The samples”

Line 125: The sentence “Total genomic DNA was extracted from the tissue of the fins using the DNeasy Blood and Tissue Kit (QIAGEN, Valencia, CA, USA).” can be the beginning of a new paragraph.

Line 127: Control region (D-loop) is not a gene! Please correct it.

Lines 128–131: Who developed the primers for PCR?

Line 132: Did you mean 1 µl of each primer of the two? If so, change “primer 1 µl” to “1 µl of each primer”.

Line 132: Remove “amplification”, because PCR = amplification.

PCR conditions were the same for both cytochrome b and D-loop?

Line 142: I propose to rephrase the title of Table 1 as follows: Sample information and genetic diversity of the cyt b + D-loop region in the genus Acrossocheilus.

Table 1: Actually, the header of the first column of the Table 1 should not be called Population, but Species.

Line 146: In the caption to the Figure 1 you write that the collection sites are circles, but in fact we see triangles, squares, and a star on this Figure.

There is no correspondence between the collection site codes in Figure 1 and Table 1. I can't find the TX code or any other codes from Figure 1 in Table 1. Conversely, I don't see the AFNB code and the other codes from Table 1 in Figure 1. Please check this and make a match.

Line 148: Why do you give a headline for this part of the text and leave the previous part about the collection of material and methods of DNA analysis without a headline?

Line 149: The D-loop is not a gene. Fix this!

Line 166: Comma before “and” and include “approaches” before “with MEGA-X”.

Line 172: Please, fix “cyt b and D-loop genes”!

Line 186: In fact, the Figure 5 shows BSPs for only two species, not for each of three.

Line 193: You write about four biogeographic units, but then in Lines 194–196 you name only three. Please, check it!

Lines 194–196: The codes AKSZ, AWLS, AWQY, AWTS, and AWFD are missing in Table 1 and Figure 1. Please unify all sample codes in the text, Table 1, and Figure 1. Why aren't the samples with codes AFLS and AFWS mentioned here?

Line 200: Comma before “and”

Line 201: Replace “cyt b + D-loop gene” by “cyt b + D-loop region”

Line 202: I think it is better: “…which was then used to discriminate the most probable scenario among…”

Line 204: Comma before “and”.

Line 204: Figure 2(A) is better, I think.

Lines 204, 206, and 208: Why do you duplicate the numbering of the Scenarios with the letters (i), (ii), and (iii)? I think they could be deleted.

Line 212: Figure 2(B) is better, I think.

Line 229: Comma before “and”.

Figure 2(B). What do N, N1, N2, N3, N4, and N5 mean? Please indicate this in the caption to the Figure.

Line 236: Replace “cyt b + D-loop gene” by “cyt b + D-loop region”

Lines 238–241: Three sentences in a row begin with the same phrase "A total...". Please rephrase this, for example, like this: “A total of 537 variable sites were observed, of which 405 were parsimony informative.  From 160 specimens of the 12 Acrossocheilus populations we indentified 124 haplotypes of which 110 were characteristic for A. fasciatus and 88 for A. wenchowensis (Table 1).”

Lines 243–245: That's not a very well-constructed sentence. Maybe that would be better: "The average haplotype diversity within A. fasciatus was high (0.994), ranging from 0.643 (AFHZ) to 1.000 (AFWL), and the average nucleotide diversity (θπ) for this species was low (0.018), ranging from 0.003 (AFHZ) to 0.0014 (AFNB) (Table 1)."

Line 245: The value of 0.0014 is less than 0.003. In addition, 0.0014 is not in the Table 1. Obviously, there is a typo here. How was the average nucleotide diversity calculated, and obtained a value of 0.018? Why does the average value exceed the maximum value detected in the populations?

Line 246–248: The same observation about sentence construction as in the lines 243–245. You can change it like this: Within A. wenchowensis the average haplotype diversity was high (0.975), ranging from 0.000 (AWTS) to 1.000 (AWWC), and the average nucleotide diversity (θπ) was low (0.013), ranging from 0.000 (AWTS) to 0.011 (AWFD) (Table 1).

Line 246: Actually, the maximum value of haplotype diversity according to Table 1 is 0.982, not 1.000.

Line 249: Actually, there are other values for A. kreyenbergii in Table 1, not 0.825 and 0.021. Check it.

Line 256: Replace “cyt b + D-loop gene” by “cyt b + D-loop region”

Line 257: There is other value in Table 2, not 0.902.

Line 258: Comma after A. fasciatus.

Lines 258–259: There aren’t values 0.491 and 0.952 in Table 2 for A. wenchowensis. Check it.

Table 2. It is very strange that at such high Fst values, the p-distances are 0.000 even between different species. How correct are these data? How were the p-distances calculated? How can you explain this situation?

Line 270: In the text, the value is -25.03% with a minus, and in the Table 2 without a minus. Fix it, please.

Line 274: Replace “cyt b + D-loop gene” by “cyt b + D-loop region”

Line 278: Replace “cyt b + D-loop genes” by “cyt b + D-loop region”

Table 3. Why are the values 63.31 and 10.15 in bold? Please explain this in the Table 3 header.

Line 281: Comma before “and”.

Line 282: Replace “cyt b + D-loop genes” by “cyt b + D-loop region”

Lines 281–282: I think you meant: “according to the haplotypes' distribution pattern from different populations”.

Line 283: Comma before “and”.

Line 284: Comma before “and”.

Line 287: Comma before “and”.

Line 289: Remove “was also”

Line 292: Replace “cyt b + D-loop genes” by “cyt b + D-loop region”

Line 293: Comma after “ML”

Figure 3. Which sequence of Spinibarbus hollandi was used? Please, give the number of the NCBI.

Line 298–300: Rephrase the sentences, for example, like it: “The spanning network confirmed the presence of the three major lineages of mtDNA (A, B, and C), with lineage A being interior and the others (lineages B and C) being located at the tip (Figure 4).”

Line 301: Comma before “and”

Figure 4. Population designations are very poorly visible.

Line 320–321: Why was not the BSP analysis perform for A. kreyenbergii?

Line 342: Comma before “and”

Line 344: Comma before “and”

Line 349–353: I think this part of text “over the “DEC model” (posterior probability = 0.0061 [0.0000,0.0265]; 0.0090 [0.0015,0.0165]), the “INC model” (posterior probability = 0.0000 [0.0000,0.0001]; 0.0000 [0.0000,0.0001]), the “INCDEC model” (posterior probability = 0.0000 [0.0000,0.0001]; 0.0002 [0.0000,0.0005]) and the “DECINC model” (posterior probability = 0.0000 [0.0000,0.0001]; 0.0000 [0.0000,0.0001])” can be removed and replaced by “over the other models in A. fasciatus and A. wenchowensis, respectively.”

Line 366: Replace “cyt b + D-loop genes” by “cyt b + D-loop region”

Line 365: Please place H. chenhsienensis with reference [4] first in parenthesis, and then S. macrops with reference [5].

Line 368: Replace “of A. fasciatus exhibited” by “of A. fasciatus also exhibited”

Line 374: Can you add in Table 1 additional column to show that lower nucleotide diversity is typical in the smaller rivers for the A. fasciatus?

Lines 382–384: I think that in this sentence you should specify the designations of the parameters that you have in mind, namely Nst and Gst.

Line 385: Replace “FST” by “F_ST”.

Lines 388–390: On what basis is the assumption made about the translocation of individuals to these reservoirs? Are there any literary or other sources for this assumption? If these populations are indeed resettled, they cannot be used when considering the natural dispersal (phylogeography) of a species! This can lead to a misinterpretation of the results. For example, in the case of AMOVA, you would combine in Scenario II the native donor population located on one side of the mountain range (AFKH) and the introduced populations located on the other side of the mountain range (AFWC and AFLS). No wonder why the contribution of the component among group turns out to be even negative.

Line 394: Add a parenthesis after [5].

Line 397: Add italics for A. fasciatus

Lines 395–397: In fact, 10.15% is the lowest value and can hardly be used to analyze results. In the Scenario III, intra-population diversity, rather than diversity among groups, has the greatest importance. Please keep this in mind when discussing your data.

Line 402: Please repleace the species in order of literature references, like this: H. chenhsienensis [4], S. argentatus [5], S. sinensis [7], and Rhynchocypris oxycephalus [38].

Line 437: Add a parenthesis after [4].

Comments for author File: Comments.pdf

Author Response

 

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	Yes	Can be improved	Must be improved	Not applicable
Does the introduction provide sufficient background and include all relevant references?	( )	(x)	( )	( )
Are all the cited references relevant to the research?	(x)	( )	( )	( )
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Comments and Suggestions for Authors

On line 27 and later in the text of manuscript, you use the term “cytochrome b (cyt-b) and control (D-loop) GENES”. However, the control region of the mtDNA is not a gene, it is a regulatory region of the molecule; it does not correspond to any protein product. Please, correct this throughout this article. In addition, when you discuss concatenated sequences cyt-b+D-loop, you can use the terms “fragment, region”, etc., but not gene. Please, check it!

Response: As requested, we revised it.

Lines 57-58: May be more successful not “According to the fundamental geohistory events and the ichthyofauna…” but “According to the fundamental geohistory events and the ichthyofauna’s structure…”

Response: As requested, we revised it.

Lines 70–71: I think that here and further it would be better to arrange the names of species that you give in parentheses, according to the order of cited works about them in the References. That is, the species Huigobio chenhsienensis should come first, because the article about which is the fourth in the list, and then all the others species.

Response: As requested, we revised it.

Line 80: change “between Acrossocheilus species” to “of Acrossocheilus species”

Response: As requested, we revised it.

Lines 81–85: Here you put a reference to the same work, namely [10] in the References, after each sentence. You can only put this reference only once – after the sentence in line 85.]

Response: As requested, we revised it.

Line 86: Is the Yangtze-Pearl River one river or two? If they are two different rivers, then the Yangtze-Pearl Rivers.

Response: As requested, we revised it.

Lines 88–90: I suggest you combine and rephrase two sentences in these lines because in lines 90–91 you start two sentences with the same word, namely “However”. This is not very good. And why do you use the past tense when describing species distribution? As I understand it, these species are still present in the discussed area. Wouldn't it be better to use the present tense, like this: “A. fasciatus is mainly distributed in the northern Zhejiang region, including the Qiantang River, Yongjiang River and Jiaojiang River, and A. wenchowensis – only in the southern Zhejiang region, mainly in the Ou River and its adjacent water systems.”?

Response: As requested, we revised it.

Lines 104: The phrase "...which is not native to the wild area where it is introduced." must be deleted, as the term "the introduced population" implies just that.

Response: As requested, we deleted it.

Lines 97–114: The paragraph is too long. It could be divided into two paragraphs: the sentence "In this study, mitochondrial....." could be the beginning of a new paragraph.

Response: As requested, we revised it.

 

Line 109: Remove “and” and replace “,” by “?”

Response: As requested, we revised it.

Lines 111–114: I suggest that the authors rephrase this part of the text like this: (3) What competing scenarios of demographic and phylogenetic population history, tested using the approximate Bayesian computation (ABC) procedure, have potentially contributed to the modern distribution of A. fasciatus and A. wenchowensis.

Response: As requested, we revised it.

Why doesn't a third species, Acrossocheilus kreyenbergii, appear in your goals? For what purpose was it included in your study?

Response: As requested, we added and revised it. “(3) What competing scenarios of demographic and phylogenetic population history, tested using the approximate Bayesian computation (ABC) procedure, have potentially contributed to the modern distribution of A. fasciatus, A. kreyenbergii and A. wenchowensis.”

In the Introduction, you touch on the level of genetic differentiation of the three species of genus Acrossocheilus. Is there any data on their differences at the level of morphology, diagnostic traits for these species? How much do they differ in morphological parameters? I think this information would also be interesting to present in the Introduction or in the Discussion when analyzing your results.

Response: Thank you for your suggestion. These three species are all good species and are very easy to distinguish based on their morphology. According to our previous research on the phylogenetic relationships based on mitochondrial DNA genomes, these three species are the closest relatives.

Hou, X. J., Lin, H. D., Tang, W. Q., Liu, D., Han, C. C., & Yang, J. Q. (2020). Complete mitochondrial genome of the freshwater fish Acrossocheilus longipinnis (Teleostei: Cyprinidae): genome characterization and phylogenetic analysis. Biologia, 75, 1871-1880.

Line 121: Replace “All specimens  “ by “The samples”

Response: As requested, we revised it.

Line 125: The sentence “Total genomic DNA was extracted from the tissue of the fins using the DNeasy Blood and Tissue Kit (QIAGEN, Valencia, CA, USA).” can be the beginning of a new paragraph.

Response: As requested, we revised it.

Line 127: Control region (D-loop) is not a gene! Please correct it.

Response: As requested, we revised it.

Lines 128–131: Who developed the primers for PCR?

Response: These primers were designed by us based on previous whole-genome studies.

Line 132: Did you mean 1 µl of each primer of the two? If so, change “primer 1 µl” to “1 µl of each primer”.

Response: As requested, we revised it.

Line 132: Remove “amplification”, because PCR = amplification.

Response: As requested, we revised it.

PCR conditions were the same for both cytochrome b and D-loop?

Response: As requested, we revised it. “PCR amplification was performed in an Eppendorf Mastercycler under the following conditions: 94°C for 4 min for initial denaturation, followed by 35 cycles of 94°C for 30 sec, 55°C (cyt b); 53°C (D-loop) for 40 sec, and extension at 72°C for 1 min and a final extension at 72°C for 10 min.”

Line 142: I propose to rephrase the title of Table 1 as follows: Sample information and genetic diversity of the cyt b + D-loop region in the genus Acrossocheilus.

Response: As requested, we revised it.

Table 1: Actually, the header of the first column of the Table 1 should not be called Population, but Species.

Response: As requested, we revised it.

Line 146: In the caption to the Figure 1 you write that the collection sites are circles, but in fact we see triangles, squares, and a star on this Figure.

Response: As requested, we revised it. “Collection sites (triangles, squares, star) correspond to locations given in the text and Table 1.”

There is no correspondence between the collection site codes in Figure 1 and Table 1. I can't find the TX code or any other codes from Figure 1 in Table 1. Conversely, I don't see the AFNB code and the other codes from Table 1 in Figure 1. Please check this and make a match.

Line 148: Why do you give a headline for this part of the text and leave the previous part about the collection of material and methods of DNA analysis without a headline?

Response: As requested, we added and revised it. “2.1. Sample collection and sequencing”.

Line 149: The D-loop is not a gene. Fix this!

Response: As requested, we revised it.

Line 166: Comma before “and” and include “approaches” before “with MEGA-X”.

Response: As requested, we revised it.

Line 172: Please, fix “cyt b and D-loop genes”!

Response: As requested, we revised it.

Line 186: In fact, the Figure 5 shows BSPs for only two species, not for each of three.

Response: As requested, we revised it. “In addition, Bayesian skyline plot (BSP) analyses were performed using BEAST v1.8.2 [29] for A. fasciatus and A. wenchowensis to estimate the change in population size over time.”

Line 193: You write about four biogeographic units, but then in Lines 194–196 you name only three. Please, check it!

Response: As requested, we revised it. “The distributions of populations were divided into three biogeographic units based on the sampling and distribution range of Acrossocheilus: (1) the Yangtze River region (AKSZ); (2) the northern Zhejiang region (AFHZ, AFNB, AFKH, AFXJ and AFWL) and (3) the southern Zhejiang region (AWLS, AWQY, AWTS and AWFD).”

Lines 194–196: The codes AKSZ, AWLS, AWQY, AWTS, and AWFD are missing in Table 1 and Figure 1. Please unify all sample codes in the text, Table 1, and Figure 1. Why aren't the samples with codes AFLS and AFWS mentioned here?

Response: As requested, we revised it. Because these two populations (AFLS and AFWS) are artificially released and not naturally distributed in Acrossocheilus fasciatus, they are not included in the analysis.

Line 200: Comma before “and”

Response: As requested, we revised it.

Line 201: Replace “cyt b + D-loop gene” by “cyt b + D-loop region”

Response: As requested, we revised it.

Line 202: I think it is better: “…which was then used to discriminate the most probable scenario among…”

Response: As requested, we revised it.

Line 204: Comma before “and”.

Response: As requested, we revised it.

Line 204: Figure 2(A) is better, I think.

Response: As requested, we revised it.

Lines 204, 206, and 208: Why do you duplicate the numbering of the Scenarios with the letters (i), (ii), and (iii)? I think they could be deleted.

Response: As requested, we deleted it.

Line 212: Figure 2(B) is better, I think.

Response: As requested, we revised it.

Line 229: Comma before “and”.

Response: As requested, we revised it.

Figure 2(B). What do N, N1, N2, N3, N4, and N5 mean? Please indicate this in the caption to the Figure.

Response: As requested, we revised it. “Time (T1-T3) and effective population size (N-N5) are not to scale.”

Line 236: Replace “cyt b + D-loop gene” by “cyt b + D-loop region”

Response: As requested, we revised it.

Lines 238–241: Three sentences in a row begin with the same phrase "A total...". Please rephrase this, for example, like this: “A total of 537 variable sites were observed, of which 405 were parsimony informative.  From 160 specimens of the 12 Acrossocheilus populations we indentified 124 haplotypes of which 110 were characteristic for A. fasciatus and 88 for A. wenchowensis (Table 1).”

Response: As requested, we revised it.

Lines 243–245: That's not a very well-constructed sentence. Maybe that would be better: "The average haplotype diversity within A. fasciatus was high (0.994), ranging from 0.643 (AFHZ) to 1.000 (AFWL), and the average nucleotide diversity (θπ) for this species was low (0.018), ranging from 0.003 (AFHZ) to 0.0142 (AFNB) (Table 1)."

Response: As requested, we revised it.

Line 245: The value of 0.0014 is less than 0.003. In addition, 0.0014 is not in the Table 1. Obviously, there is a typo here. How was the average nucleotide diversity calculated, and obtained a value of 0.018? Why does the average value exceed the maximum value detected in the populations?

Response: As requested, we revised it. “0.0142 (AFNB)”

Line 246–248: The same observation about sentence construction as in the lines 243–245. You can change it like this: Within A. wenchowensis the average haplotype diversity was high (0.975), ranging from 0.000 (AWTS) to 1.000 (AWWC), and the average nucleotide diversity (θπ) was low (0.013), ranging from 0.000 (AWTS) to 0.011 (AWFD) (Table 1).

Response: As requested, we revised it.

Line 246: Actually, the maximum value of haplotype diversity according to Table 1 is 0.982, not 1.000.

Response: As requested, we revised it.

Line 249: Actually, there are other values for A. kreyenbergii in Table 1, not 0.825 and 0.021. Check it.

Response: As requested, we revised it.

 

Line 256: Replace “cyt b + D-loop gene” by “cyt b + D-loop region”

Response: As requested, we revised it.

Line 257: There is other value in Table 2, not 0.902.

Response: As requested, we revised it.

Line 258: Comma after A. fasciatus.

Response: As requested, we revised it.

Lines 258–259: There aren’t values 0.491 and 0.952 in Table 2 for A. wenchowensis. Check it.

Response: As requested, we revised it.

Table 2. It is very strange that at such high Fst values, the p-distances are 0.000 even between different species. How correct are these data? How were the p-distances calculated? How can you explain this situation?

Response: Thank you for your suggestion. “Table 2. Matrix of pairwise FST values among twelve populations in three Acrossocheilus species based on the mitochondrial cyt b + D-loop region (below diagonal) and p values (above diagonal).” The p-value refers to the statistical significance level. We described this statistical method in the article. “Pairwise F_ST between all pairs of populations and analysis of molecular variance (AMOVA) were used to examine the spatial partitioning of genetic variation among populations in Arlequin version 3.5 [17], with tests of statistical significance per-formed with 10,000 simulation steps for each comparison.”

Line 270: In the text, the value is -25.03% with a minus, and in the Table 2 without a minus. Fix it, please.

Response: As requested, we revised it.

Line 274: Replace “cyt b + D-loop gene” by “cyt b + D-loop region”

Response: As requested, we revised it.

Line 278: Replace “cyt b + D-loop genes” by “cyt b + D-loop region”

Response: As requested, we revised it.

 

Table 3. Why are the values 63.31 and 10.15 in bold? Please explain this in the Table 3 header.

Response: Thank you for your suggestion. We remove the boldface.

Line 281: Comma before “and”.

Response: As requested, we revised it.

Line 282: Replace “cyt b + D-loop genes” by “cyt b + D-loop region”

Response: As requested, we revised it.

Lines 281–282: I think you meant: “according to the haplotypes' distribution pattern from different populations”.

Response: As requested, we revised it.

Line 283: Comma before “and”.

Response: As requested, we revised it.

Line 284: Comma before “and”.

Response: As requested, we revised it.

Line 287: Comma before “and”.

Response: As requested, we revised it.

Line 289: Remove “was also”

Response: As requested, we removed it.

Line 292: Replace “cyt b + D-loop genes” by “cyt b + D-loop region”

Response: As requested, we revised it.

Line 293: Comma after “ML”

Response: As requested, we revised it.

 

Figure 3. Which sequence of Spinibarbus hollandi was used? Please, give the number of the NCBI.

Response: As requested, we revised and added it. “Spinibarbus hollandi as an outgroup (NCBI accession No. AP012063.1).”

Line 298–300: Rephrase the sentences, for example, like it: “The spanning network confirmed the presence of the three major lineages of mtDNA (A, B, and C), with lineage A being interior and the others (lineages B and C) being located at the tip (Figure 4).”

Response: As requested, we revised it.

Line 301: Comma before “and”

Response: As requested, we revised it.

Figure 4. Population designations are very poorly visible.

Response: As requested, we revised it.

Line 320–321: Why was not the BSP analysis perform for A. kreyenbergii?

Response: Thank you for your suggestion. There is only one population in A. kreyenbergii, so we will not conduct BSP analysis.

Line 342: Comma before “and”

Response: As requested, we revised it.

Line 344: Comma before “and”

Response: As requested, we revised it.

Line 349–353: I think this part of text “over the “DEC model” (posterior probability = 0.0061 [0.0000,0.0265]; 0.0090 [0.0015,0.0165]), the “INC model” (posterior probability = 0.0000 [0.0000,0.0001]; 0.0000 [0.0000,0.0001]), the “INCDEC model” (posterior probability = 0.0000 [0.0000,0.0001]; 0.0002 [0.0000,0.0005]) and the “DECINC model” (posterior probability = 0.0000 [0.0000,0.0001]; 0.0000 [0.0000,0.0001])” can be removed and replaced by “over the other models in A. fasciatus and A. wenchowensis, respectively.”

Response: As requested, we revised it.

Line 366: Replace “cyt b + D-loop genes” by “cyt b + D-loop region”

Response: As requested, we revised it.

Line 365: Please place H. chenhsienensis with reference [4] first in parenthesis, and then S. macrops with reference [5].

Response: As requested, we revised it.

Line 368: Replace “of A. fasciatus exhibited” by “of A. fasciatus also exhibited”

Response: As requested, we revised it.

Line 374: Can you add in Table 1 additional column to show that lower nucleotide diversity is typical in the smaller rivers for the A. fasciatus?

Response: Thank you for providing very important suggestions. Due to space limitations, we will not add an additional row in the table, but we will modify the sentence in the article to express it. “In A. fasciatus, lower nucleotide diversity was found in the smaller river (e.g. Tiaoxi and Jinqing River), showing a positive relationship between the area of the drainage basin and genetic diversity.”

Lines 382–384: I think that in this sentence you should specify the designations of the parameters that you have in mind, namely Nst and Gst.

Response: As requested, we revised it.

Line 385: Replace “FST” by “F_ST”.

Response: As requested, we revised it.

Lines 388–390: On what basis is the assumption made about the translocation of individuals to these reservoirs? Are there any literary or other sources for this assumption? If these populations are indeed resettled, they cannot be used when considering the natural dispersal (phylogeography) of a species! This can lead to a misinterpretation of the results. For example, in the case of AMOVA, you would combine in Scenario II the native donor population located on one side of the mountain range (AFKH) and the introduced populations located on the other side of the mountain range (AFWC and AFLS). No wonder why the contribution of the component among group turns out to be even negative.

Response: Thank you for your suggestion. Introducing non-native species into rivers in mainland China through human activities is a serious problem. This is why we included non-native populations in our research analysis. However, it was our mistake to include these populations in phylogeographic analysis. Therefore, we conducted a new analysis using AMOVA and revised our results and discussion.

Line 394: Add a parenthesis after [5].

Response: As requested, we revised it.

Line 397: Add italics for A. fasciatus

Response: As requested, we revised it.

Lines 395–397: In fact, 10.15% is the lowest value and can hardly be used to analyze results. In the Scenario III, intra-population diversity, rather than diversity among groups, has the greatest importance. Please keep this in mind when discussing your data.

Response: As requested, we revised it. ”However, 27.03% of the total genetic variation occurred between the two geographical groups primarily divided by the Jinqing River and others in A. fasciatus.”

Line 402: Please repleace the species in order of literature references, like this: H. chenhsienensis [4], S. argentatus [5], S. sinensis [7], and Rhynchocypris oxycephalus [38].

Response: As requested, we revised it.

Line 437: Add a parenthesis after [4].

Response: As requested, we revised it.

 

peer-review-26973373.v1.pdf

Submission Date

29 January 2023

Date of this review

06 Feb 2023 15:38:14

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Dear authors,

if it's true that it was difficult to obtain the fish specimens, then I believe you.

All the best.