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Additional Editor Comments:

Dear Endashaw Terefe,

The manuscript number PONE-D-26-06464, entitled "The genomic basis of heat stress adaptation in indigenous Ethiopian cattle from arid and hot-humid climates," requires major revision to comply with the standards of PLOS One. Hence, it is recommended for submission in revised form, duly addressing all the reviewer's comments.

Regards,

Dr. Manasa. V

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

Reviewer #1: Partly

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

Reviewer #1: No

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

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Reviewer #1: Section-Wise Evaluation

Title: The title is informative but vague. “Genomic basis” does not distinguish this study from array-based genotyping work, and “indigenous Ethiopian cattle” omits the scientifically important Bos indicus (zebu) classification. A revised title is proposed: “Whole-Genome Sequencing Reveals Divergent and Shared Selection Signatures of Heat Stress Adaptation in Indigenous Ethiopian Zebu Cattle from Hot-Arid and Hot-Humid Environments.” This is more specific, methodologically precise, and avoids implying mechanistic resolution beyond what selection scans can provide.

Abstract: The abstract is generally well-structured but has three key problems. First, it implies that sequencing was conducted as part of this study, whereas all WGS data were retrieved from public repositories (NCBI SRA, CNGB, EMBRAPA). This must be corrected throughout. Second, it lacks quantitative metrics: no FST values are cited for population differentiation, and no counts of candidate regions per method or environment are reported. Third, the concluding novelty statement is generic; a more specific formulation e.g., highlighting the VEGFC vs. WIF1 vascular divergence, would better distinguish the study. Minor language issues (e.g., “remarkably adaptation,” passive-heavy constructions) also require correction.

Introduction: The Introduction is logically organized but does not clearly articulate how this study advances beyond the prior work of Terefe et al. (2023, ref. [11]), which includes some of the same authors and overlapping populations. This distinction is essential. Additional gaps include: (a) the agro-ecological description of hot-arid vs. hot-humid zones is deferred entirely to Methods; a brief overview here would better contextualize the biological rationale; (b) notable mechanisms such as epigenetic regulation, copy number variants (CNVs), and microbiome-mediated thermotolerance are unacknowledged as complementary avenues; (c) the final paragraph describing objectives should include an explicit a priori hypothesis (e.g., whether divergent or partially shared selection is expected given low FST). Language issues: “broadly into African zebu” should read “broadly classified into African zebu”; transition sentences between paragraphs on heat-stress physiology and Ethiopian cattle ecology are abrupt.

Materials and Methods:

Data sourcing: The critical concern is that all WGS data were retrieved from public repositories, yet the abstract and introduction imply original sequencing. This must be corrected throughout for transparency and compliance with PLOS ONE data policies. The inclusion of restricted-access Gir cattle data from EMBRAPA must be resolved, either by excluding these animals and assessing impact, or by obtaining documented permission for release upon acceptance. The HHETZ group has only 18 animals, and some ASZ populations have as few as two individuals per breed. The authors must discuss how these small sample sizes may inflate false-positive rates for iHS and XP-EHH, ideally with a formal power analysis or citation to relevant literature.

Bioinformatics pipeline: Several critical details are absent: (1) duplication marking (e.g., Picard/GATK MarkDuplicates) is not mentioned; (2) sequencing depth per sample and depth cutoffs for filtering are unspecified; (3) VQSR training datasets for bovine WGS are not described, nor are the hard filters applied (QD, FS, MQ, SOR thresholds); (4) LD pruning parameters for ADMIXTURE (window size and step) are incomplete, only the threshold (r² > 0.5) is stated.

Selection scan methods: The use of BEAGLE for phasing/imputation is problematic if the reference panel does not adequately represent Ethiopian zebu populations, which could introduce biases in iHS haplotype scoring. The Hp statistic, originally designed for pooled sequencing, is applied to individual-level WGS data without explanation of adaptation. For XP-EHH and XP-CLR, the sliding window rationale (100 kb window, 50 kb step) is not justified, and the specific software packages and versions used are not cited; making the analysis non-reproducible. The inconsistent significance thresholds between DHETZ and HHETZ for the same statistics are unexplained.

Functional annotation: DAVID has not been updated since 2016; more current platforms (g:Profiler, EnrichR) should be used or DAVID’s limitations explicitly acknowledged. The background gene set for GO/KEGG enrichment is not specified, which significantly affects enrichment outcomes.

Results

Variant discovery and population structure: Table 1 contains a typographic error: SUZ biallelic SNPs are listed as “29,460;635” (semicolon) should be “29,460,635.” The FST analysis states “20 samples per group” were used, but HHETZ has only 18 and DHETZ has 28 animals; any subsampling must be described and its effect discussed. The admixture CV error curve does not show a clear minimum at K=4; rather a gradual decrease followed by a plateau, and actual CV error values should be provided in a supplementary table. The PCA discrepancy between overall variance explained (PC1: 48.7%, PC2: 12.8%) and within-Ethiopian variance (PC1: 11.5%, PC2: 10.5%) is not explained; these represent separate analyses and the distinction should be made explicit.

Selection signatures: Candidate gene tables (3–5) contain errors: (1) the TTLL7 reference in Table 3 (ref. [43], Zhang et al. 2023 on Vav3 in myocardial infarction) is unrelated to polyglutamylation and appears misassigned; (2) Table 5 lists a BTA1 region with start 115.5 Mb and end 11.6 Mb; a physically impossible coordinate; (3) chromosomal coordinates in Table 3 are inconsistently presented in Mb and base pairs and must be standardized. The results do not report method overlap rates (e.g., what proportion of iHS-detected regions were confirmed by XP-EHH or XP-CLR), which would help assess statistical consistency. The detection of WIF1 by all four methods in HHETZ; the most robustly supported finding deserves greater emphasis.

Discussion: The Discussion is the strongest section, effectively integrating candidate gene biology with heat stress physiology (SESN2/antioxidant regulation, GRPEL2/mitochondrial chaperoning, XBP1/UPR, MYD88/innate immunity). However, several gaps remain: (1) No explicit comparison with Terefe et al. (2023) is made, which is a significant analytical omission given the overlapping populations. (2) Alternative explanations for selection signals population bottlenecks, founder effects, background selection are underexplored, particularly important given the low FST (0.0063) between DHETZ and HHETZ. (3) The biologically elegant contrast of VEGFC (pro-angiogenic, shared) vs. WIF1 (anti-angiogenic, HHETZ-specific) deserves a developed physiological hypothesis. (4) Practical implications for genomic selection indices or marker-assisted selection programs are vague; this should be made concrete. (5) The sentence “TNFAIP3 (TNIP3 interacting protein 3) is involved in signal transduction and immunoregulatory” is grammatically incomplete and contains a nomenclature error: TNIP3 is TNFAIP3-interacting protein 3, not TNFAIP3.

Conclusions: The Conclusions section is too brief and generic, largely recapitulating the abstract and discussion without adding specificity. It should explicitly state: (a) the most robustly supported candidate genes and their functional significance; (b) the specific arid vs. humid genomic profiles (oxidative stress/protein folding vs. immune response/anti-angiogenesis); (c) the VEGFC vs. WIF1 vascular contrast; (d) concrete recommendations for breeding or conservation application. The study’s status as a reanalysis of public data not original sequencing must be acknowledged as a scope limitation. Future directions should include population-level validation via targeted genotyping in larger cohorts, functional characterization in in vitro/in vivo heat stress models, and broader sampling across Ethiopian altitudinal and rainfall gradients.

Language, Grammar, and Style: The manuscript contains pervasive grammatical errors and stylistic inconsistencies requiring professional English editing before resubmission. Representative corrections: “exhibit remarkable adaptation” → “exhibit remarkable adaptations”; “broadly into African zebu” → “broadly classified into African zebu”; “The HP scan identifies…” → past tense throughout Results; “is involved in signal transduction and immunoregulatory” → incomplete predicate, requires revision. Overuse of formulaic phrases (“plays a crucial role in,” “is involved in,” “has been shown to”) and repetitive sentence structures suggest AI-assisted drafting; the authors should revise for originality. The manuscript should also be checked for self-plagiarism relative to Terefe et al. (2023).

Figures and Tables: Figure 1 (PCA): Font sizes are inconsistent across panels A–C; legend abbreviations (AFT, ASZ, DHETZ, EUT, HHETZ, SUZ) are undefined in the legend. Panel C’s within-Ethiopian PCA must be distinguished from the overall PCA in the legend to explain the different variance percentages. Figure 2 (Admixture): Population labels on the X-axis omit sample sizes; color assignments at each K value are not defined in the legend. Figures 3–6 (Manhattan plots): Threshold lines must be defined with exact values in figure legends, not by cross-reference to Methods. Hp plots (Figures 3B, 4B) use an inverted Y-axis that is not explained; this must be stated explicitly. Tables 3–5: Standardize all chromosomal coordinates to Mb; replace dashes in HP column with “N.D.” (Not Detected); expand column headers “Reg. Start/End” to “Region Start (Mb)/Region End (Mb)”; add footnotes specifying units for each selection statistic.

References: The reference list (109 refs.) is generally appropriate. Issues: (1) Ref. [43] cited for TTLL7 is misassigned (see Results above); (2) ref. [94] for AFAP1L1 (Wang et al. 2018 on lung cancer) lacks direct relevance to heat stress, a more pertinent reference should be sought; (3) ref. [82] (Wolf et al. 2021) carries a “[cited 6 Aug 2025]” note suggesting a preprint or online access publication status should be verified; (4) refs. [107] and [108] both appear to cite the same Chen et al. (2016) paper on WIF1, duplicate should be removed.

Data Availability and Ethics: The EMBRAPA Gir cattle dataset is described as restricted-access, which is potentially incompatible with PLOS ONE’s data sharing requirements. This must be resolved prior to acceptance. The ethics statement (listed as “N/A”) is defensible for secondary reanalysis of public data, but the Methods section must explicitly state that this is a secondary analysis and that original sampling ethics are covered by the primary data sources.

Prioritized Recommendations: Critical (must address): 1. Correct the misrepresentation of publicly available WGS data as originally generated throughout the manuscript. 2. Resolve the EMBRAPA restricted-access data issue per PLOS ONE policy. 3. Report LD pruning parameters, software/version numbers for XP-EHH and XP-CLR, duplication-marking protocol, and VQSR training datasets. 4. Correct coordinate inconsistencies and citation errors in Tables 3, 4, and 5. 5. Explicitly compare findings with Terefe et al. (2023) and clarify incremental contribution. 6. Conduct professional English language editing throughout.

Major (should address): 7. Discuss impact of small sample sizes on selection scan reliability, with a power analysis or relevant citation. 8. Justify the BEAGLE imputation approach and reference panel. 9. Replace DAVID with a current enrichment platform, or explicitly justify its use. 10. Specify the background gene set for GO/KEGG enrichment. 11. Elaborate mechanistically on the VEGFC vs. WIF1 vascular divergence. 12. Strengthen Conclusions with specific findings, limitations, and concrete breeding/conservation recommendations.

Minor (recommended): 13. Revise title to reflect whole-genome resequencing and zebu taxonomy. 14. Update abstract to accurately reflect data sourcing and include quantitative metrics. 15. Standardize units in all tables; define abbreviations in figure legends. 16. Correct grammatical errors and tense inconsistencies throughout. 17. Remove duplicate WIF1 reference and verify Wolf et al. (2021) citation status. 18. Add sample sizes to admixture plot X-axis labels and threshold definitions to Manhattan plot legends.

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Reviewer #1: Yes: Prof. (Dr.) Bharat Bhushan

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Submitted filename: REVIEW REPORT_peer_review_PONE_D_26_06464 (03-04-2026).docx

Whole genomic sequencing reveals divergent and shared selection signatures of heat stress adaptation in indigenous Ethiopian zebu cattle from hot-arid and hot-humid environments.

PONE-D-26-06464R1

Dear Dr. Terefe,

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,

Manasa Varra

Academic Editor

PLOS One

Additional Editor Comments (optional):

Dear Endashaw Terefe,

I would like to inform that the revised manuscript PONE-D-26-06464R1 entitled "Whole genomic sequencing reveals divergent and shared selection signatures of heat stress adaptation in indigenous Ethiopian zebu cattle from hot-arid and hot-humid environments, is hereby recommended for acceptance for publication in PLOS One.

Reviewers' comments: