Fungal Associates of the Moss Leucobryum candidum (Brid. ex P. Beauv.) Wilson in Southeast Queensland, Australia

Comment- 306 How were these assessed? endophytes etc It would be more useful to have the molc tree before figure 3, then one could see where the assessment came from. But in any case, it was somewhat arbitrary.

Position has changed.

These were assessed based on their molecular sequencing.

Comment- Fungi on bryophytes are generally discomycetes, such as, Lizonia, Lamprospora and Octospora. Why did you not encounter any of these? This needs to be brought into the discussion. Comment- Most of the fungi isolated are typical endophytes rather than the species we normally find on mosses and liverworts, a topic I have personally worked on. Again, there has to be a discussion of this aspect

We have now included more on this in the discussion.
We were focussed on fungi living inside bryophyte tissues. As taxonomic and molecular data fail to support the monophyly of the Discomycetes we chose to use Ascomycete. Bryosymbiotic fungi can be species specific, and our study was also region specific.

Comment- One further point: Was the tree is rooted through the basidiomycetes. These needs stating.

Yes, thank you this is stated now.

Comment- I am very concerned about the TEM study. As one who has studied fungi within plants this is a very superficial study. You say they intracellular, so how do they enter the cell? why did you not see hyphae between host cells?

Agreed and the section has been removed.

Comment- You need to revise the legends to the figures

Figures removed

Comment- you can use the word hyphal 401 (B) fungal cells that appear to have undergone mitosis. You cannot assume this at all. These are hyphae and cell division would only be possible by sequential sections. So, delete this observation.

Removed

Comment- 401 C unknown structure 401(U) near thin cell wall surely a hypha

Removed

Comment- 402 (D) fungal hyphae (H) with dark cell walls. This is rubbish as you are looking at a TEM preparation. Its only dark because the wall is thicker etc.

Removed

Comment- 409 The phylogenetic tree Figure 8 This would have been more useful earlier in the result section.

Done

Comment- 426 and not italics

Corrected

Comment- Lecanicillium brown rot or wood rot. I am not sure in what sense you use brown rot, this is a term is used for a particular type of wood decay, and not appropriate to bryophytes, where there is little lignin etc.

The text refers to the substrate suitability for the moss this is now clarified in amended text.

Comment- The following two paragraphs are highly speculative

One fungus within the Lecanicillium genus, isolated from the Mt. Mee sample, is associated with causing brown rot or wood rot, which is commonly found within rotting logs, the substrate of Leucobryum candidum in this study. This might be due to the types of fungal associates present in L. candidum are likely to be impacted by micro-environmental factors such as wood texture and substrate species. Leucobryum candidum seems to prefer a substrate of rotting logs. As brown rot consumes woody material, the substrate becomes softer and spongier, can hold more moisture and may become amenable and favourable for Leucobryum spore germination.

We have reduced the text of this paragraph to make it less speculative

Comment- The BLAST match of isolates USC-F400 and USC-F434, Ophiostoma eucalyptigena (isolated from the host genus Eucalyptus), scored <97% and thus cannot be confirmed past order level. Whilst unreportable, these isolates may be related the Ophiostoma genus, particularly as the two isolates showed phylogenetic affinities with high (100%) bootstrap confidence to members of the Ophiostomatales order, which are often found within plant debris or soil [50] and are well-known associates of bark beetles and act as parasites of some plants [51] namely eucalypts [52]. Eucalypts were dominant in the overstory vegetation composition of most collection sites and may have been the species of rotting log substrate the mosses were sampled from. If sporulating fungi residing in the overstorey release their spores they may land and germinate on moss. If so, some of the fungi cultured may have been using the moss as a transient host until they found a preferable one.

This paragraph has been reduced to make less speculative

Conclusion

Comment- You opine: This study makes a valuable scientific contribution within the field of bryomycology particularly as no known studies have yet focused on the fungal associates of L. candidum or its growth capabilities in artificial systems.

This has the making of a great paper, but it needs critical editing of deductions made, especially the two paragraphs highlighted above.

Comment- You say: Further, this study reports the first observation of fungal structures within the cells of L. candidum. I do not think your TEM work is sufficiently detailed to say this. How did the fugus get into the cell? Was it one fungus in your TEM? For real input serial sections need to be undertaken

This section has been removed.

Comment- You say: has opened pathways for future studies

I concur with this and it is why a critical editing is necessary.

Revisions are made, thank you.

4] What proportions of trophic groups of bryophilous fungi are characteristic of different stages of moss development? Perhaps a logical conclusion in the MS can be added that not only in America and Eurasia little is known about the diversity and ecology of bryophilous fungi, but the same situation in Australia.

Study prepared background for future studies and as you have pointed geo diversity has to be investigated.

5] Other corrections;

Dear Authors, after a brief review of the manuscript, I could not find the information if your newly obtained sequences from our isolates were submitted to any nucleotide database (e.g. NCBI or similar)?

Further investigations are underway, and sequence submission will be done in the near future.

6] Also, there is a discrepancy in information in results, where in the first sentence you claim to isolate 70 strains, while further only show and identify "selected" 33 (Table 3) or 22 (Table S3). Please explain and preferably show identifications for all 70 isolated strains.

Thank you for pointing out this disconnect. This was partially addressed however, a clearer explanation for why we chose these 33 has now been added in the first paragraph of the results section.

REVIEWER #2

1] Comment- The authors attempted to assess the set of fungi associated with the Australasian moss species Leucobryum candidum. This was done with the aim of studying the taxonomic spectrum of bryophilic fungi to understand the symbiotic functionality of mosses, as well as the ecosystems in which they live. The authors emphasize that in-depth studies of the taxonomic diversity, ecology and physiological functions of bryophilous fungi are still lacking. The authors identified fungi from 10 orders and 17 families in the samples of L. candidum moss. Twenty-five of the 33 isolates identified using molecular sequencing methods were unique species, confirming the high beta diversity of L. candidum fungal associates collected from coastal, forest and urban environments in south-eastern Queensland.The largest number of cultured isolates were obtained from coastal and forest sites.The smallest number of cultivated isolates were found in urban areas.

The work is interesting and relevant. However, there are a number of questions:

The authors aimed to assess taxonomic diversity, but not a single isolate was identified to the genus and species level.

Thank you for this feedback, the aims and context have been clarified to make intent of the study clearer and we have now clarified the level of diversity that was assessed.

2] Comment: The authors also point out that little is known about the ecology of bryophilous species, but the results of this study did not bring us any closer to answering this question, since the manuscript states that the identified associates have various endophytic, saprophytic and parasitic roles in vascular plants.

Such information is widely known.

As a result, not a single “new” species was added to the list of bryophilous fungi in Australia or the world, just as not a single one already known to science was found.

This is now addressed within the manuscript. Based on the rule-of-thumb <3% species delineation for fungal ITS sequences (Nilsson et al. 2008), isolates USC-F434, USC-F400, USC-F404 and USC-F433 (sequence BLAST matches <97%.) are species without reliable sequence matches. These 4 isolates could represent new species however further sequencing will need to confirm this.

3] Comment: This study did not bring us any closer to answering the question: are bryophilous species pathogens or saprotrophs? Obligate or facultative?

Actually, we found that bryophilous species associated with this particular moss can be either as some isolates were identified as saprotrophs whilst others were identified as endophytes or parasites-this is clearly stated in figure 2. (Saprotrophic fungi comprised of 48.48% of isolates. Endophytic ascomycetes formed 33.3% of total isolates and parasitic fungi accounted for 18.19%).

4] What proportions of trophic groups of bryophilous fungi are characteristic of different stages of moss development? Perhaps a logical conclusion in the MS can be added that not only in America and Eurasia little is known about the diversity and ecology of bryophilous fungi, but the same situation in Australia.

Study prepared background for future studies and as you have pointed geo diversity has to be investigated.

5] Other corrections;

Dear Authors, after a brief review of the manuscript, I could not find the information if your newly obtained sequences from our isolates were submitted to any nucleotide database (e.g. NCBI or similar)?

Further investigations are underway, and sequence submission will be done in the near future.

6] Also, there is a discrepancy in information in results, where in the first sentence you claim to isolate 70 strains, while further only show and identify "selected" 33 (Table 3) or 22 (Table S3). Please explain and preferably show identifications for all 70 isolated strains.

Thank you for pointing out this disconnect. This was partially addressed however, a clearer explanation for why we chose these 33 has now been added in the first paragraph of the results section.

REVIEWER #3

1] Page 2: please italicize

Done all along the manuscript

2] Please italicise and check if higher taxonomic ranks should be in italic.

Done all along the manuscript

3] Italicise and check thoughout the text that all Latin names are in italic. A species name is only written in full on the first mention in the text. After the first mention, in all other cases the generic name is customarily abbreviated to its first letter, unless it is in the beginning of the sentence (and then written in full), and the specific epithet is written in full. Please, check and correct throughout the text.

Done all along the manuscript

4] Always use m-dash in-between numbers.

Added

5] Please, provide suitable reference(s) to support this statement, which is otherwise certainly correct.

Done

6] There is published reference for the BLAST tool. Please, cite.

Done