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Thank you very much for your revision manuscript. I think this is ready to publish in PeerJ.
[# PeerJ Staff Note - this decision was reviewed and approved by Robert Toonen, a PeerJ Section Editor covering this Section #]
Thank you very much for your hard work on revising a manuscript. However, I have only one point that needs your response. Please see a comment from our reviewers.
t seems to me that you have significantly improved the manuscript in your results and interpretation. However, I insist that the authors should include the new mutation rate calculation results in the manuscript or in the supplementary since they do not appear in either file.
The English should be checked by an English-native speaker, as there are incorrect sentences. For example, you cannot start a sentence with: And we perform certain analyses... Nor should you start a sentence with the abbreviated species name: O. ascotanensis is....
The methods are better, but I suggest making a Bayesian Skyline plot (BSP) for the demographic history using d-loop. The analyses that have been done to calculate the time of expansion have been criticized and do not consider the population genealogies. Also, neutrality tests and mismatch distribution plots are not conclusive, they only allowed to say that populations are not in Wright-Fisher equilibrium. As the BEAST analysis has already been done, it would be very easy to do BSP with the newly calculated rate.
There are no values for the estimated rate in the results, nor in the supplementary material. I insist that this is the opportunity for other researchers to make the same analysis, and for that, the methods and results should be clear and the article could be more cited.
I suggest that they should include in a table or graphs the values of the rates obtained with their confidence intervals. In the same way, the BSP estimates the expansion time.
Dear Authors
This is an opportunity to take your article to a higher level and clearly demonstrate how these fishes have evolved in such an extreme environment.
I urge you to improve the results of the work. They should show the results of the rate estimates, otherwise, it is not possible to know what they did. We do not need more obscurity in science.
It is an easy task and should not take so much time.
Best regards
Thank you very much for your revised manuscript. However, one of our reviewers has important comments on the method that need to be addressed before making any decision.
The manuscript was improved substantially, the tables and figures are adequate, but the methods, results, and discussion are not well done yet.
I do not agree with the substitution rate of 6% used from the previous paper (Guerrero-Jimenez et al.) because is wrong. First, this rate is coming from Burridge et al. 2008, but the rate estimates in this paper are <1%.
I strongly suggest the authors calculate their own rate using geological and biogeographical information in the absence of fossils of the genus. Thus, the results and their re-interpretation could change the proposed scenario and be more striking to a wide audience.
Alternatively, the authors could be using several rates 1 - 3 - 5 %(susbst./My) and compare results. Use these rates obtained for the clade of interest and make the demographic analysis again.
I find it an interesting manuscript since it generates new findings on the phylogeography of the genus Orestias in the Altiplano. However, the analysis and results are not original or new for a large audience. However, it could be if the authors improve their data analysis. All the evolutionary inference in the discussion is based on the demographic analyses based on a very high substitution rate for a fish population (6%). I strongly suggest the authors calculate their own rate using geological and biogeographical information in the absence of fossils of the genus. Thus, the results and their re-interpretation could change the proposed scenario and be more striking to a wide audience.
Dear Authors
I insist to make your study more visible and novel, I suggest calculating a D-loop gene substitution rate and doing the demographic analyses again. This will not only generate a more accurate estimate but would also be very useful for other studies.
I will not accept the manuscript until you calculate your own rate or use several (1-5%) and compare your results. I don't know why they don't do it if it is so easy. The discussion is very appropriate to the times obtained with the rate used and they could change. It appears the authors are trying to use a rate that fits a beautiful evolutionary history of Orestias populations, but it would be properly scientific to calculate a rate, get the results and look for a correct interpretation.
Best regards
Thank you very much for your interesting manuscript, however, we need some revision. Please, respond to all comments from our reviewers carefully, point by point.
[# PeerJ Staff Note: Please ensure that all review comments are addressed in a rebuttal letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate. It is a common mistake to address reviewer questions in the rebuttal letter but not in the revised manuscript. If a reviewer raised a question then your readers will probably have the same question so you should ensure that the manuscript can stand alone without the rebuttal letter. Directions on how to prepare a rebuttal letter can be found at: https://peerj.com/benefits/academic-rebuttal-letters/ #]
The manuscript is well written, the tables and figures are adequate, but the methods, results, and discussion are not well done.
The objective of the work seems confusing since they propose to evaluate the phylogeography of Orestias, but then they say that the results will provide evidence of speciation. I suggest eliminating speciation from the objective since it cannot be evaluated with the methodology and data used.
The substitution rate of 6% used from the previous paper is wrong. First, this rate is coming from Burridge et al. 2008, but the rates estimates in this paper are <1%. Second, the rate estimated in Burridge et al. 2008 is for the Galaxiidae family a fish group belongs to another order, not related to Orestias.
I strongly suggest the authors calculate their own rate using geological and biogeographical information in the absence of fossils of the genus. Thus, the results and their re-interpretation could change the proposed scenario and be more striking to a wide audience.
Suggestion: Calculate the substitution rate in BEAST software using its D-loop sequences combined with those of Vila et al. 2013 and calibrate the split between the Titicaca clade (outgroup) with the ingroup clade. It is estimated that in 3 million years the appearance of the Titicaca or 5-2 the rise of the Andes Range.
Use the rate obtained for the clade of interest and make the demographic analysis again.
I find it an interesting manuscript since it generates new findings on the phylogeography of the genus Orestias in the Altiplano. However, the analysis and results are not original or new for a large audience. However, it could be if the authors improve their data analysis. All the evolutionary inference in the discussion is based on the demographic analyses based on a very high substitution rate for a fish population (6%). I strongly suggest the authors calculate their own rate using geological and biogeographical information in the absence of fossils of the genus. Thus, the results and their re-interpretation could change the proposed scenario and be more striking to a wide audience.
Dear Authors
To make your study more visible and novel, I suggest calculating a D-loop gene substitution rate and doing the demographic analyses again. This will not only generate a more accurate estimate but would also be very useful for other studies.
Abstract – The MS has two parts: phylogenetic analysis D loop and Microsatellite, reconstructed biogeographic histories. I think the readers would appreciate it if you add a sentence stating the aim of the study (mentioning the different parts) before presenting a summary of the results of each part.
My comments on the introduction are mainly on framing the study.
Materials and methods - Taxon sampling please make sure.
Reference - Please make sure about PeerJ format.
For research ethics purposes, cite (in acknowledgment), where applicable, permits acquired to conduct the field sampling.
- As mentioned in the previous section, give more context on current lit in the introduction.
- I enjoyed reading your MS but struggled to easily grasp the details largely because of how results were presented and having had to go back and forth from one figure to another. I think your figures are good but contain too much information and need to be explained for e.g. see sections of the discussion. With a little tweaking of how results are conveyed- I think the paper will be widely read and comprehended easily.
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