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Dear Dr. Brito, I congratulate you on the acceptance of this article for publication.
[# PeerJ Staff Note - this decision was reviewed and approved by Nigel Andrew, a PeerJ Section Editor covering this Section #]
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I commend the authors for their excellent work in significantly enhancing the clarity and rigor of the manuscript.
The topic of the article under review is relevant. The authors present an integrative taxonomic revision of the genus Oreoryzomys. Field collections were collected and an analysis was conducted based on the study of museum material. The manuscript is formatted in accordance with the requirements. The introduction provides sufficient information that relates to the topic of the study. The authors clearly described the history of the study of the Andean rodent genus Oreoryzomys. Literary sources are numbered appropriately. Comments regarding references to figures in the text have been taken into account.
The research corresponds to the subject of the journal. The research methods are described clearly, in detail and in an accessible way. The research was conducted in accordance with the prevailing ethical standards in the field.
The conclusions are clearly stated and supported by the results. In the conclusion section, the authors indicate their contribution to the research area. However, it would also be desirable to indicate unresolved issues and future directions in this research area in this section. In this section, the authors also added information regarding unresolved issues and future directions in this area of research.
Dear Dr. Brito, I kindly request you to carefully revise this manuscript in accordance with the reviewers' key comments.
**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 meets all the required points.
This manuscript addresses a well-formulated and compelling research question that fills a critical knowledge gap in the systematics of the genus Oreoryzomys, which was until now considered monotypic. The research is executed with high technical proficiency and is supported by robust and thoroughly detailed methodologies.
The submission satisfies all requirements. The manuscript is well-structured, with all necessary sections (Introduction, Materials & Methods, Results, Discussion, Conclusions) clearly presented and complete.
The manuscript presents a valuable taxonomic and systematic study of genus Oreoryzomys.
Specific Comments:
Lines 73 and 79: The citation "(Percequillo, 2015)" could not be located in the reference list.
Line 163: There is an inconsistency in the abbreviation of the “Cytb”.
Materials & Methods:
(DNA Amplification): For clarity, state the sample size (N) for the molecular analyses, specifying how many specimens were sequenced for each gene, as was done for the morphological data.
(New Section Suggestion): Adding a subsection titled "Sampling Methods" or "Field Collection". This section should detail the sampling effort, collection period, and a brief description of the study area. The collection localities could be visualized by plotting them on a map, figure 14 would be suitable for this purpose.
Figure 9: Figure 9 appears incoherence. Its citation in the text should also be verified.
Line 427: “(Fig. 18B)”
Line 516: The citation for “(Fig. 13C)” should also be verified for consistency.
The topic of the article under review is relevant. The authors present an integrative taxonomic revision of the genus Oreoryzomys. Field collections were collected and an analysis was conducted based on the study of museum material. The manuscript is formatted in accordance with the requirements. The introduction provides sufficient information that relates to the topic of the study. The authors clearly described the history of the study of the Andean rodent genus Oreoryzomys. Literary sources are numbered appropriately. There are, however, some minor comments. Not all figures are referenced in the text. For example, in the results section there is a reference to figure 3A, 3F, 3K, 3G, 3L. But there are no references to figures 3B, C, D etc. The same applies to some other figures.
The research corresponds to the subject of the journal. The research methods are described clearly, in detail and in an accessible way. The research was conducted in accordance with the prevailing ethical standards in the field.
The conclusions are clearly stated and supported by the results. In the conclusion section, the authors indicate their contribution to the research area. However, it would also be desirable to indicate unresolved issues and future directions in this research area in this section.
General Comments
Firstly, I would like to commend the authors for their excellent work. The manuscript is based on an extensive and valuable dataset, and it is written with remarkable clarity and precision. The ability to draw such a wide range of inferences from the data is a significant strength. The taxonomic and morphological descriptions are particularly thorough and well-presented.
Below, I offer suggestions aimed at strengthening the manuscript, primarily by enhancing the clarity and depth of the phylogenetic analyses and by addressing a few specific points of detail. I hope these comments are helpful for the final revision.
Major Suggestions for Improvement
A few points that I felt were not fully addressed:
o Cytogenetics: Is there any karyological data available for this genus?
o Sampling methods: Could the authors kindly clarify how exactly the collections were conducted?
Molecular Genetic Analyses and Presentation:
o Outgroup Selection and Taxa Inclusion: The phylogenetic rationale could be clearer. By definition, the outgroup should be the closest relative to the ingroup. Please clarify why several additional genera were included, especially if they are not discussed. If the goal is to provide a broader phylogenetic context or to enable future molecular dating, this should be stated explicitly. To improve figure clarity, I suggest pruning the tree to include only the ingroup (Oreoryzomys), its sister genus (e.g., Microryzomys), and one or two other closely related genera as outgroups.
o Molecular Dating: The inclusion of a molecular clock analysis would significantly enrich this study. It would allow you to explore the timing of diversification within Oreoryzomys and to correlate speciation events with past climatic or geological changes (e.g., "What was the ecological context during the speciation of the Napo region lineages?"). This would also justify the inclusion of a broader taxonomic sample for calibration purposes.
o Phylogeny Figures and Data: The current phylogeny figures are difficult to interpret due to their size and the inclusion of the distance matrix. I suggest:
Presenting the genetic distance data in a separate table in the supplementary material.
Reformatting the trees to be larger and clearer.
Providing the original alignment files (showing final dimensions, variable sites, parsimony-informative sites) and tree files as supplementary material. This is crucial for reproducibility.
o Analysis Details: The methodology for phylogenetic inference needs more precise description (see specific comments below). Key details about model application, bootstrap procedures, and software versions are currently missing or ambiguous.
Discussion of Key Patterns:
o Napo Region Endemism: The Napo region seems as a notable hotspot of diversity. This is a fascinating result. I strongly encourage the authors to expand the discussion on this aspect, e.g., examples with other taxa. What hypotheses could explain this pattern? Are there known biogeographic barriers or refugia in this area that might correlate with your findings?
Specific Comments (point by point)
• Line 65: Could you please check whether the reference to Musser et al., 1998 is correctly placed here?
• Lines 67-76: The structure of this paragraph feels somewhat confusing. Would the authors consider rephrasing for clarity? The first sentence, in particular, is potentially misleading as the genus was erected much later than the species. Perhaps a chronological narrative is not the best approach here.
• Line 106: It seems that an Oxford comma may be missing.
• Line 152: Should "analysis" be plural ("analyses")?
• Lines 154-159: PCR and Sequencing Protocols: For reproducibility, please provide more detail.
o Line 154: Clarify the primer pairs used in the PCR schemes: MVZ05 is forward, while MVZ16H (would the correct name be MVZ16?) and MVZ14 are reverse primers (e.g., MVZ05 with MVZ16 or MVZ05 with MVZ14).
o Line 157: It is not clear which protocol is being referenced here. Could you specify?
o Line 158: If libraries were constructed for Nanopore, briefly describe the method (e.g., kits used).
o Line 159: Specify which amplicons were sent. This is not entirely clear.
o Line 161: Could the authors clarify what the sequences were aligned with, and for what purpose? Also, ClustalW lacks citation (Thompson et al., 1994).
• Line 165: As above, please clarify the outgroup selection strategy in the text. It is unclear why all these genera were included, and which group was used as the outgroup.
• Lines 169-170: It is not clear whether the K80+G model was applied to each partition independently or to the entire dataset as a single partition. Please specify.
• Line 176: Could the authors please state the version of MrBayes used (e.g., 3.2.6 or 3.2.7)?
• Line 176: I believe “burn-in (0,25)” would actually be “0.25”, but if it is the same as 25%, wouldn’t it be redundant? I suggest using one or the other (e.g., " burn-in of 0.25").
• Lines 177-178: Briefly state how the ESS and PSRF were calculated (typically done on Tracer). Specify which analyses were run on the CIPRES platform (e.g., " PartitionFinder2 and MrBayes analyses were conducted on the CIPRES Science Gateway"). The term “all” feels somewhat vague.
• Lines 181-182: The sentence “The best-fit tree was selected based on 1000 bootstrap replicates” could be misleading. Bootstrap replicates are typically used to assess node support, not to select the best-fit tree. Usually, the “best-fit tree” is chosen according to the LnL score under the substitution model, and bootstrap replicates are run to measure clade support.
• Line 182: For nodal support, was Ultra-fast or standard bootstrap used? This should be clarified, since IQ-TREE offers both. And, with how many replicates?
• Line 183: The phrase “…values <70% was considered low support” could be clearer. What threshold was considered high support? Since some studies argue against fixed cutoffs (e.g., Zander, 2004), it might help to briefly justify this common but debated threshold.
• Lines 183-184: How were the genetic distances calculated (i.e., what program was used)?
• Line 185: The phrase "…based on the results of the phylogenetic analyses of this gene" is somewhat ambiguous. Please rephrase to clarify the intention (e.g., "to confirm species boundaries suggested by the phylogeny").
• Lines 217-218: The results section should include a description of the final alignment matrices: number of taxa, alignment length, number of variable sites, and parsimony-informative sites. Were ends trimmed for unmatched regions? Were poorly aligned blocks suppressed? Providing this information, along with supplementary files of the matrices, would add transparency.
• Line 220: Please, along with Fig. 1, could the authors also provide the original unedited tree files as supplementary material?
• Lines 235-239: It is unclear what the reported genetic variation values represent (mean and SD? standard error?). Could the authors specify? How do intraspecific and interspecific genetic distances compare to other genera (e.g., Microryzomys)?
• Line 676: Considering the broad variation in HBL within the genus (62–125 mm), might it be more informative to present a range rather than a single average value?
• Line 677: Similarly, since tail length averages 118% of head-and-body length in O. balneator, 117% in O. hesperus, and 138% in O. jumandi, would it perhaps be more appropriate to provide a range rather than a single percentage for the genus?
• Line 681: Considering that the coronal suture is V-shaped in O. balneator and O. jumandi, but broadly U-shaped in O. hesperus, is it correct to generalize the genus as having a markedly V-shaped coronal suture?
• Line 683: Since the incisive foramina are short in O. balneator and O. jumandi, but long in O. hesperus, is it accurate to describe the genus as having short and broad foramina?
• Line 760: The expression “classical morphological analysis” seems somewhat broad. For clarity, could the authors specify which methods or criteria are encompassed under this term?
• Figure 1:
o The figure legend must explain what the genetic distance values represent (e.g., mean pairwise distances).
o As suggested above, moving the distance matrix to a separate table will greatly improve the tree's readability.
o Consider conducting a barcode gap analysis to further explore the genetic distance data.
o Regarding outgroups, since by definition the outgroup should be the closest relative to the ingroup, would it not be more effective to include only Microryzomys (the recognized sister group), or at most Neacomys and Oligoryzomys, rooting in Oligoryzomys? This would free space in the trees and improve readability.
o Since many sequences are identical, they could be collapsed in a single haplotype in the phylogenies, further improving tree readability.
o Moreover, haplotype networks and simple phylogeographic analyses (e.g., Fst, Mantel test, Bayesian Analysis of Population Structure) could add value to the molecular genetic analyses, which otherwise feel underexplored.
o Observation: The O. hesperus sequence from El Oro appears highly divergent and poorly supported in the mt-Cytb ML tree. Could this represent another independent lineage? A haplotype network analysis for the mt-Cytb gene would be an excellent way to visualize relationships among haplotypes and investigate this further.
• Figure 2B: Which are the individuals that fall outside the ellipse of the Tungurahua + Morona Santiago clade?
• Figure 5: The legend contains a typographical error, please check: “( MECN 6140,…”
• Figure 9: The legend seems to contain untranslated words ("deûned", "ûrst"). Please correct to "defined" and "first."
• Figure 14: The map would be more informative if collection localities were indicated by numbers, with a corresponding key in the legend or supplementary material.
• Table 2: The legend should explain the meaning of the bold values (e.g., they likely represent the highest PC values).
I congratulate the authors again on a substantial piece of work and am happy to provide any further clarification if needed.
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