General Response to the Reviewers

Dear Associate Editor and Referees,

We sincerely thank the Associate Editor Dr. Pierre Amato, and the three anonymous referees for their careful evaluation of our manuscript and for their detailed and constructive comments. The reviews identified a common and fundamental concern regarding the limited, unbalanced, and temporally heterogeneous sampling design. We agree that the original manuscript overstated the inferential capacity of the dataset, particularly regarding comparisons among lakes and interpretations involving preservation status, anthropogenic pressure, lake size, spatial heterogeneity, vertical stratification, temporal variation, and predicted functional differences.

Obtaining additional contemporaneous biological replicates was not feasible during the present revision. We therefore did not attempt to defend the original statistical framework or present the six samples as representative biological replicates of the four lakes. Instead, we substantially revised the conceptual framing, analyses, figures, Results, Discussion, and Conclusions, recasting the study as an exploratory and descriptive baseline characterization of bacterial communities detected in six water samples from four Colombian Andean lakes.

Under this revised framework, we made the following major changes:

1. The individual water sample was redefined as the observational unit, rather than the lake, preservation category, or anthropogenic-pressure category.
2. Unsupported inferential analyses were removed, including lake-level and group-level statistical comparisons, Spearman correlations, co-occurrence network analyses, differential-abundance testing of predicted functions, and other analyses requiring biological replication.
3. The former temporal-comparison subsection for Fúquene and Tota was removed. Samples collected on different dates or in different years are no longer treated as temporal replicates, and no claims are made regarding seasonal change, temporal stability, or interannual variation.
4. Only descriptive analyses supported by the dataset were retained, including sequencing summaries, sample-level coverage-standardized diversity estimates, taxonomic composition, relative-abundance patterns, Bray–Curtis dissimilarities, and ordination plots presented without inferential testing or causal interpretation.
5. Statements attributing the observed patterns to preservation status, anthropogenic pressure, nutrient availability, eutrophication, seasonality, lake size, spatial heterogeneity, water-column depth, or other environmental drivers were removed or substantially weakened. These variables are now discussed only as mechanisms reported in the broader freshwater literature and as hypotheses requiring future replicated investigation.
6. The absence of concurrent physicochemical measurements is now stated explicitly. No environmental–microbiological correlation analysis was conducted because nutrient concentrations, dissolved oxygen, chlorophyll-a, pH, temperature, conductivity, organic-matter indicators, and related variables were not measured synchronously with microbiological sampling. We expanded the description of the sampling design and included all recoverable sample-specific metadata, including collection date, location, lake zone, and sequencing information. Where broader lake-level environmental information from published or institutional sources is included, it is clearly identified as contextual, non-contemporaneous information and is not used for statistical association, causal attribution, or reconstruction of conditions at the time of sampling.
7. PICRUSt2 results were reframed as descriptive predictions of potential genomic functions. Differential-abundance testing and preservation-status comparisons were removed. The revised analysis presents the most abundant predicted pathway categories, a descriptive Bray–Curtis ordination of predicted functional profiles, and sample-level weighted NSTI values. These outputs are explicitly described as predictions based on ASV phylogenetic placement and reference genomes, rather than direct evidence of gene presence, expression, metabolic activity, pathogenicity, or ecosystem processes.

The Mycobacterium analysis was substantially reduced and reframed. It is now presented only as an exploratory phylogenetic placement of genus-assigned ASVs, motivated by the occurrence of water-associated nontuberculous mycobacteria. The limited species-level resolution of the V3–V4 16S rRNA gene region is explicitly acknowledged, and no definitive species identifications, pathogenicity claims, or ecological interactions are inferred. The maximum-likelihood tree was moved to the Supplement.

The manuscript was comprehensively rewritten and reorganized. The revised title is: “Exploratory characterization of bacterial communities and predicted functional profiles in six water samples from four Colombian Andean lakes using 16S rRNA gene amplicon sequencing.”

The revised supplementary material was deposited in Zenodo and assigned a new DOI (https://doi.org/10.5281/zenodo.21647587). The manuscript’s Data Availability statement, supplementary-material references, and associated links were updated accordingly to ensure public access to the revised supporting data and files.

The Abstract, objectives, Methods, Results, Discussion, Conclusions, figures, supplementary material, terminology, and taxonomic formatting were revised so that the scope and claims are proportional to the available evidence. The revised supplementary files were deposited in Zenodo under a new DOI, and all corresponding references and data-access information in the manuscript were updated. The revised title explicitly identifies the exploratory character of the study, the number of samples and lakes represented, and the 16S rRNA gene amplicon sequencing approach.

After reviewing the manuscript categories offered by Biogeosciences, we did not reclassify the manuscript as a BG Letter merely to accommodate its reduced scope, because that category is intended for particularly important results and major advances. Subject to the Associate Editor’s assessment, we have retained the manuscript as a shorter and substantially revised Research Article focused on its value as a preliminary baseline survey of poorly characterized Colombian Andean lake bacterial communities.

We recognize that this general response does not replace the required individual replies. Accordingly, we provide separate point-by-point responses to every referee comment and indicate how each concern was addressed in the revised manuscript.

We greatly appreciate the reviewers’ recognition that the dataset may retain value as an initial characterization of microbial communities in understudied Andean lakes. We believe that the extensive restructuring has removed unsupported statistical and ecological claims, clarified the distinction between measured results and contextual hypotheses, and produced a more rigorous, transparent, and appropriately cautious manuscript.

Sincerely,

Andrés Gómez-Palacio

on behalf of all authors

General Response to the Reviewers

Dear Associate Editor and Referees,

We sincerely thank the Associate Editor Dr. Pierre Amato, and the three anonymous referees for their careful evaluation of our manuscript and for their detailed and constructive comments. The reviews identified a common and fundamental concern regarding the limited, unbalanced, and temporally heterogeneous sampling design. We agree that the original manuscript overstated the inferential capacity of the dataset, particularly regarding comparisons among lakes and interpretations involving preservation status, anthropogenic pressure, lake size, spatial heterogeneity, vertical stratification, temporal variation, and predicted functional differences.

Obtaining additional contemporaneous biological replicates was not feasible during the present revision. We therefore did not attempt to defend the original statistical framework or present the six samples as representative biological replicates of the four lakes. Instead, we substantially revised the conceptual framing, analyses, figures, Results, Discussion, and Conclusions, recasting the study as an exploratory and descriptive baseline characterization of bacterial communities detected in six water samples from four Colombian Andean lakes.

Under this revised framework, we made the following major changes:

1. The individual water sample was redefined as the observational unit, rather than the lake, preservation category, or anthropogenic-pressure category.
2. Unsupported inferential analyses were removed, including lake-level and group-level statistical comparisons, Spearman correlations, co-occurrence network analyses, differential-abundance testing of predicted functions, and other analyses requiring biological replication.
3. The former temporal-comparison subsection for Fúquene and Tota was removed. Samples collected on different dates or in different years are no longer treated as temporal replicates, and no claims are made regarding seasonal change, temporal stability, or interannual variation.
4. Only descriptive analyses supported by the dataset were retained, including sequencing summaries, sample-level coverage-standardized diversity estimates, taxonomic composition, relative-abundance patterns, Bray–Curtis dissimilarities, and ordination plots presented without inferential testing or causal interpretation.
5. Statements attributing the observed patterns to preservation status, anthropogenic pressure, nutrient availability, eutrophication, seasonality, lake size, spatial heterogeneity, water-column depth, or other environmental drivers were removed or substantially weakened. These variables are now discussed only as mechanisms reported in the broader freshwater literature and as hypotheses requiring future replicated investigation.
6. The absence of concurrent physicochemical measurements is now stated explicitly. No environmental–microbiological correlation analysis was conducted because nutrient concentrations, dissolved oxygen, chlorophyll-a, pH, temperature, conductivity, organic-matter indicators, and related variables were not measured synchronously with microbiological sampling. We expanded the description of the sampling design and included all recoverable sample-specific metadata, including collection date, location, lake zone, and sequencing information. Where broader lake-level environmental information from published or institutional sources is included, it is clearly identified as contextual, non-contemporaneous information and is not used for statistical association, causal attribution, or reconstruction of conditions at the time of sampling.
7. PICRUSt2 results were reframed as descriptive predictions of potential genomic functions. Differential-abundance testing and preservation-status comparisons were removed. The revised analysis presents the most abundant predicted pathway categories, a descriptive Bray–Curtis ordination of predicted functional profiles, and sample-level weighted NSTI values. These outputs are explicitly described as predictions based on ASV phylogenetic placement and reference genomes, rather than direct evidence of gene presence, expression, metabolic activity, pathogenicity, or ecosystem processes.

The Mycobacterium analysis was substantially reduced and reframed. It is now presented only as an exploratory phylogenetic placement of genus-assigned ASVs, motivated by the occurrence of water-associated nontuberculous mycobacteria. The limited species-level resolution of the V3–V4 16S rRNA gene region is explicitly acknowledged, and no definitive species identifications, pathogenicity claims, or ecological interactions are inferred. The maximum-likelihood tree was moved to the Supplement.

The manuscript was comprehensively rewritten and reorganized. The revised title is: “Exploratory characterization of bacterial communities and predicted functional profiles in six water samples from four Colombian Andean lakes using 16S rRNA gene amplicon sequencing.”

The revised supplementary material was deposited in Zenodo and assigned a new DOI (https://doi.org/10.5281/zenodo.21647587). The manuscript’s Data Availability statement, supplementary-material references, and associated links were updated accordingly to ensure public access to the revised supporting data and files.

The Abstract, objectives, Methods, Results, Discussion, Conclusions, figures, supplementary material, terminology, and taxonomic formatting were revised so that the scope and claims are proportional to the available evidence. The revised supplementary files were deposited in Zenodo under a new DOI, and all corresponding references and data-access information in the manuscript were updated. The revised title explicitly identifies the exploratory character of the study, the number of samples and lakes represented, and the 16S rRNA gene amplicon sequencing approach.

After reviewing the manuscript categories offered by Biogeosciences, we did not reclassify the manuscript as a BG Letter merely to accommodate its reduced scope, because that category is intended for particularly important results and major advances. Subject to the Associate Editor’s assessment, we have retained the manuscript as a shorter and substantially revised Research Article focused on its value as a preliminary baseline survey of poorly characterized Colombian Andean lake bacterial communities.

We recognize that this general response does not replace the required individual replies. Accordingly, we provide separate point-by-point responses to every referee comment and indicate how each concern was addressed in the revised manuscript.

We greatly appreciate the reviewers’ recognition that the dataset may retain value as an initial characterization of microbial communities in understudied Andean lakes. We believe that the extensive restructuring has removed unsupported statistical and ecological claims, clarified the distinction between measured results and contextual hypotheses, and produced a more rigorous, transparent, and appropriately cautious manuscript.

Sincerely,

Andrés Gómez-Palacio

on behalf of all authors

General Response to the Reviewers

Dear Associate Editor and Referees,

We sincerely thank the Associate Editor Dr. Pierre Amato, and the three anonymous referees for their careful evaluation of our manuscript and for their detailed and constructive comments. The reviews identified a common and fundamental concern regarding the limited, unbalanced, and temporally heterogeneous sampling design. We agree that the original manuscript overstated the inferential capacity of the dataset, particularly regarding comparisons among lakes and interpretations involving preservation status, anthropogenic pressure, lake size, spatial heterogeneity, vertical stratification, temporal variation, and predicted functional differences.

Obtaining additional contemporaneous biological replicates was not feasible during the present revision. We therefore did not attempt to defend the original statistical framework or present the six samples as representative biological replicates of the four lakes. Instead, we substantially revised the conceptual framing, analyses, figures, Results, Discussion, and Conclusions, recasting the study as an exploratory and descriptive baseline characterization of bacterial communities detected in six water samples from four Colombian Andean lakes.

Under this revised framework, we made the following major changes:

1. The individual water sample was redefined as the observational unit, rather than the lake, preservation category, or anthropogenic-pressure category.
2. Unsupported inferential analyses were removed, including lake-level and group-level statistical comparisons, Spearman correlations, co-occurrence network analyses, differential-abundance testing of predicted functions, and other analyses requiring biological replication.
3. The former temporal-comparison subsection for Fúquene and Tota was removed. Samples collected on different dates or in different years are no longer treated as temporal replicates, and no claims are made regarding seasonal change, temporal stability, or interannual variation.
4. Only descriptive analyses supported by the dataset were retained, including sequencing summaries, sample-level coverage-standardized diversity estimates, taxonomic composition, relative-abundance patterns, Bray–Curtis dissimilarities, and ordination plots presented without inferential testing or causal interpretation.
5. Statements attributing the observed patterns to preservation status, anthropogenic pressure, nutrient availability, eutrophication, seasonality, lake size, spatial heterogeneity, water-column depth, or other environmental drivers were removed or substantially weakened. These variables are now discussed only as mechanisms reported in the broader freshwater literature and as hypotheses requiring future replicated investigation.
6. The absence of concurrent physicochemical measurements is now stated explicitly. No environmental–microbiological correlation analysis was conducted because nutrient concentrations, dissolved oxygen, chlorophyll-a, pH, temperature, conductivity, organic-matter indicators, and related variables were not measured synchronously with microbiological sampling. We expanded the description of the sampling design and included all recoverable sample-specific metadata, including collection date, location, lake zone, and sequencing information. Where broader lake-level environmental information from published or institutional sources is included, it is clearly identified as contextual, non-contemporaneous information and is not used for statistical association, causal attribution, or reconstruction of conditions at the time of sampling.
7. PICRUSt2 results were reframed as descriptive predictions of potential genomic functions. Differential-abundance testing and preservation-status comparisons were removed. The revised analysis presents the most abundant predicted pathway categories, a descriptive Bray–Curtis ordination of predicted functional profiles, and sample-level weighted NSTI values. These outputs are explicitly described as predictions based on ASV phylogenetic placement and reference genomes, rather than direct evidence of gene presence, expression, metabolic activity, pathogenicity, or ecosystem processes.

The Mycobacterium analysis was substantially reduced and reframed. It is now presented only as an exploratory phylogenetic placement of genus-assigned ASVs, motivated by the occurrence of water-associated nontuberculous mycobacteria. The limited species-level resolution of the V3–V4 16S rRNA gene region is explicitly acknowledged, and no definitive species identifications, pathogenicity claims, or ecological interactions are inferred. The maximum-likelihood tree was moved to the Supplement.

The manuscript was comprehensively rewritten and reorganized. The revised title is: “Exploratory characterization of bacterial communities and predicted functional profiles in six water samples from four Colombian Andean lakes using 16S rRNA gene amplicon sequencing.”

The revised supplementary material was deposited in Zenodo and assigned a new DOI (https://doi.org/10.5281/zenodo.21647587). The manuscript’s Data Availability statement, supplementary-material references, and associated links were updated accordingly to ensure public access to the revised supporting data and files.

The Abstract, objectives, Methods, Results, Discussion, Conclusions, figures, supplementary material, terminology, and taxonomic formatting were revised so that the scope and claims are proportional to the available evidence. The revised supplementary files were deposited in Zenodo under a new DOI, and all corresponding references and data-access information in the manuscript were updated. The revised title explicitly identifies the exploratory character of the study, the number of samples and lakes represented, and the 16S rRNA gene amplicon sequencing approach.

After reviewing the manuscript categories offered by Biogeosciences, we did not reclassify the manuscript as a BG Letter merely to accommodate its reduced scope, because that category is intended for particularly important results and major advances. Subject to the Associate Editor’s assessment, we have retained the manuscript as a shorter and substantially revised Research Article focused on its value as a preliminary baseline survey of poorly characterized Colombian Andean lake bacterial communities.

We recognize that this general response does not replace the required individual replies. Accordingly, we provide separate point-by-point responses to every referee comment and indicate how each concern was addressed in the revised manuscript.

We greatly appreciate the reviewers’ recognition that the dataset may retain value as an initial characterization of microbial communities in understudied Andean lakes. We believe that the extensive restructuring has removed unsupported statistical and ecological claims, clarified the distinction between measured results and contextual hypotheses, and produced a more rigorous, transparent, and appropriately cautious manuscript.

Sincerely,

Andrés Gómez-Palacio

on behalf of all authors