Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study compares the diversity of cultivable fungi from different substrates and rivers. The study plan is well done, the diversity indices are well compared, and the manuscript is well prepared. Despite this, the manuscript has some writing errors, all of which are mentioned in the attached file, which require review and correction to improve the quality of the manuscript.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The manuscript is well prepared, the quality of English language is good, but some corrections are needed to improve the quality of manuscript. All are mentioned in the attached file.

Author Response

Reviewer 1

Reviewer 1 – Comment 1

Comments:

This study compares the diversity of cultivable fungi from different substrates and rivers. The study plan is well done, the diversity indices are well compared, and the manuscript is well prepared. Despite this, the manuscript has some writing errors, all of which are mentioned in the attached file, which require review and correction to improve the quality of the manuscript.

Author’s response:

We thank the reviewer for the positive assessment and for recognizing the structure and preparation of the manuscript. We carefully reviewed the manuscript and corrected writing errors identified during the revision process. The manuscript also underwent additional stylistic editing to improve clarity and readability.

Reviewer 1 – Comment 2

Comments:

The manuscript is well prepared, the quality of English language is good, but some corrections are needed to improve the quality of manuscript. All are mentioned in the attached file.

Author’s response:

We thank the reviewer for this positive comment. We revised the manuscript for grammar, style, and consistency. Minor language issues, typographical errors, duplicated words, and unclear expressions were corrected throughout the revised version.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript investigates the diversity and distribution of cultivable fungal communities associated with water, sediment, and submerged wood across three major Amazonian river systems representing distinct hydrochemical conditions. By combining culture-based isolation with morphological identification and standard diversity metrics, the study provides useful baseline information on an understudied component of freshwater microbial biodiversity. The work highlights clear differences in fungal richness, abundance, and community composition among substrate types and river systems, emphasizing the influence of both substrate characteristics and environmental conditions in structuring cultivable fungal assemblages. Overall, the study contributes valuable ecological insights and expands our understanding of fungal diversity in tropical freshwater ecosystems.

However, I found that some of the descriptions of the paper were neglected, and the description of some important points was inadequate or completely missing. As a result, I have little confidence in some facts and came away with questions. I cannot recommend this paper for publication as it stands. I explain my concerns in more detail below. I ask that the authors specifically address each of my comments in their responses. If the authors could arrange these responses in an appropriate way, it would provide an invaluable contribution to this timely and needed cost-effective approach.

Major comments:

1. The arrangement is well-structured and effectively supports the manuscript's topic. The explanations are appropriate and relevant to the manuscript title, including the introduction section.

2. Material and Methods: The provided methodologies and explanations align with the scope and aim of the manuscript. However, authors would have been providing each morphological picture plate for further confirmation of the found species in a detailed manner, separately at least for rare species found from the isolates. In addition, I am wondering why authors have not sought further molecular confirmation of species, at least at the genus level, using ITS regions. Moreover, I am suggesting that authors make further confirmation of found species, as the majority of islands are considered as contaminant species, such as Aspergillus, Penicillium, Cladosporium, and Fusarium. Please go with further confirmation of the found species in the correct manner. Avoid summarising the content in the middle of the body as I have noticed it on Page no. 08, lines 265-268. 

3. The data representations are informative and well-presented. However, I found that the data is not arranged correctly. Please arrange the data and readings from the instrument used for the physicochemical characteristics to decimal places, equal to the instrument's error decimal (Table 01).  

4. The highlights, abstract, and conclusion sections are organized well and aligned with the paper's content.

Minor comments:

1. Please carefully review the author guidelines. I have noticed some errors in the reference citations and bibliography list.

2. Images without scale bars, and please give your attention to those. 

Comments on the Quality of English Language

The manuscript is generally well written and the English language is of a good standard. The text is clear, concise, and scientifically appropriate, allowing the reader to follow the objectives, methodology, results, and conclusions without difficulty. Only minor grammatical and stylistic revisions may be considered during the final editing stage to improve readability, but no substantial language revision is required.

Author Response

Reviewer 2

Reviewer 2 – Comment 1 — General positive assessment

Comments:

This manuscript investigates the diversity and distribution of cultivable fungal communities associated with water, sediment, and submerged wood across three major Amazonian river systems representing distinct hydrochemical conditions. By combining culture-based isolation with morphological identification and standard diversity metrics, the study provides useful baseline information on an understudied component of freshwater microbial biodiversity. The work highlights clear differences in fungal richness, abundance, and community composition among substrate types and river systems, emphasizing the influence of both substrate characteristics and environmental conditions in structuring cultivable fungal assemblages. Overall, the study contributes valuable ecological insights and expands our understanding of fungal diversity in tropical freshwater ecosystems.

Author’s response:

We thank the reviewer for the positive evaluation of the study and for recognizing its contribution to the knowledge of fungi recovered from Amazonian freshwater environments. We maintained the core structure of the manuscript while revising the text to make the scope and limitations of the culture-dependent approach clearer.

Reviewer 2 – Comment 2 — General concern about missing or inadequate descriptions

Comments:

However, I found that some of the descriptions of the paper were neglected, and the description of some important points was inadequate or completely missing. As a result, I have little confidence in some facts and came away with questions. I cannot recommend this paper for publication as it stands. I explain my concerns in more detail below. I ask that the authors specifically address each of my comments in their responses. If the authors could arrange these responses in an appropriate way, it would provide an invaluable contribution to this timely and needed cost-effective approach.

Author’s response:

We thank the reviewer for this general comment. We revised the manuscript to provide more complete methodological and interpretative descriptions. In particular, we expanded the sections related to morphological characterization, morphotype definition, treatment of taxa that may also occur as contaminants, limitations of the absence of ITS sequencing, scale bars, decimal formatting of physicochemical data, and interpretation of diversity metrics based on cultivated strains.

Reviewer 2 – Comment 3 — Manuscript arrangement and introduction

Comments:

The arrangement is well-structured and effectively supports the manuscript's topic. The explanations are appropriate and relevant to the manuscript title, including the introduction section.

Author’s response:

We thank the reviewer for this positive assessment. The Introduction was widely maintained because the reviewer considered it appropriate and relevant to the topic. Only minor wording adjustments were made elsewhere in the manuscript to align the overall interpretation with the revised scope of the study.

Reviewer 2 – Comment 4.1 — Morphological picture plates

Comments:

Material and Methods: The provided methodologies and explanations align with the scope and aim of the manuscript. However, authors would have been providing each morphological picture plate for further confirmation of the found species in a detailed manner, separately at least for rare species found from the isolates.

Author’s response:

We agree with the reviewer. We added information indicating that representative images of agar plates used for the isolation of colonies, purified colonies, and micromorphological structures were organized as Supplementary Figure S1. These images include representative morphotypes from the most frequent genera and selected low-frequency morphotypes when diagnostic structures were available. We also revised the wording to avoid referring to these isolates as confirmed species and instead used the terms 'morphotypes' or 'genus-level assignments'.

Reviewer 2 – Comment 4.2 — Molecular confirmation using ITS regions

Comments:

In addition, I am wondering why authors have not sought further molecular confirmation of species, at least at the genus level, using ITS regions.

Author’s response:

We acknowledge this limitation. Molecular confirmation using ITS sequencing was not performed for the full isolate set in the present study. We clarified this explicitly in the methods and discussion and stated that taxonomic assignments should be interpreted conservatively, mainly at the morphotype or genus level. We also added that future studies derived from this collection will include ITS sequencing and additional markers when necessary to resolve species-level diversity.

“Finally, taxonomic assignments were based on macro- and micromorphological traits, without ITS sequencing for the full isolate set. Because phenotypic plasticity, morphological convergence, and cryptic diversity are common among fungi, the results should be interpreted primarily at the morphotype or genus level rather than as definitive species-level identifications. Future studies should combine synchronized seasonal sampling, standardized processing times, ITS sequencing of representative isolates, additional taxonomic markers when necessary, and high-throughput sequencing to validate taxonomic assignments and provide a broader estimate of fungal diversity in these Amazonian river systems.”

 

 

Reviewer 2 – Comment 4.3 — Confirmation of taxa commonly considered contaminants

Comments:

Moreover, I am suggesting that authors make further confirmation of found species, as the majority of islands are considered as contaminant species, such as Aspergillus, Penicillium, Cladosporium, and Fusarium. Please go with further confirmation of the found species in the correct manner.

Author’s response:

We agree that the detection of genera such as Aspergillus, Penicillium, Cladosporium, and Fusarium require careful interpretation, because they are widespread environmental fungi and may also occur as laboratory contaminants. We added a paragraph explaining that these isolates were included only when recovered from processed environmental samples, purified, and supported by reproducible macro- and micromorphological features. We now interpret them conservatively as cultivated environmental morphotypes rather than as definitive species-level records.

Reviewer 2 – Comment 4.4 — Avoid summarizing content in the manuscript body

Comments:

Avoid summarising the content in the middle of the body as I have noticed it on Page no. 08, lines 265-268.

Author’s response:

We agree with the reviewer. The summarizing paragraph at the end of the morphological identification section was removed to avoid a conclusion-like statement within the materials and methods section.

Reviewer 2 – Comment 5 — Decimal places in physicochemical data

Comments:

The data representations are informative and well-presented. However, I found that the data is not arranged correctly. Please arrange the data and readings from the instrument used for the physicochemical characteristics to decimal places, equal to the instrument's error decimal (Table 01).

Author’s response:

We agree. Table 1 was revised so that measured physicochemical data are presented with decimal places consistent with the resolution of the measurements.

Reviewer 2 – Comment 6 — Highlights, abstract, and conclusion

Comments:

The highlights, abstract, and conclusion sections are organized well and aligned with the paper's content.

Author’s response:

We thank the reviewer for this positive comment. The highlights, abstract, and conclusion were retained in structure, but we made cautious wording adjustments to align them with the editor's request. In particular, we avoided broad claims about total fungal diversity and clarified that the results refer to cultivated fungal strains and morphotypes recovered under the conditions used in this study.

Reviewer 2 – Comment 7 — Reference citations and bibliography

Comments:

Please carefully review the author guidelines. I have noticed some errors in the reference citations and bibliography list.

Author’s response:

We agree. We carefully reviewed the references and in-text citations according to the journal guidelines. We corrected formatting inconsistencies, checked citation order and bibliography style, and revised author initials and punctuation where needed.

Reviewer 2 – Comment 8 — Scale bars in images

Comments:

Images without scale bars, and please give your attention to those.

Author’s response:

We agree. We revised the figure legends and supplementary figure descriptions to indicate the presence of scale bars. Micromorphological images in the revised figure set and supplementary material are described with scale bars whenever applicable.

Reviewer 2 – Comment 9 — English language quality

Comments:

The manuscript is generally well written and the English language is of a good standard. The text is clear, concise, and scientifically appropriate, allowing the reader to follow the objectives, methodology, results, and conclusions without difficulty. Only minor grammatical and stylistic revisions may be considered during the final editing stage to improve readability, but no substantial language revision is required.

Author’s response:

We thank the reviewer for the positive assessment of the language quality. We performed an additional round of minor grammatical and stylistic editing to improve readability while preserving the scientific meaning of the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

The article“Substrate and Water Type Shape the Diversity of Cultivable Fungi in Amazonian Black-, White-, and Clear-Water Rivers” has been submitted to the journal of Biology, This study addresses the topic—the diversity of culturable fungi from water, sediment, and submerged wood in three major hydrochemically contrasting Amazonian rivers . The dataset is substantial, and the study provides useful ecological comparisons across substrates and river types. However, several methodological and analytical limitations reduce the robustness and generalizability of the conclusions. Major concerns include inconsistent sampling timing, variable sample processing protocols, reliance on morphological identification alone, and insufficient handling of limitations. So it need major revision.

1. Samples were collected in different months: Negro River in April, Tapajós in May, and Solimões in September 2025. Seasonal differences in temperature, rainfall, hydrology, and organic matter input can strongly influence fungal community structure.The authors should explicitly state this as a critical limitation.  I suggest that the discussion must temper any claims of “river-specific” differences, as these may partly reflect temporal variation.
2. Tapajós samples were transported refrigerated and processed after ~24 hours, whereas Negro and Solimões samples were processed within <2 hours at room temperature. This discrepancy may have reduced recovery of viable propagules from Tapajós, potentially biasing diversity and abundance comparisons.Please discussed this as a limitation.
3. Fungal identification was based solely on morphological traits (macroscopic and microscopic). No molecular confirmation (e.g., ITS sequencing) was performed. Given the high phenotypic plasticity and convergence in fungi, genus-level assignments and morphotype definitions are uncertain. I suggest to provide ITS sequencing for representative isolates of each morphotype to validate identification.
4. Section “4.1” is nested under “2.4”, but later sections is 2.5, I suggest combine the 2.4 section and delete 2.4.1 number.
5. Fig.3 is not clear ,I suggest to give the high absolution picture, there are several Penicillium such as A, E, and F, please give the picture of other genera like Cladosporium or Phoma.
6. The data in the results have great variation in statistical analysis, such as  200±447 in table 5？and there are several the same number, Please explain these data why are the same.
7. The format of references are inconsistant, for example, some authors had “-”, some haven’t, such as H.-P has the “-”,  Please carefully check and revise them.

Author Response

Reviewer 3

Reviewer 3 – Comment 1 — Overall assessment and need for major revision

Comments:

The article“Substrate and Water Type Shape the Diversity of Cultivable Fungi in Amazonian Black-, White-, and Clear-Water Rivers” has been submitted to the journal of Biology, This study addresses the topic—the diversity of culturable fungi from water, sediment, and submerged wood in three major hydrochemically contrasting Amazonian rivers . The dataset is substantial, and the study provides useful ecological comparisons across substrates and river types. However, several methodological and analytical limitations reduce the robustness and generalizability of the conclusions. Major concerns include inconsistent sampling timing, variable sample processing protocols, reliance on morphological identification alone, and insufficient handling of limitations. So it need major revision.

Author’s response:

We thank the reviewer for the careful and constructive assessment. We agree that the original manuscript needed a clearer announcement of methodological limitations. The revised version now explicitly addresses the non-synchronized sampling months, differences in sample transport and processing, the reliance on morphological identification, the absence of ITS sequencing for the full isolate set, and the limits of generalizing from a culture-dependent study. We have also clarified that the study should be interpreted as an exploratory, hypothesis-forming assessment, designed to provide an initial comparative overview of cultivable fungal assemblages across contrasting Amazonian river types and substrates, rather than as a fully replicated seasonal or inferential survey.

Reviewer 3 – Comment 2.1 — Samples collected in different months

Comments:

Samples were collected in different months: Negro River in April, Tapajós in May, and Solimões in September 2025.

Author’s response:

We agree. The manuscript already reported the sampling months, but we revised the interpretation to make clear that the study should be considered as an exploratory cross-sectional comparison rather than a temporally synchronized survey. This clarification was added to the methods and reinforced in the discussion.

Reviewer 3 – Comment 2.2 — Seasonal influence on fungal community structure

Comments:

Seasonal differences in temperature, rainfall, hydrology, and organic matter input can strongly influence fungal community structure.The authors should explicitly state this as a critical limitation.

Author’s response:

We agree. We added an explicit limitation statement explaining that seasonal variation in rainfall, hydrology, water level, temperature, and organic matter input may influence fungal propagule availability, substrate colonization, and recovery of cultivated fungi. We also state that temporal variation cannot be fully separated from river type effects with the chosen study design of this hypothesis-forming analysis.

Reviewer 3 – Comment 2.3 — Tempering claims of “river-specific” differences

Comments:

I suggest that the discussion must temper any claims of “river-specific” differences, as these may partly reflect temporal variation.

Author’s response:

We agree with the reviewer. We weakened claims on river-specific differences throughout the manuscript. Stronger statements were replaced by more cautious wording such as 'apparent differentiation', 'patterns observed among river systems', 'may contribute to', and 'exploratory results'. We also added text explaining that some differences among rivers may partly reflect temporal variation.

Reviewer 3 – Comment 3.1 — Different transport and processing conditions among rivers

Comments:

Tapajós samples were transported refrigerated and processed after ~24 hours, whereas Negro and Solimões samples were processed within <2 hours at room temperature.

Author’s response:

We agree. The revised manuscript more clearly reports the different transport and processing conditions: Tapajós samples were transported under refrigeration conditions and processed after approximately 24 h, whereas Negro and Solimões samples were processed within less than 2 h. We also explain that this difference resulted from logistical constraints associated with sampling distant Amazonian locations and hope that our readers will understand these very specific issues.

Reviewer 3 – Comment 3.2 — Possible bias in recovery of viable propagules from Tapajós

Comments:

This discrepancy may have reduced recovery of viable propagules from Tapajós, potentially biasing diversity and abundance comparisons.Please discussed this as a limitation.

Author’s response:

We agree. We added this issue as a limitation in the discussion. The revised text states that, although refrigeration was used to minimize biological changes during transport, delayed processing may have affected the recovery of viable propagules, particularly for sensitive or slow-growing fungi. Therefore, abundance and richness comparisons involving the Tapajós setting should be interpreted cautiously.

Reviewer 3 – Comment 4.1 — Reliance on macroscopic and microscopic morphology

Comments:

Fungal identification was based solely on morphological traits (macroscopic and microscopic).

Author’s response:

We agree that the identifications were based on macroscopic and microscopic morphology. We clarified this in the methods and discussion sections and emphasized that the purpose of the study was to compare cultivated morphotypes and genus-level assignments, not to provide definitive species-level identification. Representative images supporting the morphological interpretation were included as supplementary material S1.

Reviewer 3 – Comment 4.2 — Uncertainty in genus-level assignments and morphotype definitions

Comments:

No molecular confirmation (e.g., ITS sequencing) was performed. Given the high phenotypic plasticity and convergence in fungi, genus-level assignments and morphotype definitions are uncertain.

Author’s response:

We agree. We added text acknowledging that phenotypic plasticity, morphological convergence, and cryptic diversity are common among filamentous fungi and may limit the resolution of morphology-based assignments. Therefore, the revised manuscript states that taxonomic assignments should be interpreted mainly at the morphotype or genus level.

Reviewer 3 – Comment 4.3 — Suggestion to perform ITS sequencing

Comments:

I suggest to provide ITS sequencing for representative isolates of each morphotype to validate identification.

Author’s response:

We agree that ITS sequencing would strengthen species level identification. However, ITS sequencing of representative isolates from all morphotypes was beyond the scope of the present revision. We clarified this limitation and added that future studies derived from this collection should prioritize ITS sequencing of representative morphotypes, complemented by additional taxonomic markers when necessary.

Reviewer 3 – Comment 5 — Section numbering problem

Comments:

Section “4.1” is nested under “2.4”, but later sections is 2.5, I suggest combine the 2.4 section and delete 2.4.1 number.

Author’s response:

We agree with the reviewer. The subsection numbering was corrected. The previous '2.4.1. Spectrophotometric Characterization of Water Samples' heading was removed, and the spectrophotometric characterization was incorporated directly into section 2.4.

Reviewer 3 – Comment 6.1 — Figure 3 resolution

Comments:

Fig.3 is not clear ,I suggest to give the high absolution picture,

Author’s response:

We agree. Figure 3 was revised for the resubmission package, and a high-resolution version will be provided with the revised manuscript files. The figure legend was also updated to better describe the representative morphotypes and to indicate the scale bars.

Reviewer 3 – Comment 6.2 — Repetition of Penicillium images and need to include other genera

Comments:

there are several Penicillium such as A, E, and F, please give the picture of other genera like Cladosporium or Phoma.

Author’s response:

We agree with the reviewer. The figure 3 legend and composition were revised to reduce repetition of Penicillium and to include other genera such as Cladosporium and Phoma. The revised figure is intended to better represent the morphological diversity of cultivated fungal morphotypes recovered in the study.

Reviewer 3 – Comment 7.1 — High variation in statistical analysis

Comments:

The data in the results have great variation in statistical analysis, such as  200±447 in table 5？

Author’s response:

We thank the reviewer for pointing this out. We added an explanation in the results section clarifying that high standard deviations arise because morphotype abundance values were calculated across five subsamples and many morphotypes occurred in only one or a few subsamples. Zero values from subsamples in which a morphotype was not detected were retained in the calculation.

Reviewer 3 – Comment 7.2 — Repeated identical numbers

Comments:

and there are several the same number, Please explain these data why are the same.

Author’s response:

We agree that repeated values required clarification. We added notes to tables 4-6, explaining that repeated mean ± standard deviation values occur when different morphotypes show the same distribution pattern among the five subsamples after correction for dilution. For example, a value such as 200 ± 447 reflects detection in only one subsample and absence from the remaining subsamples.

Reviewer 3 – Comment 8 — Inconsistent reference format

Comments:

The format of references are inconsistant, for example, some authors had “-”, some haven’t, such as H.-P has the “-”,  Please carefully check and revise them.

Author’s response:

We agree. We carefully checked and revised the reference list for formatting consistency according to the journal style. Particular attention was given to author initials, including the use of hyphens in compound initials such as H.-P. when appropriate, as well as punctuation, journal abbreviations, and DOI formatting.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

According to the revised version, the manuscript has successfully achieved all its objectives. Furthermore, the authors have diligently addressed all the comments that were raised during the review process.

Comments on the Quality of English Language

The manuscript is generally well written and the English language is of a good standard. The text is clear, concise, and scientifically appropriate, allowing the reader to follow the objectives, methodology, results, and conclusions without difficulty. Only minor grammatical and stylistic revisions may be considered during the final editing stage to improve readability, but no substantial language revision is required.