Review Reports

Round 1

Reviewer 1 Report

The Whole Genome Sequencing, Comparative Genome Analysis and Biotechnological Potential of Emericellopsis alkalina and E. fimetaria (Bionectriaceae, Ascomycota) from the Sediment of Alkaline, Saline Lakes contributes to the fields of extremophiles and natural antimicrobial by providing the full length genomes of two fungi isolated from extreme environment and analyze their gene content. It is noted that in fungi complete genome sequences and basic functional annotation is a rate limiting step and therefore the manuscript does advance the field.

Nevertheless, I would like to see in a revised version a broader and deeper analysis.

Major points:

1. Test at least of of the prediction proposed for the activity of the CAZymes.
2. It is suggested that some biosynthetic gene clusters are actually not functional. Because the expression of the these clusters is often limited to very specific conditions, sometimes functional clusters are identified by the presence of resistant genes (often the target). Did the authors found these genes in the clusters? How many?
3. A functional analysis of RNAseq of Emericellopsis alkalina in 10 and 7 pH and E. fimetaria in high vs low salinity
4. The genomes are small and encode relatively low amount of proteins, what are the families of protein loss in comparison with other Hypocreales?

Minor points:

1. The abstract should not include technical aspects of the sequencing results (# of contigs etc).
2. The introduction should be revised and shorten historical facts

Major points:

1. Test at least of of the prediction proposed for the activity of the CAZymes.
2. It is suggested that some biosynthetic gene clusters are actually not functional. Because the expression of the these clusters is often limited to very specific conditions, sometimes functional clusters are identified by the presence of resistant genes (often the target). Did the authors found these genes in the clusters? How many?
3. A functional analysis of RNAseq of Emericellopsis alkalina in 10 and 7 pH and E. fimetaria in high vs low salinity
4. The genomes are small and encode relatively low amount of proteins, what are the families of protein loss in comparison with other Hypocreales?

Minor points:

1. The abstract should not include technical aspects of the sequencing results (# of contigs etc).
2. The introduction should be revised and shorten historical facts

Author Response

Response to the reviewer’s comments

Thank you for taking the time to review our manuscript, as well as your valuable comments and suggestions. We position our research as a presentation of previously non sequenced genomes of species from the genus Emericellopsis along with their functional annotation, including some preliminary conclusions regarding the potential biotechnological applications of these species. We plan to further develop hypotheses raised in this study and will certainly take into account all your remarks and proposals.

Major points

1. Test at least of the prediction proposed for the activity of the CAZymes.

Response: Determination of enzyme activity that depolymerizes various natural polysaccharides is undoubtedly necessary for a more complete understanding of fungal ecology. In relation to the genus Emericellopsis, isolated from saline/alkaline lakes, it would be interesting to additionally assess the stability of such enzymes at high pH values. We believe that this topic deserves a separate paper.

2. It is suggested that some biosynthetic gene clusters are actually not functional. Because the expression of these clusters is often limited to very specific conditions, sometimes functional clusters are identified by the presence of resistant genes (often the target). Did the authors find these genes in the clusters? How many?

Response: We fully agree with the reviewer's view that some biosynthetic gene clusters are actually not functional. In the predicted biosynthetic gene clusters (BGCs), no genes were identified that could unequivocally be interpreted as responsible for self-resistance against their possible products. This may be due both to limitations of the databases and algorithms used for annotation, as well as alternative tolerance mechanisms of the producer organism. The activation of silent biosynthetic clusters indeed sometimes requires specific conditions, but we consider this an issue deserving separate investigation.

3. A functional analysis of RNAseq of Emericellopsis alkalina in 10 and 7 pH and E. fimetaria in high vs low salinity.

Response: Analysis of expression under different conditions, including extreme ones, would indeed allow us to better evaluate possible adaptation mechanisms. However, we believe that such analysis would be more informative when using several strains of one species, which makes it a subject for an additional study.

4. The genomes are small and encode relatively low amounts of proteins, what are the families of protein loss in comparison with other Hypocreales?

Response: Indeed, the genomes of Emericellopsis contain fewer protein-coding genes compared to some other representatives of Hypocreales. There are several reasons for this phenomenon, and we have some hypotheses about them. However, they require additional verification, e.g., by assessing evolutionary dynamics within families of orthologous genes among species of Emericellopsis and other members of Hypocreales belonging to different ecological groups (parasites, soil saprotrophs, marine fungi, entomopathogenic species, coprophilic species). Nevertheless, this is quite an extensive task, and we consider it a subject worthy of separate research.

Minor points

The abstract should not include technical aspects of the sequencing results (# of contigs etc).

Response: We took note and removed technical details from this section.

The introduction should be revised and shorten historical facts

Response: We took into account and removed historical facts from the introduction. Indeed, such facts would fit better in a review article. The major revisions have been made to the introduction section:

We have carefully revised and polished the language of the entire manuscript

Reviewer 2 Report

I recommend Major Revision with the following suggestions: 1.  Clarify the genome sequencing coverage and, if necessary, discuss any limitations this might impose.  2.  Consider adding targeted experimental data (e.g., gene expression analysis or chemical profiling) to support a key claim.  3.  Refine the interpretation to avoid overstatement and to address the interesting paradoxes raised by the data.  4.  Streamline the Discussion to focus on the most significant findings and their implications.  5.  Improve the presentation, including language, figure quality, and a more focused conclusion.

Summary

This study presents the whole-genome sequences of two extremophilic fungi, Emericellopsis alkalina and E. fimetaria, and provides a comparative genomic analysis focusing on carbohydrate-active enzymes (CAZymes), transporters, and biosynthetic gene clusters (BGCs). The generation of new genomic resources for this understudied genus is certainly valuable, and the authors should be commended for their efforts to explore the biotechnological potential of these fungi. However, the manuscript in its current form is primarily descriptive, and several methodological concerns, along with some instances of overinterpretation, limit the strength of the conclusions. With significant revisions to address these points, the work could become a more impactful contribution to the field.

Major Concerns

1. Scientific Contribution and Opportunities for Deeper Analysis

The core findings are largely descriptive, focusing on cataloging genomic features. While this provides a useful foundation, the manuscript would benefit from deeper, hypothesis - driven insights. Key questions remain partially addressed: How might these fungi specifically adapt to high pH and salinity at a molecular level? Could any of the numerous "unknown" BGCs be linked to potential bioactive compounds? The speculation regarding the higher BGC count in E. fimetaria as an adaptation to competition on dung is interesting, but would be strengthened by experimental support.

2. Methodological Points That Would Benefit from Clarification or Strengthening

Sequencing Coverage

Table 1 reports the genome coverage as "<10x". For a study aiming for precise comparative genomics and BGC prediction, this coverage is lower than typically expected, as it could potentially lead to incomplete assemblies or fragmented gene predictions. Could the authors clarify whether this is a typographical error? If the coverage is indeed below 10x, a brief justification or discussion of how this might affect the interpretations would be helpful.

Phylogenetic Detail

The methods section mentions using the "GTR + IG model" but could provide additional details to enhance reproducibility. For instance, explaining how the model was selected and whether parameters were treated independently across the different gene partitions (ITS, LSU, tef1a, rpb2, tub2) would strengthen this section.

Gene Prediction Accuracy

The use of AUGUSTUS with a heterologous model ("Fusarium") is understandable in the absence of RNA - seq data. However, the authors might acknowledge that this approach could introduce some inaccuracies in gene structure predictions, particularly for genes unique to extremophilic fungi. This is not a fatal flaw, but a brief discussion of this limitation would demonstrate scientific rigor.

3. Suggestions for Strengthening the Interpretation of Results

CAZyme - Ecotype Relationship

The observation that E. alkalina encodes numerous plant cell wall - degrading enzymes despite inhabiting an environment with presumably limited plant material is intriguing. The current explanation ("saprotrophic lifestyle") feels somewhat simplistic given this paradox. A more nuanced discussion—perhaps considering alternative carbon sources or the possibility of relic metabolic capabilities—would add depth. Additionally, the negative finding regarding chitinase genes (despite the presence of Artemia) is interesting and deserves more thorough exploration.

Transporters and Adaptation

The increased number of Na+/H+ antiporters in E. alkalina is a suggestive finding. However, presenting this as definitive evidence of an adaptation mechanism may be premature, as the current data show correlation rather than causation. The authors could temper this conclusion or, alternatively, strengthen it by including a more rigorous comparative analysis with a closely related, non - alkaliphilic species.

BGC Functional Assignments

The suggestion that E. fimetaria might produce specific compounds (e.g., antiamoebins) based solely on BGC sequence similarity is reasonable as a hypothesis. However, the language used sometimes implies a more definitive conclusion than the evidence supports. The manuscript would be strengthened by acknowledging the speculative nature of these assignments, particularly given that "the majority of BGCs lack any known similarity." Including chemical profiling data (e.g., LC - MS/MS) would, of course, provide much stronger support.

4. Discussion Section

The Discussion section is somewhat lengthy and, in places, reiterates the Results. The authors might consider focusing it more tightly on the most important findings and their implications. For example, the point about E. alkalina lacking an increased number of known "pH sensors" is interesting, and the suggestion that "uncharacterized proteins" might fill this role could be expanded into a more thoughtful discussion about the limitations of homology - based annotation or alternative pH adaptation strategies.

Minor Comments

Typographical errors

In Figure 5, "non-rybosomal" should be corrected to "non-ribosomal". In the Figure 3 legend, "Gene clusters encoding of" is grammatically incorrect.

Vague Conclusions

The concluding section reads like a list of future experiments ("OSMAC approach," "gene knockouts") rather than summarizing the actual, tangible discoveries of the present study.

 

Author Response

Response to the reviewer’s comments

We sincerely appreciate the time you devoted to reviewing our work and value your constructive comments. We present this study as a showcase of Emericellopsis species' genomes that had not been published before, along with their functional annotations, serving as a minimal basis for comprehending their biotechnological potential and adaptability to harsh environmental conditions. We acknowledge the constraints imposed by interpreting genome data alone without chemical profiling or gene expression analyses across diverse environments, taking these limitations into consideration in our conclusions. We intend to continue working with these organisms, aiming to gain deeper insights into their capacity as producers of antimicrobial compounds, and will definitely incorporate your feedback and recommendations.

Major Concerns

Scientific Contribution and Opportunities for Deeper Analysis.

Many thanks for the comments and suggestions. You are absolutely right. We position our study as a presentation of previously non sequenced genomes of Emericellopsis species along with their functional annotation, accompanied by some preliminary conclusions concerning the potential biotechnological application of these species. Certainly, a more thorough analysis involving chemical profiling of extracts and RNA-seq would broaden the understanding of issues raised in the manuscript, and we do plan to conduct such studies separately.

Methodological Points That Would Benefit from Clarification or Strengthening.

Sequencing Coverage, Gene Prediction Accuracy.

We fully agree with the reviewer's view that the sequencing coverage is indeed low, at approximately 10x. We understand that this can significantly affect the quality of structural annotation and consequently the quality of subsequent functional annotation. Besides low coverage, prediction accuracy of gene boundaries, especially those of lineage-specific genes in Emericellopsis, might also be influenced by the use of non-optimal models for AUGUSTUS. Nonetheless, results obtained through completeness evaluation of genomes in terms of predicted protein sequences enable us, in our opinion, to regard these assemblies as suitable foundations for functional annotation and subsequent cautious interpretation of its outcomes.

Phylogenetic Detail

Phylogenetic analysis was conducted using Bayesian inference via MrBayes based on a concatenated matrix. However, model parameters (such as transition/transversion rates) for each locus were independent because they evolve at different speeds but share common tree topology. Although we evaluated models using IQTree, since MrBayes supports only a limited number of models, we chose GTR+I+G as the most comprehensive option. It’s likely that overparameterization affects analysis speed, but for such short matrices, this effect is negligible.

Suggestions for Strengthening the Interpretation of Results.

CAZyme - Ecotype Relationship

Habitats typical for E. alkalina — saltwater alkaline lakes — are relatively poor in higher plant residues. However, during spring, water levels rise substantially, flooding areas where halophytic plants grow, and occasionally plant debris enters the lake ecosystem. These lakes are densely populated by Artemia salina, leading us to hypothesize that E. alkalina might utilize chitin as a carbon source. Additionally, we have another hypothesis suggesting gradual adaptation of Emericellopsis to aquatic habitats (including marine and continental salty waters), resulting in reduced need for carbohydrate-active enzymes (CAZymes) required for degradation of higher plant residues compared to strictly terrestrial members of Hypocreales. Since this hypothesis demands additional bioinformatics and biochemical investigations, we refrain from presenting it in this paper.

Transporters and Adaptation

It is true that current data show correlation rather than causality. The number of certain Na+/H+ antiporters in the genome of E. alkalina is indeed higher than in other Emericellopsis genomes, particularly in comparison to E. terricola. However, regulation plays no less important role. Determining expression levels of corresponding genes under varying conditions, ideally comparing multiple species, would provide a more comprehensive assessment of their roles in adaptation to increased NaCl concentrations. We agree that this should be addressed in future studies. We do recognize the importance of softening our conclusions by stating explicitly that these are merely assumptions requiring experimental validation.

BGC Functional Assignments

Our hypothesis that E. fimetaria p24 produces amebicidal compounds is based on the following premises: Strains of this species have indeed been reported to produce amebicidals, as reflected in literature. Furthermore, we possess data demonstrating antimicrobial activity of E. fimetaria p24, presented in the appendix. While the biosynthetic gene cluster (BGC) responsible for synthesizing amebicidals has yet to be described and thus remains absent from available databases, there exists a description of related and structurally similar compounds—emericillins—for which a matching BGC has been found in two detected clusters of E. fimetaria p24. We acknowledge that confirming this hypothesis necessitates additional research, potentially involving chemical profiling of extracts and/or experiments with targeted gene knockouts, possibly complemented by RNA-seq. Nonetheless, we deem this matter appropriate for a distinct study. Consequently, in the revised version of the manuscript, we added clarification emphasizing the speculative nature of our assertions.

Discussion Section

We have revised the "Discussion" section according to your recommendations, reducing its length and removing fragments that duplicated information already provided in the "Results" section. Moreover, we have moderated the confidence level of our conclusions, clearly indicating that they require mandatory experimental validation.

Conclusions

We have slightly modified the "Conclusion" section, placing greater emphasis on the achieved results rather than on directions for future research. However, we still believe that specifying prospective research paths is beneficial.

Typographical errors

We have rechecked the manuscript for any possible typos and improved the quality of figures. Additionally, language clarity has been enhanced, which should contribute to better overall presentation of the manuscript.

Reviewer 3 Report

This manuscript reports the genome sequencing and comparative genomic analyses of two saline alkaline tolerant fungi, Emericellopsis alkalina E101 and E. fimetaria p24. The authors identified numerous transporters and several poorly characterized biosynthetic gene clusters (BGCs), which may provide a useful foundation for future biotechnological or pharmaceutical applications. However, the manuscript requires substantial revision in terms of formatting, language, and methodological detail before it can be considered for publication.

1. The genome sequences of E. alkalina E101 and E. fimetaria p24 should be deposited in a public database such as GenBank, and accession numbers should be provided in the manuscript.
2. The title is somewhat lengthy and could be revised to make it more concise.
3. In the Materials and Methods section, the formatting is not standardized. Subsections should be clearly numbered to improve structure and readability.
4. In the sentence “A phylogenetic tree has been reconstructed based on the sequences of (ITS1-5.8S-ITS2), DNA (LSU), (tef1α), (rpb2), and (tub2),” the parentheses are unnecessary and should be removed.
5. The phrase “Gene clusters encoding of carbohydrate-active enzymes (CAZymes) in Emericellopsis genomes” is grammatically incorrect and should be revised. e.g., modified to “Gene clusters encoding carbohydrate-active enzymes……”
6. The sentence “Genomes play an important role in understanding the antimicrobial activity……” appears incorrect. It would be more appropriate to state, “Genomics plays an important role in understanding antimicrobial activity……”
7. The sentence “We found that E. fimetaria p24 two BGCs showing similarity to the BGC of emerimicins from E. tubakii” is grammatically incorrect and requires revision for clarity.
8. In the Supplementary Materials, figures and tables should be presented separately rather than mixed together.
9. In Table S3, the meaning of numerical ranges (e.g., 9–13) is unclear. An explanatory note should be added below the table to clarify these values.

Author Response

Major comments

This manuscript reports the genome sequencing and comparative genomic analyses of two saline alkaline tolerant fungi, Emericellopsis alkalina E101 and E. fimetaria p24. The authors identified numerous transporters and several poorly characterized biosynthetic gene clusters (BGCs), which may provide a useful foundation for future biotechnological or pharmaceutical applications. However, the manuscript requires substantial revision in terms of formatting, language, and methodological detail before it can be considered for publication.

Thank you for taking the time to review our manuscript, as well as your valuable comments and suggestions.

1. The genome sequences of  alkalinaE101 and E. fimetaria p24 should be deposited in a public database such as GenBank, and accession numbers should be provided in the manuscript.

 

Response: The genome sequences were submitted to GenBank, underwent automatic verification, and were assigned accession numbers. Unfortunately, due to funding-related delays, NCBI and GenBank in particular are currently experiencing processing backlogs; as a result, the genome sequences of E. alkalina E101 and E. fimetaria p24 are not yet publicly available. We have updated the manuscript by replacing the previously listed BioSample IDs with the corresponding accession numbers, and the sequences will be made publicly accessible as soon as the verification process is complete.

2. The title is somewhat lengthy and could be revised to make it more concise.

 

Response: The title of the paper most comprehensively reflects the essence of the work.

 

3. In the Materials and Methods section, the formatting is not standardized. Subsections should be clearly numbered to improve structure and readability.

Response: We have adjusted the manuscript text to standardize formatting and correct typographical errors.

 

4. In the sentence “A phylogenetic tree has been reconstructed based on the sequences of (ITS1-5.8S-ITS2), DNA (LSU), (tef1α), (rpb2), and (tub2),” the parentheses are unnecessary and should be removed.

Response: The necessary adjustments have been made to the text.

 

5. The phrase “Gene clusters encoding of carbohydrate-active enzymes (CAZymes) in Emericellopsis genomes” is grammatically incorrect and should be revised. e.g., modified to “Gene clusters encoding carbohydrate-active enzymes……”
6. The sentence “Genomes play an important role in understanding the antimicrobial activity……” appears incorrect. It would be more appropriate to state, “Genomics plays an important role in understanding antimicrobial activity……”
7. The sentence “We found that  fimetariap24 two BGCs showing similarity to the BGC of emerimicins from E. tubakii” is grammatically incorrect and requires revision for clarity.

 

Response: Thank you for your comments; we have edited the manuscript to improve the English language.

 

8. In the Supplementary Materials, figures and tables should be presented separately rather than mixed together.

Response: We have also corrected the layout of the Supplementary Material in accordance with your comments.

 

 

9. In Table S3, the meaning of numerical ranges (e.g., 9–13) is unclear. An explanatory note should be added below the table to clarify these values.

Response: We also clarified the meaning of the numerical ranges of antimicrobial activity for E. fimetaria p24 extracts in Table S3. The antimicrobial activity was influenced by the carbon source in the culture medium.

 

Round 2

Reviewer 1 Report

The authors did nothing I requested

No need for a detailed comments

Author Response

Response: We sincerely appreciate the time you devoted to reviewing our work and value your constructive comments. We present this study as a showcase of Emericellopsis species' genomes that had not been published before, along with their functional annotations, serving as a minimal basis for comprehending their biotechnological potential and adaptability to harsh environmental conditions. The additional studies you proposed, such as determining the enzymatic activity of Emericellopsis alkalina E101 and Emericellopsis fimetaria p24 cultures in relation to natural polysaccharides, functional analysis of RNA-seq under various conditions, and comparison of the genomes we presented with other representatives of Hypocreales, are undoubtedly useful and necessary for a better understanding of the ecology of the genus Emericellopsis. However, we believe that such extensive additional studies would be more appropriate as a topic for a separate publication.

We have taken into account your comments regarding technical details in the Abstract and the historical background in the Introduction.

The abstract should not include technical aspects of the sequencing results (# of contigs etc).

Response: We took note and removed technical details from this section.

The introduction should be revised and shorten historical facts

Response: We took into account and removed historical facts from the introduction. Indeed, such facts would fit better in a review article. The major revisions have been made to the introduction section:

We have carefully revised and polished the language of the entire manuscript

Reviewer 2 Report

Accept with Minor Revisions

1.Complete phylogenetic model description: Ensure the justification for selecting the GTR+I+G model is fully written into the phylogenetic analysis part of the manuscript.

2. Maintain cautious tone: Keep the prudent narrative style for BGC functional prediction throughout the manuscript.

3. Final language polishing: Conduct a full-text proofread to fix occasional minor grammatical or expression issues. eg.  In the abstract: "..." encoding various enzymes, including carbohydrateactive enzymes such as endoxylanases, "which are useful for many ecological adaptations." the "which" here is not clear.  
Page 15: "..." warrant additional investigation to explore their potential...” It is suggested that "additional" be changed to "further" to better conform to academic writing habits.

Author Response

We sincerely thank the Reviewer for their constructive and insightful comments. All suggestions have been carefully considered and incorporated into the revised manuscript, which has significantly improved the clarity, scientific quality, and overall presentation of our work.

1. Complete phylogenetic model description: Ensure the justification for selecting the GTR+I+G model is fully written into the phylogenetic analysis part of the manuscript.

Response: We conducted a search for the optimal nucleotide substitution model for each locus separately using the Model Finder module (IQTree). However, MrBayes does not support most of these models directly (e.g., TN or TIM2). Nevertheless, the GTR+I+G model encompasses these models as special cases with fewer parameters. We did not wish to artificially simplify the model for Bayesian inference, so we selected GTR+I+G as the most comprehensive option. We have added this rationale to the Results section, where we describe the phylogeny reconstruction results.

2. Maintain cautious tone: Keep the prudent narrative style for BGC functional prediction throughout the manuscript.

Response: We have reviewed the text and revised the wording to maintain a cautious narrative style when describing the functional prediction of BGCs.

3. Final language polishing: Conduct a full-text proofread to fix occasional minor grammatical or expression issues. eg.  In the abstract: "..." encoding various enzymes, including carbohydrateactive enzymes such as endoxylanases, "which are useful for many ecological adaptations." the "which" here is not clear.  
   Page 15: "..." warrant additional investigation to explore their potential...” It is suggested that "additional" be changed to "further" to better conform to academic writing habits.

 

Response: We also performed a thorough proofreading of the text to improve the language.

Reviewer 3 Report

the authors have satisfactorily addressed all my questions and concerns. Therefore, the manuscript can be accepted in its present form.

the authors have satisfactorily addressed all my questions and concerns. Therefore, the manuscript can be accepted in its present form.

Author Response

We sincerely thank the Reviewer for their constructive and insightful comments. All suggestions have been carefully considered and incorporated into the revised manuscript, which has significantly improved the clarity, scientific quality, and overall presentation of our work.