Phylogeny, Distribution, and Biology of Pygmy Whitefish (Prosopium coulterii) in the Beringia Region (Chukotka)

Round 1

Reviewer 1 Report

The authors aimed to explore the phylogeny of pygmy whitefish Prosopium coulterii using three mtDNA loci from 25 samples collected from the lakes of Chukotka. The MS is well written. However, the major concern in the MS is there is no phylogenetic analysis. The authors emphasized phylogenetic reconstruction of the species in the title as well as the introduction, but the same is lacking in the methods and results. There are only haplotype networks that can not provide sufficient information about phylogeny.

Interestingly, there are some morphological and anatomical measurements included which are not relevant to the objectives of the MS. 

I suggest authors run phylogenetic analysis (ML or BI) using the concatenated sequences of those three loci. It would really improve the quality of the MS.   

I have made some suggestions in the PDF. 

 

Comments for author File: Comments.pdf

Author Response

Dear Reviewer, 

First of all, we would like to thank you for sparing the time to write detailed and useful comments.

Please find the point-by-point replay for your comments below

Major comments:

1. I suggest authors run phylogenetic analysis (ML or BI) using the concatenated sequences of those three loci. It would really improve the quality of the MS

 

We have made the phylogenetic analysis and embed the corresponding paragraphs to the methods, result and discussion sections. We have also undertaken the phylogeny analysis for the connected sequences and discussed the result throughout the manuscript. All minor corrections were accepted and included into the text.

 

2. Interestingly, there are some morphological and anatomical measurements included which are not relevant to the objectives of the MS

 

We would like to preserve the basic biological features sections, since we believe it could be useful for future studies and for specialists in the similar fields. We have rather restricted data to publish it in a separate manuscript, so we think that this manuscript is the only place where that data could be presented. To make the manuscript more relevant for basic biological features data we slightly reformulated the manuscript title, abstract and aims of the study. We hope that the manuscript now reads better.

 

Minor comments

3. please rephrase

 

corrected

 

4. How many samples?

 

Specified

 

5. I could not see any phylogenetic analysis in results

 

Please see comment #1

 

6. It is a family name, no need to italicize

 

corrected

 

7. Use en-dash for indicating a range

 

Corrected throughout the text

 

8. Does this have any relation to this MS?

 

Please see comment #2

 

9. WHat are these underlines meant for?

 

Table is corrected and simplified

 

Thank you once more time for you corrections

Best regards,

Grigorii Markevich

 

 

 

 

Reviewer 2 Report

 

​​ The authors have done a great job. A unique material was collected in a remote area. As the authors rightly point out, the northeastern border of Asia is known as one of the several hardest-to-reach regions of the World, therefore defining the extremely low exploration degree of the region. No Asian populations have been previously found eastward or westward from this region making this habitat unique and remarkable beyond the rationale of zoogeographical explanation. Moreover, the phylogeny of the pygmy whitefish of Chukotkan lakes as well as the relation of Asian and North American populations have not been investigated yet.

​​ The manuscript is worthy of publication, relevant to the field of research, presented in a well-structured form, scientifically sound. The design of the experiment is suitable for testing the hypothesis. The authors made maximum use of the information provided in the NCBI database and literature. The cited references are up to date, although they are mostly not recent publications (only 5 out of 40 in the last 5 years). No excessive self-citations were noted.​

​However, there are some important notes:

​​ - The results of the manuscript cannot be reproduced based on the details provided in the Methods section because haplotype IDs are missing.

​- There are no ethical statements.

​- There are errors, inaccuracies, etc. in the Supplementary section.

​- It is desirable to detail the given map of the studied territory (Figure 1).

​- The conclusions from the given networks (Figure 2) are not quite legitimate. According to networks Western and Eastern groups are not reliably separated.

​- In addition, the emerging trends of networks are rather contradictory and not indicate that the Eastern group is a derivative of the Western one or vice versa. Probably, but not necessarily, additional network based on the combined sequence of all three fragments separately for the analyzed territory of Chukotka will partially clarify the picture.

​- Accordingly, it is desirable to partially revise the discussion. Including section 4.4. “Global phylogenetic history and colonization pathways”. Rather, it is reasonable to discuss two penetrations of the pygmy whitefish from the territory of Chukotka/Beringia (it is not clear) to the North American continent as well as the time of these penetrations.

​​​ Specific comments relating to tables, figures and text are given in edit mode in the respective files.

 

 

Comments for author File: Comments.zip

Author Response

Dear Reviewer, 

We would like to thank you for careful reading of the manuscript and providing detailed and useful comments.

Please find the point-by-point replay for your comments below

Major comments:

1. ​​ The results of the manuscript cannot be reproduced based on the details provided in the Methods section because haplotype IDs are missing.

 

The haplotypes IDs are added into table s4

 

2. ​- There are no ethical statements.

 

Added, please find it in the end of the text

 

3. ​- There are errors, inaccuracies, etc. in the Supplementary section.

 

Supplement is corrected according the recommendations. Sorry for the misprints in the haplotype IDs.

 

4. ​- It is desirable to detail the given map of the studied territory (Figure 1).

 

The figure is corrected according the recommendations i.e. (i) the borders of eastern and western Chukotka are added; (ii) the lakes that are habitat by pygmy whitefish are signed; (iii) some geographic object are also signed.

 

5. ​- The conclusions from the given networks (Figure 2) are not quite legitimate. According to networks Western and Eastern groups are not reliably separated.

 

​- In addition, the emerging trends of networks are rather contradictory and not indicate that the Eastern group is a derivative of the Western one or vice versa. Probably, but not necessarily, additional network based on the combined sequence of all three fragments separately for the analyzed territory of Chukotka will partially clarify the picture.

 

Since we embed ML-tree into the manuscript, we believe that conclusions about Western and Eastern groups of Pygmy Whitefish became more convincing yet they were slightly rephrased as well.

 

6. ​- Accordingly, it is desirable to partially revise the discussion. Including section 4.4. “Global phylogenetic history and colonization pathways”. Rather, it is reasonable to discuss two penetrations of the pygmy whitefish from the territory of Chukotka/Beringia (it is not clear) to the North American continent as well as the time of these penetrations.

 

We corrected “Global phylogenetic history and colonization pathways” sections and added the paragraph about the possible colonization waves from Chukotka to North America.

 

Minor comments:

7. It is written about 5 lakes, but 6 sites are marked on the map (2 districts with 3 points each)

 

We improved the fig.1 map. In the current version all lakes are marked by numbers which allow to clarify which lakes were sampled and where the pygmy whitefish was found

 

8. Nothing is written about the choice of primers. Usually, if the authors are not indicated, then the design was done by the authors. At least the primer sequences for the ATP synthase F0 subunit are known from the literature (Witt 2011 after Giuffra et al., 1994).

Corrected the information about primers is embed into the text.

 

9. It would be logical to detail this information on the map (figure 1). Please, indicate localities, rivers, basins, bays; and in Table S3 designate WC and EC.

 

Done

 

10. Supplementary must be carefully corrected

 

Corrected

 

11. Witt et al 2011 indicated a total of 16 localities. Explain in the supplementary how the number of localities in figure 2 in the text of the article correlates with the data in table S2 or at least indicate in the table S2 which ATP6 haplotypes were used in the network

 

The COX and ATP haplotypes used for network are marked by bold.

 

12. According to networks Western and Eastern groups are not reliably separated. The emerging trends of networks are rather contradictory and not indicate that the Eastern group is a derivative of the Western one or vice versa. Probably, but not necessarily, additional network based on the combined sequence of all three fragments separately for the analyzed territory of Chukotka will partially clarify the picture.

 

The ML-tree demonstrates the separation of the WС and EC groups with the high support, so we believe that our conclusions will be more evidential in the current version of manuscript. Looking forward for the new corrections if it would be necessary.

 

13. It would be usefool to indicate localities, rivers, basins, bays; and WC and EC.

 

Done

14. It would be logical to build additional network based on the combined sequence of all three fragments separately for the analyzed territory of Chukotka.

 

ML-tree is build on the base of combined sequences is that enough or the network based on the combined sequences is still necessary?

15. Designate WC and EC.

 

Corrected

 

16. 6A is missing! Were individuals from Ravkergytgyn (Amguema) analyzed?

 

Corrected

 

17. Since the division of groups from the Chukotka territory was not confirmed by genetic analysis, probably it makes no sense to talk about this.

 

Please see comment #5

 

18. Rather, we can talk about two penetrations of the pygmy whitefish from the territory of Chukotka to the North American continent and about the time of these penetrations.

 

Please see comment #6

 

19. It would also be logical to compare with the colonization history of the of North America by the lake whitefish Coregonus clupeformis, right?

 

It seem that the distribution of Coregonus clupeformis probably supports that data as well. Thank you for the right idea!

 

20. This is not supported by reliable facts, although it seems plausible. The above networks do not support the withdrawal of resettlement from Chukotka to Alasca or America, not vice versa! This conclusion may be supported by additional sampling.

 

Yes, the genetic data do not allow to support the hypothesis about the colonization of Alaska from Chukotka. However, the new data on the glaciation history demonstrates that the lakes that are dwelled by the Pygmy Whitefish at the Alaskan range were covered by the ice-shield during the LGM. So, that data makes the colonization scenario suggested in the discussion section more plausible

 

21. Haplotype ID (accession numbers) should be given in the peer-reviewed manuscript so that the reviewer has the opportunity to check the consistency of the received data, information inGenBank and the analysis performed.

 

Please find the Haplotype ID in Table S4. We apologize that we have not included that information in the very first version of manuscript.

 

Supplements

 

22. reference: does not correspond to that given in the list of references (year of publication) - did you want to indicate the date the sequences were placed in the databank? Then what about clade1 and clade2? Probably it is better to make a footnote to the table? For example: Region* * - regions SA and NA are indicated in accordance with …Or just use your own text from the Materials and Methods section without reference to Witt et al 2011: Southern Alaska (= SA, waterbodies of the Alaska Range), continental part of North America (= NA, waterbodies of the Mackenzie, Yukon and Lake Superior basins)

 

corrected

 

23. Errors in Haplotype ID (accession numbers)

 

Corrected. Sorry for misprints.

 

24. Designate belonging to WC or EC

 

done

 

25. Haplotype ID (accession numbers) should be obtained and given in the peer-reviewed manuscript so that the reviewer has the opportunity to check the consistency of the received data, information inGenBank and the analysis performed.

 

Please see comment #21

26. Data on haplotype frequencies are not given (indicate in parentheses next to the haplotypes)

 

The data on the haplotype frequencies is added into the table 1

 

Looking forward for additional corrections if they are necessary.

Best regards,

Grigorii Markevich

 

 

Reviewer 3 Report

The manuscript “ Phylogeny and distribution of pygmy whitefish (Prosopium  coulterii) IN THE Beringia region (Chukotka), by Grigorii et al. provides interesting data about distribution of pigmy whitefish, which should be of interest for many researchers. I read the manuscript and I think inclusion of a phylogenetic three, a molecular clock calibration, and nucleotide diversity indices beside a language check by a native English speaking colleague can improve the manuscript.

Here I have provided a few minor corrections which should be considered all over the text, where ever such cases exist.

Line 22 delete of

Line 25: drainages?

Line 28: identified?

Lines 103-104: which primer was used for sequencing? Please provide details.

Lines 117-119: data about haplotype and nucleotide diversity in each population or geographic region will be interesting. Such data can be easily extracted using DNasp software.

Line 132: the authorship of scientific names at first mention should be presented.

Line 157: please provide statistical diversity indices! Like the ones produced using DNasp!

Line 162: may be better to use “common haplotype” instead of “basal haplotype”?

Lines 196 and 197 …: Arctic draining and Pacific darining  maybe better to be replaced by Arctic drainages and Pacific drainages or more precisely to indicate Arctic and Pacific basins.

Line 207: “registered” = “recorded”?

Line 209: it is better to use “drainage” for a river system rather than “basin”

Line 222: haplotype network instead of haplotype net.

Line 312: Accession Nos?

Author Response

Dear Reviewer, 

We would like to thank you for sparing the time to write detailed and useful comments.

Please find the point-by-point replay for your comments below

1. I think inclusion of a phylogenetic three, a molecular clock calibration, and nucleotide diversity indices beside a language check by a native English speaking colleague can improve the manuscript.

We embed the ML-tree according your recommendation and recommendation of reviewer #1. We also calculated and integrated into the standard indexes of Nucleotide and haplotype diversity. Please see the supplements to find the table with the corresponded data. All minor corrections were accepted and included into the text.

We do not like to add to the manuscript molecular clock estimations, since the different sequences of mtDNA are defined by the drastically different mutation rate (could be noted on the haplotype nets for different genes under analysis). In this regard, we think that molecular cloсk estimations will demonstrate the errors comparable with the timing of population disjunction. Therefore, it would be better to keep that kind of estimations for future NGS studies.

2. Line 22 delete of

 

Done

 

3. Line 25: drainages?

 

Corrected

 

4. Line 28: identified?

 

Corrected

5. Lines 103-104: which primer was used for sequencing? Please provide details.

 

The information about primers was embed to the text

 

6. Lines 117-119: data about haplotype and nucleotide diversity in each population or geographic region will be interesting. Such data can be easily extracted using DNasp software.

 

Done the table with the indexes was added to the supplement

 

7. Line 132: the authorship of scientific names at first mention should be presented.

 

Done

 

8. Line 157: please provide statistical diversity indices! Like the ones produced using DNasp!

 

Done

 

9. Line 162: may be better to use “common haplotype” instead of “basal haplotype”?

 

Accepted and corrected

 

10. Lines 196 and 197 …: Arctic draining and Pacific darining  maybe better to be replaced by Arctic drainages and Pacific drainages or more precisely to indicate Arctic and Pacific basins.

Corrected

 

11. Line 207: “registered” = “recorded”?

 

Corrected

 

12. Line 209: it is better to use “drainage” for a river system rather than “basin”

 

Corrected throughout the text

 

13. Line 222: haplotype network instead of haplotype net.

 

Corrected

 

14. Line 312: Accession Nos?

 

The reference to the table with haplotype IDs is added to the supplement

 

Thank you once more time for you corrections

Best regards,

Grigorii Markevich

 

 

 

Reviewer 4 Report

 

This manuscript represents a nice phylogeographic analysis of the pygmy whitefish in Chukotka, which is an interesting region for boreal fish biogeography due to its association with the Bering land bridge. It is exciting to see this work being made more broadly available in an English-language journal. I did not find anything major to critique about the analyses. I have a few suggestions for how some of the information could be presented better in the figures and tables, and the rest of my comments are just related to idiomatic use of English (note that I did not do a full proofread/edit).

 

Figure 1 could use more geographic context. It is not absolutely necessary, because with the N compass marker and the “Bering Strait” label, it is possible to determine where in the world this map is situated. But if possible, it would be helpful to have an inset or adjacent map, at larger scale, that shows where this map is located with respect to the Pacific Rim region.

 

Figure 1: For the smaller Arctic basins, which is where the interesting samples are, it’s hard to see what is going on. It would help to have a finer-scale map that shows the location of each of the named lakes within those basins (i.e., the lakes named in Table 1). On the current map, basins 4 and 5 are very small so they’re hard to see, and basins 1 and 3 have very similar colors, so that could be hard for readers to see, depending on their screen/print quality.

 

Figure 1 caption: add “(PC)” after coulterii to define its use in the map label. For red and green circle legends, the wording “have been” (or have not been) suggests that this is information that existed prior to your study. I would reword as “investigated sites where PC were [not] found”

 

Table 1. Is it possible to show the haplotype frequencies for each basin and mitochondrial marker? Or better yet, show the number of individuals in which a given haplotype was found for each marker and basin - that way you can also convey sample size information.

 

Table 2: It took me some effort to figure out Table 2. I wonder if it would be easier to understand if, rather than stacking the different pairwise difference values on top of each other, if they were presented in a single comma-delimited cell, e.g., for Southern Alaska vs. North America, 0.028, 0.036, 0.032. Also, in the manuscript text the Alaska region is termed “southern Alaska” rather than “South Alaska”; be consistent.

 

“brownification” - I wasn’t entirely clear on the intended usage. Is this meant to describe eutrophic lakes, turbid lakes, or lakes that are stained brown from tannins/peat? The authors might want to consult with a native English speaker to find the best word to describe what they are referring to. (e.g., lines 131, 202, 208)

 

In supplementary Table S2, it would be great to provide the geographic coordinates for each of the sampling locations referenced, or make a supplementary figure that shows the locations.

Minor comments on wording:

 

In some places “pygmy” is misspelled as “pigmy”; fix throughout (e.g., lines 75-76)

 

“haplotype network” would be the more common English wording than “haplotype net”; similarly, when writing about connected waterways “network” would be more appropriate (e.g., line 283)

 

Abstract:

lines 20-21, suggest “a highly disjunct distribution ranging from…”

line 22: “.. dispersal history due to limited information…”

line 25: “belong to rivers draining into the Arctic”

line 30: replace “net” with “network”

 

line 33: don’t italicize family name Coregonidae. Also in the USA the whitefishes are considered to be a sub-family of the family Salmonidae, Coregoninae, but I’m not sure if that is accepted in Russian taxonomy.

 

Introduction:

line2 41-42: add commas: “The Beringian refugium, considered as a vital…. easternmost Asia, is one of the….”

 

line 45 - add “and” before Prosopium coulterii; also Prosopium is misspelled

 

line 45: suggest “...are known to have trans-Beringian distributions…”

 

line 55: “disjunct” rather than “disjoint”

 

line 62: don’t capitalize “world”, suggest “...hardest-to-reach places in the world, resulting in limited exploration of the region.”

 

lines 63-64, suggest “... the Amguema River basin, which flows from the central part of Chukotka down to the Chukchi Sea…”

 

line 66 - I don’t quite understand this statement; you are applying a rational zoogeographical explanation in your interpretation of the data… reword

 

line 69-70 - “ancient” seems too old a word to use for a Pleistocene era fish, plus I usually think of “phylogeny” as referring to the relationships among species, not lineages within species, so I would reword.

 

line 75: “could be distributed more broadly”

 

line 80: “a dozen” means exactly 12, “dozens” means roughly something between 12-100. I’d just report the exact number of lakes you sampled (from the map it looks like 19?)

 

Line 85 - unclear what is meant by “totally fished” (I’d interpret that as you caught everything in the lake but I doubt that is what you meant)

 

line 90: suggest “fork length, sex, and the numbers of pyloric caeca and gill rakers…”

 

line 134: do you mean “covered with perennial ice until the Holocene…”?

Lines 154-156: the use of the dashes is hard to read. In lines 153-154 they can just be removed without changing the meaning of the sentence. In line 156, I would reword: “c) eight and four in EC, seven and six in WC, respectively.”

 

line 162: replace “distanced” with “differed”

 

lines 196-197: suggest “...all lakes populated by this species drain into the Arctic, and no populations were found in Pacific basins”; similarly, in line 199 you can just say “but not in the Arctic [17].”

 

line 201: “only deep, clear lakes”

Figure 2. the correct spelling for “Klark lake” on middle haplotype network is “Lake Clark”

 

Line 234 - do you mean “several hundreds of thousands” of years? Also, it is not clear what era of climate change you are referring to - the warming since the last glacial maximum or the very recent anthropogenic warming? I’m guessing the former if you’re talking about time frames that can be estimated with the molecular clock.

 

Line 239 - I think you mean “Copper River”

 

Line 240, Figure 3 - I wonder if you mean “temporary waterway connections” rather than “temporal water spillways”

 

Figure 3 is great! But as you reference a very specific location in line 241, I would place that location on the map in Figure 3. And I would also change the wording on “temporal water spillways” (per previous comment).

 

Lines 260-261, suggest “identical regional distribution … S. alpinus [36-38] provides support for this refugium.”

Author Response

Dear Reviewer, 

We are grateful to you for sparing the time to write so many detailed and useful comments.

Please find the point-by-point replay for your comments below

1. Figure 1 could use more geographic context. It is not absolutely necessary, because with the N compass marker and the “Bering Strait” label, it is possible to determine where in the world this map is situated. But if possible, it would be helpful to have an inset or adjacent map, at larger scale, that shows where this map is located with respect to the Pacific Rim region.

Figure 1: For the smaller Arctic basins, which is where the interesting samples are, it’s hard to see what is going on. It would help to have a finer-scale map that shows the location of each of the named lakes within those basins (i.e., the lakes named in Table 1). On the current map, basins 4 and 5 are very small so they’re hard to see, and basins 1 and 3 have very similar colors, so that could be hard for readers to see, depending on their screen/print quality.

Figure 1 caption: add “(PC)” after coulterii to define its use in the map label. For red and green circle legends, the wording “have been” (or have not been) suggests that this is information that existed prior to your study. I would reword as “investigated sites where PC were [not] found”

 

The corrections of the fig. 1 were provided following your recommendations i.e. (i) the location of the region in Pacific Rim is added; (ii) the coordinated table of the each lake location is added to supplement; (iii) the dots displaying the small basins are set aside from the basins; (iv) the colors were corrected; (v) the correction of the legend was also done.

2. Table 1. Is it possible to show the haplotype frequencies for each basin and mitochondrial marker? Or better yet, show the number of individuals in which a given haplotype was found for each marker and basin - that way you can also convey sample size information.

 

Corrected. Number of individuals included into analysis is now given after haplotype name

3. Table 2: It took me some effort to figure out Table 2. I wonder if it would be easier to understand if, rather than stacking the different pairwise difference values on top of each other, if they were presented in a single comma-delimited cell, e.g., for Southern Alaska vs. North America, 0.028, 0.036, 0.032. Also, in the manuscript text the Alaska region is termed “southern Alaska” rather than “South Alaska”; be consistent.

 

The table 2 was simplified according the recommendations. Wording is also corrected

 

4. “brownification” - I wasn’t entirely clear on the intended usage. Is this meant to describe eutrophic lakes, turbid lakes, or lakes that are stained brown from tannins/peat? The authors might want to consult with a native English speaker to find the best word to describe what they are referring to. (e.g., lines 131, 202, 208)

 

The “brownification” is accepted term, which is used in some papers. We add the explanation in the place we use it for the first time.

 

5. In supplementary Table S2, it would be great to provide the geographic coordinates for each of the sampling locations referenced, or make a supplementary figure that shows the locations.

 

The geographic coordinates were embed into the text in Table S2 and S3

 

6. In some places “pygmy” is misspelled as “pigmy”; fix throughout (e.g., lines 75-76)

 

Corrected

 

7. “haplotype network” would be the more common English wording than “haplotype net”; similarly, when writing about connected waterways “network” would be more appropriate (e.g., line 283)

 

Corrected

 

8. lines 20-21, suggest “a highly disjunct distribution ranging from…”

 

accepted

 

9. line 22: “.. dispersal history due to limited information…”

 

accepted

 

10. line 25: “belong to rivers draining into the Arctic”

 

accepted

 

11. line 30: replace “net” with “network”

 

corrected

 

12. line 33: don’t italicize family name Coregonidae. Also in the USA the whitefishes are considered to be a sub-family of the family Salmonidae, Coregoninae, but I’m not sure if that is accepted in Russian taxonomy.

 

Corrected to sub-family Coregoninae

 

13. line2 41-42: add commas: “The Beringian refugium, considered as a vital…. easternmost Asia, is one of the….”

 

done

 

14. line 45 - add “and” before Prosopium coulterii; also Prosopium is misspelled

 

done and corrected

 

15. line 45: suggest “...are known to have trans-Beringian distributions…”

 

accepted

 

16. line 55: “disjunct” rather than “disjoint”

 

corrected

 

17. line 62: don’t capitalize “world”, suggest “...hardest-to-reach places in the world, resulting in limited exploration of the region.”

 

accepted

 

18. lines 63-64, suggest “... the Amguema River basin, which flows from the central part of Chukotka down to the Chukchi Sea…”

 

accepted

 

19. line 66 - I don’t quite understand this statement; you are applying a rational zoogeographical explanation in your interpretation of the data… reword

 

reworded

 

20. line 69-70 - “ancient” seems too old a word to use for a Pleistocene era fish, plus I usually think of “phylogeny” as referring to the relationships among species, not lineages within species, so I would reword.

 

Also reworded

 

21. line 75: “could be distributed more broadly”

 

accepted

 

22. line 80: “a dozen” means exactly 12, “dozens” means roughly something between 12-100. I’d just report the exact number of lakes you sampled (from the map it looks like 19?)

 

corrected

 

23. Line 85 - unclear what is meant by “totally fished” (I’d interpret that as you caught everything in the lake but I doubt that is what you meant)

 

Corrected the word totally was excluded

 

24. line 90: suggest “fork length, sex, and the numbers of pyloric caeca and gill rakers…”

 

accepted

 

25. line 134: do you mean “covered with perennial ice until the Holocene…”?

 

yes, corrected

 

26. Lines 154-156: the use of the dashes is hard to read. In lines 153-154 they can just be removed without changing the meaning of the sentence. In line 156, I would reword: “c) eight and four in EC, seven and six in WC, respectively.”

 

Corrected

 

27. line 162: replace “distanced” with “differed”

 

corrected

 

28. lines 196-197: suggest “...all lakes populated by this species drain into the Arctic, and no populations were found in Pacific basins”; similarly, in line 199 you can just say “but not in the Arctic [17].”

 

Accepted

 

29. line 201: “only deep, clear lakes”

 

corrected

 

30. Figure 2. the correct spelling for “Klark lake” on middle haplotype network is “Lake Clark”

 

Corrected

 

31. Line 234 - do you mean “several hundreds of thousands” of years? Also, it is not clear what era of climate change you are referring to - the warming since the last glacial maximum or the very recent anthropogenic warming? I’m guessing the former if you’re talking about time frames that can be estimated with the molecular clock.

 

Corrected. We were referring to the end of LGM

 

32. Line 239 - I think you mean “Copper River”

 

Corrected, sorry

 

33. Line 240, Figure 3 - I wonder if you mean “temporary waterway connections” rather than “temporal water spillways”

 

Corrected

 

34. Figure 3 is great! But as you reference a very specific location in line 241, I would place that location on the map in Figure 3. And I would also change the wording on “temporal water spillways” (per previous comment).

 

The additional figure was added to the supplement

 

35. Lines 260-261, suggest “identical regional distribution … alpinus [36-38] provides support for this refugium.”

 

accepted

 

I’d like to believe that our corrections made the tables more visible. Thank you very much for the minor corrections of English wording. All your suggestions were accepted.

 

Best regards,

Grigorii Markevich

 

Round 2

Reviewer 1 Report

Thank you for incorporating the comments and suggestions. All the best!

Author Response

Dear Reviewer,

Thank you once again for your corrections.

All the best,

Grigorii

Reviewer 2 Report

Кey notes:

Support for branch nodes on ML tree is incorrectly interpreted. From this, the rest of the comments basically follow (all comments are given in the text in edit mode).

Since the sequence of appearance of haplotypes is roughly visible across networks, it is possible that a combined network (COI + CytB + ATP6 for Chukotka samples) would clarify the situation (in addition to the tree).

Unfortunately, the sequences obtained by the authors, although they were provided with access numbers, remained unavailable for the reviewer (authors' decision).

In connection with the foregoing, the authors will probably correct the results and discussion once again.

Comments for author File: Comments.pdf

Author Response

Dear reviewer,

Thank once again for your corrections, which allows us significantly, improve the quality of analysis and interpretations. We apologize for misinterpretations of some data sections and misprints. We believe that the new version of manuscript will be more appropriate for publication. Please find the point-by-point replay for your comments below.

 

Point-by-point answers for major comments

Support for branch nodes on ML tree is incorrectly interpreted. From this, the rest of the comments basically follow (all comments are given in the text in edit mode).

The interpretation in result and discussion sections are corrected according the recommendations

Since the sequence of appearance of haplotypes is roughly visible across networks, it is possible that a combined network (COI + CytB + ATP6 for Chukotka samples) would clarify the situation (in addition to the tree).

The networks combining COI + CytB + ATP6 for Chukotkan samples as well as COI + CytB combination we build and analyzed throughout the result and discussion sections

Unfortunately, the sequences obtained by the authors, although they were provided with access numbers, remained unavailable for the reviewer (authors' decision).

The access for the sequences in GeneBank is opened

In connection with the foregoing, the authors will probably correct the results and discussion once again.

The result and discussion sections are corrected according the recommendations. The new block discussing the step-by-step scheme of pygmy whitefish disjunction is added into conclusion part of discussion

 

Response for line-by-line corrections

Line 4

Bochkarev A. Nikolai3 -  4!!!Institute of Systematics and Ecology of Animals, SB RAS, Novosibirsk 630091, Russian 11 Federation

 

Corrected

Line 123

How were the non-Chukotian sequences combined???

 

complete deletion is inappropriate here

 

Formally, only sequences obtained from the same individual can be combined into one. In your case this is only possible for Cytb and CO1. Then only Cytb and CO1 will be used when building the tree. The ATP6 sequences were obtained from completely different fish. Accordingly, you can only use the pairwise deletion option!!!

And in the tree in the figure, you need to indicate the corresponding access numbers in the data bank. For example:

Kenai peninsula KT630728 + KT630730

 

The new section explaining how the sequences we combined is added to the methods section. The sequences combinations are also specified for each case in the figures footnotes.

Since the complete deletion option is more strict comparing to pairwise deletion option we tend to leave this block as it was. The addition exploantions are embed into the methods section.

 

Line 173

Since you will not be able to compare the combined sequences of non-Chukotian origin, here you need to compare not the absolute number, but the % of nucleotide substitutions.

 

All taken sequences were trimmed and aligned to our shortest sequence, thus absolute number and percentage of nucleotide substitution have the same meaning.

 

Line 174

Well, you can’t exactly call them populations, neither in terms of sample size, nor geographically. Rather, analyzed groups?

 

Corrected

 

Line 186

Support for branch nodes in a tree is misinterpreted

+

Probably, an external group is needed (for example, another species - Pr.cylindr?).

 

Corrected, sorry for wrong interpretation. The ML-tree is recomposed according the recommendations.

 

Line 188

It is not clear from the figure, site 7 (Lake Vykvyrkapgytgyn) belongs to basin IV or V)? If V, then, probably, there is no need to single out basin IV at all?

In the table of Appendix S3, another name for the basin is indicated - Koluchinskaya ??? which, judging by the results on the ML tree, is quite logical (samples from lakes 7 and 8 form a monophyletic group with 83% support).

 

Figure 1 was reconstructed one more time to make it clearer. The two parts of footnotes are switched around and the Latin numeration of the basins is now associated with the particular lakes.

The wrong spelling of Koluchinskaya is corrected in S3.

 

Line 191

Since the sequence of appearance of haplotypes is roughly visible across networks, it is possible that a combined network (COI + CytB + ATP6 for Chukotka samples) would clarify the situation (in addition to the tree).

And a common network for all sequences would not hurt (COI + CytB, excluding ATP6, since it is impossible to combine with ATP6 due to the different origin of the sequences). And in order not to clutter up the main text of the article, the superfluous can be taken out in the appendix.

 

The new combined networks are embed into the text and supplement, describe throughout the result sections and discussed.

 

Line 199

1. Support for branch nodes on ML tree is incorrectly interpreted:

The 93% support only indicates that all Chukchi accessions form a monophyletic group with such support, and not "Separation of EC and WC subclusters was supported with 93% bootstrap". Within the EC/WC cluster, only the Kokanlegytgyn/ Vykvyrkapgytgyn group has high support (the value of 83% may indicate the isolation of lakes Kokanlegytgyn and Vykvyrkapgytgyn and, in turn, 92%, the isolation of lake Vykvyrkapgytgyn).

2. For sequences borrowed from the data bank, access numbers must be specified.
3. The sequences borrowed from the data bank, combined for three mtDNA regions, must be from the same individuals.

 

1. Corrected in the right way. Sorry for the wrong interpretation.
2. Specified in footnote
3. Specified in footnote
4.  

 

Line 202

Table 1 and Table S3 are actually duplicates of each other!

I didn't pay attention to this in my first review.

 

The most part of information is transferred into table 1. Table S3 (S1 in current version) now contains the information about the lakes position only and mentioned in the beginning of methods section.

 

Line 261

According to the tree and networks, it turns out the opposite: eastern populations are derived from western ones!

The section is majorly corrected according the combined sequences data. We believe that new data interpretation will be more relevant

 

Line 278

Judging by networks (and the tree only reinforces this), the proposed distribution scheme does not correspond to them. As a first approximation, the sequence of events is as follows:

1. Separation of groups of North America and Alaska;
2. Separation of Alaska and Chukotka (moreover, the Western group);
3. And then, in Chukotka, separation occurs in the Koluchinskaya basin

 

The section describing the step-by-step scheme of pygmy whitefish disjunction is added into conclusion part of discussion. We also corrected the section of ‘Global phylogenetic history and colonization pathways’ according new data and recommendations. Several corrections are also done for figure 3 (figure 5 in current version).

 

Line 288

But then the conclusion follows that no traces have been preserved in Asia, right?

 

I think we cannot see the whole picture since the populations of Kabuk River and North Slope are not discovered still.

Line 462

38

 

Corrected

Round 3

Reviewer 2 Report

 Accept after minor revision. Please see attached file.

By the way, I would use to the maximum  the ATP 6 synthase F0 sequences known for Alaska (not only obtained by Witt et al. 2011, but also obtained by Gowell et al. 2012). But it's probably better to leave that for another time.

 

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

Thank you once again for your corrections. We apologise for the typographical errors in the table and appreciate your pointing them out. Please find a point-by-point response below.

All the best,

Grigorii Markevich

Major comments

 

By the way, I would use to the maximum the ATP 6 synthase F0 sequences known for Alaska (not only obtained by Witt et al. 2011, but also obtained by Gowell et al. 2012). But it's probably better to leave that for another time.

 

The addition haplotype network is embedded in the supplement. We have not used the haplotypes following Gowell et al. 2012 as they are shorter than ours. The paper is primarily focused on the Chukotkan samples, so we prefer to analyse the longer haplotypes from the Chukotkan sites rather than the more diverse network of short haplotypes across the region.

 

Minor comments

 

Line 87

Table 1 shows 9 haplotypes for these 8 individuals

The mistake was found and corrected in the Table 1

Line 170

Not indicated how many sequences were received in total?

It is probably easiest to add sample sizes by lakes and a summary line at the bottom to Table 1 (how many individuals were analyzed in total (25) and how many for each sequenced fragment (19, 25 and 22, respectively)?

Table 1 was reorganized and corrected

 

Line 190

Form -à from

corrected

 

Line 224

Actually, for a reader unfamiliar with the details, it would be nice to somehow highlight where North America is, where Alaska is, and where Chukotka is (for example, by combining brackets on the right, as was done in your previous version of the manuscript)

 

The brackets with addition explanations are embed to the figure

 

Line 232

In the materials and methods section, it is indicated that 8 individuals were caught in this lake, but here we see 9? (I did not pay attention to this in the second version of the manuscript).As already indicated above, it would probably be logical to add a row in the table summarizing the number of analyzed individuals in general and for each fragment, in particular.

 

Corrected