-->PONE-D-25-58380-->-->The evolution of the control region in the mitochondrial genome of vertebrates-->-->PLOS One

Dear Dr.--> -->Prada Quiroga,

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Additional Editor Comments:

Dear Author,

Thank you for submitting your manuscript to PLOS ONE.

After careful consideration and evaluation of the reviewers’ comments, we believe that the manuscript addresses an interesting and important topic. However, the current version of the manuscript cannot be accepted for publication at this stage. We invite you to revise the manuscript substantially and resubmit it for further consideration.

Please carefully address all reviewers’ comments and provide a detailed response explaining how each point has been addressed in the revised version.

We appreciate your contribution to the journal and look forward to receiving your revised manuscript.

Kind regards,

Amaal.

Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Yes

Reviewer #2: Yes

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-->2. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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-->5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: This manuscript presents a large-scale comparative analysis of the mitochondrial control region (CR) across vertebrates using 5,235 complete sequences retrieved from GenBank. The authors investigate variation in CR length, repetitive elements, conserved sequence blocks (ETAS and CSBs), nucleotide composition, and duplication patterns across major vertebrate groups. The study aims to provide a comprehensive framework for understanding the evolution and structural variability of the mitochondrial control region.

The topic is relevant and timely, and the dataset analyzed is impressively large. The comparative perspective across multiple vertebrate classes provides useful insights into lineage-specific patterns in mitochondrial genome architecture. The manuscript contains extensive statistical analyses and attempts to link structural variation with evolutionary mechanisms.

However, despite the potential significance of the study, the manuscript currently suffers from several methodological and presentation issues that need clarification before it can be considered for publication. In particular, the bioinformatic workflow, sequence selection criteria, repeat detection methods, and motif identification procedures are insufficiently described, making it difficult to reproduce the analyses. Some interpretations are also overstated relative to the evidence presented, and the manuscript would benefit from clearer structure and improved figure interpretation.

Overall, the manuscript has scientific merit, but major revisions are required to improve methodological transparency, strengthen the interpretation of results, and enhance the clarity of the presentation.

The Methods section lacks sufficient detail to ensure reproducibility. Important information that should be clarified includes:1) How the 5,235 mitochondrial control regions were selected from GenBank. 2) Criteria used to determine “complete CR sequences”. 3) Whether sequences were manually curated or automatically extracted. The manuscript reports detection of repetitive elements and conserved motifs (ETAS and CSBs), but the algorithms or software used for identifying these motifs are not clearly described. The dataset contains 2,861 fish sequences and 2,374 tetrapod sequences, but the manuscript does not explain whether this distribution reflects: true biological diversity, availability of sequences in GenBank and deliberate sampling strategy. Because the dataset relies on public database sequences, the authors should discuss potential biases caused by uneven taxonomic representation or incomplete assemblies. The authors report a strong correlation between CR length and repeat abundance (r = 0.69). However, the manuscript sometimes implies causation, suggesting that repeat expansion drives CR evolution. The authors should be more cautious and clarify that: 1) correlation does not demonstrate causation. 2) other processes (e.g., recombination, replication dynamics) may contribute to CR length variation. Additional analyses (e.g., phylogenetically controlled tests) would strengthen the argument. Although the study includes many vertebrate taxa, the analyses are largely descriptive rather than phylogenetically explicit. For example: correlations and statistical comparisons are performed across classes, but phylogenetic non-independence is not considered. Because mitochondrial genome features may reflect shared ancestry, the authors should consider:1) phylogenetic comparative methods. 2) controlling for phylogenetic signal. Without this, some observed patterns may reflect phylogenetic clustering rather than evolutionary processes. The discussion frequently proposes adaptive explanations for CR variation (e.g., metabolic demands, thermoregulation, life-history traits). However, these interpretations are largely speculative and not supported by direct tests in this study. For instance: the hypothesis linking CR repeat expansion to metabolic plasticity in amphibians or flight energetics in birds should be clearly framed as hypotheses rather than demonstrated conclusions. The manuscript references numerous supplementary tables and figures (S1–S5), but their descriptions in the text are limited. The authors should: 1) ensure that all supplementary materials are clearly explained. 2) improve figure captions to describe statistical tests and sample sizes. 3) clarify which datasets correspond to each analysis. The Discussion is very long and sometimes repetitive. Several sections restate results already presented earlier. Condensing this section would improve readability.

The manuscript presents an interesting and potentially valuable comparative analysis of vertebrate mitochondrial control regions. However, substantial revisions are required to improve methodological transparency, clarify statistical interpretations, and moderate speculative explanations.

Addressing these issues would significantly strengthen the manuscript and improve its contribution to the field of mitochondrial genome evolution.

Reviewer #2: Comments to authors:

Main comments

- I would congratulate to authors for performing such a broad study with thousands of CRs sequences. I believe the manuscript will benefit much from the provided comments and well fit for the publication after revision. The provided text lacks a dedicated, detailed Methods section. Key elements are missing or only referred to: exact search strategy and filters used in GenBank/NCBI to retrieve the 5,235 "complete" CR sequences; criteria for defining "complete" CRs (critical given known assembly difficulties with repeats); bioinformatics pipeline for extraction, annotation of TAS/ETAS, central domain (CSB-A to F), and Domain III (CSB1-3); tools and parameters for motif discovery, consensus sequence generation, maximum-likelihood motif analysis (S5 Fig.), repeat detection (e.g., tandem repeat finder settings), statistical tests (Kruskal-Wallis, Dunn's with Bonferroni), and correlation analyses.

- How were partial or ambiguous CRs excluded? How were duplicates or heteroplasmy handled? Describe quality filtering, multiple sequence alignment (if used), and any custom scripts for domain identification. Without this, results are not reproducible.

- The manuscript's primary strength is its ambitious scale—analyzing over 5,000 vertebrate CR sequences—which enables robust documentation of inter- and intra-class variation in length, repeat content, nucleotide composition, and conserved motif copy number/position. This provides a valuable empirical foundation for understanding how tandem repeat proliferation drives CR expansion in tetrapods while core regulatory elements (TAS, CSBs) show differential conservation, revealing lineage-specific evolutionary trajectories (e.g., elongation in anurans/reptiles, streamlining in birds/mammals). When methods are fully detailed, this work could serve as a useful reference for mitogenomic researchers. Therefore, major weaknesses include insufficient methodological transparency (no detailed pipeline for sequence retrieval, CR annotation, or motif detection), numerous numerical inconsistencies and typographical errors suggesting the manuscript is not yet polished, and limited reproducibility due to incomplete data sharing. Analytical descriptions for custom motifs and phylogenetic motif analyses need expansion, the novelty claim requires better contextualization against existing literature, and the text contains structural/grammatical issues. These issues currently compromise rigor and clarity.

Require thorough proofreading and verification:

- Abstract: "5,235 complete vertebrate CRs spanning 12 classes"; "tetrapods (1,283 ± 239.5 bp) than in fishes (969 ± 489.6 bp)". Later text reverses or varies SDs (e.g., fishes 969.25 ± 239.5; tetrapods 1,283.27 ± 489.6). Fish vs. tetrapod numbers shift slightly across pages.

- "11 vertebrate taxonomic classes" in one place, "12" in another; "8 taxonomic classes" for fishes.

- Minimum length 329 bp in Percopsis transmontana (note spelling: transmontana).

- Duplication frequencies: Reptiles 35.3% (108/306), Aves 20.16% (158/767), Amphibia 7.1% (17/241)—verify totals against overall 2,374 tetrapod sequences.

- Check the correct spelling of the scientific name and pay attention to the inconsistent capitalization and italics for taxa.

Analytical rigor and interpretation:

- Phylogenetic context for motif evolution (S5 Fig., ML analysis of consensus motifs) is promising but requires clearer methods (alignment, model selection, tree inference) and discussion of limitations (e.g., short motifs, saturation).

- Nucleotide skews and compositional biases: Discuss potential artifacts from replication asymmetry or sequencing/assembly biases in repeat-rich regions.

- Duplications: Clarify detection method and functional implications (e.g., concerted evolution in snakes/birds).

- Strengthen the "why these matters" section: Link findings more explicitly to broader questions in mitochondrial evolution, compensatory evolution, or implications for phylogenomics/barcoding (CR is often excluded due to variability).

Writing and presentation:

- Title is clear but could specify "large-scale comparative analysis" or similar for precision. Claims of "first comprehensive comparative framework" need tempering—while the scale is large, prior comparative mitogenomic work exists (including on CR in specific clades or broader vertebrate mitogenomes). Position the study more accurately as a large-scale synthesis rather than wholly unprecedented.

- Please write all the terms in full form when they appear at the first time; e. g ETAS: Extended Termination-Associated Sequences in Abstract

- The provided text is too lengthy, fragmented with repetitive phrasing or long sentences. Please improve its fluency, use concise scientific English and avoid redundancy.

• Provide a complete, standalone ‘Methods section’ with all bioinformatics details, parameters, and validation steps. Deposit data/code publicly and update the Data Availability statement with specific links/DOIs.

• Systematically verify and standardize all numerical results, and statistical reporting across abstract, results, tables, and figures. Include sample sizes (n) for all means ± SD and post-hoc tests. Several times ‘±’ is missing when average (mean)±SD are reported. E.g. Page 9, Line 57: ‘between 1,028 900 to 1,581 bp in Aves’/ Page 22, Line 345: and 2.01 1.0477 copies per…

• Please be consistent with format when reporting standard deviation: use +/- OR ± throughout the text; e.g. Page 12, Line 122: CR length was 969.25 (+/- 239.5) bp/ replace Fig 1a ----> Fig 1A

• Proofread thoroughly for grammar, typos, consistency (e.g., GC- vs. CG-, CSB- vs. CBS, fish vs. tetrapod stats), and flow. Consider professional editing if needed. E.g. Page 15, Line 193: statistically significant lower GC content...; replace 'significantly' with 'significant'/ Page 17, Line 239: delete ‘the’ a posteriori… and I would reword it as follow: “… and a posteriori Dunn's test indicated significant variation among all four compared classes..”

• Page 18, Line 250: Testudines

• Page 19, Line 290: the CR length was moderately correlated …

• Page 23, Line 372: frequently detected (74%)…

• Page 24, Line 381: Delete "in", also "ing": ...are lacking CSB-F to CSB-B within their CRs.

• Page 32, Line 579: These partial copies retain an important? Please check the sentence for its structure and meaning.

• Ensure all figures/tables are complete, high-resolution, and self-explanatory with proper legends! In current version, provided figures are in low quality I cannot read the words.

• Address limitations explicitly (e.g., biases in public databases toward certain taxa, challenges with repeat-rich assemblies, potential homoplasy in short motifs).

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Reviewer #1: No

Reviewer #2: No

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Submitted filename: Comments to PONE-D-25-58380.pdf

Evolutionary patterns of the mitochondrial control region in vertebrates: a large-scale comparative analysis

PONE-D-25-58380R1

Dear Dr. Dr. Quiroga,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Amaal Gh. Yasser, Ph.D.

Academic Editor

PLOS One

Additional Editor Comments (optional):

Dear Dr. Quiroga,

Thank you for submitting the revised version of your manuscript and for the considerable effort invested in addressing the reviewers' comments and recommendations.

After careful evaluation of the revised manuscript and your detailed responses to the reviewers, I am pleased to inform you that your manuscript has been accepted for publication.

The revisions have substantially improved the quality and clarity of the manuscript, and the concerns raised during the peer-review process have been satisfactorily addressed.

On behalf of the editorial team, I would like to congratulate you on the acceptance of your work and thank you for choosing PLOS ONE for the dissemination of your research.

We appreciate your contribution and look forward to seeing your article published.

Congratulations once again, and we wish you continued success in your future research endeavors.

Kind regards,

Amaal Yasser

Academic Editor

Reviewers' comments: