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The authors have done a great job addressing all the concerns and comments of the reviewers and myself, and I am happy to accept this current version for publication.
[# PeerJ Staff Note - this decision was reviewed and approved by Patricia Gandini, a PeerJ Section Editor covering this Section #]
General comments
I think you should be a little more reserved in your interpretations, as you are using a single gene region with a variable number of samples per location (some being quite small). These are limitations that really need to be stressed. You should be more clear on which sequences are ~400 bp. This is quite short and may not contain many mutations, therefore, in some areas which you have found no differences from others, this could just be a product of the data (eg. line 407 of discussion). Please be more clear on this point throughout. I think the analysis section would benefit from STRUCTURE plots (either using STRUCTURE or admixture).You need some better tests to work out the correct number of haplogroups.
One of the reviewers suggested adding a section to your introduction on mito-nuclear discordance. That is an entire other issue that is not addressed in this study and I do not think you should add it in.
Specific comments:
Abstract: The abstract needs some more numbers and references to the analysis used
Line 25: “We found moderate genetic variation within New Mexico…” using what analysis? Add in brackets after ‘New Mexico’ with some numbers from Fst or other analysis that supports this explanation.
Line 28: “Additionally, sex of the eight…” Please change sentence to the following and fill in the X’s with appropriate information: “Based on X analysis, we found a total of X haplotypes, of which six haplotypes found across…”
Line 29-30: Again, please add in some values to corroborate your explanation. This comment is the same for line 32 as well.
Introduction
Line 49: Is Pravalie in bold?
Lines 103-106: What part of the world were these studies? Right next door or in other countries?
Line 112: Please change sentence to: “…Specifically, we examined a region of cytochrome b in the mitochondrial genome of X individuals, sampled…”
Line 113: Did you reanalyze all the data from all regions around the world or did you follow what they did for your own region. Please make this clear.
Methods
Line 124: Either put the scientific name in italics or use the common name. I would use the common name for consistency (you used the common name for the entire introduction).
Line 139: “As the second euthanasia step…” This sounds like a backup plan if the first step didn’t kill completely. Please reword to “Following euthanasia, we removed…”
Lines 153-160: You need more info on the PCR. Please include the PCR profile from the thermal cycler. Also include the model of thermal cycler. It needs to be clear so that someone could follow along and do what you did.
Line 199: You should have a haplotype network (using PopART or other similar software) instead of or alongside the ML phylogeny. You will better see the different connections. I also think you should add in a structure analysis (either using structure or admixture). They will help you to see how many haplo-groups you have. SAMOVA is another one that could be beneficial if you don't want to add in a structure plot. It is like an AMOVA but adds in the geographic data (latitude and longitude) and therefore can point out the best number of haplo-groups. You need some better tests to work out the correct number of haplogroups.
Line 212-214: I don’t think this is the conclusion you should make with Fst tests alone. I think you need to amend this to “In conjunction with haplogroups..”
**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.
I added some technical corrections to the main text. You can skim the corrected file. I wonder why authors do not follow the updated systematic review. Frost et al. (2024) and AmphibianWeb suggest the name "Aquarana catesbeiana" for the Bullfrog. I recommend the use of "Rana (Aquarana) catesbeiana" for this species. All the species and genus names must be italicised throughout the text. Also, the use of "P" (significance index) must be standardised throughout the text.
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Researchers sometimes used the abbreviation of the location, but sometimes did not. Please keep an eye on uniformity throughout the text.
In this manuscript, the authors explore the population genetics and invasion history of the American Bullfrog (Rana catesbeiana or Lithobates catesbeianus).
With a few exceptions, writing was clear and unambiguous, citations were appropriate and sufficient, and figures looked professional. I have noted in my comments below exceptions to this.
All DNA data checks, vertebrate animal usage checks, and field study checks were passed.
The authors specifically sought to examine the genetic diversity within and between sites, compare genetic diversity between native and introduced populations, and elucidate the number and source(s) of introduction. These are well-defined and relevant questions.
Limitations to the experimental design include the fact that the authors used only a single gene to explore these questions, cytochrome b, and sample sizes were both small and variable.
This paper contributes to the literature by not only exploring the genetic diversity of some understudied New Mexican populations but also by reviewing and incorporating data from previous studies that allow for a broader picture of bullfrog invasion.
I would argue that the investigation was performed to a high standard, given the limitations of the methodology, and is replicable.
The authors found little differentiation between sites in New Mexico, supporting a singular introduction event, with similarities to haplotypes in other western, invasive populations.
This study is limited in two main aspects: 1) Using only cyt b for population genetic analysis (especially in 2025) is an extremely limited way to explore genetic diversity and most population genetic measures. Further, 2) the sample sizes at some sample sites are extremely small. Small sample size may be less of an issue if using multiple genes, or genome-wide SNP data, but the problems with small sample size are only exacerbated by using a singular mitochondrial gene.
Though the authors have already included a section on limitations, I would advise for even more cautious language throughout to limit overinterpretation.
Abstract: I would include in the abstract that the methodology involved using a single mitochondrial gene. This will help readers immediately understand the methodology on which the results are based.
Line 42: the native eastern United States. Consider “their native range in the eastern US.
Line 54: Hayes and Jennings 1984. Is there not a more recent citation?
Line 64: I strongly suggest staying away from language like “it is apparent”, apparent to whom? Just say they are not native there and provide a citation.
Line 75: True, but perhaps a bit ambitious given the specific methodologies used in this paper.
Line 124: Sticking with Rana throughout is fine, but if using Lithobates, it is catesbeianus.
Line 133: There are some very low sample sizes in here (1-4). While historic standards for sample sizes are above 10 or even 20, smaller sample sizes can still be informative, but typically only when using a large number of markers (Nazareno et al. 2017). I understand the difficulties of sampling and am not suggesting that these data are therefore uninformative, but I would increase language throughout, highlighting this limitation. Lastly, though the authors report that they collected individuals from a variety of life stages. Were there any sampling protocols followed to reduce the sampling of full sibs within the same cohort?
Line 177: Editors fix the broken symbol.
Line 267: Full sibs?
Line 277: Clarify the meaning of the best-fit model here in text.
Line 294: Is it worth reporting the results without the Bonferroni correction? I don’t understand why this is reported.
Line 304: leave interpretation to the discussion
Line 355: Great point, I think this is something to highlight!
Lines 365-367: Good to caution here, but should also caution before now.
Line 421: This paragraph is needed, and I would argue that more emphasis should be placed on the small (and variable) sample sizes and how that might affect your results.
The manuscript is clearly written in professional English and follows a logical, coherent structure. The introduction provides sufficient background and establishes the ecological relevance of Rana catesbeiana as an invasive species. References are generally appropriate and up to date, and the figures and tables are relevant and properly referenced. Overall, the manuscript meets the general standards of PeerJ for clarity and structure. However, some improvements are recommended to enhance clarity and completeness.
I suggest the background section should be expanded to include more recent genomic or multilocus studies on amphibian invasions and mitochondrial–nuclear discordance, which would strengthen the conceptual framework.
The regional context of bullfrog introductions in New Mexico should be elaborated, particularly with reference to hydrological connections and human-mediated dispersal routes. Some redundancy in the early paragraphs describing intentional and unintentional introductions should be removed to improve conciseness.
The resolution and labeling of Figures 1 and 2 should be improved.
The methods are generally appropriate and conducted to a high ethical and technical standard, with field collections covered by institutional animal care protocols. I have a question about whether, in addition to the protocols, collection permits are required; if so, should they be included?
Several aspects of the experimental design require clarification to improve methodological transparency and interpretability. The reliance on a single mitochondrial locus (cytochrome b) limits the resolution for detecting multiple introductions or gene flow, and this should be emphasized as a key limitation of the study. Sample sizes are uneven across sites, with some localities represented by very few individuals (e.g., Mora River, n = 1), which could bias estimates of genetic diversity and differentiation.
The authors should explicitly discuss how they accounted for this imbalance or whether certain sites were excluded from specific analyses. In addition, it should be clarified whether comparative data from previous studies were re-analyzed from raw sequences or derived from published summary statistics, as this affects reproducibility.
Including more details on the number of sequences included in phylogenetic and AMOVA analyses would also strengthen the methodological section.
Overall, the experimental framework is sound, but greater transparency in data provenance, sample representation, and analytical scope will enhance the rigor and reproducibility of the study.
Generally, the findings are clearly presented and consistent with the objectives of the study. The analyses are methodologically sound, and the data appear robust and well controlled. The results appropriately show reduced genetic diversity in invasive populations relative to native ones, and this pattern is interpreted within an established theoretical framework.
However, the strength of some conclusions should be moderated to better reflect the limitations of the dataset. The inference of a single introduction into New Mexico, for instance, should be presented as being “consistent with” rather than definitively demonstrating one introduction, given the limited resolution of the single mitochondrial marker and uneven sampling.
The phylogenetic results show only moderate support for some nodes (bootstrap values around 80%), and this uncertainty should be discussed more explicitly in the interpretation.
While the statistical methods are appropriate (e.g., AMOVA, FST, Mann–Whitney tests), the small sample size in certain localities reduces the power of these tests and should be acknowledged.
Overall, the conclusions are coherent and supported by the data, but a more cautious tone and explicit discussion of methodological limitations would strengthen the validity and transparency of the findings.
This is a well-written and valuable contribution that advances our understanding of invasive amphibian populations in a region with limited prior genetic data. The integration of field and museum samples is commendable, and the authors have presented their analyses clearly and thoughtfully. With the major clarifications and improvements suggested above, the manuscript will provide a strong addition to the literature on invasion genetics and amphibian biogeography.
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