Fungal Diversity in Korean Caves and Cave-Inhabiting Bats with Attention to Pseudogymnoascus Species

Round 1

Reviewer 1 Report

The manuscript presents interesting data on fungal diversity in three Korean caves. Although the authors found a rich fungal diversity, they focused on the genus Pseudogymnoascus.

In general, the manuscript is well written and organized, but there are some points, especially in the methodology, that need to be improved and/or corrected before a possible acceptance for publication. These corrections are especially important for the study's replicability. Below I list all my comments.

 

L54: “bat habitat cave”

I don't think this is the best, most widely held term. I suggest an adaptation. Perhaps, "cave-dwelling bats" or "cave-inhabiting bats", and "bat cave" when referring to the environment. Please review throughout the text.

 

L55: “isolated Pseudogymnoascus species, and further confirmed Pd.”

I find it fearful to assert the occurrence when, in fact, the phylogenetic analysis revealed that no isolate belonged or was closely related to the clade with Pseudogymnoascus destructans.

 

L62-63: “A few years ago, Dr. Chung who is a bat ecologist, provided information on bats showing WNS-like symptoms in their habitat.”

Where is this information available? Citation is missing.

 

L64-65: “Environmental and bat samples were collected from three karst caves.”

How the samples were collected needs to be better described. Swabs on bats were done on which body parts? And in the cave environment, which substrates were sampled (from the air, ceiling, wall, floor...)?

Why specifically were these caves chosen?

How big are these caves (e.g., linear development, volume, area)? And about the number of entries, is there any information?

This information is important, for example, to provide an overview of the cave environment and its susceptibility to influences from the external environment.

 

L68-69: “The temperature of the caves was 6–15 °C, and the humidity was 50–83%.”

How were temperature and humidity measured and how often or time interval?

 

Figure 1

The map can be enhanced by including coordinates, scale, and north arrow, for example.

 

Figure 2

Ascomycota's pie chart is difficult to interpret because there is a very subtle difference between some of the colors used, in addition to being many. It would facilitate the understanding of the figure if the authors directly associated the names of the fungal families with their respective portion of the graph.

In the legend, italicize “Pseudogymnoascus”.

Author Response

Thank you for your kind comments. I attached revision note

Author Response File: Author Response.pdf

Reviewer 2 Report

In the last decades, the interest in caves and show caves has increased exponentially. As reported by the authors there is a lack of information about the Korean caves and so this research could be of interest to the readership. Anyway, the manuscript suffers several weaknesses in form and substance in all sections. The reference list should be improved by taking into account the broad data available on fungal diversity in cave environments, such as Varderwolf et al, 2013 (https://digitalcommons.usf.edu/kip_articles/5660/); and papers from Cesareo Saiz-Jimenez/ Jurado, Moenne- Loccoz and Aleya, Mulec, Ogorek, and Whang and Ma (e.g. Mogao Grottoes) or Zhang et al 2014 (https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0108714) only to give some examples. English revision is required.

Below is reported a detailed, not exhaustive, list of comments on the amendments required.

TITLE: the title should be improved because it does not reflect the manus’ content. It could be better to do something like Fungal diversity in Korean caves and cave-inhabiting bats with attention to Pseudogymoascus species

KEYWORDS. The majority of KW chosen by authors are already present in the title and then are not fruitful in improving the article finding. Here you can find some suggestions for choosing KW: https://falconediting.com/en/blog/6-tips-for-choosing-keywords-for-your-scientific-manuscript

https://blog.wordvice.com/choosing-research-paper-keywords/

ABSTRACT. This section should be rewritten following the basic rules of scientific communication. The reason why this investigation becomes necessary should be introduced as the research aim will be more evident.

L10 Fungi are the second-largest kingdom of eukaryotes. In the abstract, this sentence is not necessary and too general

L10-11 “They are present on most substrates at various locations”. This sentence sounds confusing. there are two words, “substrates” and “locations”, misused. Substrates are mainly used to indicate that microorganisms feed them. A location is a fixed place or position in space. If you are lost, you don't know your location. (please, see at https://dictionary.cambridge.org/dictionary/english/location)

L11-12 “Several vectors influence the inflow…” the term vector is improperly used. A vector is a living organism that transmits an infectious agent from an infected animal to a human or another animal.

L14 the authors did not explain why it was necessary to focus on Pseudogymnoascus. This background information Is necessary to justify and give relevance to this research and the results achieved.

L17 Thirty-one… did you mean: “One hundred sixty fungal strains belonging to 31 different genera were isolated”?

L17 of these… Please, rework. Now it seems that  20 are Pseudogymoascus species, and in table 2 are the isolates.

L22 belonging to clades A, B, .. this sentence is more appropriate for a result that could be supported by pictures. Here it is not, rework or remove it.

INTRODUCTION

L27-28 the fungi….estimated Hawksworth, et al., 2017. Here is more recent esteem of fungal biodiversity: Wu, B.; Hussain, M.; Zhang, W.; Stadler, M.; Liu, X.; Xiang, M. Current insights into fungal species diversity and perspective on naming the environmental DNA sequences of fungi. Mycology 2019, 10, 127–140. Please modify the sentence consistently. 

L29 substrate, please rework

L29 from deep-sea to animal skin. The citation is not so appropriate to describe the variety of ecological niches occupied by fungi nor to describe their lifestyles.

L31 caves /underground environments are considered extreme because of the low availability of carbon sources, they are indeed oligotrophic environments. Otherwise, the high relative humidity and constant temperature favour microbial growth. Darkness and low photosynthesis sounds are a repeat being strictly connected to each other. The citation is not updated (2001) many papers are reporting the underground environmental features, such as Barton, H.A., 2006. Introduction to cave microbiology: a review for the non-specialists. J. Cave Karst Stud. 68, 43–52.

Barton, H.A., Northup, D.E., 2007. Geomicrobiology in cave environments: past current and future perspectives. J. Cave Karst Stud. 69, 163–178.

 L32 “In these harsh environments” instead of harsh it could be better severe/stringent/challenging 

L33 the fungal diversity is generally considered to be low. This is not true, many authors underlined as cave/underground environments are characterized by unexpected diversity and a reservoir for undescribed species.

L39 Among several fungi. What does it mean? Please modify

L44-46 attention to Pseudogymonoascus soared in 2009… was discovered. This is confusing because in 2009, the species Geomices destructans was described and reported as the etiologic agent of the WND. In 2013 Minnis and Linder proposed the new combination after a phylogenetic analysis of Geomyces species and allies. L46-47 “This discovery of fungal pathogen has led to in-depth phylogenetic research of fungi living in bat caves [22]”. The sentence is misleading, and the citation is not appropriate. As before, the cause-effect relation between Geomyces destructans and WND has been clear since 2009. Broad investigations on caves and bats have started since then. In this light, the sentences at L39-51 should be reorganized respecting the timeline and fact succession.

L52-55 the aim should be better reported

Figure 1. The sampling sites are not visible on the map (increase their size). The relative caption should be modified, indicating the meaning of yellow crosses and grey squares and circles. Both figure and caption should be reported after section 2.1. Please modify

L72 Is MBcell the supplier of Chloramphenicol? What about the supplier of the culture medium (Sabouraud) and gentamycin

L70-82 the section should follow the operative timeline. How were samplings performed?

How many bats from which sites? etc. How were supernatants obtained? What dilution? Of the 83 samples, how many were from bats (oral, skin etc.), and how many were from caves? Please provide a detailed description.

 L79-80 to obtain pure cultures, the single-spore culture method has been used

L86-89 Genomic DNA was amplified using primers for 5 molecular markers: ribosomal internal transcribed spacers (ITS), 28S large subunit rRNA gene (LSU) … please correct consistently.

 L89-91 please add the PCR program used in the text or in the Table where the reference of primers should be added.

L90 I addition…please add the reason why you used species-specific primers.

Table 1. The column should be Molecular markers (ITS is not properly a gene), primer name, Primer sequences, and References.  In the caption, use the term species-specific primers. Numbering on molecular markers is not necessary to disclose in the Table notes the acronym meaning.

 https://journals.sagepub.com/doi/pdf/10.1177/104063871002200208 five primers have been proposed. Please explain your choice.

 L98 and their accession numbers are listed…

 L104 the title of section 2.4 should be changed. It’s more appropriate Strains identification and Phylogenetic analysis

Anyway, there is a discrepancy between the described methods and results. Indeed, reading the materials it seems that all isolates have been processed in this way. Otherwise, the phylogenetic analysis has been limited to strains belonging to the genus Pseudogymnoascus.  Please amend.

L105 are not the sequences to be identified but the fungal isolates.

 L125 Fungal colonies were…this sentence contains the missing info of the materials and methods (M&M).

L126-127 All bats showed negative results for the UV light test. No mention of the UV light test in the M&M section.

 L129 Pd-diagnostic test should be separated from the other results.

L130 Ascomycota, Basidiomycota and Mucoromycotina. These three are not consistent with each other, being the first two divisions and the last (Mucoromycotina), a subphylum. To be consistent, it should be Mucoromycota.

L133 Family Davidiellaceae was changed to Cladosporiaceae years ago, please amend it along with the text and legends.

 L139 Classification of cultured fungi at the phylum and family levels. Figure 2 doesn’t report a “classification”. It is most appropriate to change the title to Fungal diversity frequencies by phylum, family, and genera.

The sequences of all isolated strains are missing. Which molecular marker has been used?

The figure 2 legend is unreadable, please amend it.

Table 2. Please correct Davidiellaceae and Mucoromycotina as above. More important, it is not clear how these data were achieved. Namely, which molecular marker was used and

Figure 4 should be improved. Along with the isolate nr, it should be added the species names and ex-type indicated. the authors are required to indicate the names of the non-Pseudogymnoascus species and outgroup. This figure is a carbon copy of the one reported by Villanueva et al., 2021 in which 20 Pseudogymnoascus species have been added. the figure has poor readability, I suggest proportionally reducing the long external branches (indicating, for example, x3, x4 as needed) as the species and strain number will be readable 

Table S1 in the notes should be added the acronyms of the culture collection of strains included in the phylogenetic analysis.

L151-153 this sentence should be removed because M&M.

L155 were classified using … rework the whole sentence …something like “ the clades achieved by BI were named as defined by Minis and Lindner

L156-157 Twenty Pseudogymnoascus… please modify to something like “the Pseudogymonoascus isolates fall in clades A (4), …..respectively

L158 No Pseudogymnoascus species was located… Please improve the sentence. Something like “ None of the isolates fall in the Pd clade (F)”

L167-171 is not a discussion, but a repeat of M&M. Please improve the discussion section. It cannot be a recap of M&M, nor a description of the achieved results. This section should compare the results achieved with other worldwide pieces of evidence. Try to explain, for example, the reasons for the false positives in Pd diagnostics….

L173 were – please change to belong to

L174-175 rework the sentence removing repeats

L176-177 These two genera are….these genera also are the most frequent in indoor and outdoor environments…not only in caves.

L187-189 Please explain the possible reason for the false positives. The most probable is that the authors based the primer design on Pd strains, but no sibling species were included.

L194-195 It is presumed that it is difficult to identify the Pseudogymnoascus species by testing only a single gene region. This sentence is a nonsense because diagnostic is based always on a single target. Otherwise being proved that the 156 intron is common in Pseudogymnoascus species, another target region is needed.

L197 were located please change to ..fall in clade A

L200 they are new and unknown species. Please change to they represent a new undescribed species

L202 "Pseudogymnoascus are biased by the region".  What does it mean? 

Authors' contributions should be rewritten following the instruction for authors' template.

L210-211 Nevertheless, they were confirmed to be distributed in a wide range, belonging to clades A, B, C, D, H, and J. – this is a description, not a discussion

L211-212, this sentence is not supported by the data given. No comparison with other researches

Does WNS  given by Pd only? What is the genetic variability with the Pd species?

Author Response

Thank you for your kind comments. I attached revision note

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The authors partially addressed the concerns raised in the previous run, but others are still pending. The English still needs attention. The abstract still misses the standard requirements of scientific communication as the reason for species-specific primers use has yet to be disclosed before the results. The same is in the introduction, where the PCR-based identification is mentioned in the last rows without explaining the reason for its use. Please amend the discrepancies in the text about, for example, the number of samples taken 83+53/83+64.

 The sentence about the Fungi Kingdom's wideness is still in the abstract opening, contrary to the authors' declaration. Moreover, now it also conflicts with the information given at L28. Please remove it.

 L11 “Among these, caves are unique environments with various limitations” this sentence crashes with the previous one because changes the subject…most substrates. Among these (substrates), caves are unique environments. Please rework. Moreover, it is not clear why the caves are unique. Reading the sentence, it seems that caves are unique because they have various limitations. Many environments have various limitations, so the sentence should be reworked.

 L12-13 “the causative agent of white-nose syndrome (WNS) that is a fungal emerging disease of hibernation bats”. This subordinate sentence is too long and contains grammar errors.

 L15 “…investigated to confirm the presence of Pd and the diversity of cave fungi including Pseudogymnoascus genera”. The sentence is redundant. Rewrite, simplifying it.

 L18. Twenty Pseudogymnoascus species were identified. Twenty isolates belong to the Pseudogymnoascus genus.

 L28 Fungi are the second-largest eukaryote and organotrophic microorganisms…second largest what? Something missing. Rework the sentence correcting the grammar errors.

L30-31 every substrate on Earth, from deep-sea deposits to animal skin. The authors still create confusion between the substrate with the environment.

L33 varied physicochemical micro-gradients… please elucidate.

 L33 is generally considered to be low. As previously stressed, several papers underline as caves harbour unexpectedly high biodiversity (see Tomczyk-Żak and Zielenkiewicz, 2016; Hershey and Barton, 2018). Please correct.

 L34 fungal biodiversity is redundant, the prefix bio- is not necessary because it is well known that fungi are organisms and then BIO

 L39 Among these, cave-inhabiting bats are estimated to be the most important factor in the inflow of fungal spores [18,19]. This sentence cannot be applied to all caves, even because there are many kinds of caves.

 L43 commonly found in cold areas. Not clear please rework

L46 “..soared in 2009” change to soared since 2009

L48 This discovery has led to in-depth phylogenetic research of fungi living in bat caves. This sentence could be discarded because implicit in the previous one. References 24 and 27 can be included in the previous citation.

L49-53 Minnis and Lindner reorganized Geomyces species and allies through phylogenetic analyses using five locus gene sequence concatenations….species have been reported until recently. Please, change to "Minnis and Lindner reorganized Geomyces and allied species after a multi-locus analysis. Geomyces destructans has been included in the Pseudogymnoascus genus [27], and many other species have been described in the last years [24,27-29]"

 L64-86 In general, this section should be synthesized, removing the exceeding info out of the scope of this research.

 L64-65 In 2018, we were provided a picture of a bat carcass from Geum cave showing WNS- 64 like symptoms (Dr. Chung CU, personal communication; Figure 1). Rework the sentence

L65 Figure 1 is not necessary please remove it or move to supplementary material

 L68-70 Environmental and bat samples were collected from three karst caves (Geum: 67 36°59′ N, 128°21′ E; Eun: 37°00′ N, 128°21′ E; and Handemy: 36°59′ N, 128°26′ E) inhabited by Vespertilionidae and Rhinolophidae bats in Danyang, South Korea, from September 2019 to April 2020 (Figure 2). Please reorganize the sentence giving the right order.

 L70-71 The sentence is unclear and can be removed since the temperature and RH ranges are given immediately after.

 L75 The specialist in bat ecology verified the species and gender of the captured bats. This info is not fruitful for the research aim. Remove it

 L76-77Skin swabs were stroked approximately 10 times, focusing on the bat's wing membrane, ears, nose, and side of the mouth. In this order, the sampling swab could be the vehicle for infection, potentially moving Pd spores from the skin to target mucosae such as the mouth and nose. Please explain.

 L81-82 “All bats in the caves were examined for the presence of Pd using ultraviolet (UV) light at a wavelength of 365 nm (Analytik Jena, Jena, Germany) [32,33]. All bats showed negative results for the UV light test.” I suppose this test has been performed before sampling. For this reason, it should be moved up there.

 L98 …”83 environmental samples and 64 bat samples” this information competes with the previous section (2.1 Sample collection). Moreover, it is too general. Describe the environmental samples, how many from each cave, how many bats were taken, etc. Please check the number’s consistency.

 L 99 As previously, the supplier of Sabouraud medium should be added

L100  swab swabs please rework the sentence avoiding repeats

 L110 Total DNA of single fungal colonies was extracted using please change to Genomic DNA from pure fungal colonies was extracted…

 L112 The extracted genomic DNA was primarily classified at the… the isolates were first identified at genus level by ITS sequencing and BLASTn comparison.

 L113-116 The fungal DNA identified as corresponding to the Pseudogymnoascus genus was amplified using a primer for five molecular markers: ITS, 28S large subunit rRNA gene (LSU), translation elongation factor 1 alpha (TEF1-α), minichromosomal maintenance protein 7 (MCM7), and RNA polymerase II second largest subunit (RPB2). The sentence should be improved as, for example, The strains belonging to the Pseudogymnoascus genus were furtherly processed for 28S large subunit rRNA gene (LSU), translation elongation factor 1 alpha (TEF1-α), minichromosomal maintenance protein 7 (MCM7), and RNA polymerase II second largest subunit (RPB2).

 L119 As before https://journals.sagepub.com/doi/pdf/10.1177/104063871002200208 five primers have been proposed. Please, explain your choice.

 L124 After purification, all samples were sent for barcode-tagged sequencing (BTSeqTM; 124 CELEMICS, Seoul, South Korea)- amplicon purification is common and generally skipped in M&M descriptions. More generally, the sentence could be changed to Sequencing has been performed by…

 Table S2 and Figure 5. One ex-type only is indicated in table S2. Each ex-type strain should have indicated in both figure and table.

 L152 ITS sequencing doesn’t allow “classification” but closest identification. Please amend this and the analogues along with the text.

 L152 the proportions of isolated fungi were as follows: / they are not proportions but occurrence frequencies!

 L154 The family most isolated – please correct

L155-156 The most 155 dominant genera were – please correct

 L207-212 The explanation proposed is far to be solid. The false positives recorded are quite obvious since the primers were designed on a well-conserved molecular marker. The authors used only Pd species without siblings since most weren’t described yet. It should be investigated if the WND is caused by Pd only or if siblings can be involved too.

Table S1 the hold step at 4°C should be removed from all the PCR protocols because it is not functional to the reaction success.

Author Response

Dear Editor

 

Thank you for inviting us to submit a revised draft of our manuscript entitled, "Fungal diversity in Korean caves and cave-inhabiting bats with attention to Pseudogymnoascus species" to Journal of fungi. We also appreciate the time and effort you and each of the reviewers have dedicated to providing insightful feedback on ways to strengthen our paper. Thus, it is with great pleasure that we resubmit our article for further consideration. We have incorporated changes that reflect the detailed suggestions you have graciously provided. We also hope that our edits and the responses we provide below satisfactorily address all the issues and concerns you and the reviewers have noted.

 

To facilitate your review of our revisions, the following is a point-by-point response to the questions and comments.

 

Reviewer #2:

The authors partially addressed the concerns raised in the previous run, but others are still pending. The English still needs attention.

RESPONSE: We got English revision 2 times from a professional language editing company "Editage". We described this in the "Acknowledgments" section.

 

The abstract still misses the standard requirements of scientific communication as the reason for species-specific primers use has yet to be disclosed before the results.

RESPONSE: We conducted a diagnostic test using a Pd-specific primer to check whether Pseudogymnoascus destructans was present among the isolated Pseudogymnoascus genera. We described this in lines 17–19.

 

The same is in the introduction, where the PCR-based identification is mentioned in the last rows without explaining the reason for its use.

RESPONSE: The information regarding why PCR-based identification was used has been added in lines 50–56.

 

 

Please amend the discrepancies in the text about, for example, the number of samples taken 83+53/83+64.

RESPONSE: The discrepancy in the text about the number of collected samples was corrected to 83+53. We corrected in line 94.

 

 The sentence about the Fungi Kingdom's wideness is still in the abstract opening, contrary to the authors' declaration. Moreover, now it also conflicts with the information given at L28. Please remove it.

RESPONSE: We removed sentence that “Fungi are a large kingdom of eukaryotes with an estimated 12 million species.”

 

L11 “Among these, caves are unique environments with various limitations” this sentence crashes with the previous one because changes the subject…most substrates. Among these (substrates), caves are unique environments. Please rework. Moreover, it is not clear why the caves are unique. Reading the sentence, it seems that caves are unique because they have various limitations. Many environments have various limitations, so the sentence should be reworked.

RESPONSE: We removed existing sentence and replaced it with a new one “Pseudogymnoascus is a psychrophilic fungus, which is a genus widely distributed in cold regions around the world.” in lines 10–11.

 

L12-13 “the causative agent of white-nose syndrome (WNS) that is a fungal emerging disease of hibernation bats”. This subordinate sentence is too long and contains grammar errors.

RESPONSE: We changed sentence from “the causative agent of white-nose syndrome (WNS) that is a fungal emerging disease of hibernation bats“ to “the causative agent of white-nose syndrome (WNS), belonging to Pseudogymnoascus“ in line 11–12.

 

L15 “…investigated to confirm the presence of Pd and the diversity of cave fungi including Pseudogymnoascus genera”. The sentence is redundant. Rewrite, simplifying it.

RESPONSE: We changed sentence from “investigated to confirm the presence of Pd and the diversity of cave fungi including Pseudogymnoascus genera” to “investigated the diversity of cave fungi, and tried to confirm the presence of Pd” in line 14–15.

L18. Twenty Pseudogymnoascus species were identified. Twenty isolates belong to the Pseudogymnoascus genus.

RESPONSE: The sentence was changed from “One hundred and fifty-four fungal strains belonging to 31 different genera were isolated. Twenty Pseudogymnoascus species were identified.” to “One hundred and fifty-four fungal strains belonging to 31 different genera were isolated, and 20 of 154 were confirmed to belong to Pseudogymnoascus.” in lines 16–17

 

L28 Fungi are the second-largest eukaryote and organotrophic microorganisms…second largest what? Something missing. Rework the sentence correcting the grammar errors.

RESPONSE: The sentence was revised from “Fungi are the second-largest eukaryote and organotrophic microorganisms…second largest” to “Fungi are organotrophic microorganisms and the second-largest eukaryote” in lines 28–29

 

L30-31 every substrate on Earth, from deep-sea deposits to animal skin. The authors still create confusion between the substrate with the environment.

RESPONSE: The word was revised form “substrate” to “environment” in line 30.

 

 

L33 varied physicochemical micro-gradients… please elucidate.

RESPONSE: This sentence was extracted from "Phylogenetic diversity of culturable fungi in the Heshang Cave, central China (Man et al. 2015)”, and modified, but this part was deleted.

 

L33 is generally considered to be low. As previously stressed, several papers underline as caves harbour unexpectedly high biodiversity (see Tomczyk-Żak and Zielenkiewicz, 2016; Hershey and Barton, 2018). Please correct.

RESPONSE: This sentence was corrected form “In these stringent environments, fungal biodiversity is generally considered to be low. However, many authors underlined that cave/underground environments are characterized by unexpected diversity and are a reservoir for undescribed species” to “In these stringent environments, fungal biodiversity is generally considered to be low. However, several papers underline as caves harbor unexpectedly high diversity” in lines 33–35.

 

 

 L34 fungal biodiversity is redundant, the prefix bio- is not necessary because it is well known that fungi are organisms and then BIO

RESPONSE: The prefix “bio” was removed in line 33.

 

 L39 Among these, cave-inhabiting bats are estimated to be the most important factor in the inflow of fungal spores [18,19]. This sentence cannot be applied to all caves, even because there are many kinds of caves.

RESPONSE: This sentence was revised from “estimated to be the most important factor” to “estimated to be one of the most important factors” in line 38.

 

 

L43 commonly found in cold areas. Not clear please rework

RESPONSE: This sentence was changed from “The genus Pseudogymnoascus is widely distributed worldwide and reportedly present on various substrates. Furthermore, they are psychrophilic fungi, commonly found in cold area.” to “In addition, the genus Pseudogymnoascus is a keratolytic and psychrophilic fungus that had wide geographic distribution in cold regions worldwide” in lines 40–42.

 

L46 “..soared in 2009” change to soared since 2009

RESPONSE: This sentence was corrected from “soared in 2009” to “soared since 2009” in line 45.

 

L48 This discovery has led to in-depth phylogenetic research of fungi living in bat caves. This sentence could be discarded because implicit in the previous one. References 24 and 27 can be included in the previous citation.

RESPONSE: This sentence was removed, and references was included in the previous citation (line 46).

 

 

L49-53 Minnis and Lindner reorganized Geomyces species and allies through phylogenetic analyses using five locus gene sequence concatenations….species have been reported until recently. Please, change to "Minnis and Lindner reorganized Geomyces and allied species after a multi-locus analysis. Geomyces destructans has been included in the Pseudogymnoascus genus [27], and many other species have been described in the last years [24,27-29]"

RESPONSE: The sentence was changed from “Minnis and Lindner reorganized Geomyces species and allies through phylogenetic analyses using five locus gene sequence concatenations….species have been reported until recently.” to “Minnis and Lindner reorganized Geomyces and allied species after a multi-locus analysis. Geomyces destructans has been included in the Pseudogymnoascus genus, and many other species have been described in the last years.” in lines 47–49.

 

L64-86 In general, this section should be synthesized, removing the exceeding info out of the scope of this research.

RESPONSE: We removed Fig1, bat gender information, and Measurement cycle information for cave environmental conditions.

 

L64-65 In 2018, we were provided a picture of a bat carcass from Geum cave showing WNS-like symptoms (Dr. Chung CU, personal communication; Figure 1). Rework the sentence

RESPONSE: This sentence was reworked from “In 2018, we were provided a picture of a bat carcass from Geum cave showing WNS-like symptoms (Dr. Chung CU, personal communication; Figure 1).” to “In 2018, we were provided information about bat showing WNS-like symptoms in Geum cave by a bat ecologist (Dr. Chung CU, personal communication).” In lines 67–68.

 

L65 Figure 1 is not necessary please remove it or move to supplementary material

RESPONSE: Figure1 was removed.

 

 

 

L68-70 Environmental and bat samples were collected from three karst caves (Geum: 67 36°59′ N, 128°21′ E; Eun: 37°00′ N, 128°21′ E; and Handemy: 36°59′ N, 128°26′ E) inhabited by Vespertilionidae and Rhinolophidae bats in Danyang, South Korea, from September 2019 to April 2020 (Figure 2). Please reorganize the sentence giving the right order.

RESPONSE: This sentence was reworked to “Environmental and bat samples were collected from three karst caves (Geum: 36°59′ N, 128°21′ E; Eun: 37°00′ N, 128°21′ E; and Handemy: 36°59′ N, 128°26′ E) in Danyang, South Korea, during September 2019 to April 2020 (Figure 1).” in lines 70–72.

 

L70-71 The sentence is unclear and can be removed since the temperature and RH ranges are given immediately after.

RESPONSE: “Temperature and humidity measurements were performed every 10 min for approximately 2 h when samples were collected; furthermore, these parameters were measured every three months.” This information was prepared at the request of another reviewer. We reflecting your opinion, and deleted this information.

 

L75 The specialist in bat ecology verified the species and gender of the captured bats. This info is not fruitful for the research aim. Remove it

RESPONSE: This information was removed.

 

L76-77 Skin swabs were stroked approximately 10 times, focusing on the bat's wing membrane, ears, nose, and side of the mouth. In this order, the sampling swab could be the vehicle for infection, potentially moving Pd spores from the skin to target mucosae such as the mouth and nose. Please explain.

RESPONSE: Fungal culture and/or polymerase chain reaction (PCR) of skin swabs are effective diagnostic methods(https://www.woah.org/app/uploads/2022/02/pseudogymnoascus-destructans-in-bats-white-nose-syndrome-infection-with.pdf, p3). WNS also occurs during the hibernation of bats. Bats captured during hibernation did not have fluorescence reactions under UV and did not show clinical symptoms. Considering that bats live in groups to maintain their body temperature, the movement of pathogens caused by cotton swabs is considered negligible.

 

 

 

L81-82 “All bats in the caves were examined for the presence of Pd using ultraviolet (UV) light at a wavelength of 365 nm (Analytik Jena, Jena, Germany) [32,33]. All bats showed negative results for the UV light test.” I suppose this test has been performed before sampling. For this reason, it should be moved up there.

RESPONSE: This information was moved to lines 76–77, before information about sampling.

 

L98 …”83 environmental samples and 64 bat samples” this information competes with the previous section (2.1 Sample collection). Moreover, it is too general. Describe the environmental samples, how many from each cave, how many bats were taken, etc. Please check the number’s consistency.

RESPONSE: The discrepancy in the text about the number of collected samples was corrected to 83+53. We corrected in line 94.

 

L 99 As previously, the supplier of Sabouraud medium should be added

RESPONSE: Supplier of Sabouraud medium was added in line 95.

 

L100  swab swabs please rework the sentence avoiding repeats

RESPONSE: The sentence was revised from “skin swabs, and oral swabs” to “skin and oral swabs” in lines 96–97

 

 L110 Total DNA of single fungal colonies was extracted using please change to Genomic DNA from pure fungal colonies was extracted…

RESPONSE: The sentence was changed from “Total DNA of single fungal colonies was extracted” to “Genomic DNA from pure fungal colonies was extracted” in line 106.

 

 L112 The extracted genomic DNA was primarily classified at the… the isolates were first identified at genus level by ITS sequencing and BLASTn comparison.

RESPONSE: The sentence was revised from “The extracted genomic DNA was primarily classified at the” to “the isolates were first identified at genus level by internal tran-108 scribed spacer (ITS) sequencing and BLASTn comparison.” in lines 108–109.

 L113-116 The fungal DNA identified as corresponding to the Pseudogymnoascus genus was amplified using a primer for five molecular markers: ITS, 28S large subunit rRNA gene (LSU), translation elongation factor 1 alpha (TEF1-α), minichromosomal maintenance protein 7 (MCM7), and RNA polymerase II second largest subunit (RPB2). The sentence should be improved as, for example, The strains belonging to the Pseudogymnoascus genus were furtherly processed for 28S large subunit rRNA gene (LSU), translation elongation factor 1 alpha (TEF1-α), minichromosomal maintenance protein 7 (MCM7), and RNA polymerase II second largest subunit (RPB2).

RESPONSE: The sentence improved from “The fungal DNA identified as corresponding to the Pseudogymnoascus genus was amplified using a primer for five molecular markers: ITS, 28S large subunit rRNA gene (LSU), translation elongation factor 1 alpha (TEF1-α), minichromosomal maintenance protein 7 (MCM7), and RNA polymerase II second largest subunit (RPB2).” to “The strains belonging to the Pseudogymnoascus genus were furtherly processed for 28S large subunit rRNA gene (LSU), translation elongation factor 1 alpha (TEF1-α), minichromosomal maintenance protein 7 (MCM7), and RNA polymerase II second largest subunit (RPB2).” in lines 109–112

 

 L119 As before https://journals.sagepub.com/doi/pdf/10.1177/104063871002200208 five primers have been proposed. Please, explain your choice.

RESPONSE: Information of used Pd-specific primer were added in lines 112–114.

 

L124 After purification, all samples were sent for barcode-tagged sequencing (BTSeqTM; 124 CELEMICS, Seoul, South Korea)- amplicon purification is common and generally skipped in M&M descriptions. More generally, the sentence could be changed to Sequencing has been performed by…

RESPONSE: Amplicon purification sentence was removed. Sentence was changed from “After purification, all samples were sent for barcode-tagged sequencing” to “Sequencing has been performed by barcode-tagged sequencing” in lines 117–118.

 

Table S2 and Figure 5. One ex-type only is indicated in table S2. Each ex-type strain should have indicated in both figure and table.

RESPONSE: The word “ex-type” was removed in Table S3 (former Table S2).

 

 

 L152 ITS sequencing doesn’t allow “classification” but closest identification. Please amend this and the analogues along with the text.

RESPONSE: The text was amended from “In the classification of isolated” to “In the identification of isolated” in line 147.

 

 L152 the proportions of isolated fungi were as follows: / they are not proportions but occurrence frequencies!

RESPONSE: The text was amended from “proportion” to “occurrence frequencies”

 

L154 The family most isolated – please correct

RESPONSE: The sentence was corrected from “The family most isolated was” to “The most abundantly isolated family was” in lines 151–152.

 

L155-156 The most dominant genera were – please correct

RESPONSE: The sentence was changed from “The most dominant genera were” to “The highest-frequency isolated genera were” in lines 153–154

 

L207-212 The explanation proposed is far to be solid. The false positives recorded are quite obvious since the primers were designed on a well-conserved molecular marker. The authors used only Pd species without siblings since most weren’t described yet. It should be investigated if the WND is caused by Pd only or if siblings can be involved too.

RESPONSE: Only Pseudogymnoascus destructans causes white-nose syndrome. It has never been reported that sibling species in the same genus cause white-nose syndrome. References were added to support the hypothesis in lines 218–224.

 

Table S1 the hold step at 4°C should be removed from all the PCR protocols because it is not functional to the reaction success.

RESPONSE: All hold steps at 4°C have been removed from Table S2 (former Table S1).

Author Response File: Author Response.docx

Round 3

Reviewer 2 Report

Dear Authors,

Please change the first sentence, "Pseudogymnoascus is a psychrophilic fungus, which is a genus widely distributed in cold...", to "Pseudogymnoascus is a psychrophilic fungal genus, whose members are widely distributed in cold regions.."

regions around the world.

regions around the world. 

Author Response

Reviewer #2:

Please change the first sentence, "Pseudogymnoascus is a psychrophilic fungus, which is a genus widely distributed in cold...", to "Pseudogymnoascus is a psychrophilic fungal genus, whose members are widely distributed in cold regions.."

regions around the world.

RESPONSE: The sentence was changed from "Pseudogymnoascus is a psychrophilic fungus, which is a genus widely distributed in cold regions around world", to "Pseudogymnoascus is a psychrophilic fungal genus, whose members are widely distributed in cold regions around world " in lines 10–11.

Author Response File: Author Response.docx