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Thank you for making the changes.
[# PeerJ Staff Note - this decision was reviewed and approved by Nigel Andrew, a PeerJ Section Editor covering this Section #]
Thank you for your explanation. Just because something is published it does not mean it is correct. Please use the word "tissue" in its scientific meaning and not colloquial meaning. If the kit has the word "tissue" in its commercial name obviously it has to be used. If it is only in the kit's instructions, it is used incorrectly and should not be used. Please replace "tissue samples" with "samples" or "organ samples" or "biological samples".
Thank you for making the changes. Could you please correct terminology you are using? Tissue is composed of cells of a common origin performing the same function, for example epithelial tissue or connective tissue or muscle tissue or nervous tissue. Liver is an organ, made up of many tissues. The samples collected were organ samples. Better to use “sample” instead of “tissue”, so “tissue sample” should be changed to “sample” and "liver tissue" to "liver".
Please explain the implementation of evolutionary models and partition analysis. Please provide the phylogenetic analysis log files as supplementary materials. Please address all comments from the reviewers.
**PeerJ Staff Note:** Please ensure that all review, editorial, and staff comments are addressed in a response letter and that any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
The manuscript is written clearly and easy to follow. References are appropriate and sufficient background is provided. Raw data has been made available.
The overall research question is well-defined and highly relevant. Methods are described in sufficient detail.
All analyses appear valid. Conclusion are well-stated.
This is a highly relevant study given the the historical problems associated with extracting useable DNA from formalin fixed specimens. I appreciate the author undertaking this analysis on a rarer group of fishes which are excluded from previous phylogenetic analyses. Furthermore, the confirmation that we can still extract useful data from very old, formalin preserved specimens using modern-day methods (and integrate it into modern day phylogenetic analyses) is a very nice result. I enjoyed the inclusion of CT scans of the skull and the report on the morphology of these fishes.
Overall, I think this manuscript is in excellent shape. I just have a few minor suggestions which I think will improve the text:
Lines 170-172: Is Melanotaeniidae not included here for a reason? It is listed in Table S1.
Figure 1: it would be helpful to have an icon or something to differentiate between the historic DNA specimens vs the modern DNA specimens. Maybe include an asterisk after Leptomelanosoma macrophthalmus and Filistriatus bifurcus gen. nov. in the figure panels? It just makes it easier to tell which are historic without having to refer back to Table 2.
Can Supplemental Table 1 be a .docx or .xlsx file instead of a .png image? It makes it harder to copy accession numbers if its an image.
Is it possible to include the consensus tree file (.tre) in the Supplement?
Figure 3: What do the asterisks denote? Are they the excluded bootstrap values? This should be noted in the caption.
Figures 4 and 5: I see scale bars but nothing saying how long they are/units.
I felt the article was very clearly written, the number of references was appropriate and the article followed a good logical order. I really struggled to find issues with any aspect of this paper, which is quite unusual relative to many papers I review. The author has done a great job.
I had no issues with the experimental design.
Seemed like a solid study which balanced using a novel technique along with some good traditional systematic approaches.
One thing that could be improved upon is including some information in the introduction about what tissue types are recommended for these extractions. Is liver best (I presume), but are there other options which maybe only slightly less good? In the more distant past I think people have most often tried fin or muscle tissue, but I’m not sure how successful that has been (I haven’t really followed stuff around extracting formalin preserved material). Maybe only good for short fragments, but not enough DNA to get enough fragments to allow assembly? Anyway, be good to provide a little background on this point as most people will benefit from this information.
Line 59, perhaps make it clear at the start of this section that you are talking about formalin preserved specimens, i.e. change to “museums house millions of formalin preserved samples"
Line 129, would be good to include the length of the specimens you sampled. One advantage you have is that these are larger fish, which makes the sampling easier to do, if they were small specimens, <5 cm then it’s much harder to access their liver without more damage to the specimen.
Line 485, use methods, not methodology, the latter means the study of methods (many people use it incorrectly).
The manuscript "Unlocking the genomic potential of historical and formalin-fixed specimens: Phylogenetic insights from museum preserved threadfin fishes (Teleostei: Polynemidae)" presents a clear structure, and incorporates relevant references.
The supplementary material includes the raw data, necessary to carry the analysis. However, I was surprised that the author did not provide the log file of the phylogenetic analysis. Without it the process of reviewing the steps followed during the analysis, as well as the implementation of the parameters (those stated by the authors and those implemented by the program by default) becomes impossible. Additionally, details such as the evolutionary model or whether the partitions employed linked or unlinked models, are absent from the paper. This lack of information raises concerns regarding the transparency of the findings.
The results are relevant to the hypothesis and questions stated in the manuscript.
The manuscript "Unlocking the genomic potential of historical and formalin-fixed specimens: Phylogenetic insights from museum preserved threadfin fishes (Teleostei: Polynemidae)" aligns with the aims and scope of PeerJ. The research question and results are highly relevant and address a significant knowledge gap concerning the role of historical material in phylogenetic analysis. As noted in the previous comment the alignment of the research question and results suggest a well-defined research question.
The investigation is of high technical value and is undoubtedly relevant for future research. However, as stated before, the absence of log files from the phylogenetic analysis makes it difficult to evaluate the rigour of the phylogenetic protocol. Similarly, in my opinion the author should provide an additional phylogenetic criteria for evaluating the data. Bayesian inference, which does not necessarily produce a unique tree and could reflect any phylogenetic uncertainty associated with the data.
Furthermore, the paper lacks clear statements regarding the implementation of evolutionary models. It is unclear whether the partitions used employed uncorrelated models or if they were all evaluated under the same model. This lack of detail hinders the ability to replicate the phylogenetic analysis accurately.
As previously stated, the investigation is undoubtedly relevant for future research and of high technical value. However, the light upon which the results are presented is not ideal. Clear explanations of the implementation of evolutionary models and partition analysis are lacking. Additionally, the phylogenetic analysis log files are not provided, which makes it impossible for readers to verify the procedures. These issues raise concerns about the rigour of the phylogenetic protocol, which ultimately reflects on the relevance of the results.
The conclusion is well-stated, and linked to the original research question. However, this is not the main issue with the paper.
In my opinion, I strongly suggest that both author and the editor should strive to include the missing pieces of the phylogenetic protocol and incorporate the log files. This will help to avoid transparency and reproducibility issues, which are particularly important in today's scientific discourse.
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