Evaluation of the Bacterial Diversity in the World’s Deepest Cave—Veryovkina, Arabika Massif, Western Caucasus

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript addresses microbiological diversity in Veryovkina Cave, the world’s deepest known cave system, using 16S rRNA amplicon sequencing. The study is potentially valuable, given the cave’s uniqueness and the scarcity of microbiological data from extreme subterranean environments. However, the current version presents several conceptual, methodological, interpretative, and stylistic weaknesses that limit its clarity, rigor, and overall scientific contribution. Substantial revisions are needed before the manuscript can be considered further.

-The Introduction provides an informative but overly descriptive compilation of cave microbiology background. Yet, it does not sufficiently articulate why studying depth as an ecological driver is scientifically relevant, nor does it clearly express the specific research goals and hypotheses. Lines 54–64, in particular, lack clarity and use narrative rather than scientific language. The manuscript would benefit from explicit research questions and well-defined hypotheses, ideally placed after line 74, that connect existing cave microbiome literature with what is novel about the Veryovkina Cave case study. Without clearly defined expectations, the Discussion later feels disconnected from the aims.
-Regarding sampling design and methodology, the manuscript does not provide adequate justification for the limited number of samples or explain how these locations were selected relative to substrate, moisture, depth, or human access. Lines 91–99 describe the sampling process but do not discuss whether single samples per site are representative, nor do they mention spatial replicates, which are particularly important in such heterogeneous environments. The authors should acknowledge the limitations imposed by low replication, low biomass, and the inability to perform robust statistical testing, especially since three of the ten samples were sterile. Furthermore, although a negative control is mentioned (lines 128 and 135), the manuscript lacks critical information about contamination control strategies, which are essential in low-biomass cave microbiology studies. Details such as field blanks, reagent controls, or post-sequencing decontamination procedures (e.g., Decontam or similar) are not provided, raising concerns about the reliability of results.
-A recurrent concern throughout the Results and Discussion sections is the tendency to over-interpret descriptive patterns without adequate statistical support. In lines 280–301, the manuscript correctly reports that the Kruskal–Wallis and PERMANOVA tests showed no significant differences among depth, substrate, moisture, or human access. Yet, both sections go on to draw strong ecological conclusions about “functional redundancy,” “balanced communities,” and “nutrient cycling hotspots.” These interpretations must be toned down or properly substantiated with more robust analyses. Presently, statements such as “substrate and moisture exerted stronger influence than depth” (lines 27–29) are not analytically supported.
-The Discussion often uses subjective or metaphorical wording without quantitative or functional validation. The manuscript would benefit from incorporating more precise ecological terminology supported by diversity metrics (e.g., Shannon index, Pielou’s evenness, dominance), or by complementing the composition-based analysis with predictive functional profiling tools, if appropriate. Additionally, although interesting, the discussion of flooding's influence on microbial composition remains largely speculative. It should be clearly identified as a hypothesis rather than interpreted as a plausible mechanism without direct evidence.
-Stylistically, the manuscript shows recurrent issues with inconsistent nomenclature, particularly concerning taxonomic formatting. Scientific bacterial names must follow established conventions: phyla and classes are not italicized but capitalized, genera and species are italicized, and ecological guilds are explained when used.
-Finally, the manuscript would benefit from improvements in figure clarity, particularly Figure 2. The multi-panel figure is overcrowded, and the legends tend to narrate rather than explain, making interpretation difficult. Some panels (e.g., family- and genus-level distributions) could be moved to the supplementary material, as they contain fragmented and noisy data, as the authors acknowledge in lines 251–260. The argument that the class-level resolution offers the optimal balance between interpretability and ecological relevance is interesting, but it should be supported by citations and be more convincingly integrated.

Author Response

We thank the reviewer for the thorough and constructive evaluation. All comments were accepted and fully addressed, resulting in substantial improvements in clarity, methodological transparency, interpretative caution, and overall scientific rigor of the revised manuscript.

Comments 1: Overall conceptual clarity and contribution

Response 2: We agree with this comment. The Introduction has been substantially revised to explicitly articulate the scientific relevance of depth as an ecological gradient and to clearly state the study objectives and hypotheses. Explicit research questions and hypotheses were added after the background section, linking prior cave microbiome studies to the novelty of the Veryovkina Cave system (Introduction, page 3, paragraph 4, lines 86–112).

Comments 2: Sampling design, replication, and contamination control

Response 2: The limitations imposed by low sample number, lack of spatial replication, and low biomass typical for deep caves are now explicitly acknowledged (Materials and Methods, page 6, paragraph 1; Discussion, page 17, paragraph 2). The rationale for site selection based on depth, substrate, moisture, and accessibility has been clarified (Materials and Methods, Section 2.1). Negative controls are now described in detail, including reagent-only controls and subtraction procedures, and the constraints of low-biomass contamination control are explicitly discussed (Sections 2.2–2.3).

Comments 3: Over-interpretation without statistical support

Response 3: All statements implying causality or strong ecological effects unsupported by statistics were toned down or rephrased as descriptive patterns or hypotheses. Interpretations such as “functional redundancy,” “hotspots,” and claims about depth effects were revised to explicitly reflect the non-significant Kruskal–Wallis and PERMANOVA results

Comments 4: Subjective language and speculative mechanisms

Response 4: We agree. Subjective and metaphorical language has been removed throughout the Results and Discussion. Ecological interpretations are now supported by diversity metrics and framed descriptively. The role of flooding is now explicitly presented as a testable hypothesis rather than an inferred mechanism, with clear statements about the lack of direct evidence (Discussion, page 18, paragraph 3).

Comment 5. Taxonomic nomenclature and formatting

Response 5: We agree and have corrected all taxonomic nomenclature to comply with accepted conventions. Phyla and classes are capitalized and non-italicized, while family, genera and species are italicized throughout the manuscript.

Comment 6. Figures and data presentation

Response 6. Figure 2 was simplified and clarified, and family- and genus-level distributions were moved to the Supplementary Materials (Figures S1). Figure legends were rewritten to be explanatory rather than narrative. The rationale for focusing on class-level resolution is now explicitly justified and supported by literature (Results, page 13; Discussion, page 16).

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have designed and carried out a valuable study characterizing the microbial population and identifying the dominant communities in the world’s deepest Veryovkina Cave. I have some minor comments and suggestions (see below for specific comments) before the manuscript can be considered further for publication.

Specific Comments and Suggestions for the authors

Line 80: Could you revise this sentence and include “moisture content” also?

Line 82: Please include the full forms of “PCoA and PERMANOVA”.

Line 92: The title of this section is “Sample Collection” therefore you may move this sentence to the later section (Section 2.2). Please include the full form of “NGS”.

Lines 94-95: You may move this sentence also to Section 2.2. Please provide quantitative information about DNA (e.g. concentration).

Line 98: Three different substrate types (clay, sand, and silt) were mentioned in this section. However, silt was discussed infrequently in the results and discussion parts of the manuscript. Please incorporate a discussion of this substrate type into the relevant sections (results or discussion) as appropriate.

Line 101: Could you provide specific reasons for using 70% ethanol?

Lines 106-107: Please include the figure citation specifically related to the text “yielded no microbial reads”.

Lines 110-115: Please move this paragraph to the results section.

Lines 118-125: Please provide citations.

Line 128: Could you elaborate on the “negative-reactive control”?

Line 137: Include the full form of “OTU”.

Lines 147-152: Please provide citations.

Lines 158-161: Could you include possible reasons for this difference between sites? Provide Figure citations at the end of sentences to guide the reader.

Line 160: In Figure 1 caption, you mentioned that A005, A007, A010 sites were sterile. Please verify.

Line 169: Please make sure the site numbers (e.g. A004 and A009) are consistent throughout the manuscript.

Lines 192-216: Please revise this paragraph and include figure citations to guide the reader.

Lines 241-243: Provide citations.

Lines 265-276: Include figure citation to guide the reader.

Lines 319-331: Provide citations.

Figures

Figure 1: The figure caption included clay, sand and silt substrates however the silt legend was not included in the figure.

Figure 2A: Include a description of the red line and the depth in the figure caption.

Figures 2B and 2C: Which site/sample numbers do these figures represent?

Ensure all figures are cited appropriately in the text.

Author Response

We thank the Reviewer for these helpful minor suggestions, all of which were addressed in the revised manuscript.

Reviewer 2 – Minor comments

Line 80
Response. We agree and revised the sentence to explicitly include moisture content (Introduction, page 3, line 80).

Line 82
Response. We agree and added the full forms of Principal Coordinate Analysis (PCoA) and Permutational Multivariate Analysis of Variance (PERMANOVA) at first mention (Introduction, page 3, line 82).

Line 92
Response. We agree. The sentence was moved to Section 2.2, and the full form of Next-Generation Sequencing (NGS) was added (Materials and Methods, page 5).

Lines 94–95
Response. We agree. This information was moved to Section 2.2 and quantitative DNA concentration data were added (page 6).

Line 98
Response. We agree. The role of silt as a substrate was clarified and explicitly addressed where relevant in the Results and Discussion, noting its limited representation by a single site (A010) (Results, page 10; Discussion, page 18).

Line 101
Response. We agree. A justification for using 70% ethanol for sample preservation was added with appropriate citation (Materials and Methods, page 5).

Lines 106–107
Response. We agree and added explicit figure citations related to samples yielding no microbial reads (Materials and Methods, page 6).

Lines 110–115
Response. We agree. This paragraph was moved to the Results section (Results, page 9).

Lines 118–125
Response. We agree and added appropriate citations (Materials and Methods, page 6).

Line 128
Response. We agree and expanded the description of the negative-reactive control, including its purpose and processing (Section 2.2).

Line 137
Response. We agree and included the full form Operational Taxonomic Unit (OTU) at first use (Section 2.3).

Lines 147–152
Response. We agree and added the relevant citations (Section 2.4).

Lines 158–161
Response. We agree. Possible ecological explanations were added and figure citations included to guide the reader (Results, page 11).

Line 160
Response. We agree and verified that A005, A007, and A010 were consistently described as sterile throughout the manuscript and figure captions.

Line 169
Response. We agree and corrected site numbering to ensure consistency across the manuscript.

Lines 192–216
Response. We agree. The paragraph was revised for clarity and appropriate figure citations were added (Results, page 13).

Lines 241–243
Response. We agree and added supporting citations (Discussion, page 16).

Lines 265–276
Response. We agree and added figure citations to guide the reader (Discussion, page 17).

Lines 319–331
Response. We agree and included appropriate references (Discussion, page 19).

Figures

Figure 1
Response. We agree. The silt substrate was included in the sand legend in the figure to match the caption.

Figure 2A
Response. We agree. The red line and corresponding depth information are now explained in the figure caption.

Figures 2B and 2C
Response. We agree. The captions were revised to clarify that these panels represent summed data across all samples.

All figures
Response. We agree and have verified that all figures are now properly cited in the main text.