Evolutionary Conservation and Diversification of Five Pax6 Homologs in the Horseshoe Crab Species Cluster
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
Dear Author/s,
Manuscript ID: Arthopods-2615360- "Evolutionary conservation of five Pax6 homologs under strong purifying selection in the horseshoe crab species cluster" contributes valuable insights into the evolutionary diversification of a transcription factor Pax6 gene family (paralogs) in ancient arthropod belongs to Xiphosura taxon. The manuscript's (research) includes comprehensive genomic data collection and phylogenetic analysis of 4 genomic species and provide a strong foundation for its relationships with other arthropods. Addressing the suggested improvements and major revisions would enhance the manuscript's impact and readability. Overall, this study makes a significant contribution to understand the significance of evolution of gene and genomes.
Strengths:
1. Relevance and Significance: The study addresses an important topic, particularly in the context of the gene evolution. The manuscript's emphasis on mechanism of evolution derived from purifying selection which is relevant, and its findings contribute to our understanding of the factors that impacted in this challenging environment millions of years ago.
2. Comprehensive Data Collection: The authors have employed a comprehensive approach for data collection of genome sequences and annotation, involving a wide variety of models/software to validate its performance. The use of an anonymous question and collateral mechanisms adds robustness to the study's findings.
Suggestions for Improvement:
1. Limitations and Generalizability: Manuscript should acknowledge some limitations, such as the lack of other insect’s species genome data inclusion in your analysis. Although, this would be helpful to check the potential for accuracy of bias on the same phenomenon in other ancient arthropods. Further inclusion in discussion on the applicability and generalizability of the findings to other species like mosquitoes would enhance the manuscript's completeness (see minor comment #4).
2. Ethical Considerations: The manuscript should include a section that highlights the ethical considerations taken into account, particularly with respect to data accession/collection date and availability of public domain data, as the assembly used to get change periodically.
3. Literature Review and Context: The manuscript has done comprehensive/exhaustive review of the existing literature on different dynamics. Authors should have presented them in well-verse format (although line numbers are missing to point out). This would help the reader to better understand how the current study contributes to the phylogenetic field and differentiates itself from previous research.
Minor Revisions:
1. Too many key words, don’t use the same which already present in the title. Two times repetition of same word doesn’t look good.
2. Methods of the paper need more description and explaining the default setup of used software would be helpful to other user to replicate the same exercise elsewhere.
3. The figures and tables provided including supplementary are essential for understanding the results. To enhance clarity, consider providing more descriptive figure legends that explain the main findings depicted in each figure. Each figure legends should have description of terms as has been used in Fig 1. R1, R2, R3 and R1-3.
4. Citing in discussion papers like PMID: 27664587; DOI: 10.1016/j.gene.2016.09.022 and PMID: 34578158 DOI: 10.3390/pathogens10091127 would be useful to understand the gene duplication is central phenomenon in other ancient insect species too.
5. The manuscript's language and writing style are generally clear and concise. However, some sentences appear to be lengthy and could be restructured for better readability.
Overall, I would recommend justify all these minor mistakes at your end. Above are some examples; authors should take care of rest likewise and proof-read (English and Grammar) at your end.
All the best.
Comments on the Quality of English Language
The manuscript's language and writing style are generally clear and concise. However, some sentences appear to be lengthy and could be restructured for better readability. Authors should take care and proof-read of English and Grammar at your end.
Author Response
- Limitations and Generalizability: Manuscript should acknowledge some limitations, such as the lack of other insect’s species genome data inclusion in your analysis. Although, this would be helpful to check the potential for accuracy of bias on the same phenomenon in other ancient arthropods. Further inclusion in discussion on the applicability and generalizability of the findings to other species like mosquitoes would enhance the manuscript's completeness (see minor comment #4).
Response: We appreciate the reviewer’s concern and suggestion. It is true that we did not test the singleton homologs of eyeless and twin of eyeless for purifying selection in other arthropod species. However, the consistent conservation of both genes in any arthropod sequenced or studied today, that the two genes are functionally highly conserved in the arthropods outside the Xiphosura and hence evolving under purifying selection.
- Ethical Considerations: The manuscript should include a section that highlights the ethical considerations taken into account, particularly with respect to data accession/collection date and availability of public domain data, as the assembly used to get change periodically.
Response: We agree with the reviewer that genome sequence assemblies undergo regular updates and improvements. However, this is a problem when genes that are expected to be recovered from the genome of a species can not be detected which may be due to a true lack or due to incomplete genome coverage. In our case, all expected homologs were detected. So we think this concern does not apply to our study.
- Literature Review and Context: The manuscript has done comprehensive/exhaustive review of the existing literature on different dynamics. Authors should have presented them in well-verse format (although line numbers are missing to point out). This would help the reader to better understand how the current study contributes to the phylogenetic field and differentiates itself from previous research.
Response: We appreciate the reviewer’s concern and examined the possibility of a better arrangement to discuss the findings of our study with previous relevant studies. In the end, we felt that keeping the introduction as short as possible while pointing out how our findings compare to other whole genome duplication studies was the best way to present findings and implications that we could think of.
Minor Revisions:
- Too many key words, don’t use the same which already present in the title. Two times repetition of same word doesn’t look good.
Response: We agree with the authors and removed “Pax6” and “horseshoe crab” from the keyword list.
- Methods of the paper need more description and explaining the default setup of used software would be helpful to other user to replicate the same exercise elsewhere.
Response: In agreement with the reviewer, we expanded the information in the Methods&Results section.
- The figures and tables provided including supplementary are essential for understanding the results. To enhance clarity, consider providing more descriptive figure legends that explain the main findings depicted in each figure. Each figure legends should have description of terms as has been used in Fig 1. R1, R2, R3 and R1-3.
Response: In agreement with the reviewer, we expanded the information in the figures and table legends including those in the Supplementary data.
- Citing in discussion papers like PMID: 27664587; DOI: 10.1016/j.gene.2016.09.022 and PMID: 34578158 DOI: 10.3390/pathogens10091127 would be useful to understand the gene duplication is central phenomenon in other ancient insect species too.
Response: We agree that gene duplication has been found to be of major significance in insect genome evolution. However, unlike in the Chelicerata, gene duplications in insects are generally not caused by whole genome duplications. We therefore abstained from including these otherwise very deserving studies.
- The manuscript's language and writing style are generally clear and concise. However, some sentences appear to be lengthy and could be restructured for better readability.
Overall, I would recommend justify all these minor mistakes at your end. Above are some examples; authors should take care of rest likewise and proof-read (English and Grammar) at your end.
Response: We appreciate the reviewer’s comments and conducted another round of editing review on the manuscript.
Reviewer 2 Report
Comments and Suggestions for Authors
The data that the study provided are useful, and have potential in further researches. However, many improvements are needed.
My concerns,
1. Does background clades used in analyses? They should be provided in methods.
2. The figures and legends in the ms are under expressiveness, including figure 1 to figure 3.
Author Response
Comments and Suggestions for Authors
The data that the study provided are useful, and have potential in further researches. However, many improvements are needed.
My concerns,
- Does background clades used in analyses? They should be provided in methods.
Response: In agreement with this comment, we added the choice of Pax6 homologs from mouse, common house spider, and red flour in the gene tree reconstruction section to the Methods and Materials section.
- The figures and legends in the ms are under expressiveness, including figure 1 to figure 3.
Response: We revisited figure 1 to figure 3 and added important details to the legends.
Reviewer 3 Report
Comments and Suggestions for Authors
Authors reported comparative sequence evidence for the functional conservation of the five L. polyphemus Pax6 transcription factor homologs. They revealed that all paralogs are conserved in the approximately 135 million-year-old horseshoe crab species cluster and that they evolve under strong purifying selection. These findings identify subfunctionalization as the likeliest post-WGD outcome for the five Pax6 homologs. In a word, it is a really cool research.
Author Response
Authors reported comparative sequence evidence for the functional conservation of the five L. polyphemus Pax6 transcription factor homologs. They revealed that all paralogs are conserved in the approximately 135 million-year-old horseshoe crab species cluster and that they evolve under strong purifying selection. These findings identify subfunctionalization as the likeliest post-WGD outcome for the five Pax6 homologs. In a word, it is a really cool research.
Response: We appreciate the reviewer’s enthusiasm for our study.
Reviewer 4 Report
Comments and Suggestions for Authors
This is a well written and structured article focused on the evolutionary conservation and diversification of five Pax6 and I recommend the publication of this manuscript
Only have a couple of brief comment:
1. Although the paper is focused on current horseshoe crab, I miss some reference which I believe are important for the story of this work, at least the authors should mention one of these two papers below. We cannot forget that to understand evolution of current arthropods we have to look at deep times.
Harzsch, S. & Hafner G. 2006. Evolution of eye development in arthropods: phylogenetic aspects. Arthropod Struct. Dev., 35, 319-340.
Strausfeld et al. 2016. Arthropod eyes: The early Cambrian fossil record and divergent evolution of visual systems. 45, 152-172.
2. The authors introduce the term “living fossil” in the abstract and because the great discussion around the term “living fossil” I was waiting a brief discussion at the end of the paper, but nothing show up. Please can you add something to such discussion, nothing long at the end of your discussion. Look some of the discussion published recently:
Turned, D.D. 2019. In defense of living fossils. Biology & Philosophy, 34:23 https://doi.org/10.1007/s10539-019-9678-y
Lidgan S. & Love, A.C. 2021. The living fossil concept: reply to Turner. Biology & Philosophy, 36, 1-16. https://doi.org/10.1007/s10539-021-09789-z
Bennett, Dominic J., Sutton, Mark D., and Turvey, Samuel T. 2018. Quantifying the living fossil concept. Palaeontologia Electronica 21.1.14A 1-25. https://doi.org/10.26879/750 palaeo-electronica.org/content/2018/2194-quantifying-the-living-fossil
Author Response
Reviewer 4
- Although the paper is focused on current horseshoe crab, I miss some reference which I believe are important for the story of this work, at least the authors should mention one of these two papers below. We cannot forget that to understand evolution of current arthropods we have to look at deep times.
Harzsch, S. & Hafner G. 2006. Evolution of eye development in arthropods: phylogenetic aspects. Arthropod Struct. Dev., 35, 319-340.
Strausfeld et al. 2016. Arthropod eyes: The early Cambrian fossil record and divergent evolution of visual systems. 45, 152-172.
Response: We fully agree and added both references to the introduction.
- The authors introduce the term “living fossil” in the abstract and because the great discussion around the term “living fossil” I was waiting a brief discussion at the end of the paper, but nothing show up. Please can you add something to such discussion, nothing long at the end of your discussion. Look some of the discussion published recently:
Turned, D.D. 2019. In defense of living fossils. Biology & Philosophy, 34:23 https://doi.org/10.1007/s10539-019-9678-y
Lidgan S. & Love, A.C. 2021. The living fossil concept: reply to Turner. Biology & Philosophy, 36, 1-16. https://doi.org/10.1007/s10539-021-09789-z
Bennett, Dominic J., Sutton, Mark D., and Turvey, Samuel T. 2018. Quantifying the living fossil concept. Palaeontologia Electronica 21.1.14A 1-25. https://doi.org/10.26879/750 palaeo-electronica.org/content/2018/2194-quantifying-the-living-fossil
Response: We also agree on this comment. However, this manuscript focusses on just one of the seven subfamilies of the Pax gene family. We are currently finalizing a followup manuscript that surveys all Pax genes in Limulus. The discussion section of this paper will expand on the contrast between relatively slow pace phenotypic change in contrast to the dramatic genomic evolution in this lineage. The references you provided will be very useful to include there. So thank you for the comment.
Round 2
Reviewer 1 Report
Comments and Suggestions for Authors
I appreciate that authors have done the tremendous job in revising manuscript. Thay have extended the scope of study and increased the number of figures in the revised version. Somehow, they have answered the reviewer’s comments although, I have found some noteworthy mistakes in the present form. Line numbers has not been introduced hence it is difficult to point-out everything.
In method section, Gene model annotation (Liao et al. 2019), has not been mentioned in the bibliography.
Authors has not cited the suggested references (mentioned in previous report) to enrich their discussions.
Conclusive remarks are missing.
Figure quality should be improved.
References are not uniformly written.
These are some suggestions which need to incorporate in the manuscript. As I said that line numbers have not been introduced hence it is difficult to point-out everything.
Overall, I found that manuscript is good in shape now but do not overlook these minor mistakes. Proof-read the article carefully one more time.
All the best.
Comments on the Quality of English Language
Minor spelling check and formatting required.
Author Response
Suggestions for Improvement:
- In method section, Gene model annotation (Liao et al. 2019), has not been mentioned in the bibliography.
Response: Thank you for catching this. Has been fixed and is now reference [30]
- Authors has not cited the suggested references (mentioned in previous report) to enrich their discussions.
Response: As we pointed out in our previous response, we agree that gene duplication has been found to be of major significance in insect genome evolution. However, unlike in the Chelicerata, gene duplications in insects are generally not caused by whole genome duplications. We therefore abstained from including these otherwise very deserving studies. We hope this is understandable.
- Conclusive remarks are missing.
Response: Concluding remarks are not mandatory in MDPI arthropoda. However, the two final paragraphs of the discussion address broader themes, i.e. the relation of our study to that of other gene family studies in the Xiphosura and the logic expansion of our study to other members of the Pax gene family.
- Figure quality should be improved.
Response: We agree on this point and will pay attention to figure quality during the production process.
- References are not uniformly written.
Response: We reviewed the references and corrected:
- Lamsdell, J.C. The Phylogeny and Systematics of Xiphosura. PeerJ 2020, 8, e10431.
- Ohno, S. Evolution by Gene Duplication; Springer Science+Business Media New York, 1970.
- Liberles, D.A.; Kolesov, G.; Dittmar, K. Understanding Gene Duplication through Biochemistry and Population Genetics. In Evolution After Gene Duplication; Dittmar, K., Liberles, D.A., Eds.; John Wiley & Sons, Inc. Hoboken, NJ, USA, 2010; pp. 1–21.
- Kimura, M. The Neutral Theory of Molecular Evolution. Scientific American 1979, 241, 98–129.
- Baudouin-Gonzalez, L.; Harper, A.; McGregor, A.P.; Sumner-Rooney, L. Regulation of Eye Determination and Regionalization in the Spider Parasteatoda Tepidariorum. Cells 2022, 11, 631.
- Peterson, K.J.; Beavan, A.; Chabot, P.J.; McPeek, M.A.; Pisani, D.; Fromm, B.; Simakov, O. MicroRNAs as Indicators into the Causes and Consequences of Whole-Genome Duplication Events. Molecular Biology and Evolution 2022, 39, msab344.
Moreover, we decided to delete references 55 and 56 which do not directly speak to the evolution of Pax genes
Reviewer 2 Report
Comments and Suggestions for Authors
1. The revised version should provide the updated sequence data. They could not be find in the supplimentary files.
2. The vertebrates used in the present analyses should be recosidered, the species sampled, such as Mus musculus, is inapprooriate. The other ecodysozoan taxa might be better.
Author Response
Reviewer 2
- The revised version should provide the updated sequence data. They could not be find in the supplimentary files.
Response: Thank you for catching this. In the previous submission, Text document S1 was a zipped folder within the zipped Supplementary data folder. We now saved the annotated genomic sequences in a regular folder as Text document S1. We hope this resolves the issue.
- The vertebrates used in the present analyses should be recosidered, the species sampled, such as Mus musculus, is inapprooriate. The other ecodysozoan taxa might be better.
Response: We agree that Mus musculus on its own would not be too distantly related. This is the reason why we also included the house spider Parasteatoda tepidariorum (Arachnopulmonata: Araneae), the Arizona bark scorpion Centruroides sculpturatus, and the red flour beetle Tribolium castaneum in our analyses.
Round 3
Reviewer 1 Report
Comments and Suggestions for Authors
I appreciate that authors have done the tremendous job in revising manuscript.
Comments on the Quality of English Language
Chek again.
Author Response
Reviewer 1
I appreciate that authors have done the tremendous job in revising manuscript.
Response: We appreciate the compliment. We also made a few more language edits.
