Characterization of Bacterial Communities in Volcanic Soil from Northern Patagonian Area of Chile

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article presents comprehensive analysis of microbial populations of volcanic soils. The work includes analysis of microbial compositions and soil samples, as well as analysis of the factors affecting microbial communities.

In the same time, the main conclusion is doubtful.

Authors postulated that the results provide clear evidence that anthropogenic factors are key drivers shaping bacterial community structure. Despite this, the soils, which were analyzed, differed significantly in the composition and vegetation. These factors can be of natural origin and their relationships with human impact is not clear. Therefore, the main conclusions of the work should be revised.

Questions and recommendations:

1. Line 26. 41,1°S; 72W à Check coordinate spelling.
2. Lines 34-35 [hereinafter]. Italicize taxon names.
3. Line 110. A total of 18 soil samples – 17 samples are listed in Table S1 and Figure S2.
4. The H soil samples ….. characterized by esplanades of volcanic soil without vegetation. The NI soil was sampled from areas with the presence of vegetation.

According to the description of the goal of the study, the main difference between the sites should be the difference in human impact.

In the same time, according to the description of the sites, they significantly differed in the vegetation. Also, significant differences were observed in organic matter, N, C, and trace element content.

Thus, the difference in soil composition and vegetation can be the main reason of the main difference between microbial communities. These differences seem to be caused by natural differences between soils.

5. Line 230 Candidatus udaeobacter à “Candidatus Udaeobacter”
6. Line 341. Table S5 – supplementary materials contain the file “Figure S5”. Check file names and numbers.
7. Line 348. The bacterial communities from the Osorno volcano were predominantly enriched in pathways… Common pathways, which are pivotal for all microbial communities are listed. Could you describe crucial specific pathways determined in the studied sites? For example, carbon and nitrogen fixation, primary production, etc.

Author Response

Comment 1: The article presents comprehensive analysis of microbial populations of volcanic soils. The work includes analysis of microbial compositions and soil samples, as well as analysis of the factors affecting microbial communities. In the same time, the main conclusion is doubtful. Comment 2: According to the description of the goal of the study, the main difference between the sites should be the difference in human impact. At the same time, according to the description of the sites, they significantly differed in the vegetation. Also, significant differences were observed in organic matter, N, C, and trace element content. Thus, the difference in soil composition and vegetation can be the main reason for the main difference between microbial communities. These differences seem to be caused by natural differences between soils.

Response to Reviewer:

We thank the reviewer for this valuable comment and fully agree that natural factors such as soil composition and vegetation cover can also strongly influence microbial community structure. To address this point, we have revised the text and adjusted the main conclusions to clarify that, although marked differences were observed between humanized and non-intervened soils, these differences cannot be attributed solely to anthropogenic activities. In the revised version, we now specify that the observed patterns are consistent with the combined influence of natural environmental factors (e.g., vegetation and soil composition) and anthropogenic pressures (e.g., tourism, surface compaction, and waste deposition). Accordingly, we have replaced absolute statements such as “clear evidence that anthropogenic factors are key drivers” with a more nuanced formulation: “In this study, we identified the structure of bacterial communities in volcanic soils in northern Chilean Patagonia. Overall, the results of the study revealed significant differences in alpha and beta diversity indices between areas with and without anthropogenic activities. Specifically, four phyla and 105 families exhibited substantial shifts in their relative abundance between the two regions, indicating that members of these taxa may exhibit particular associations with the abiotic (physicochemical soil composition) and biotic (vegetation composition) conditions associated with increased anthropogenic activity. However, it must be recognized that changes in abiotic or biotic factors could also be natural in origin, thus obscuring the impact of anthropogenic activity. Additional field studies are necessary to differentiate between these possibilities”(Lines 625-635).

 

Comment 3: Questions and recommendations.

1. Line 26. 41,1°S; 72W à Check coordinate spelling.
2. Lines 34-35 [hereinafter]. Italicize taxon names.

Response: We thank the reviewer for these valuable corrections. All suggestions were duly corrected in the text.

Comment 4: Line 110. A total of 18 soil samples – 17 samples are listed in Table S1 and Figure S2.

Response: We thank the reviewer for these valuable corrections. We corrected Line 110. A total of 18 soil samples – 17 samples are listed in Table S1 and Figure S2. When performing a preliminary cluster analysis of samples (data not shown), sample NH1.3 from site NH1 clustered with samples from site NH3. To avoid further problems, we decided to discard this sample. Therefore, site NH1 was analyzed with only two sample replicates. This was added in lines 245-248.

Comment 5: The H soil samples ….. characterized by esplanades of volcanic soil without vegetation. The NI soil was sampled from areas with the presence of vegetation.

Response: We thank the reviewer for these valuable corrections The paragraph was reformulated (Lines 123-130): “A total of 18 soil samples were collected in January 2022 from six distinct sites on Osorno Volcano, categorized into two main groups based on presumed human impact: three Humanized (H) sites near tourist facilities and three Non-Intervention (NI) sites with restricted access. While the study was designed to assess the effects of human activity, the H and NI areas exhibited significant inherent natural differences, particularly in vegetation cover (largely absent in H) and underlying soil composition (e.g., organic matter, N, C, and trace elements) (Figure S1, Table S1)”.

Comment 6:

Line 230 Candidatus udaeobacter à “Candidatus Udaeobacter”

Line 341. Table S5 – supplementary materials contain the file “Figure S5”. Check file names and numbers.

Response: We thank the reviewer for these valuable corrections. All suggestions were duly corrected in the text and supplementary materials.

Comment 7:

Line 348. The bacterial communities from the Osorno volcano were predominantly enriched in pathways… Common pathways, which are pivotal for all microbial communities are listed. Could you describe crucial specific pathways determined in the studied sites? For example, carbon and nitrogen fixation, primary production, etc.

Response: We thank the reviewer for this constructive suggestion. In the revised manuscript we now explicitly highlight ecologically relevant pathways inferred by PICRUSt2 and report site-specific differences between non-intervened (NI) and humanized (H) soils. These additions are reported between Lines 379–391 and lines 401-415 in the Results section and Lines 577–598 in the Discussion; KO-level statistics are provided in Table S6 (with pathway summaries in Figure S3).

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The study is well conducted with an appropriate experimental approach, and the results are stated clearly and are relevant to the scientific community. I suggest a few edits to improve manuscript quality:

1. The introduction could benefit from a clear objective being stated, and the environmental relevance of a comparative study on bacterial communities in a site with and without human intervention.
2. Please clarify in the methodology what type of human intervention characterizes the H sites and what the authors are considering as human settlements.
3. I suggest adding a map with the geographical locations of H and NI sampling points. Readers may not be familiar with the area, and a map could help guide that aspect. Also, it will illustrate the distance between the sampled sites, giving a better idea of whether the differences found have any interference from the spatial variation.
4. Add references for the primers used. Add details about the sequencing.
5. Figure 6: I think the CCA should not show the taxa information, and the sampling points should be presented separately, plotting the triplicates separately and not as only one point.

Author Response

Comments and Suggestions for Authors

The study is well conducted with an appropriate experimental approach, and the results are stated clearly and are relevant to the scientific community. I suggest a few edits to improve manuscript quality:

Comment 1: The introduction could benefit from a clear objective being stated, and the environmental relevance of a comparative study on bacterial communities in a site with and without human intervention.

Response: We sincerely thank the reviewer for this valuable suggestion. Accordingly, the introduction was revised to include a clear statement of the study objective and to emphasize its environmental relevance. The main goal of this study is now explicitly presented as evaluating how anthropogenic disturbance influences the composition, diversity, and interaction networks of bacterial communities in volcanic soils. In the revised version, we now specify in the introduction the following text:

“This comparative approach between humanized and non-intervened sites highlights the ecological significance of understanding how tourism and other human activities may affect microbial diversity and, consequently, the resilience and functioning of volcanic ecosystems. In addition, other comparative studies on soil bacterial communities in a site with and without human intervention, such as agricultural practices, have shown significant and complex changes in soil bacterial communities (Fang et al., 2022; Hua et al., 2024). A common effect is the reduction in bacterial diversity (alpha index), often linked to factors like pesticide application or soil management. However, frequent anthropogenic disturbance can, paradoxically, increase total bacterial abundance. Crucially, this intervention alters the community composition, changing the relative abundance of dominant phyla and potentially enriching pathogens, reflecting how land use directly modifies nutrient levels and soil structure”(lines 102-113).

 

Comment 2: Please clarify in the methodology what type of human intervention characterizes the H sites and what the authors are considering as human settlements.

Response: We appreciate the reviewer’s insightful comment. We have now clarified in the Materials and Methods section the specific characteristics of the human intervention (H) sites. “These sites correspond to areas near tourist facilities and parking zones located at the base of the Osorno Volcano, which experience frequent human activity, vehicle movement, and soil compaction, but lack vegetation cover. In contrast, the non-intervened (NI) sites were selected from restricted-access areas within the national park, covered by native vegetation and showing no visible signs of anthropogenic disturbance”(lines 130-134). This clarification has been incorporated to better define what is meant by “human settlements” and the type of human intervention considered in this study.

Comment 3: I suggest adding a map with the geographical locations of H and NI sampling points. Readers may not be familiar with the area, and a map could help guide that aspect. Also, it will illustrate the distance between the sampled sites, giving a better idea of whether the differences found have any interference from the spatial variation.

Response. According to your suggestions a map with the graphical location H and Ni samples pools was added as a supplementary figure (Figure S1).

Comment 4: Add references for the primers used. Add details about the sequencing.

Response: We sincerely thank the reviewer for this valuable suggestion. We added the primers reference in Lines 165-168. “To amplify DNA, the primers 341F (5’-CCTAVGGGRBCCASCAG-3’) and 806R (5’-GGACTACNNGGGTATCTAAT-3’) were used, and 16S rRNA gene sequencing was done using Illumina PE250 at Novogene, UK (Dawson et al., 2025)”.

Comment 5: Figure 6: I think the CCA should not show the taxa information, and the sampling points should be presented separately, plotting the triplicates separately and not as only one point.

Response: We appreciate the reviewer’s thoughtful observation regarding the ordination diagram and sampling representation. We respectfully clarify that each point in the CCA corresponds to a composite soil sample, obtained by homogenizing three spatially close soil replicates within each site. This approach was chosen to minimize microscale heterogeneity and to represent the physicochemical and microbial characteristics of each site more robustly. Therefore, plotting triplicates separately would not be statistically appropriate in this case, since the heavy metal concentrations and microbial data correspond to a single integrated composite sample per site rather than three independent measurements. We also consider it relevant to retain the taxa information in the ordination diagram, as the objective of this analysis was to explore the relationship between key bacterial families and specific heavy metals. Including these associations provides valuable ecological insight, especially given that volcanic soils naturally contain various metals that influence microbial community structure. For example, in our dataset, manganese (Mn) is closely related to the taxon P2-11E, while chromium (Cr) is associated with 0319-7L14. These relationships reveal how particular bacterial lineages may adapt to or be influenced by metal availability in volcanic environments. Given that scope of this study focus on the Bacterial Communities in Volcanic Soil, we believe that preserving this taxon–metal interaction information significantly enhances the environmental relevance and interpretative depth of the CCA.

 

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors have improved the article. It may be accepted.