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Thank you for your thorough attention to the reviewers' comments and suggestions. Your paper is much improved because of your efforts.
[# PeerJ Staff Note - this decision was reviewed and approved by Robert Toonen, a PeerJ Section Editor covering this Section #]
I received three thoughtful reviews of your paper. The general consensus is that only minor changes are needed; however, please pay close attention to the suggested revisions and comments of reviewer #2. I look forward to your reading your revised manuscript.
[# PeerJ Staff Note: Please ensure that all review and editorial comments are addressed in a response letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate. #]
The language is clear and unambiguous throughout the manuscript. The article provides a strong introduction and background to demonstrate how the work fits into the broader field. Relevant prior literature is appropriately referenced. The structure of the article is appropriate. Figures are relevant to the content of the article, of sufficient resolution, and appropriately described and labeled. All appropriate raw data have been made available in accordance with our Data Sharing policy.
All methods are standard for the field. The research topic is similar to other papers published in the journal. The research question is well defined, relevant and meaningful. The investigation was and conforms to high technical and ethical standards. The methods are all described with sufficient information to be reproducible by another investigator.
The results are all reasonably interpreted within light of the methods, and the discussion is consistent with the results.
NA
Text mostly clear and unambiguous. Few grammar and structure problems that can be easily addressed by proof reading.
Literature references can be improved, mostly in the methods, introduction and discussion regarding ecological and biogeographical inferences.
Figures can be improved, especially the haplotype networks. I think the authors could also provide an additional phylogeny, resulting from their RAxML analysis. Tables can be added to complement the materials.
Results can be edited for clarity and cohesiveness.
Methods lack proper details.
Materials presented in a confusing way. Can be edited for clarity.
Findings seem to be par with results, and can be improved by improving some of the methods used. Comments below.
## Title: I would suggest choosing either Negro or Amazon, since it can be confusing for a reader that is not familiar with these basins
## Abstract
l23-24: The way this sentence is presented, it can be interpreted that there are assemblages of fishes shared between the Orinoco and Amazon that are not found in the Neotropics. Edit for clarity
l31: Please standardize the nomenclature of the genes: COI // MYH6 or CoI // Myh6 or coI // myh6. There are also inconsistencies throughout the text. Change pb to bp.
## Introduction
l72-75: Provide references for water chemistry properties and river origins. I suggest Sioli, 1984 and Crampton, 2011
l101-112: This is a good literature review of molecular studies of P. axelrodi, however it is disconnected from the previous sessions and the concluding paragraph below. I would suggest edits to highlight the knowledge gap that exists and is going to be addressed in this paper.
Materials and Methods:
l124: "DNA muscle tissue samples" makes no sense. Edit for clarity
L126: Is samples here referring to the fish, to the muscle tissue or the DNA datasets? Edit for clarity.
l123-135: The reader would benefit from a table that shows vouchers used, which of them are represented in each dataset and if there is any overlap.
l199: Change pb to bp.
l200: Where the sequences aligned manually? Or was an alignment algorithm used?
l201: How were haplotypes determined and how was the network inferred?
l207: As a GTR+GAMMA evolution model usually is fit for complex datasets, it can also over parameterize analyses. I do not believe choosing this model because the manual of a program suggest is a good approach. Evolution models are easily inferred by programs such as JModelTest, and are computationally fast, especially for datasets that are not massive. I recommend the authors to perform a proper inference of evolution model for their datasets. Also, were these analyses performed on a concatenated dataset or for individual genes?
l223: For the tree prior, was the tree rooted? And where?
l249: Mantel tests are tools that investigate correlations between two distance matrices, and can be used to infer isolation by distance. Please edit this session providing the rationale of using a mantel test, how it was performed and the steps.
l255: What is the rationale of k=1 to 6?
l261: While the Evanno method for deltaK inference is a thorough approach used in population genetics, it has its caveats. I would suggest the authors investigate further its applicability, especially referring to Puechmaille, 2016; Janes et al. 2017 and Cunningham et al. 2020. I think this is a necessary step once the optimal K recovered is 2, and k=2 can be related to algorithm biases.
l264: Please provide details on the steps taken in the DAPC analyses.
## Results
l288: How have the haplotypes been inferred and how was the network generated?
l289: How was reciprocal monophyly deliniated here? Lineages have to be properly defined for this inference to be made. Please provide more details.
l290-291: I would suggest providing a figure and a table with more details for the haplotype network. The numbers presented do not show the claims made by the authors in text.
l292-300: Since all these ages are based on an a priori evolutionary rate prior, I am curious if both markers would present similar results if analysed individually. I would also suggest the authors investigate paleogeographic patterns of connections of these basins for the recovered dates.
l314: Fst = 0.010 is not the same as Fst = 0.00, which would mean no differentiation. Please refine text to accurately reflect the results obtained. Are these values for microsat only?
l315-318: What test has been used to access significancy of the differentiation here? I would suggest straightforward tests like G-statistic permutations and bootstrapping permutation over loci. Why did the authors consider the Negro populations to be all different with an Fst of 0.03 but not the Orinoco ones with 0.01?
l331-333: Please refer to my previous comment regarding the use of the Evanno method for deltaK determination and the possible caveats of this approach.
## Discussion
l362-380: Please provide geological dates for the hypothesized paleogeographic events that could explain the patterns observed.
l370: If the results suggest K=2 (see my comment above about this inference) as the best K for the data, further exploration of the K3-K6 is rather redundant and somewhat circular. By forcing extra partitioning of the dataset, it is expected that independent groups would appear. Do they mean anything, if your tests show that the extra partitioning is not the best explanation for your dataset? The authors should rely on their results, or explain why this extra (and in theory not optimal) partitioning is relevant.
l384: Mantel is not a hypothesis, it is a test. Please edit for clarity.
l386: Any literature on this possible ecological isolation for P. axelrodi? It would benefit the manuscript if the authors could include references on the biology of this fish that could be linked to ecological isolation.
l395: Provide references on the geological and topographical information provided.
l402: This information about the ecology of P. axelrodi could be presented earlier to back previous claims about ecological isolation.
l460: This paragraph contradicts many claims made by the authors beforehand.
l467-469: Provide references
No comment.
No comment
No comment
Dear authors,
The review of the manuscript, with comments and suggestions, is in the pdf file.
This is a good contribution for Neotropical freshwater fishes.
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