Review Reports

Round 1

Reviewer 1 Report

The overall experimental method and technology of the manuscript are relatively simple. It is suggested to increase the analysis of the correlation between environment and fungal community.

Author Response

Reviewer#1

Comments and Suggestions for Authors

The overall experimental method and technology of the manuscript are relatively simple. It is suggested to increase the analysis of the correlation between environment and fungal community.

 

Response:

Thank you very much for reviewing our manuscript and offering valuable advice.

Unfortunately, we have very little environmental data from this north polar location, which is extremely remote (at the northern limit of North America) and rarely visited. We did measure pH, carbon and nitrogen content, but found no correlative relationships with the fungal communities. More intensive sampling and measurement of a wider range of environmental parameters will be required, and we hope to be able to do so in the future.

Reviewer 2 Report

Comment: I reviewed the manuscript and represented the readers; please respond and add the text to the manuscript

 

Introduction:

Line 38-40, how to measure in loss of fungal diversity?

How is the benchmark for an ecosystem not to lose its diversity? Any sample in some references?

 

Materials and Methods

-Why did the authors use the Potato Dextrose Agar (PDA) for fungal isolation? Some fungi could not be culturable in the general medium like PDA.

- How determine of loss or stability of fungal diversity? Any control for fungal richness diversity in this experiment?

 

Results

According to the finding and identification of all fungi, Could the authors describe the dominant fungal group: Decomposer, Saprophytes, Pathogen, Endophytes, fungal Symbiosis.

Did the authors find some fungal unidentified?

Could the authors make Principal Component Analysis (PCA) based on fungal species' presence or absence data in your study?

Discussion:

Is there some potential for the fungal collection to become bioprospection? Please have a brief discussion.

 

 

 

Author Response

Reviewer#2

Comments and Suggestions for Authors

Comment: I reviewed the manuscript and represented the readers; please respond and add the text to the manuscript

 

Response: We wish to express our appreciation for this insightful advice which has helped us helped us significantly improve the paper.

 

Introduction:

Comment:Line 38-40, how to measure in loss of fungal diversity?

 

How is the benchmark for an ecosystem not to lose its diversity? Any sample in some references?

 

Response: There is no uniform approach to assessing the loss of fungal diversity. However, in recent years there has been a movement to create a Red Data Lists of fungi (i.e.;https://www.iucnredlist.org/,https://www.pref.kyoto.jp/kankyo/rdb/en/bio/fungi.html). If this Lists can be expanded to different countries and regions, we believe that in the future we will be able to assess the diversity of fungi and the impact of their loss on the environment in a unified manner. In this manuscript, our measure of diversity is total number of cultures taxa. We also note in the revised manuscript the opportunity to improve these estimates by way of metagenomics surveys (please see comment below on this subject).   

 

・Materials and Methods

 

Comment:-Why did the authors use the Potato Dextrose Agar (PDA) for fungal isolation? Some fungi could not be culturable in the general medium like PDA.

 

Response: Thank you very much for indication of this point.

We conducted culture tests with YMA, YpDA, and Czapek dox agar media as well as PDA before conducting this experiment. These tests showed that PDA supported the greatest variety of fungal species (colors and shapes) that could be cultured, and we therefore used this medium for our subsequent experiments.

We have now added this important point (lines 78-82) in the revised manuscript:

Preliminary culture tests of Walker Glacier samples with potato dextrose agar (PDA), yeast malt extract agar (YMA), yeast peptone dextrose agar (YpDA), and Czapeck Dox Agar showed that PDA produced the best growth of the largest number of taxa, including those that grew on the other media. We therefore used PDA for all subsequent fungal culture experiments.

 

Comment:- How determine of loss or stability of fungal diversity? Any control for fungal richness diversity in this experiment?

 

Response: In the glacier retreat area, the distance of each sampling point from the glacier terminus indicates different times since the glacier retreated. We investigated the fungal diversity at each sampling site, thus stage in the chronosequence, and considered the presence or absence of a taxon at each point as indicative of stability or loss of that taxon. The results showed that many of the fungi on the glacier could not be found in the glacier retreat area at all, implying an ice-specific microbiome, with loss of its habitat as the glacier retreats. We showed that fungal diversity (presence or absence of taxa, community richness) was not related to the pH, carbon or nitrogen content in the sample.

 

 

・Results

 

Comment: According to the finding and identification of all fungi, Could the authors describe the dominant fungal group: Decomposer, Saprophytes, Pathogen, Endophytes, fungal Symbiosis.

 

Response: This is a very interesting point. We can were able to classify the fungi in this study as in terms of the dominant fungal group. However, we have not been able to confirm whether these fungi are acting as decomposers, saprophytes, pathogens (seems unlikely, but not impossible for some taxa), symbionts or endophytes in this experiment, and are therefore reluctant to speculate further with out, for example metagenomics information about functional diversity (please see below).

 

Comment: Did the authors find some fungal unidentified?

 

Response: We note that in this study, 52 fungal strains were unidentified (line 114-116) and five potentially new fungal species were found (line147).

 

 

Comment: Could the authors make Principal Component Analysis (PCA) based on fungal species' presence or absence data in your study?

 

Response: In general, the number of data obtained in culture experiments often does not provide clear results even when PCA analysis is performed. The same result was obtained in this study. Instead we used cluster analysis to determine the similarity in fungal diversity among sampling sites, and believe that this shows a clear separation of communities (please see Figure 2).

 

 

・Discussion:

 

Comment: Is there some potential for the fungal collection to become bioprospection? Please have a brief discussion.

 

Response: This is another interesting point, and we have now added a paragraph on this subject in the revised manuscript (Lines 193-201):

 

Fungi inhabiting the High Arctic must survive the extreme polar conditions of persistent cold temperatures, continuous bright light exposure and winter-freeze-up, and are likely to have many unusual biochemical properties. Studies elsewhere in the cryosphere have drawn attention the value of low-temperature fungi in the search for cold active enzymes, raw materials for new drugs, bio-surfactants and other biotechnological applications (as described for Antarctic isolates [32]). These fungi represent a new bioresource that could contribute to sustainable development goals. The present study took place in the Inuit homeland of Nunavut, Canada, and any such commercial development would require close attention to Indigenous rights and benefit sharing.

Reviewer 3 Report

The study conducted by Tsuji et al. is well-written and seems of practical significance with regard to cryopreservation of glacier microbes.

However, it would be great if the authors present the complete picture of the fungal species by providing fungal metagenomics (shotgun metagenomic sequencing) in addition to culture methods.

Author Response

Reviewer#3

 

Comments and Suggestions for Authors

The study conducted by Tsuji et al. is well-written and seems of practical significance with regard to cryopreservation of glacier microbes.

 

However, it would be great if the authors present the complete picture of the fungal species by providing fungal metagenomics (shotgun metagenomic sequencing) in addition to culture methods.

 

Response: We very much appreciate your insightful comments on our manuscript. This is a very good point, and although we have no metagenomics data at this stage, it should be a priority for the future, at our site and throughout the cryosphere. We have now developed this point in a new paragraph in the Discussion, with reference to high-latitude studies elsewhere (lines 180-192):

 

Although the PDA medium used this study supported the growth of a wide range of species, it likely selected for only a subset of the total fungal community. More dilute media might favor more oligotrophic species, and determination of the true total diversity will require the application of next-generation molecular methods. In particular, metagenomic analysis would allow resolution of the entire glacier microbiome, including algae, bacteria, archaea and viruses, in addition to fungi, and may also reveal their functional diversity. Molecular analyses of other cryo-habitats have shown a rich fungal diversity living on ice. For example, amplicon analyses revealed 184 fungal taxa in samples from glacial ice in maritime Antarctica [30], and 697 fungal taxa from algal associated communities on the surface of the Greenland Ice Sheet [27]. Metagenomic analyses are likely to pick up additional species that elude even amplicon analysis (as found for prokaryotes in High Arctic lakes [31]), and full molecular surveys are urgently required throughout the fast changing cryosphere.

Round 2

Reviewer 1 Report

Although the author has increased the discussion on fungal community, the analysis is still slightly simple.

Place table S2 in the text.

The manuscript needs to be polished by native English speaking professionals.

Author Response

Reviewer#1

Comments and Suggestions for Authors

Although the author has increased the discussion on fungal community, the analysis is still slightly simple.

Place table S2 in the text.

 

Response:

Thank you very much for reviewing our manuscript and for these additional comments.

We have moved Table S2 into the text as requested (now Table 2). This has strengthened the manuscript, and draws attention to the large number of isolates (273 strains) that were successfully cultivated and sequenced in this study. We have considered other ways to develop the analysis, but are limited by our sample size from this extreme North Polar location.

 

Comment: The manuscript needs to be polished by native English speaking professionals.

 

Response:

As per the reviewer's comments, the text has now been edited by a native English speaker, and improvements have been made throughout the manuscript.