PONE-D-20-37420

Phylogeography of Aphyocypris normalis Nichols and Pope, 1927 at Hainan Island and adjacent areas based on mitochondrial DNA data

PLOS ONE

Dear Dr. Jang-Liaw,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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We look forward to receiving your revised manuscript.

Kind regards,

Tzen-Yuh Chiang

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript by IS Chen and NH Jang-Liaw reports the mtDNA phylogeography of a cyprinid fish, Aphyocypris normalis, commonly distributed in the southern part of China including Hainan Island, based on an extensive geographical sampling. This seems to be the first report of “detailed” phylogeography and population structuring of freshwater fish within Hainan Island, and will contribute to our understanding of geographical formation of fish fauna under the climate (sea level) oscillation in Asian regions. On the basis of the potential importance of this work, it is worth to be published.

However, unfortunately I should say that the present form of the manuscript is still immature and includes several points to be improved much. I would be happy if my comments will help the authors improve their manuscript for its publication.

Major points:

(1) The scope, originality, and emphasis of this paper should be clarified and limited.

First of all, the authors must recognize and adequately value the closely relevant work, Huang et al. (2019: ZooKeys) [ref.21], which studied on the same species and geographic region and the similar topics with longer mtDNA sequences, but with lower sampling density. The previous study has already reached the same conclusions as parts of the present study. They should not be fully repeated in this paper. Huang et al. (2019) has already revealed the wide range distribution of a part of haplotypes across the Qiongzhou (Hainan) Strait and the interspecific relationships. I think the detailed population structure should be the main, original results in the present study.

Also, extra parts apart from the main purposes are found in Discussion; i.e., parts about the nomenclature of genera (L393–), Aphyocypris arcus (L522), and Aphyocypris moltrechti in Taiwan. I understand the authors’ mind, but they are not the discussion from this work.

(2) Some recent references relevant to this work should be referred to and cited.

I believe some recent references as exemplified below should be cited, and some data should be updated.

Kang et al. (2014) Mapping China’s freshwater fishes: diversity and biogeography. Fish Fish 15: 209–230. https://doi.org/10.1111/faf.12011

Xing et al. (2016) Freshwater fishes of China: species richness, endemism, threatened species and conservation. Diversity Distrib 22:358–370. https://doi.org/10.1111/ddi.12399

Xiong et al. (2018) Factors influencing tropical Island freshwater fishes: species, status, threats and conservation in Hainan Island. Knowl Manag Aquat Ecosyst 419:6. https://doi.org/10.1051/kmae/2017054

He et al. (2020) Diversity, pattern and ecological drivers of freshwater fish in China and adjacent areas. Rev Fish Biol Fisheries 30:387–404. https://doi.org/10.1007/s11160-020-09600-4

(3) The examination of population divisions using AMOVA should be more clearly explained in Methods (L182–) and Results (L302–). Quite complicated. Different (contrasted) models should be shown clearly in text, or, as Tables or Figure.

Also, why do not you consider the use of SAMOVA?

(4) Nested clade analysis is not actually used here and also should not be used.

There is a section titled “Nested clade analysis and variations among clades” in Results (L351–). However, the authors do not actually conduct this analysis. The nested clade analysis (NCPA) is not only a nesting method of a haplotype network but a series of procedures to identify past demographic events by calculating some summary statistics and inferring histories of clades based on a set of criteria (see, e.g., Templeton 1998: Mol Ecol 7:381–397). This method is presently not recommended to be used. See Panchal and Beaumont (2010: Syst Biol 59:415–432) and the cited references on debates on NCPA. The section is inadequate in double meaning.

(5) Limitation of the conclusions due to use of mtDNA (effectively one “gene”) should be adequately mentioned in Discussion.

Medium problems:

(6) L36, “The relationships of A. normalis between Hainan and main land China populations are also discussed in this report.”: Readers want to know the contents of the discussion.

(7) L177, “the Kimura 2-parameter model (K2P)”: Why do not the authors use the best model selected by jModeltest? Should validate the use of the model.

(8) L220, “under a fixed clock pairwise evolutionary rate of 1.0 %/Myr following Ketmaier et al. [54] and Zhang et al. [55] with a strict clock model.”: Explain why the fixed clock model and the evolutionary rate were adopted. The results of divergence time are basically determined by the latter value. A range of estimates of the cytb evolutionary rate has been reported in literature.

(9) L240–: The diversity indices should be compared only among populations with not very few specimens. I do not think that the values for those with one, two, or three specimens are meaningful.

(10) L253, “A high number of haplotypes is characteristic of one and only populations.”: I cannot understand this sentence.

(11) L291–: Move the explanation of methods to “Methods”.

Also, what is “Noncoding” (L294) here? The data consist of only cytb coding region, don’t it?

(12) L298–302, Is this part replicated with that from L240? I cannot understand the relationship between these parts.

(13) L324–, “suggesting that the differences in climates among biogeographic regions constitute important barriers to gene flow”: Not fully logical. Even if the boundary is based on climates, it does not mean that the gene flow was prevented due to the differences in climates.

(14) L382–, “0.6344±0.0011 Ma (mean ±s.e.)...”: Should show 95% HPD instead of standard error for the estimate. Estimation errors of the divergence time are represented by the former.

(15) L395–: Readers cannot find any information what species once belonged to those synonymous genera solely in this paper. Also, as mentioned above (1), this part is not the conclusion from this study (Huang et al. 2019 have already shown the tree), but just taxonomic comments based on the phylogenetic tree. Nevertheless, the paragraph includes a nomenclatural statement. It should be valuable but be done elsewhere.

(16) L423–: The comparison of the results of AMOVAs only indicates that genetic diversity that cannot be explained by drainage differences is still large. This does not mean that the drainage differences do not contribute to geographical isolation. In fact, an extent of geographic cohesion of haplotypes are found in Figure 5. The expression in L434 (not only by...) seems fine.

(17) L429–: Is there no possibility that the distribution of haplotypes was partly caused (disturbed) by artificial transportation?

(18) L437–: Figure 5 (especially 5b) should be placed in Results because of easy recognition of the distribution of haplotypes (clades).

(19) L510, “except for the west central mountains southwestern mountains, and lowlands of Hainan Island.”: From where does this inference come?

(20) L513, “the river systems within Hainan Island did not create efficient isolation barriers similar to the strait”: This should be explained only by the short period after the last isolation. The drainage and strait undoubtedly work as barriers for strictly freshwater fish.

(21) L516–, “this research demonstrates that vicariant events have not yet generated interregional population differentiation in P. normalis.”: Same as above.

(23) L533–561: As mentioned in (1), this part is out of range of this paper. It should be worth to discuss but be done elsewhere.

Minor/Trivial points:

(22) L27, “based on molecular data and modifications based on climate patterns”: Difficult to understand here in Abstract.

(23) L32, “(W).The”, and other places (L276...): Insert a space between two sentences.

(24) L36, “main land” --> “mainland”

(25) L40, “It is”: What is “It”?

(26) L43, “how factors such as population structure ...”: Is the “population structure” a factor? I do not think so.

(27) L125, “were” --> “are”

(28) L173, “20,000” --> “15,000”?

(29) L212, legend of Fig. 3, “HKY+I+C” --> “HKY+I+G”?

(30) L219, “cytb”: Italicized.

(31) L267, “the Lou River”: Where is it?

(32) L290–: A too long paragraph. Change the paragraph at least at L302 (For...).

(33) L295, “Among” --> “Within”? Also, the correspondence between clades and average haplotype differences values should be clarified.

(34) L299, “among sampling locality populations”: What is this?

(35) L320, “the last model”: What is this?

(36) L343–, “and other two species of Aphyocypris. Like A. normalis and A. moltrechti, the two Aphyocypris species”: Show the species names of the two species.

(37) L352 and other places: TCS is the name of a computer software, not a name of method or algorithm.

(38) L381, “MCVRA”: What is this?

(39) L385, “in the middle to late Pleistocene”: The range (at least of means) does not seem to include the late Pleistocene.

(40) L419, “is” --> “were”

(41) L462, “Subclade...”: Change the paragraph.

(42) L487, “no obvious differentiation from Hainan Island populations.” --> “... from parts of Hainan Island populations”?

(43) L498, “These geographic events”: Not very precise. Sea level rising in the Holocene did not provide the opportunity.

(44) L504, L506, “Subclade Ia separated from other Clade I populations”, “Subclade Ic separated from remaining Clade I populations”: A clade cannot separate from a population.

(45) L511, “Clades” --> “The major clades”?

(46) Fig. 1: In the panel a, “b” and “c” in white are less visible.

(47) Fig. 5: Indicate names of major river systems.

(48) Table 2: “evolutionary divergence”: Should show which index was used.

--- end of comments

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PONE-D-20-37420R1

Phylogeography of Aphyocypris normalis Nichols and Pope, 1927 at Hainan Island and adjacent areas based on mitochondrial DNA data

PLOS ONE

Dear Dr. Jang-Liaw,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by May 22 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Tzen-Yuh Chiang

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript by IS Chen and NH Jang-Liaw is the revised version of PONE-D-20-37420, which reports the mtDNA phylogeography of a cyprinid fish, Aphyocypris normalis, commonly distributed in the southern part of China including Hainan Island, based on an extensive geographical sampling. Responding to several critical opinions on the original version, the manuscript has been largely revised and at least partly improved.

However, unfortunately I should again point out several flaws in the present form of the manuscript, which should be further improved. Also, the manuscript is still immature in terms of structure, explanation, and sentences. I would be happy if my comments will help the authors improve their manuscript for its early publication.

Major points:

(1) Divergence time estimation:

L245–: Because the divergence time estimation is directly affected by the evolutionary rate, the validity of 1.0%/Myr should be explained, or some excuse is required for the resultant estimates. Also, in the Discussion part (L517–), more careful discussion is required for the divergence time.

Furthermore, I’m not sure whether the authors notice it or not, Huang et al. (2019) present about twice values for the corresponding estimates (e.g., tMRCA of A. normalis = 2.33 mya, 1.92–2.74). Huang et al. (2019) mentioned as “A molecular clock was calibrated using a divergence rate of 2% per million years (Yang et al. 2016)” and the basis of Yang et al. (2016) is “A molecular clock was calibrated using the divergence rate of 2% per million years, as previously postulated by Brown et al. [44] and Bermingham and Martin [45]”, i.e., very famous, classic papers’ values. Namely, Huang et al. (2019) should use the evolutionary rate of 1% (not 2% of the “pairwise” value, which should be half for one lineage), as same in this study. The discrepancy (twice values of the estimates) is quite strange. I simply guess that the Huang et al. (2019) misused the pairwise divergence value (2%/Myr) as the evolutionary rate directly, if the present study is correct. Similar mistakes can be found in not few papers. This can be mentioned to emphasize the value of the present revised analysis.

(2) L344–, “suggesting that the differences in climates among biogeographic regions constitute effective isolation mechanisms to gene flow”:

This “suggestion” is not satisfactory. Correlation does not necessarily mean causality. Also, there are many differences between the boundaries in Fig. 3a and 3b.

Also in the Discussion part (L457–), I cannot understand the degree of congruence between climate division by He and Zhang [45] and phylogeographic division by this study, although I agree that geographic and climatic settings (topographical features, frequency of floods, etc.) have affected the population structure by disturbing the separation of basins that is an important basis of isolation. I believe that the latter claim has general value and should be emphasized in this paper. However, the evidence for the claim in this manuscript is still not sufficiently convincing.

(3) “isolation by distance” is not well evidenced.

The authors contrast an isolation by distance scenario with a vicariance scenario (L501, L520–523, L531–). But they do not provide the evidence distinguishing these scenarios. Peripheral populations can be isolated also by “small” vicariance factors.

(4) The structure of the “Discussion” part is confusing.

Obviously, the paragraphs from L508–L536 should be included the preceding section (“Genetic diversity and structuring in Aphyocypris normalis”), since the paragraph is the discussion deeply relating to those just above. Only the paragraph from L537 may be separated (but see the below comment).

(5) Discussion of A. arcus (L537–546) should be shortened or removed. In the latter case, the results should be also removed from the main text and figures.

The phylogenetic relationships of Aphyocypris spp. have already shown in Huang et al. (2019) with the same type (mtDNA, but longer) of data. The only new knowledge is that the authors could not collect the A. arcus in Hainan Island. Nobody can conclude that A. arcus is not distributed in Hainan from this result of the sampling. A possible way to do it is to reexamine the specimens used in Du et al. [76] with comparative specimens and to show their morphological key is not effective to identify different species. In this paper, at least the expression of the suggestion should be weakened more.

(6) Discussion of A. moltrechti and A. amnis (L548–580) should be much shortened or removed, because no new information is given in this study for this matter.

I agree that any taxonomic problems should be resolved as soon as possible. However, the present data do not provide any new information to resolve this problem. This part is just the authors’ claim.

That is, (1) the low genetic diversity in the Zhoushui R. population and its small divergence from other populations have already been reported in Chiang et al. [77] using mtDNA and microsatellite data; (2) Liao et al. [62] provide the morphological diagnostic characters for A. amnis, which seem not to be distinct and hence controversial; (3) generally, taxonomic ranking (intraspecific variation, ESU, subspecies, or species) of allopatric populations with small morphological/genetic differences could be often subjective; and (4) there are biological species with no genetic differences from relatives in neutral markers. A possible way to conclude this problem is to publish a nomenclatural article to synonymize the A. amnis with integrating morphological and genetic data.

However, some taxonomists may consider that if a population can be distinguishable from others in any characteristics or combination of characteristics, it can be treated as a “species” and the species rank is more beneficial for conservation action than just a local population.

I do not dare to discuss this matter with the authors, but I only like to say that the part should be shortened or removed because no new information is given in this study and the discussion is not satisfactory to lead to resolution.

Medium points:

(7) L36, “The relationships of A. normalis between Hainan and mainland China populations are also discussed in this report.”

Again, I should say that the readers of Abstract want to know what is discussed.

(8) L161–: The section of “Data analysis” is better to be divided. The next section “Divergence times among major clades” is also “data analysis”; so they are unbalanced. The section of “Data analysis” includes two topics; i.e., “Phylogenetic analysis” (including two datasets) and “Population structure” (AMOVA and SAMOVA).

Also in the “Results” part, the results of AMOVA/SAMOVA (L321–377) should not put in “Phylogenetic analysis” section because they are not phylogenetic analysis. A separate section should be established.

(9) L185–: In the explanation of AMOVA/SAMOVA, the authors should first explain all the models include two hierarchies of groups (i.e., groups of population samples and population samples). Some (schematic or realistic) figure for each model (even as supplementary) will be effective. Consider presenting it. It is not easy to understand the models only by text.

(10) around L246: The tree prior should be explained here. According to Table 5, the constant population model seems to be used. I strongly recommend that the analysis with other models, especially the Birth–Death model (see Ritchie et al. 2017: Syst Biol, DOI:10.1093/sysbio/syw095), should be also added and mentioned for showing the robustness of the results. This is because coalescent processes for the geographical samples of animals with low dispersal ability (like freshwater fish) should differ from those in a constant population.

(11) L247, “for each partition”: There is no explanation on the partition of sequence data. Should be explained.

(12) L295, “NJ and ML trees (S1 Figure)”: The information of the branch length of the ML tree should be kept and shown. Also, bootstrap values should be shown also in S1 Figure.

(13) L321–L345: Why does not the authors refer to the number of hypotheses here? What are the “two hypotheses” (L336) and “the last hypothesis” (L339)? The explanation here is difficult to understand. L321–323: This should not be a main topic (i.e., relation to hypotheses is unclear). Gene flow (L323) between which? It will much better to start to explain important issues.

(14) L359–: The result of SAMOVA should be shown as a figure (even if in supplementary).

(15) L396–: The term “Clade” is not suitable if it is defined in the haplotype network, because some are not monophyletic. The “Clade” should be those defined in Figure 2, shouldn’t it? Explain it first. Relating to this, not only Clade II but also Clade I is NOT monophyletic (L404).

(16) L483, “Subclade Ia is currently limited mainly to the west-central mountain area and shows clear differentiation between nearby populations in ranges NW and SW. “

I cannot understand this part. The Subclade Ia is distributed in NW. Probably it’s Subclade Ib in C, according to Fig. 3b, isn’t it?

(17) L496–, L519–: No figure is provided for haplotype (clade) distribution in the continental area (only explained in text). I wonder if such a figure also should be put in Fig. 3(c?).

Minor/Trivial:

(18) General: A blank line should be inserted between paragraphs and between figure legend/table footnote and text, if no indent is used. It is difficult for reviewers to identify the paragraphs and to distinguish the text from explanation of figures/tables in this manuscript.

(19) General: The name of a river is usually accompanied by “the” (definite article). There are inconsistencies in many places.

(20) L91, “with much lower sampling density”: Compared what? That does not yet appear. --> e.g., “with too low sampling density to clarify....”

(21) L96: “within” --> “with”?

(22) L98: “a freshwater fish” --> “this freshwater fish”, “this species”.... Should be restricted.

(23) L102: “proposed” --> “propose”

(24) L106: “were” --> “are”

(25) L109: “Sample used” --> “Samples used”

(26) L175, “15,000,000 trees”: This should be “15,000 trees”. (i.e., 2,000,000/100*0.75=15,000)

(27) L177–, “The genetic distances ... were calculated using MEGA5 with the Kimura 2-parameter model (K2P)”: In the response letter, the authors explaine the reason why the best model was not used as it (HKY+I) cannot be available in MEGA. There should not be large differences between the K2P and HKY because they are similar models (ti, tv, 2-parameter model). However, I wonder why the authors do not use other software that can use HKY+I to calculate the distances (e.g., PAUP*).

(28) L181, “which was carried out using ANeCA v1.2”: Should be deleted. TCS, which was actually used in this study, is an independent program. If ANeCA can automatically nest the network, the explanation should be changed. However, the result of the nesting is only partly shown in the Results (Fig. 5). Readers cannot see full results of the nested clades.

(29) L185, “The relative contribution...”: Change the paragraph. AMOVA/SAMOVA are quite different approaches from tree analyses.

(30) L193, “as (hypothesis 3) and (hypothesis 4)”: Should remove parentheses.

(31) L205: “suggests” --> “suggest”

(32) L206: “the hypothesis” --> “the relevant <or corresponding=""> hypothesis”

(33) L206, “SAMOVA 2.0 [48] to confirm that the grouped populations are geographically homogeneous and maximally differentiated from each other or not,...”: This explanation should be insufficient (inadequate) and reconsidered.

(34) L211, “We set the population of a single river system as a grouping unit (17 counts),”: What is the “population”? “population samples”?

(35) L213: “exam” --> “examine”?

(36) L214–, “However,... pattern).”: I think this sentence should be removed. Why “However”?

(37) L216–222: Redundant. This part can be shortened.

(38) L228: “are” --> “is”

(39) L269–, “Estimates of genetic variability differed accordingly.”: Not enough words, and not necessary. Should be removed

(40) L270–274, “Within-population ....(Table 1).”: Put this general description above the specific description (--> L264).

(41) L286: The section title “Phylogenetic analysis” seems not suitable in the Results. For example, “Phylogenetic relationships” seems better.

(42) L290: “including” --> “including those in”

(43) L318, “Distances”: Which distances? Should be clearly shown.

(44) L383–: “Like A. normalis and A. moltrechti, the two Aphyocypris species also formed...” --> “Aphyocypris kikuchii and A chinensis also formed...”

(45) L392, “Haplotype Network and variations among clades”: “Network” --> “network”. “variations among clades” --> What variation?

(46) L394, “within haplotypes”: Impossible.

(47) L396, “MP tree”: No such analysis in this paper.

(48) L399, “a fourth”: “one of four sampling locations”? But there are eight localities on the mainland. Ununderstandable.

(49) L400, “However”: Ununderstandable.

(50) L402: “, and included” --> “, which included” (h1 cannot include five haplotypes.)

(51) L405, L406, “the two haplotypes”, “these two haplotypes”: Clearly show them.

(52) L414, “Numbers inside circles are haplotypes.”: Should not be shown in bold.

(53) L426: “Pleistocene” --> “the Pleistocene”

(54) L428: “tMRCA” and “tmarca” should be consistent.

(55) L439, “These two groups”: There are three groups (clades). What are the groups here?

(56) L455: “resulting” --> “resulted”?

(57) L476, “highly supported phylogenetic subclade”: Show the subclade in parentheses.

(58) L484: “Upstream Changhua River (range SW) populations show...” --> “Sharing Clade III haplotypes, the upstream Changhua River (range SW) populations show...”. (to add an explanation of the “close relationship”)

--- end of comments</or>

**********

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PONE-D-20-37420R2

Phylogeography of Aphyocypris normalis Nichols and Pope, 1927 at Hainan Island and adjacent areas based on mitochondrial DNA data

PLOS ONE

Dear Dr. Jang-Liaw,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Reviewer #1: (No Response)

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Reviewer #1: Yes

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6. Review Comments to the Author

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Reviewer #1: This manuscript by IS Chen and NH Jang-Liaw is the second revised version of PONE-D-20-37420, which reports the mtDNA phylogeography of a cyprinid fish, Aphyocypris normalis, commonly distributed mainly in the southern part of China including Hainan Island, based on an extensive geographical sampling. Responding to several critical opinions on the original version, the manuscript has been revised and at least partly improved.

However, again, I should say that the manuscript is still immature in many points including problems of structure, descriptions, and a huge number of minor points.

I hope that the authors will carefully consider the below comments and revise the manuscript thoughtfully. Also, I hope that you will forgive me for adding many new comments that I failed to point out last time. Also, my comments at this time will not be complete ones. There are too many points... At the very least, please check the English before resubmitting.

Major points:

(1) Structure of the “Materials and methods” part

I strongly recommend that the authors reconsider the structure of “Materials and Methods”. Specifically:

(1-1) The paragraph of L229–242 is for the interspecific phylogenetic analysis, so it should be placed in the “Phylogenetic analysis” section. Move to L190.

(1-2) The “Divergence times among major clades” section (L244–260) should be placed before the “Population structure” section (L191–). This is because the divergence time estimation is the analysis next to “Phylogenetic analysis”.

(2) Structure of the “Results” part

I strongly recommend that the authors reconsider the structure of “Results”, too.

After the “Phylogenetic relationships” section (L256–), the order of contents will be better to be [1] “Phylogenetic relationships” (including that among the related species), [2] “Haplotype network”, [3] “Divergence times among major clades” (but also see below), and [4] “Population structure” (L338–404, AMOVA, SAMOVA section).

Specifically, L338–404 should be moved to the new (last) section of [4] “Population structure”, as in Methods, because this part is not about “Phylogenetic relationships”.

The results of phylogenetic relationships among the related species (L407–412) should be moved to L338. However, if I were an author, [3] Divergence time estimation would be placed before the part of phylogenetic relationships among the related species, without establishing an independent section. This is because of the tight connection between the phylogeny and divergence time.

(3) Structure of the Discussion part

The structure of the Discussion part should be also reconsidered. Specifically,

(3-1) The newly added part to examine Huang et al.’s [24] dating (L489–496) should be moved to the place after the discussion based on the divergence time estimated in the present analysis (L585?). The place in the present manuscript is quite inadequate.

(3-2) The section starting from L550 (Comments on distribution of...), at least its first half is one of the main topics of this study. I strongly suggest changing the heading, e.g., “Distribution processes of Aphyocypris normalis in Hainan and southern China”. And, if the authors like, add “, with a comment on Aphyocypris arcus in Hainan”. Again, the former is one of the main topics of this paper, not just a comment.

(4) L501–, and Results: It is not well explained how the final phylogeographic pattern was derived. It should be made clear how the climate zones were modified. Does it differ from the pattern solely based on the cytb data?

L502, “we have constructed...”: Where? Probably here the authors “propose” the modified divisions.

Medium points:

(5) L35–: The sentence from L35 (The divergence events of...) is better to be moved to L27, before the sentence “Additionally...” with small modifications as:

“The divergence events of these clades were estimated to have occurred between 1.05–0.16 Ma.”

This is because the explanation of the divergence times is not well linked with the explanation of the population divisions.

(6) L36–: The two sentences from L36 (Furthermore..., The strait between them...) should be removed, because these two sentences are a repetition of the above part and hence are redundant.

(7) L82–83: The distribution of Aphyocypris normalis should include Vietnam and some Indochina regions, according to the Results and Discussion parts.

(8) L115–: If the (most of) present samples were collected by the authors, they should be declared in this paragraph. At present, it reads as if it were simply a study using museum specimens without any fieldwork.

(9) L144: “which...”--> “Specimens that have been treated as Aphyocypris amnis.”

(10) L184, Issue of genetic distances and “PAUP*”:

I believe that the evolutionary distance is better to be calculated under the best evolutionary model that is shown in the same paper. However, I do not dare to claim this again because the authors have already denied it twice.

Now the only thing I like to say is that the author’s explanation does not make sense. The authors indeed used PAUP* in this paper. Also, PAUP* has been continually updated until now (see https://paup.phylosolutions.com/), although the authors write that the software has stopped updating in the reply letter.

Again, I do not dare to request the change because the models will not largely change the results (distances) and the usage of K2P is not incorrect (only it is not the best). However, only the thing I want to say is the authors’ explanation did not convince me.

(11) L247, L462, Issue of the tree prior:

In the previous comments, I recommend that the authors should try to use also the “Birth-Death” model as a tree prior (see Ritchie et al. 2017: Syst Biol, DOI:10.1093/sysbio/syw095) because the constant population size model is the coalescent model for a practically single population. The use of the latter simple model seems to be inconsistent with the population differentiation claimed in this paper. The authors explain something in the reply letter, which did not convince me. Because this is the authors’ paper, I do not dare to repeatedly comment on the same thing. However, I only want to say that the use of the constant population model alone may not be the best choice.

(12) L256, “The parameters of the substitution model GTR were estimated independently for each haplotype data.”: Although the authors note that they estimate the model parameters independently for each haplotype, I have completely no idea what they mean by that. Since this study uses only one gene (cytb), there must be only one set of parameters for GTR. Or, since it is a protein-coding region, it is possible to give three different models for each codon site. The authors should provide a clear explanation.

(13) L320–330, Figure S1: The branch length should be reflected in the ML tree. Why did the authors remove the length information only from the ML tree? Strange.

(14) L361–, “, suggesting... AMOVA results”: Should be removed because this is a discussion, not a result from the analysis. Should more carefully discuss this in the Discussion part.

(15) L391–396: This part is a discussion, which should be moved to the “Discussion” part. Also, readers are surprised when Indochina is suddenly mentioned. It should be properly explained in the “Introduction” and the “Materials and methods” parts.

(16) L561, “In summary”: It’s not “in summary”. Should start summarizing the results and depicting the history of this fish. --> For example, “According to our divergence time estimation of the major clades of A. normalis, the order of phylogenetic isolation events began with....”

(17) L605–, “since only four haplotypes were identified from 22 sequences that were 1,091 bp long and only one haplotype was found in the Dajia and Wu Rivers’ population”:

The number of haplotypes neither supports nor denies a different species. Should be rewritten.

(18) L627–629, “We suggest that both....in the future.”: What’s happened? Is this “suggestion” for A. amnis issue? How related? Should be rewritten.

Minor/Trivial points:

(19) L38: “We suggest that...” --> “The present results indicate that...”

(20) L44, “closely to....closely and”: Remove the latter “closely”.

(21) L73: “animal diversification” --> “intraspecific diversification”. Should be more specific.

(22) L86: Remove “the major river systems of”, because the same phrase appears at the end of the sentence again and it is not necessarily needed in the first part.

(23) L88: “major river systems.” --> “major river systems in these regions.”

(24) L93, “had”: Remove.

(25) L94: “same fish” --> “the same fish”

(26) L94, “longer”: Remove. No comparison can be done, because the present data have not yet appeared.

(27) L96, “specific”: Remove? Obscure.

(28) L99, “Same as the study of Huang et al. [24],”: Remove. No need to refer to here. It gives an only negative effect. --> “The objective of....”

(29) L100, “mitochondrial”: Remove. No need.

(30) L101, “more”: Remove.

(31) L102: “comprehensive sampling efforts across the species’ entire range” --> “comprehensive geographic sampling”. Redundant and not very precise (how about Vietnam?)

(32) L102–104, “is the first...sampling localities,”: Remove. Repetitive and redundant.

(33) L105: “with mitochondrial cytochrome b gene” --> “with the mitochondrial cytochrome b gene”

(34) L105, “combining the molecular data and the evolutionary time to estimate the evolution or dispersal...”: Should be rephrased, because the sentence is strange in some respects. --> For example, “estimating evolutionary time to infer the distribution history of A. normalis with respect to....”

(35) L107: “We also propose...” --> “On the basis of our results, we propose...”

(36) L110: “the nomenclature status” --> “its taxonomic status” (Any nomenclatural statement should not be given in this paper.)

(37) L111: “(comments...) proposed” --> “(comments...) provided”

(38) L167–168, “Preliminary phylogenetic... DnaSP v5 [33].”: Removed. No relation to the Results.

(39) L193: “were” --> “was”, or (L192) “contribution” --> “contributions”

(40) L251, “a fixed clock pairwise evolutionary rate”: Remove “clock” here.

(41) L264–, “A total...specimens were ... deposited in the GenBank database”: Inaccurate statement.

(42) L285–, “from Changhua River”: Add “the”. Check it throughout the text again.

(43) L290: “that stream. Copious haplotype” --> “that stream; copious haplotype”. The second sentence is the specific description of the first sentence. Some connection (logical words or style) is necessary.

(44) L319, “sequence.”: Of what? The caption should be understandable on its own.

(45) L321, and Figure 2, “>90%”: As the authors may know that the Bayesian posterior probability is usually expressed as 0.00–1.00 as probability, not in %, although I do not dare to request to change it.

(46) L325–, “Abbreviations beside clades or sub-clades correspond to the sampling sites listed in Table 1.”: I cannot find such abbreviations.

(47) L338, “the two a priori phylogenetic hypotheses that we examined”: There are four hypotheses. Also, the part “, isolated by the strait (Scenario 1 in Table 3) and drainage by river system (Scenario 2 in Table 3),” seems not clear. Please rephrase this sentence.

(48) L339–363: Replace “scenario” by “hypothesis”, as is in “Methods”. Confused.

(49) L339–341: Two “in Table 3” should be removed and put “(Table 3)” at the end of the sentence (i.e., “gene flow (Table 3).”). Redundant.

(50) L341–343, “we found in both hypothesis... of the Hainan Island vs. mainland hypotheses”: Contradicted. The last part should be for only Hypothesis 1, not both.

(51) L343: Remove “see”.

(52) L348–, “Our second set of experiments...”: In fact, the part below describes scenarios (hypotheses) 2 and 3. What’s happened?

(53) L352: “are” --> “were”. These are the results of a specific analysis. Also should correct the tense in L354 (are) and L356 (do).

(54) L354, “low”: Are the 0.32773 and 0.51140 really low? Strange.

(55) L358, “(Scenario 4 in Figure 3)”: In Figure 3, no word of “Scenario 4” appears. Strange citation.

(56) L360: “We found...” --> “That is, we found...” This sentence is a specific explanation of the former sentence. Some connection is needed.

(57) L365– (Table 2): K2P values are shown as two orders of magnitude larger.

(58) L372–379: This part is a footnote of Table 3, isn’t it?

(59) L378, “ FST values correspond to among individuals within populations.”: This is quite wrong.

FST in AMOVA is the variation component assigned to populations (without group division). So, 1–FST is the value that corresponds to the variation component among individuals within populations, isn’t it?

(60) L381: “can be” --> “could be” or simply “was”

(61) L382, “population Lou River”: Strange. --> “the Lou River population”?

(62) L385: Cite “(Figure S2)” after “Hainan Island”

(63) L385: “the AMOVA” --> “the results of the AMOVA”

(64) L398: “climate” --> “Climate”

(65) L415: “its sister species” --> “its closely related species”. Check the meaning of “sister species”.

(66) L416, “of the MrBayes program”: I do not think that the software name is needed to be described in the legend of the figure.

(67) L417: “Bayesian inferences” --> This should be “Bayesian posterior probabilities”

(68) L422, “A haplotype network is helpful in understanding the structure of phylogenetic relationships between haplotypes of Aphyocypris normalis among populations”: Should be rephrased. Strange. What is “the structure of phylogenetic relationships”? Phylogenetic relationships of what? “Between haplotypes”, or “among populations”?

(69) L424, “two groups were erected”: What are these? Specify. First, these two groups should be defined (explained) based on the network.

(70) L425, “only the small group of haplotypes h7, h8, h10, h11 is agreed with the Bayesian haplotype tree and ML as the Clade III”: Should be rephrased. See the above comment.

(71) L428, “network. . ”: Delete the second colon.

(72) L437, “In the small group of network result”: Reader cannot understand what this is. Rephrase, e.g., as “In the smaller group of the whole network (Clade III), the variation among the haplotypes was low...”

(73) L440, “occurred”: Should be removed.

(74) L441: “haplotype” --> “specimen”

(75) L444, “Parsimony haplotype network”: More precisely, a “Statistical parsimony haplotype network” is obtained by TCS. Not a “parsimony” network.

(76) L445: “haplotypes” --> “haplotype codes”?

(77) L453–456: Reconsider the number of significant digits to be shown. Also, make the number of significant digits the same among places

(78) L452: “MRCA divergence times for the three major clades were estimated at...” --> “MRCA divergence time for each of the three major clades was estimated at...” I think that the authors want to explain NOT “the time of MRCA of the three clades” but “that of each of the three clades”. Also, remove “, respectively” at the end of the sentence.

(79) L461, “significant”: Unnecessary?

(80) L462 (Table 5): What are the numbers in parentheses? No explanation.

(81) L467: “reveals” --> “revealed”

(82) L475: “the strait” --> “the Qiongzhou Strait”. This is the first place about this strait in the Discussion part.

(83) L477: “the major clade, Clade I, ” --> “one of the major clades, Clade I, ” or simply “Clade I”

(84) L482: “according to the phylogenetic analyses” --> “according to the present comprehensive phylogenetic analyses”. Why don’t you emphasize this here?

(85) L486: “were” --> “was”

(86) L489: “the corresponding estimates” of WHAT???

(87) L492: “which previously postulated” --> “which was previously postulated”

(88) L493, “Nevertheless”: Rephrase. This word does not make sense in this context.

(89) L493–495, “Nevertheless, Huang... value.”: This is a guess (inference), not a fact. Should be rephrased so that it can be understood to be a guess. Also, the sentence from L495 (The inconsistency...) should be placed before the previous one.

(90) L529: “valid” --> “distinct” or other adequate words?

(91) L540: “with the Pearl River System” --> “with that of the Pearl River system”

(92) L543, “(the major)”: Should be Removed. No information.

(93) L557, “77].With”: Insert a space.

(94) L564: “by vicariance and by distance” -> “by vicariance or by distance”?

(95) L565: “three subclades:” --> “three subclades of Clade I, i.e., ”

(96) L562–570: “separating” or “separated”?

(97) L568–: “in which show no efficient geographic barrier between them” --> “in which no efficient geographic barrier is shown between them” (no subject)

(98) L571: “that run northwestward.” --> “that run northwestward in Hainan.”

(99) L574: “land bridge existed...” --> “land bridge that existed...”

(100) L577: “vicariance” --> “isolated”

(101) L579–, “In general, this research demonstrates that vicariant events have not yet generated interregional population differentiation in A. normalis.”: Strange. Should be rephrased, as, e.g., “This research demonstrates that apparent vicariance events do not necessarily explain interregional population differentiation in A. normalis.”

(102) L582, “original”: What?

(103) L585: The author and description year should be shown for A. arcus anywhere, if any taxonomic discussion is done.

(104) L592, “We suggest that A. arcus...” --> “We suggest a high possibility that A. arcus...”

(105) L594–, “More comprehensive comparative studies of A. arcus on Hainan Island both on DNA and morphology are necessary in the future.”: Strange. In the sentence immediately above, the authors state the possible absence of this species in Hainan, so a comprehensive comparative study may not be possible.

(106) L605, “however”: Should be removed.

(107) L609: “0.0035” --> “0.0035 on average”

(108) L608, “Aphyocypris moltrechti and A. amnis” --> “Aphyocypris moltrechti (only the specimens from the Dajia R. and Wu R.) and A. amnis (the specimens from the Zhoushui R.)”. The authors do not support the validity of the latter species, don’t they? Also, in L610, “these two species” will be better to be “these two groups”.

(109) L615, “Their”: Whose? Describe.

(110) L618: “Zhoushui River population” --> “the Zhoushui River population”

(111) L620: “population” --> “populations”

(112) L620: “Compare” --> “Comparing”/“Compared”

(113) L622: “remarkable” --> “remarkably”

(114) L625: “the conclusion comes with...” --> the conclusion that comes with...”

(115) L626: “mitochondrial data as the only marker for inferring population histories may...” --> “only mitochondrial data may...”

--- end of comments

**********

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Reviewer #1: No

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PONE-D-20-37420R3Phylogeography of Aphyocypris normalis Nichols and Pope, 1927 at Hainan Island and adjacent areas based on mitochondrial DNA dataPLOS ONE

Dear Dr. Jang-Liaw,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jan 03 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

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Tzen-Yuh Chiang

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: No

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: N/A

Reviewer #3: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The manuscript titled “Phylogeography of Aphyocypris normalis Nichols and Pope, 1927 at Hainan Island and adjacent areas based on mitochondrial DNA data” investigated the phylogeographic structure of A. normalis, in Hainan Island and mainland china. Unfortunately, I found the paper is poorly organized with some mistakes as well as concept misunderstanding, not suitable for publication at current form. The aim and the abstract are not well clarified. The Introduction did not focus on clear background and miss some important references, in need of further revision on previous studies of freshwater fishes in Hainan island. Discussion and Results must be improved by removing general statements and explaining the results obtained and comparing these results with genetic studies for the structure of mitochondrial DNA. In reading the manuscript, I encountered several problems that required the author's most careful attention. Some of the issues are semantic/conceptual, others technical. I try to make my comment as constructive as possible and hope the author finds it useful.

L108-L111, The title of manuscript is “Phylogeography of Aphyocypris normalis Nichols and Pope, 1927 at Hainan Island and adjacent areas based on mitochondrial DNA data”. The author mentions the phylogeographic structure of A. normalis in Hainan Island and mainland China. However, the authors gave an interesting conclusion as “We suggest that more appropriate nuclear markers should be used to evaluate the validity of A. amnis in the future（L644-L646）”. As the specimen of Aphyocypris normalis Nichols and Pope, 1927 were from Hainan Province, and “A. amnis” were sampled in Taiwan Province, what is the evidences? And is this relevant to the topic of this MS.

L129, Why does the author combine populations into 17 groups as AMOVA analysis? L236-L238 “we combined populations into 17 groups based on drainage basins or close geographic proximity (i.e., the following populations were combined: sites 1–2, 4–5, 7–8, 12–14, 15–17, 19–25, and A2–A5; see Fig. 1).”

L285, “15 (1.283%) on the Zhubi River, which was represented by only two specimens”, why not use all the 7 specimens in the Zhubi River, and the author use 7 individuals in the AMOVA analysis.

L348-L349, Table 2, Clade Ⅰ is an unresolved tree. Clade Ⅰa, Ⅰb and Ⅰc are unresolved trees and inappropriately defined as monophyletic group. This is quite doubtful.

L418-L423, The author shows the phylogenetic relationships between A. normalis and closely related cyprinid species. But the author does not mention the phylogenetic relationships in discussion.

L485-L488, The reference (71) mention the phylogeography of Rana sauteri (Ranidae). R. sauteri is an amphibian, not freshwater fish.

L488-L491, The freshwater fish and the amphibian are the same in ecology?

L496-L512 According the phylogeny and network, the results do not support the eight phylogeographic ranges of A. normalis. The author must present enough evidence to prove it and explain in detail.

L517-L519, The author must explain why the same river system can be divided into two groups. What is the reason for the distinction?

L527, Subclade Ib is unresolved tree.

L534-L542, There are many studies of freshwater fish focus on the phylogeographic role of Changhua River. Needs to added the latest refs.

L571, Subclade Ia is unresolved tree and not a distinct monophyletic Clade. How about the h46?

L572-L573, Subclade Ic is a distinct monophyletic Clade. How about the h1 and h3? Why didn't the author combine the h1 and h3 in Subclade Ic?

L616-L646, Why does the author discuss the other A. species in Taiwan province? not concerning to this MS.

Reviewer #3: I had access to the previous MS, to the reviewer points, and to the answers given to the reviewer.

Generally, it was not easy to follow what has happened during the review process. Nevertheless I tried to keep up.

I see the effort to adequately answer the points, but I also feel, that sometimes the answers are quite elusive. For example the authors are asking for 'more time to reconsider' suggestions.

The reviewer was asking to use up-to-date softwares, or models. As I see, the authors are willing to use updated software, still they did not use it. The journal is PlosOne having high standards. So, if I were the lead author of this manuscript I would immediately recompute the results using better model even if the data is 8 years old. I would also try the suggested models to see what is the difference of the outcomes of the reported and the suggested models.

Additionally, rewriting of the sentences sometimes remained as confusing as previously were. I suggest to consult with more colleagues, go through the entire manuscript. Involve new eyes into the quest for such sentences.

I suggest to do the polishing work.

**********

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Reviewer #2: No

Reviewer #3: No

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PONE-D-20-37420R4Phylogeography of Aphyocypris normalis Nichols and Pope, 1927 at Hainan Island and adjacent areas based on mitochondrial DNA dataPLOS ONE

Dear Dr. Jang-Liaw,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by May 27 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
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• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Tzen-Yuh Chiang

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Partly

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: N/A

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: No

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The authors made an interesting topic in this MS, and I especailly apprecaite their detailed samling work and analysis on Wuzhi & Yinggeling, beyond the formers' study. However, the authors did not give more discussion on their differences and possible explanation. Here is another work focus on the same topic, pleased read it and discuss. Wei et al. 2021. Population genetic structure and phylogeography of Aphyocypris normalis on Hainan island and its neighboring regions. Chin J Appl Environ Biol, 2021, 27 (1): 191-199.DOI: 10.19675/j.cnki.1006-687x.2020.01025

The authors gave interesting workd by proposing hypothesis in explaining the phylogenetic pattern of A. normalis. As the resecrch focus in this MS,the details of the hypothesis as well as why giving such a hypothese are need, e.g., in hypothesis 2, the ciatations on why draniage or climate could be used as the controlled factor; the chracters of 8 climatic zone and its relation to freshwater fauna; what are the 12 drainages and their geological developmental traits; either in introduction or in discussion.

Line 487. what does the logic of this sentence "Nonetheless, .. fit...". Thus, did the results by Cytb fit/unfit the division of climate? This was a really problem need the authors to clarify seriously.

The result of 8 pholographic ranges was one of the two importants conclusion of this MS, seen from Abstract. Unluckily, I am puzzled by the authors work and explanation.

First, the details of samling sites needed illustration: climatic zone, river, corresponding to hypothesis 2b as sites (Just seen from figure 1 and figure 5a, I am not sure whether sample sites cover all the 8 climatic zones). Maybe still one more info, why the significant differences in site number in different climatic zones (only 1 site in a climate zone but many in another).

Second, seen from the figure 5a,b, the climate zone in Hainan Island was not concistent with the result of eight phylogeographic divisions. For example, climate 1 was divided into E and SE, climate zone 6 covered W and west part of SW; that means,the climatic division and the phylographic divisions intersected. As the authors suggested, the results in eight pholograhic ranges were moderated from climatic zone, so how to mederated, and the criteria? For example, the authors cancaelled the Subcalde (but in subgroup) in Clade I, then would it be suitabale to used subgroup to determine phylographical division?

The anthors took the results granted, too far-fetched without a equal criteria,and seemed eliberated to give such a result only aiming to match the 8 climatic zones for a possible explanation.

Third, the as an important conclusion, the author needed to give direct and clear result on the eight ranges by cytb, possibly directly marked in figure 2 or figure 3.

Moreover, adjacent areas were also inportant in this paper (the word also in the title), so the spatial pattern of A. normalis (clade II) also needed clarified and explanation, a 9th range or more? also controlled by climate or other factors? Should the factors controlling the phylographic range of A. nomalis be same or different in Hainan Island or adjacent areas?

In clade I, according to answers to the comments, the authors cancelled the validity of subclade and used subgroup, please giving personal difination on subgroup; otherwise, it is still cause confusion, as "group is monophyletic".

Moreover, in discussion the author still kept Subclade(since Line 562). I do think the author need to revised the paper in a throughout instead of only anwere the reviewers' comments and just simple corrections.

Why add a part of comment on A. moltrechti, and discussed a validity of a new species A. amins? It is non busisness of the thesis of this paper. It needs another complicated work in another paper, using works following by taxonomic standarss, including morphology, molecular,evolotion,as well as possible geological and ecological evidences, but not only in discussion.

Reviewer #3: The authors answered the fellow researchers' questions as well, they rewritten the manuscript.

I suggest the manuscript for publication.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #2: No

Reviewer #3: No

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PONE-D-20-37420R5Phylogeography of Aphyocypris normalis Nichols and Pope, 1927 at Hainan Island and adjacent areas based on mitochondrial DNA dataPLOS ONE

Dear Dr. Jang-Liaw,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Oct 16 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Tzen-Yuh Chiang

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #4: (No Response)

Reviewer #5: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #4: Partly

Reviewer #5: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #4: Yes

Reviewer #5: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #4: Yes

Reviewer #5: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #4: No

Reviewer #5: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #4: I think this is a very interesting system with excellent regional sampling. The broad questions are compelling, and I think the methods and sampling are appropriate to answer some of these broad questions of population structure across the region. I think the results that the authors found are interesting and a valuable contribution. However, I do have some serious concerns regarding the clarity of this paper and the over-interpretation of the findings.

General comments

1: The writing needs a thorough editing for brevity and clarity.

2: Additional natural history of the species is needed, what's the life-cycle, what's the global distribution, habitat preferences, etc.

2: Overall I think the paper needs to be simplified, it will have a cleaner and more compelling result and conclusion that way and the readership will be able to follow the flow of the paper better. After a number of reads through the paper I see the following flow of information:

- We provide thorough geographic sampling of a freshwater fish to address two questions

1: what, if any, genetic structuring is there among populations/drainages?

2: does genetic structuring follow biogeographic expectations? (and outline these clearly)

- We used the following methods to identify genetic structure among populations

1: phylogenetic analysis

2: haplotype network analysis

3: clustering analysis

- We conclude that one drainage on Hainan Island forms a distinct genetic group (very interesting result!), but little differentiation/structure was identified across the other samples and between Hainan and the mainland.

I think keeping a clear and clean flow of information in mind would significantly increase the strength of this paper. In doing so, I think the authors need to be exceedingly careful to not read into the genetic patterns too much and to not over interpret their results.

3: Following on the end of the above comment, I think the genetic data is over interpreted. The authors do no explicit tests of gene flow but talk extensively about evidence for gene flow. Note that there are other processes that result in low divergence (recent colonization, large population sizes and slow/low genetic drift, conserved genetic regions under stabilizing selection, and yes, ongoing or recent gene flow). The data are analyzed solely to assess population level genetic structure, the interpretation of results should be restricted to this. It is an interesting question and the authors have good sampling to ask the question of what is the genetic structure of the species across the sampling, it will be a much stronger paper and argument if the results and discussion remain focused on describing the patterns of genetic structure of the sampling. Interpretation of gene flow and historical biogeography should be limited, if not completely removed. The interpretation of these data, based on the data and the analyses, should be restricted to commenting on population-level patterns of genetic divergence/similarity. The results, in my opinion, have two compelling conclusions, there is structure within Hainan Island with samples from Clade III (SW region, Hainan) showing a distinct genetic background suggestion historical isolation, and there is little structure between populations on the remaining drainages of Hainan and the nearby mainland of China. These are very interesting and well-supported results, and it's the strength of the authors' sampling to identify these patterns. In my opinion, over-interpretation of these patterns does nothing but weaken the argument and conclusions.

4: There needs to be more historical geological information in the introduction. In order to formulate the questions that the authors are asking, the reader needs to have a description of the geological history of the island and of patterns found in other organisms, both terrestrial and freshwater. This needs to be included in the introduction in order to support the questions being asked. Moving the information from lines 492 to 499 into the introduction would make a huge difference, expanding on this geologic history and providing evidence shown in other organisms would be extremely helpful.

5: I found it very difficult to interpret the haplotype-based tree figure. I understand what was done, and that it is essentially a polarized network with phylogenetic-focused analyses of node support. However, in my opinion, it provides very little information additional to the haplotype network. I would suggest combining the haplotype tree and network into one figure with two panels as these two visuals are very complementary. I found it extremely difficult to interpret the results in a geographic context without a phylogenetic analysis where individual samples were the tree tips, then some geography (maybe the group identities) were overlaid onto this tree. I got there eventually through reading the haplotype distribution portion and interpreting the clade distribution from the maps. However, this should be a result that is clearly presented visually. Running the same phylogenetic analyses that you did, but where individual samples represented tips would be extremely helpful. One could code tip colors to correspond with the map colors etc., for ease of interpretation. I think this addition would provide much needed clarity and strength to the conclusions.

Specific Comments

Throughout: be consistent on how you refer to Cytb, I suggest using "Cytb" throughout

Throughout: need to clearly outline early on how you define group/site/population, and ensure consistent use

Title: add some higher-level classification to the title, e.g., "Teleostei: Cyprinidae"

Here on, line numbers refer to lines in version 1

Line 21: replaces populations with sites

Lines 40–46: I'd remove this paragraph, I think starting at line 48 is perfectly fine

Line 97: remove "thoroughly" but add "with dense geographic sampling across the island" following "Hainan Island"

Methods: need to add exactly how the F-statistics were calculated

Table 1. Move to supplemental

Lines 245 & 246: remove, end paragraph with "Zhubi River."

Line 232: Sentence starting with "Eighty-three..." belongs in methods, not results

Line 253: Sentence starting with "A high number..." seems incomplete

Figure 2 caption, line 281: Be clear what the numbers at the nodes refer to, bootstrap support values is good, but the numbers presented by the Bayesian inference should be posterior probabilities (I believe)

Line 291: Sentence starting with "Standard error" should be moved to methods

Line 393: move this section to the end of Discussion, as such it breaks the flow up notably

Line 492: as noted above, this section (492 to 499) would fit much better in introduction

Reviewer #5: The authors of the manuscript entitled Phylogeography of Aphyocypris normalis Nichols and Pope, 1927 at Hainan Island and adjacent areas based on mitochondrial DNA data sequenced mtDNA from 127 specimens and created a phylogenetic tree. The results indicated 3 major clades- one with wide geographic distribution and 2 that were more limited, however no obvious geographic barriers exist between them. There also appeared to be 8 biogeographic regions of A. normalis that correspond to climate. Based on results of the phylogenetic tree, The authors conclude that A. normalis diverged between 1.05-0.16 Ma, before the temporary land bridge existed across the Qiongzhou Strait.

Line 184- can you please clarify- were the separate runs combined? Did they converge?

Line 207- can you provide more information about parameters of the model were selected- tree prior, clock model, etc. Was any model selection performed? Are you using a model that was reported previously.

Line 212- please check the references throughout. This reference was not correct.

Line 544 subclade Ib AND Subclade III

Line 554 BY geographic barriers

Line 557-577- how do your phylogeographic regions differ by the ones described by He and Zhang.

Line 605- can you suggest any alternative explanations?

Line 637- and BECOME isolated

**********

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Reviewer #4: No

Reviewer #5: No

**********

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PONE-D-20-37420R6Phylogeography of Aphyocypris normalis Nichols and Pope, 1927 at Hainan Island and adjacent areas based on mitochondrial DNA dataPLOS ONE

Dear Dr. Jang-Liaw,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Dec 29 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
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Reviewer #4: All comments have been addressed

Reviewer #6: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #4: Partly

Reviewer #6: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #4: Yes

Reviewer #6: Yes

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Reviewer #4: Yes

Reviewer #6: Yes

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Reviewer #4: Yes

Reviewer #6: Yes

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6. Review Comments to the Author

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Reviewer #4: In general I think the authors present a well-sampled dataset that provides additional knowledge of population structuring of this freshwater species. Below I provide two sets of comments. I do have two general concerns both revolve around the fact that this is a single-marker mitochondrial dataset. With that, I'd like to see more discussion on the limitations of such a dataset (i.e., it's not appropriate to look for gene flow using this type of data), and a bit more caution with how "deep" into the methods and analyses the authors go. I think restricting analysis to patterns of differentiation, haplotype patterns, dated phylogeny, etc., is appropriate. Inferring gene flow, or lack thereof with a single marker mitochondrial dataset is difficult if not inappropriate. Further, some discussion on how mitochondrial capture or sweeps could have affected the conclusions would be useful.

This group of comments is primarily focused on wording and sentence flow, not content.

line 39: First sentence is too broad and nebulous in my opinion, I think you can combine the first two sentences to be a bit clearer: "Biogeography is tied closely to both ecology and phylogenetics and is a key topic..." The rest of this paragraph is very nice.

General: throughout, and this is likely a type-setter comment, the dash between a span of reference numbers should probably be an n dash, unless there is something special about a span of reference numbers.

Line 50: it'd be great to provide the maximum depth of Hainan Strait right off the bat here... "Strait (maximum depth ####; Fig. 1)"

Line 52: change to "se level (ASL) near its center, with Wuzhizhan Mountain being the highest point on the island...."

Line 70: I'm having a tough time linking the first and second sentences... maybe to increase flow you could add the following to the first sentence "among species and by general ecology (e.g., aquatic vs. terrestrial; [12]). In general, freshwater aquatic animals on islands, such as freshwater fishes, should share a similar evolutionary history to terrestrial species, and provide excellent opportunities for...."

Line 82: remove "commonly"

Line 86: change to "fish on the Indochina Peninsula is very limited, for example, the first formal record of A. normalis on this peninsula was in 2011 from northern and central Vietnam..."

Line 91: change to "conducted on terrestrial species on Hainan Island, including: earthworms....."

Line 94: change to "single sampling source"

Line 97: i'd remove the information in parentheses, the statement and reference are sufficient in my opinion

Line 99: replace comprehensive with "densely sampled"

Line 100: replace "natural history" with "historical biogeography"

Line 105: replace "at" with "based on data from"

Line 106: item ii reads like two items...i'd change this list to include three items with current item ii split into two

Line 116: Does "tissue" mean frozen in this example? Or were these also ethanol-fixed, if so, you could just say "utilized ethanol-fixed tissue samples from...." if not, i'd change to "frozen and ethanol-fixed tissue samples from...."

Line 118: Split this up...(44 specimens). This sampling encompasses almost...."

Line 122: collected at...not in

Line 128: I think this would read smoother as follows "All of the samples used in this study are specimen-vouchered, coming from collections established in 2009–2012 as part......"

Line 129: replace dash with n dash

Line 150: replace crude with "genomic"

Line 154: replace "an" with "the" and remove "segment of the"

Line 155: OK, I believe that abbreviations in the abstract don't follow through to the main text...so...right here I'd say you need to define the mtDNA abbreviation and cytb (as you did)....

Line 164: replace "with the aid of" with "using"

Line 165: "calibrated" seems like an odd word here? Maybe replace with "edited" or "trimmed"?

Line 271: add period after length

Line 403: Fst...need to clearly define in methods how this was calculated as there are multiple different ways to calculate Fst

Line 407: I'm having a tough time with this interpretation, there are major issues with using a single mitochondrial marker to infer anything about geneflow...the mitochondrion doesn't recombine... Further, low genetic divergence between regions in general can be explained by just low divergence (neutral or selective) or gene flow. And when talking about a mitochondrial marker....gene flow would either produce no change or entirely erase an evolutionary history due to the fact that these regions don't recombine...they either don't interact or replace.

Line 434: this is a poor definition of Fst, need to include at least some mention of relative...

Line 473: insufficient isolation barrier or just recent isolation...without sufficient time to build up differential mutations even if completely isolated.

Line 574: I think adding some clarity and discussion here would be good. First, a landbridge only provide connectivity for terrestrial animals...it may be a stronger barrier for freshwater species if there are not connecting drainages than saltwater may be. I think some commentary on this should be made...given unknown connectivity of freshwater drainage systems between mainland china and hainan island during terrestrial continuity, we are uncertain if there was freshwater connection or not, and if not, this terrestrial barrier may have been stronger than a saltwater aquatic barrier....

Line 642: replace variation with divergence...however, as eluded to above...I'd restrain from making too strong claims on taxonomy and species status based on a single mitochondrial marker. Mitochondrial capture/sweeps could have occurred, erasing the mitochondrial history even with very limited gene flow, and the nuclear genome could retain very large differences, not detectable with cytb sequencing along

Line 644: this sentence doesn't make sense...yes mtDNA is maternal, but comparing maternal origin with phylogenies at the species level or higher seems to not be a valid comparison. Comparing mitochondrial data with nuclear genomic data may provide different conclusions, using mtDNA data at population or species levels should change much...

Line 646: I'd suggest using reduced representation genome-wide markers

General comments:

Gene-flow: As mentioned above, I have strong reservations with inference of gene-flow, or lack thereof, from a single mitochondrial marker (cytb), see above for reasons. For this reason, I think it's important to either reduce the amount of discussion on gene flow or include a cautionary section on why a single mitochondrial marker and mitochondria in general are not good for detecting gene flow. Bottomline, I think there needs to be much more circumspection with inferring population structure and gene-flow from a single mitochondrial marker.

Landbridges: I'd appreciate more discussion on land bridges and how they affect freshwater aquatic organisms...as mentioned above, a landbridge between Hainan and China may have been a stronger barrier to gene flow than the current saltwater barrier, and without known patterns of freshwater drainages on this landbirdge (I assume), it's difficult to accurately infer the expectation for degree of this barrier to a freshwater species.

Reviewer #6: The manuscipt of phylogeography of Aphyocypris normalis at Hainan Island present a rather interesting study, that after these 5 rounds of revision has been considerably improved. There are only a few minor points that, in my opinion need revision, abd thus I suggest the manuscript to be accepted right after the authors proceed with these corrections, without the need for a further revision round.

Detailed points:

Line 105: the possible isolation mechanism of freshwater...

Lines 358 and forth: the terms used to describe the network (group, clade, subclade) are at points relatively confusing as they mix with the terms of the phylogenetic structure. For that I suggest the authors to name the compoenents of this network with a precise name (f.e. Cluster, Super-clusters), and follow that terminology with accuracy.

Line 439: SAMOVA indeed showed that K=3 has the highest value in terms of grouping options ; however, as K=4 was also very very close to this value, I would like the authors to say a few things more about this clustering option as well. What was this fourth cluster that exhbited such close possiblity?

Line 482: which might separate Subclade Ib...

Line 645-648: this sentence is indeed very important, as the authors rightfully acknowledge the fact that additional markers should be employed to understand the identity and phylogeny, as using only mtDNA marker might exhbiti only the single-organelle point of view.

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Reviewer #4: No

Reviewer #6: Yes: Dimitrios Avtzis

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PONE-D-20-37420R7Phylogeography of Aphyocypris normalis Nichols and Pope, 1927 at Hainan Island and adjacent areas based on mitochondrial DNA dataPLOS ONE

Dear Dr. Jang-Liaw,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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PLOS ONE

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Reviewer #7: All comments have been addressed

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #7: Yes

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Reviewer #7: Yes

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Reviewer #7: Yes

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Reviewer #7: Yes

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Reviewer #7: The article describes the values of genetic diversity, as well as its spatial distribution and ancestry-descent relationships based on a single gene. This is a limitation, but the information is undoubtedly relevant and provides a coherent explanation for the observed patterns. This limitation of using a single gene is identified by the authors, who suggest interesting lines of research proposals for the future.

212 I suggest the use of mode of previously reported molecular substitution rate instead mean

234 Combining the 19-25 populations that have intermediate to the Songtao Reservoir seems like a good idea, but can't a lentic system be a barrier for organisms that inhabit lotic systems? Explain

241-242 Define what values of Fst are considered High and low and references if corresponds ej Freeland 2005

243 Mention if the Fst values were computed using the information of sequences or just the haplotype frequency as the reviewer 4 suggested previously

272 Include the transition transversion ratio and mention the number of private haplotypes

278-283 It would be a good idea to explain in the discussion that the pattern of lower diversity in islands is common with respect to the continent and can be used to approximate the historical Ne of the populations. It can be suggested that in Zhubi river there is a higher Ne, i.e. how can it be explained that with an N of 2 it is the highest value? Considering that there are sites such as Wanquan river with n =10 with zero h and pi

285. Explain the average number of differences between these 12 haplotypes to get an idea of whether they are single nucleotide substitutions or involve several substitutions.

286-287 Indicate the number of differences of the shared haplotype between the Wanquan and Nandu, with respect to their closest haplotype. I.e. both systems are close but it is possible that it is the same haplotype by convergence?

292 haplotype 4 the ancestral one? Explain

366-369 h4 and h1 are the ancestral ones? Explain

419-420 Are there data on the characteristics of the rivers? Oxygen, temperature, which may be more relevant than what happens out of the water?

475-479 Mention that in addition to isolation by distance, there are other alternatives (e.g. isolation by environment).

491-496 Since the strait is not so relevant is it worth considering the depth of the strait, that space was land at glacial maximum?

497-498 Water characteristics of lotic systems this may be more relevant than just precipitation or temperature.

S2 please change the order of the two below figures d and c

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Reviewer #7: No

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Phylogeography of Aphyocypris normalis Nichols and Pope, 1927 at Hainan Island and adjacent areas based on mitochondrial DNA data

PONE-D-20-37420R8

Dear Dr. Jang-Liaw,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #7: All comments have been addressed

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #7: Yes

**********

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Reviewer #7: Yes

**********

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #7: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #7: Yes

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Reviewer #7: All my concerns about the manuscript was attended. The response of authors was according the theory as far as I know

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Reviewer #7: No

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