Fungal Diversity in the Phyllosphere of Pinus heldreichii H. Christ—An Endemic and High-Altitude Pine of the Mediterranean Region

Round 1

Reviewer 1 Report

The author investigated the fungal community on and within Pinus heldreichii  needles in Montenegro. The approached used NGS methodology, specifically the ITS2 and PacBio sequencing.

There are some major issues with the interpretation of the results as it is currently presented.

1) Was there a DNA extraction kit control?

2) There were 150 samples collected: 6 sites, 5 trees per site, and 5 samples per tree. Was the data analyzed to see how consistent the per tree samples were to each other? how per site trees were consistent to each other? This there a lot of per needle variation? per site variation?

If the objective of the paper is to do a species inventory than this is not needed, but to make assertions beyond an inventory paper this should be completed. I would suggest completing some cluster analyses at the per sample basis to see if each per tree sample clusters closest to each other and that per site samples cluster closest to each other. In addition, dropping singleton sequences  has the potential to remove relevant micro fungi (if this is meant to be an inventory paper). Was the remove of singleton sequences warranted in the community analyses? A cluster analysis with and without singleton sequences should be conducted to see if they should be remove.

3) No section on the limitations of using next generation sequencing. The authors use count data for abundance, DNA presence for community association, and database match for their taxonomic assignments which form the basis of their conclusions but make no mention of the limitations associated with each of these steps. I'm sure they are aware of these limitation but the readers may not be aware.

Minor concerns:

1) NCBI is not a curated database and contains misidentified sequences. Were the sequences also compared against UNITE or RDP to determine if there was consensus in taxonomic assignment?

Additional comments below:

Abstract:

Line 24-25: .....lower altitudes (milder growth conditions), indicating that environmental conditions were among major determinants of fungal communities...

There is not sufficient support for this statement. Suggest altering it to: ...lower altitudes (milder growth conditions), suggesting that environmental conditions were among major determinants of fungal communities

Keywords:

Line 31: Words found in the title are not usually included within the Keywords. Please review the instructions to authors or inquire with editor. It is likely that Pinus heldreichii will need to be removed from the key words.

Introduction:

Lines 34-37: This paragraph has only two sentences and the information is contained within the paragraph below. Suggest removing these two sentences.

Line 38: Although in the past P. heldreichii formed a continuous forest belt in the Balkans, nowadays its forests are scattered and largely isolated. Suggest the following changes: change nowadays to currently

Lines 52-55: This paragraph is two sentences. Usually paragraphs are more than two sentences. Authors should integrate these sentences into another section or make a better connection to the following paragraph.

Fungal paragraphs at and below line 56: Need a better connection to the first part of the paper.     Paragraph Line 56: Fungal and tree interactions introduction

            Paragraph Line66: current fungi identified

            Paragraph Line 77: New fungi found

            Paragraph line 87: pathogenic fungi

Suggestion: Perhaps move the paragraph that starts on line 56 to the beginning of the introduction. In addition, the connections between paragraphs needs further work or shorten the introduction and integrate some of the more detailed information into the discussion to relate the current results with what is already known.

Line 59: Suggest changing the sentence to the following:

determine forest health and sustainability as they modulate stress tolerance, enhance growth...

Line 60-61: The sentence needs further refinement.

Line 65: Suggest changing scare to limited.

Line 70: Morchella esculenta is edible so not sure why it was placed in this list and not the edible list.

Paragraph Line 77: New fungi found. This information may not be needed in the introduction. Introduction typically contains background information needed by reader to understand the investigation. May be more appropriate in the discussion as a way to integrate current results with previously known data.

 

Methods and Materials:

1) This sections should be written as much as possible in the past tense. Currently, most of this section is written in the present tense. Should be specific for 2015 or May 2015. For example, (Line 127: The mean daily summer maximum is between 7 and 11 C......This sentence should reflect the information that occurred when the study was conducted so it should state:....... The mean daily summer maximum was between 7 and 11 C

2) Was there a negative control? No extraction kit control was mentioned?

Lines 143-145: Consider removing these sentence as this information seems to be presented already in Lines 118-119.

Line 172: Consider changing done to completed

Results:

Lines 217-218: The chi-square test showed the largest difference in richness of fungal taxa was between KKO and the remaining sites. Is this KKO or KKN?  Is this appropriate given that the rarefaction showed that fungal taxa detected in all sites did not reach the species saturation (line 219-220)?

Lines 233-240: How are these species defined as pine needle pathogens, pathogenic species, fungal endophytes? The surface of the needles were not sterilized so it is possible that some of the fungi identified were from the surrounding attached soils or inactive. Next generation sequencing approaches amply all DNA within the sample regardless of state (active or inactive), it does not provide information on the life-style of the fungus. It would be more appropriate to state what was found in the results and make a case for the life-style in the discussion based on previous literature.

 

Figure 5:

This figure should contain all 150 data points or minimum the 5 data points from each site.

Discussion:

Line 295-296: If N. germanicum and A. pseudotsugae are the only two taxa recorded for the first time, than just state that. There is no need for e.g.

Line 318: ...was reported previously.  Consider revising to previously reported.

Line 323:.. and Pinus yunnanensis. Consider revising to P. yunnanensis.

Line 325: consider removing e.g.

Line 352: Capitalize the word Cyclaneusma to be consistent with previous capitalization.

Line 354: Similar applies to D. septosporum. Consider revising as it seems awkward as written.

Line 387: The study have also detected.... Consider revising to: This study has also.....

Author Response

Please see atachment , 

GENERAL (additional explanation of study design) (For all reviewers)

The aim of our work was to characterize fungal community associated with needles of P. heldreichii:

• to make an inventory of species associated with this tree species
• to evaluate abundance and composition of fungal communities
• to compare fungal communities among different sampling sites

We were particularly interested in fungal pathogens, which can potentially affect growth and survival of the host tree. Until our study, there was no information about fungal communities associated with needles of this pine species.

In order to gather potentially broader information on fungal community, we selected different sampling sites (6 sites). They were:

• in different geographical areas i.e. covering different regions of Montenegro
• situated under different environmental conditions; we are later referring to these as different growing condition (as harsh or moderate)
• at one locality trees were additionally stressed by presence of biotic enemies/insects..

This approach enabled comparison of fungal communities developed on the same host tree under different environmental pressures. 

• In order to gather potentially higher fungal diversity (including different functional groupos of fungi), for the lab work we used randomly selected parts of both symptomatic and asymptomatic f needles, which were used without surface sterilization.

Regarding the experimental design, fungal community was assessed per each site. In order to get a representative information on fungal communities at each site, multiple samples were taken: 5 needle samples from 5 twigs that were collected from 5 different trees, resulting in 25 needle samples per site. All samples per each site were amplified using primers with the same barcode i.e. 25 needles samples x 1 barcode per site. For the whole study, 150 needles samples x 6 barcodes. This resulted that the variation in fungal communities was not assessed within each site, but only among different sites.

About geographical/species range of P. heldreichii in Montenegro and in our sampling:

Different sampling sites were selected in geographically different regions of Mne. They represent characteristic and “representative” sites of species distribution on western, central and eastern areas of Mne with a geographical distance of ca 120 km among most distant sites. The sites were also at the different distance from the Adriatic sea, which is important “factor” influencing P. heldreichii distribution. Sampling was done on localities were host populations/ forests are relatively large, “important”and “representative”. It was not intention or idea to sample on all possible sites of P. heldriechii distribution in Montenegro.

It is also important to mention that the chosen localities, were monitored for tree health and general crown conditions for a long period of time i.e. more than 10 years. As it is written in the beginning of M&M section, our forest stands were generally (not ideally, but) healthy-looking, thereby representing in most cases “typical” host trees and environmental condition for each site.

Moreover, regarding the comment by R2, localities, which are mentioned in the report as Tara canyon, Durmitor NP and Hajla (Rožaje), were not  representative for P. heldreichii (although some group of trees can exist on Hajla, that it is a typical spruce habtat; in NP Durmitor P. nigra, not P. heldreichii is present). Discussion about range / distribution of P. heldreichii in Mne is not under the scope of this paper, but we acknowledge the comment about wording “whole” range, which was changed in the revised version of the manuscript. Please observe, the data presented on a P. heldreichii distribution map is not completely accurate, and thus, we added two larger areas, which previously been missing.

Further, we have localities under different environmental pressures (referred as “harsh” and “moderate”).

About climate data and soil:

General characteristics of the climate and soil of (mountain) P. heldreichii sites are given in the Methodology section. In the Table 1, the climate type on different sampling sites according to Koppen climate classification [23] is given. In the text (lines 107-114) the data is used from climate Atlas of Montenegro [22]. The values were calculated based on 30 year series of observations and measurements (officially available data).

In Montenegro, measurements of climatic parameters are provided only from the limited number of meteorological stations, usually from lower altitudes. We do not have precise measurements of climatic parameters for sampling sites, and these were not measured by ourselves.

We are using those parameters to characterize overall climate and growing conditions for P. heldreichii trees (and associated biodiversity) at the sites, and not to present the situation at each locality during the sampling season. The same is for soil, a short general description of soil types and processes were provided.

Work with needles and needle fungal communities:

All samples collected within each site were used as pseudo-replicates. We analysed fungal community “as a whole” on each site, and compared it with fungal communities present on other sites.  

The fungal community at each site was represented by 25 needle/DNA samples (please also see above the description of experimental design). We have selected up to 5 (representative) needles per twig, with or without disease symptoms, and cut into 0,5-1 cm long segments, which were randomly selected for DNA extraction (in amounts that can fit into a 2 ml tube for grinding and extraction).

Needles were from the part of the twig which was 2-4 year old. Current /one year old needle are mainly green and rarely with symptoms, and thus, in order to get a good representation of both pathogens and other fungal species, these needles were excluded. It is not difficult to distinguish the age of needles for the last few seasons in pines. It is even more straightforward in the case of P. heldreichii as needles are grouped for each year, likely due to a short growing season. On figure 2, please see difference between 1 and 2 years old needles, and on the bottom, some fragments of 3 years old needles.

According to Editor recommendation, changes in Bioinformatic section to avoid duplications.

A map in Figure 2 with P. heldreichii distribution range has been changed because of copyrights but includes similar content.  

 

There are some major issues with the interpretation of the results as it is currently presented.

• Was there a DNA extraction kit control?

There was no DNA extraction control, butwe used the best practices of DNA isolation. This does not supplement the control, but according to Lindahl et al., 2013, extraction control is not necessarily required. To avoid possible cross-contaminations, DNA extractions were done per each site separately, in sterile UV hoods, using pipette tips with filters and new sets of chemicals.

Lindahl, B.D.; Nilsson, R.H.; Tedersoo, L.; Abarenkov, K.; Carlsen, T.; Kjøller, R.; Kõljalg, U.; Pennanen, T.; Rosendahl, S.; Stenlid, J., et al. Fungal community analysis by high-throughput sequencing of amplified markers--a user's guide. New Phytol. 2013, 199, 288–299.

2) There were 150 samples collected: 6 sites, 5 trees per site, and 5 samples per tree. Was the data analyzed to see how consistent the per tree samples were to each other? how per site trees were consistent to each other? This there a lot of per needle variation? per site variation?

Briefly, in the present study we did not assess within site variations. Please also see out explanation about experimental design on how the fungal community from one locality was treated. This also provides other information, and answers on separate questions by Reviewevers.

Some necessary explanation were missed in our previous text/ manuscript. Now more descriptions were  provided in material and method section (Lines 133-135, 160-163, 170-171)

If the objective of the paper is to do a species inventory than this is not needed, but to make assertions beyond an inventory paper this should be completed. I would suggest completing some cluster analyses at the per sample basis to see if each per tree sample clusters closest to each other and that per site samples cluster closest to each other. In addition, dropping singleton sequences has the potential to remove relevant micro fungi (if this is meant to be an inventory paper). Was the remove of singleton sequences warranted in the community analyses? A cluster analysis with and without singleton sequences should be conducted to see if they should be remove.

According to Lindahl et al., 2013, singletons should be removed.

It is true that many singletons may represent rare fungal taxa, but also, they can be a consequence of sequencing biases, and can be unreliable.  In our data set, we detected 375 fungal taxa.  Most abundant taxa were represented with almost 4000 sq, but 86 taxa were represented by only 2 sequences.  Having in mind limitations of the NGS, in order to increase the number of detected species associated with P. heldreichii, we could either increase the sequencing depth or change the strategy for sampling, i.e. do the sampling few times during the season, and to include more samples per locality.  

3) No section on the limitations of using next generation sequencing. The authors use count data for abundance, DNA presence for community association, and database match for their taxonomic assignments which form the basis of their conclusions but make no mention of the limitations associated with each of these steps. I'm sure they are aware of these limitation but the readers may not be aware.

To address this issue, the following was included with references: “While using high-throughput sequencing, we need to learn how to solve potential limitations that can include methodological biases, limitations of markers and bioinformatics challenges”(Lines 293-295) .Regarding using the read data to estimate the relative abundance, Porazinska et al. (2010) have showed that both qualitative and quantitative data of high-throughput sequencing is consistent and highly reproducible.

Porazinska DL, Sung W, Giblin-Davis RM, Thomas WK (2010) Reproducibility of read numbers in high-throughput sequencing analysis of nematode community composition and structure. Mol Ecol Resour 10:666-676. 10.1111/j.1755-0998.2009.02819.x

Lindahl, B.D.; Nilsson, R.H.; Tedersoo, L.; Abarenkov, K.; Carlsen, T.; Kjøller, R.; Kõljalg, U.; Pennanen, T.; Rosendahl, S.; Stenlid, J., et al. Fungal community analysis by high-throughput sequencing of amplified markers--a user's guide. New Phytol. 2013, 199, 288–299.

Tedersoo, L.; Tooming-Klunderud, A.; Anslan, S. PacBio metabarcoding of Fungi and other eukaryotes: errors, biases and perspectives. New Phytol 2018, 217, 1370–1385.

Minor concerns:

1) NCBI is not a curated database and contains misidentified sequences. Were the sequences also compared against UNITE or RDP to determine if there was consensus in taxonomic assignment?

That is true, NCBI is not a curated database and contains misidentified sequences. For species identification we did not obligatory use sequences with highest possible matches but those (still with highest, or almost highest rang (>98 %)), which are based on type material or culture collections by CBS, where it was possible. We have also checked the publication status where sequence belongs. Environmental sequences were avoided, when it was possible. 25 most common species, presented in Table 1 mainly have good  and recently published sequences, provided by eminent experts, and not rarely in publications where taxonomical position of species were discussed. UNITE is a database initially established for ectomycorrhizal fungi, and later expanded with other fungal species. We tested both UNITE and RDP, but in our case there was much lower sequence coverage, mainly because out fungal community is mainly composed of non-mycorrhizal Ascomycetes.

Additional comments below:

Abstract:

Line 24-25: .....lower altitudes (milder growth conditions), indicating that environmental conditions were among major determinants of fungal communities...

There is not sufficient support for this statement. Suggest altering it to: ...lower altitudes (milder growth conditions), suggesting that environmental conditions were among major determinants of fungal communities

Changed as suggested. (NOW Line 25)

 

Keywords:

Line 31: Words found in the title are not usually included within the Keywords. Please review the instructions to authors or inquire with editor. It is likely that Pinus heldreichii will need to be removed from the key words.

It seems this is not required in case with this journal. We checked also with other already published articles, where words repeats, in many cases. We accept the comment. New key words: DNA metabarcoding and Montenegro

Introduction:

Lines 34-37: This paragraph has only two sentences and the information is contained within the paragraph below. Suggest removing these two sentences.

This first paragraph was joined with next paragraph.

Line 38: Although in the past P. heldreichii formed a continuous forest belt in the Balkans, nowadays its forests are scattered and largely isolated. Suggest the following changes: change nowadays to currently

Word nowadays is changed to currently . (NOW Line 41)

Lines 52-55: This paragraph is two sentences. Usually paragraphs are more than two sentences. Authors should integrate these sentences into another section or make a better connection to the following paragraph.

We would like to keep the paragraph in lines 52-55 separately. But the following text in introduction is reorganized (55-58)

Fungal paragraphs at and below line 56: Need a better connection to the first part of the paper.     Paragraph Line 56: Fungal and tree interactions introduction

Part of text between lines 59 and 87 (previously) has been moved from Introduction to Discussion. (Now in lines  413-443)

Some parts of sentences were deleted for better fit. Information about edible mushroom species were deleted. Only information about red-listed and protected species, as well as new described species of Ascomycota retained. Those species are relevant to demonstrate a high value of P. heldreichii habitats as being unique and biodiversity hotspots in high-altitude mountain areas.

            Paragraph Line66: current fungi identified

            Paragraph Line 77: New fungi found

            Paragraph line 87: pathogenic fungi

Suggestion: Perhaps move the paragraph that starts on line 56 to the beginning of the introduction. In addition, the connections between paragraphs needs further work or shorten the introduction and integrate some of the more detailed information into the discussion to relate the current results with what is already known.

Accepted. Explanation is already given above.

Line 59: Suggest changing the sentence to the following:

determine forest health and sustainability as they modulate stress tolerance, enhance growth...

This suggestion is accepted, but now is moved to discussion. Line 413  

Line 60-61: The sentence needs further refinement.

It is now in discussion and changed, line 414-415

Line 65: Suggest changing scare to limited.

 Word scarse is changed to limited. Line65 

Line 70: Morchella esculenta is edible so not sure why it was placed in this list and not the edible list.

Morchella escullenta is edible species. But Morchella esculenta is protected in Montenegro because it is rare and its collection is officially forbidden. The other Morchella species are endangered due to excessive collection and exploitation. This part is now moved to discussion.

Paragraph Line 77: New fungi found. This information may not be needed in the introduction. Introduction typically contains background information needed by reader to understand the investigation. May be more appropriate in the discussion as a way to integrate current results with previously known data.

We acknowledge this observation. The text between lines 59 and 87 (in initial text ) has been moved to discussion and slightly modified. Information about edible mushroom species were deleted. Only information about red-listed and protected species, as well as new described species of Ascomycota retained. Those species are relevant to demonstrate a high value of P. heldreichii habitats as being unique and biodiversity hotspots in high-altitude mountain areas. Now in lines: 421-427, 427-433

 

Methods and Materials:

• This sections should be written as much as possible in the past tense. Currently, most of this section is written in the present tense. Should be specific for 2015 or May 2015. For example, (Line 127: The mean daily summer maximum is between 7 and 11 C......This sentence should reflect the information that occurred when the study was conducted so it should state:....... The mean daily summer maximum was between 7 and 11 C.

Section about climate is written based on official meteorological /climate data. In the Table 1, climate type on different sampling sites according to Koppen climate classification, adjusted for Montenegro [23] is given, and later in description (lines 107-114) based on data from climate Atlas of Montenegro [22]. This data was calculated based on 30 year series of observations and measurements (and those are officially available data). Measurements of climatic parameters were provided only from limited number of meteorological stations in Montenegro, usually from lower altitudes. We do not have precise measurements of climatic parameters for sampling sites, and we were not measuring by ourselves. As it is a continuous data it was written in the present tense.

• Was there a negative control? No extraction kit control was mentioned?

There was no negative control in our experiment, but we used the best practices of DNA isolation (This does not supplement the control, of course). According to Lindahl et al., 2013, extraction control is not necessarily required. We will use such controls in future studies.

Lindahl, B.D.; Nilsson, R.H.; Tedersoo, L.; Abarenkov, K.; Carlsen, T.; Kjøller, R.; Kõljalg, U.; Pennanen, T.; Rosendahl, S.; Stenlid, J., et al. Fungal community analysis by high-throughput sequencing of amplified markers--a user's guide. New Phytol. 2013, 199, 288–299.

Lines 143-145: Consider removing these sentence as this information seems to be presented already in Lines 118-119.

In lines (now) 116-122 short general description of belonging soil types and processes are given for soil/s.  It is not exactly the same as in 93-95 and we would like to keep it intact.

Line 172: Consider changing done to completed.

Done as suggested. Done is changed by completed. Now line 155

Results:

Lines 217-218: The chi-square test showed the largest difference in richness of fungal taxa was between KKO and the remaining sites. Is this KKO or KKN?  Is this appropriate given that the rarefaction showed that fungal taxa detected in all sites did not reach the species saturation (line 219-220)?

It is the KKO site, with highest absolute number of detected fungal taxa and the highest number of generated sequences. Rarefaction showed that fungal taxa detected in all sites did not reach the species saturation, but it allow to compare the same number of sequences vs. the number of fungal species detected at each site.

Lines 233-240: How are these species defined as pine needle pathogens, pathogenic species, fungal endophytes? The surface of the needles were not sterilized so it is possible that some of the fungi identified were from the surrounding attached soils or inactive. Next generation sequencing approaches amply all DNA within the sample regardless of state (active or inactive), it does not provide information on the life-style of the fungus. It would be more appropriate to state what was found in the results and make a case for the life-style in the discussion based on previous literature.

The identification of the life-style of each fungus was based on the literature. References are provided in the discussion.

Figure 5:

This figure should contain all 150 data points or minimum the 5 data points from each site.

Please see the explanation regarding experimental design. Adding more data to Fig. 5 is not possible.

Discussion:

Line 295-296: If N. germanicum and A. pseudotsugae are the only two taxa recorded for the first time, than just state that. There is no need for e.g.

Not only N. germanicum and A. pseudotsuga were recorded for the first time. Some other species we are discussing were also recorded for the first time (Microsphaeropsis olivaceum, Ranoconidiophora, Geastrumia), as well as some species in Suplemmentary table). All of those species are recorded for the first time on the Balkans. Some are recorded for the first time for the Balkans, but also for the first time on Pines/ conifers. We have a lot of “first” records, but this is something what we expect, or what is provided due to high troughtput “methodology”.

1. germanicumand A. pseudotsugae are abundant in our dataset, and can potentially present important pathogens (even for wider region, not only for P. heldreichii). So, it was important to “emphasize” them here and highlight their new presence. On the other hand, we have many more new records, which are presented only with “e.g.”.

Line 318: ...was reported previously.  Consider revising to previously reported.

Construction was changed. (NOW Line 331)

Line 323:.. and Pinus yunnanensis. Consider revising to P. yunnanensis.

Not changed. This is the first (and only mentioning of P. yunnanensis in the text. Names of fungi are now also written with full Genus name.  Line 336

Line 325: consider removing e.g.

Removed. Line 338

Line 352: Capitalize the word Cyclaneusma to be consistent with previous capitalization.

Done. Line 364

Line 354: Similar applies to D. septosporum. Consider revising as it seems awkward as written.

Here, new arrangement was made. Part of the text was transferred from the introduction, where it was deleted. Now it is written: “

Investigation of Dothistroma septosporum accomplished using PCR and species-specific primers, has demonstrated the presence of this potentially invasive pathogen across the P. heldreichii distribution range in Montenegro [17]. (Lines 368-370)

Line 387: The study have also detected.... Consider revising to: This study has also.....

Changed as suggested. Line 400

Reviewer 2 Report

Review of the manuscript entitled "Fungal Diversity in the Phyllosphere of Pinus heldreichii H. Christ - An Endemic and High-altitude Pine of the Mediterranean Region".

The main aim of the paper is to characterize fungal diversity in living needles of Pinus heldreichii based on high-throughput sequencing of ITS2 rDNA marker gene. This is the first study which aims to describe fungal community in needles of this endemic pine species using DNA metabarcoding methods. Sampling was done at six different sites throughout Montenegro. The authors found 376 fungal taxa in total. Around 80% of fungal species found belong to the phylum Ascomycota and 20% to Basidiomycota. The most common fungi were Lophodermium pinastri, L. conigenum, Sydowia polyspora, and Cyclaneusma niveum. The community composition varied between sites, but two sites at higher attitudes were statistically separated from three sites at lower altitudes, thus indicating that environmental conditions strongly influence composition of fungal communities. Among all functional groups of fungi, pathogens appeared to be an important component of fungal communities. The most abundant fungal pathogens and endophytes present in phyllosphere community were discussed together with their ecological roles.

The article is written in prtetty good English but has a lot of typographical errors and some parts need to be somewhat changed. I find it suitable for publication after minor revision by the authors. My changes and comments on the text are attached in additional word doc file.

Best,

Reviewer

 

 

Changes and comments on the text:

 

 

17 ITS -> ITS2 rDNA

23 attitudes -> altitudes

23 growth conditions -> growing conditions

24 growth conditions -> growing conditions

31 Keywords: delete -> Pinus heldreichii, phyllosphere fungi

31 Keywords: add -> DNA metabarcoding, Montenegro

69 The species recorded were - > The characteristic species recorded were

70 glyocyclus -> gliocyclus

70 hypotejus -> hypothejus

70 Morchella esculenta -> Morchella esculenta sensu lato (or s.l.)

71 S. collinituse -> S. collinitus

69-72 Comment: Hygrophorus hypothejus, H. gliocyclus, Morchella esculenta are also edible species (but maybe protected under the law in Montenegro?). So it is better to omit the sentence on line 71 Edible mushroom species were... and list all fungal species characteristic for Pinus heldreichii forests in one sentence (The characteristic species recorded were...).

72 Comment: Then you need to erase the sentence In seasons when these mushrooms are abundant, they are collected for food by locals [19,20]. And transfer references [19,20] into the sentence on line 66: To date, about 50 mushroom species were recorded in P. heldreichii forests [14-16].

81 and Trichophaea flavobrunnea comb. nov. -> and rare species Trichophaea flavobrunnea

94 the later indicated -> it indicated

97-98 Comment on sentence: Needles were sampled across the whole range of P. heldreichii natural distribution in Montenegro... This statement is not true, since sampling was not carried out in some parts of P. heldreichii range e.g. Northwestern MN (Canyon of Tara river in Durmitor Nat. Park), around Rožaje in Northeastern MN (Hajla mountain in Rožaje municipality), Southeastern MN (Rumija mountain near Bar), Southern MN (Lovćen mountain). Please rephrase the sentence.

105-106 Applies the same as for 97-98

120 harsh growth conditions -> harsh growing conditions

120 while other sites with moderate growth -> while other sites were with moderate growing

124 by humid -> by a humid

129 Comment on sentence: The mean annual precipitation is highest at ORJ with ca. 3800 mm... mean annual precipitation of 3800 mm - It is multi-year average, but for which years?

145 in needles -> of needles

146 Question: How can you know that needles are 2-4 years old? Give an explanation.

153 Question: In abstract and in line 146 you mention that you sampled 2-4 years old needles, and here you discuss "The current-year needles". How can you distinguish between the current-year needles and needles of 2 years old? Explain.

165 DNA work -> DNA extraction and sequencing

167 mashine -> machine

209 Apllies the same as for 97-98

240 Mollisia ligni(0.7 %) -> Mollisia ligni (0.7 %)

241 Which reference or database you are using for nomenclature of fungal names in Table 3. and throughout the text? It is not an Index Fungorum database (http://www.indexfungorum.org/Names/Names.asp). Please cite the reference used for fungal nomenclature.

Table 3. Dothideomycetes -> Dothideomycetes

245 Leotyomycetes -> Leotiomycetes

255 needles of P. heldreichii showed -> needles of P. heldreichii (Fig. 5) showed

257 proximity (Fig. 4) -> proximity (Fig. 5)

260 growth -> growing

261 A. pseudotsugae L. calyciformis -> A. pseudotsugae, L. calyciformis

261 Phaeomoniella sp. - > Phaeomoniella sp.

262 M. lighni -> M. ligni

262 Comment: Collophorina species - You have only one member of the genus Collophorina in table 3., C. euphorbiae. You have Collophora capensis but not Collophorina.

263 growth -> growing

264 C. niveum, , P. punctiformis -> C. niveum, P. punctiformis

266 Uncultured sp. 2814_10 -> Uncultured sp. 2814_10

266 Chaetothyriales 2814_18, and -> Chaetothyriales 2814_18, and

288 teada -> taeda

289 the diversity of fungal -> the species diversity of fungal

303 revieled -> revealed

305 growth -> growing

306 growth -> growing

307 (ORJ; KMT) -> (ORJ, KMT)

308 mainy -> mainly

316 dominat -> dominant

317 Tsuga , -> Tsuga,

319 damged -> damaged

328 Behnke-Borowxzyk -> Behnke-Borowczyk

330 dominat -> dominant

341 Cyclaeusma -> Cyclaneusma

343 compated -> compared

344 Phomopsis pseudotsugae ) -> Phomopsis pseudotsugae)

349 heldreichii grown under harsh growth conditions -> heldreichii growing under harsh conditions

352 cyclaneusma -> Cyclaneusma

357 suspectile -> susceptible

367 to needle decomposition -> to a needle decomposition

369 colloniser -> coloniser

370 comparisin -> comparison

378 oliveacea -> olivacea

378 Coniothyrium olivaceaum  -> Coniothyrium olivaceum

380 oliveacea -> olivacea

390 Phaemoniella -> Phaeomoniella

392 Caluna -> Calluna

395 Phaemoniella -> Phaeomoniella

400 largely depend -> largely dependent

412 All -> Both

458 Low -> Law

495 phisiological -> physiological

 

In many cases, authors shorten fungal genus (e.g. N. instead Neocatenulostroma etc.) when they mention taxa names in the text. It is sometimes hard to read the text because of that. So, I propose that they use full names of genera as musch as possible throughout the text.

 

 

 

Author Response

Please see the atachment, 

GENERAL (additional explanation of study design) (For all reviewers)

The aim of our work was to characterize fungal community associated with needles of P. heldreichii:

• to make an inventory of species associated with this tree species
• to evaluate abundance and composition of fungal communities
• to compare fungal communities among different sampling sites

We were particularly interested in fungal pathogens, which can potentially affect growth and survival of the host tree. Until our study, there was no information about fungal communities associated with needles of this pine species.

In order to gather potentially broader information on fungal community, we selected different sampling sites (6 sites). They were:

• in different geographical areas i.e. covering different regions of Montenegro
• situated under different environmental conditions; we are later referring to these as different growing condition (as harsh or moderate)
• at one locality trees were additionally stressed by presence of biotic enemies/insects..

This approach enabled comparison of fungal communities developed on the same host tree under different environmental pressures. 

• In order to gather potentially higher fungal diversity (including different functional groupos of fungi), for the lab work we used randomly selected parts of both symptomatic and asymptomatic f needles, which were used without surface sterilization.

Regarding the experimental design, fungal community was assessed per each site. In order to get a representative information on fungal communities at each site, multiple samples were taken: 5 needle samples from 5 twigs that were collected from 5 different trees, resulting in 25 needle samples per site. All samples per each site were amplified using primers with the same barcode i.e. 25 needles samples x 1 barcode per site. For the whole study, 150 needles samples x 6 barcodes. This resulted that the variation in fungal communities was not assessed within each site, but only among different sites.

About geographical/species range of P. heldreichii in Montenegro and in our sampling:

Different sampling sites were selected in geographically different regions of Mne. They represent characteristic and “representative” sites of species distribution on western, central and eastern areas of Mne with a geographical distance of ca 120 km among most distant sites. The sites were also at the different distance from the Adriatic sea, which is important “factor” influencing P. heldreichii distribution. Sampling was done on localities were host populations/ forests are relatively large, “important”and “representative”. It was not intention or idea to sample on all possible sites of P. heldriechii distribution in Montenegro.

It is also important to mention that the chosen localities, were monitored for tree health and general crown conditions for a long period of time i.e. more than 10 years. As it is written in the beginning of M&M section, our forest stands were generally (not ideally, but) healthy-looking, thereby representing in most cases “typical” host trees and environmental condition for each site.

Moreover, regarding the comment by R2, localities, which are mentioned in the report as Tara canyon, Durmitor NP and Hajla (Rožaje), were not  representative for P. heldreichii (although some group of trees can exist on Hajla, that it is a typical spruce habtat; in NP Durmitor P. nigra, not P. heldreichii is present). Discussion about range / distribution of P. heldreichii in Mne is not under the scope of this paper, but we acknowledge the comment about wording “whole” range, which was changed in the revised version of the manuscript. Please observe, the data presented on a P. heldreichii distribution map is not completely accurate, and thus, we added two larger areas, which previously been missing.

Further, we have a localities under different environmental pressures (referred as “harsh” and “moderate”).

About climate data and soil:

General characteristics of the climate and soil of (mountain) P. heldreichii sites are given in the Methodology section. In the Table 1, the climate type on different sampling sites according to Koppen climate classification [ref 23] is given. In the text (lines 108-115) the data is used from climate Atlas of Montenegro [22]. The values were calculated based on 30 year series of observations and measurements (officially available data).

In Montenegro, measurements of climatic parameters are provided only from the limited number of meteorological stations, usually from lower altitudes. We do not have precise measurements of climatic parameters for sampling sites, and these were not measured by ourselves.

We are using those parameters to characterize overall climate and growing conditions for P. heldreichii trees (and associated biodiversity) at the sites, and not to present the situation at each locality during the sampling season. The same is for soil, a short general description of soil types and processes were provided.

Work with needles and needle fungal communities:

All samples collected within each site were used as pseudo-replicates. We analysed fungal community “as a whole” on each site, and compared it with fungal communities present on other sites.  

The fungal community at each site was represented by 25 needle/DNA samples (please also see above the description of experimental design). We have selected up to 5 (representative) needles per twig, with or without disease symptoms, and cut into 0,5-1 cm long segments, which were randomly selected for DNA extraction (in amounts that can fit into a 2 ml tube for grinding and extraction).

Needles were from the part of the twig which was 2-4 year old. Current /one year old needle are mainly green and rarely with symptoms, and thus, in order to get a good representation of both pathogens and other fungal species, these needles were excluded. It is not difficult to distinguish the age of needles for the last few seasons in pines. It is even more straightforward in the case of P. heldreichii as needles are grouped for each year, likely due to a short growing season. On figure 2, please see difference between 1 and 2 years old needles, and on the bottom, some fragments of 3 years old needles.

According to Editor recommendation, changes in Bioinformatic section to avoid duplications.

A map in Figure 2 with P. heldreichii distribution range has been changed because of copyrights but includes similar content. 

 

Changes and comments on the text:

17 ITS -> ITS2 rDNA

Changed as suggested. (NOW Line 18)

23 attitudes -> altitudes

Changed as suggested. (NOW Line 24)

23 growth conditions -> growing conditions

Changed as suggested. (NOW Line 24)

24 growth conditions -> growing conditions

Changed as suggested. (NOW Line 24)

31 Keywords: delete -> Pinus heldreichii, phyllosphere fungi

Please, see in comment below next comment

31 Keywords: add -> DNA metabarcoding, Montenegro

Suggestion is accepted. Key words DNA metabarcoding and Montenegro were added .instead  Pinus heldreichii and phyllosphere fungi

69 The species recorded were - > The characteristic species recorded were

This part of text is now moved to discussion (lines 422-435), and part of text is changed. See with 69-72 comment.

70 glyocyclus -> gliocyclus

Changed as suggested. Moved to discussion, line 424

70 hypotejus -> hypothejus

Changed as suggested . Moved to discussion, line 424

70 Morchella esculenta -> Morchella esculenta sensu lato (or s.l.)

Changed as suggested. . Moved to discussion, line425

71 S. collinituse -> S. collinitus

Edible fungi were removed from discussion. Explanation comes with next comment.

69-72 Comment: Hygrophorus hypothejus, H. gliocyclus, Morchella esculenta are also edible species (but maybe protected under the law in Montenegro?). So it is better to omit the sentence on line 71 Edible mushroom species were... and list all fungal species characteristic for Pinus heldreichii forests in one sentence (The characteristic species recorded were...).

Our initial idea was to divide those two groups. We would like to present characteristic species, but to underline those which are protected because (at least here) they are consider as unique. The whole habitat could consider as unique, with some “different species”. Edible fungi were also mentioned, because they are characteristic, and abundant. Now, in new rearrangement, information about edible mushroom species were deleted. Only information about red-listed and protected species, as well as new described species of Ascomycota retained. Those species are relevant to demonstrate a high value of P. heldreichii habitats as being unique and biodiversity hotspots in high-altitude mountain areas. This part was moved to discussion and slightly modified.

72 Comment: Then you need to erase the sentence In seasons when these mushrooms are abundant, they are collected for food by locals [19,20]. And transfer references [19,20] into the sentence on line 66: To date, about 50 mushroom species were recorded in P. heldreichii forests [14-16].

This part of text is now deleted

81 and Trichophaea flavobrunnea comb. nov. -> and rare species Trichophaea flavobrunnea

Changed as suggested Moved to discussion, line430

94 the later indicated -> it indicated

Now construction is different, line 68.

97-98 Comment on sentence: Needles were sampled across the whole range of P. heldreichiinatural distribution in Montenegro... This statement is not true, since sampling was not carried out in some parts of P. heldreichii range e.g. Northwestern MN (Canyon of Tara river in Durmitor Nat. Park), around Rožaje in Northeastern MN (Hajla mountain in Rožaje municipality), Southeastern MN (Rumija mountain near Bar), Southern MN (Lovćen mountain). Please rephrase the sentence.

Suggestion is acceptedand the WHOLE is deleted, line 72.

But explanation is provided above, in long introductory part.

105-106 Applies the same as for 97-98.

Changed as suggested, line 81

120 harsh growth conditions -> harsh growing conditions

moderate growth is replaced by moderate growing. Line 98

120 while other sites with moderate growth -> while other sites were with moderate growing

 moderate growth is replaced by moderate growing line 99

124 by humid -> by a humid

Changed as suggested line 105

129 Comment on sentence: The mean annual precipitation is highest at ORJ with ca. 3800 mm... mean annual precipitation of 3800 mm - It is multi-year average, but for which years?

Please see explanation above. Those are climatic data, and average values for 30 years periods. It is not possible to provide different data for mountain sites, especially they are very variable. The other possibility is direct measurements of climatic parameters in the field, but we do not have possibilities to support and complete that.

145 in needles -> of needles

Changed as suggested, line 127

146 Question: How can you know that needles are 2-4 years old? Give an explanation.

It is easy to make difference between needles from last vegetation and previous vegetation. I think it is not complicated with pine. In case of P. heldreichii it is even more easy because needles usually have some appearance as skirt (part of clothes). In our language, the name of this pine is also skirt pine, as different “needle triangles” overlapping. As growth from different years. It is also visible on branches, and according to positions of lateral branches and cones. On figure 2, it is possible to distinguish last year needles, and mainly 2 years old needles. Tree year old needles are present, and little visible on the bottom (lateral branches also starts on the bottom, on the left.

153 Question: In abstract and in line 146 you mention that you sampled 2-4 years old needles, and here you discuss "The current-year needles". How can you distinguish between the current-year needles and needles of 2 years old? Explain.

Please see the explanation above; question for line 146.

165 DNA work -> DNA extraction and sequencing

Changed to: DNA isolation, amplification and sequencing (line 153)

167 mashine -> machine

Changed as suggested, line 155.

209 Apllies the same as for 97-98

Changed as suggested The word whole is deleted. New constriction is: “represented by six sampling sites across the distribution range, Now line 203.

240 Mollisia ligni(0.7 %) -> Mollisia ligni (0.7 %)

Changed as suggested, line 239.

241 Which reference or database you are using for nomenclature of fungal names in Table 3. and throughout the text? It is not an Index Fungorum database (http://www.indexfungorum.org/Names/Names.asp). Please cite the reference used for fungal nomenclature.

We used NCBI based on criteria explained in bioinformatics part (Lines  185-189).

Names of fungi as given as closest match from NCBI databases. Whenewer it was possible we used type material, or culture collections, and not environmental samples. Fungal names is later compared with those in index fungorum, for the first 25 species in table 3.

In discussion we put the synonyms in brackets , when we find that the “old” names were more usual/common to readers.

NAMES OF FUNGI IN TABLE 3.

During the revision, we repeated Blast analysis for for fungi listed in the Table 3.

1) The sequence for Collophorina euphorbiae, (MG592739) was submitted by Nasr et al., in 2018. (Reference: Nasr,S., Bien,S., Soudi,M.R., Alimadadi,N., Fazeli,S.A.S. and Damm U. Novel Collophorina and Coniochaeta species from Euphorbia polycaulis, an endemic plant in Iran, Mycol. Prog. 17 (6), 755-771 (2018). The sequence was based on a type_material="culture from holotype of Collophorina euphorbiae"

During the work on our manuscript, we have used this one for species identification.

From the literature [69], it was obvious that species name is changed in the meantime, but because we were useing  Genbank for species identification (lines 185-190), we kept the name Collophorina as in Genbank.

It happened that in March 2020 Genbank corrected this accession  to the recent nomenclature as Ramoconidiophora (according to the reference:  Bien, Kraus & Damm, Persoonia 45: 61 (2019), what is now a valid species name.

The same has happened with Collophora capensis , which is now renamed to Collophorina.

In the revised version of the manuscript, we have changed to recently published names and cite respective references.

 

2) For species Rhizoctonia carotae, Athelia arachnoidesis is a valid species name according to Index fungorum. (Athelia arachnoidea (Berk.) Jülich, Willdenowia, Beih. 7: 53 (1972).

The sequence of Rhizoctonia carotae- MH861139 was submitted by Vu,D., Groenewald,M., de Vries,M., Gehrmann,T., Stielow,B.,Eberhardt,U., Al-Hatmi,A., Groenewald,J.Z., Cardinali,G.,Houbraken,J., Boekhout,T., Crous,P.W., Robert,V. and Verkley,G.J.M., in press: Large-scale generation and analysis of filamentous fungal DNA barcodes boosts coverage for kingdom fungi and reveals thresholds for fungal species and higher taxon delimitation, Stud. Mycol. 92, 135-154 (2019. From the same publication, there were sequences for Microsphaeropsis olivaceae (MH871969), Allanthomopsiella pseudotsugae (MH857222) and Lachnellula calciformis (MH858771.Our sequence MT241929.1 have 100 % similarity to the sequence MH861139 belonging to Rhizoctonia carotae., and that is why it was named and later discussed as R. carotae.  But, our sequence has also 99 % similarity to the species Athelia acrospora, which is, as corticoid fungus already recorded in pine forests, and ecologically better fits in our research. Hence, during the revision,  we have change  Rhizoctonia solani to Athelia  acrospora. (This sequence was submitted by Rosenthal,L.M. and Bruns,T.D., under the title: Survey of corticioid fungi in North American pine forests reveals hyperdiversity, widespread distribution and potential ectomycorrhizal taxa (Unpublished) (July 2016)Consequently, the following changes were done in discussion:Lines 237- name of species is changed; deleted from  line 402., and 408. New sentence *410-411 about Athelia is added, with corresponding references [71, 72]

3) Because of sequence similarity being lower than 98 %, we referred to Collophorina and Gaestrumia  only to genus level. Due to low sequence similarity, we have also named Unidentified sp. 2814_11 instead of  Pezicula rubi. The name of Unidentified sp. 2814_22 is now changed to Rhitismataceae 2814_22

Table 3. Dothideomycetes -> Dothideomycetes

Changed as suggested

245 Leotyomycetes -> Leotiomycetes

Changed as suggested , line 247

255 needles of P. heldreichii showed -> needles of P. heldreichii (Fig. 5) showed

Changed as suggested , (Fig. 5 )is added,line 261

257 proximity (Fig. 4) -> proximity (Fig. 5)

Changed as suggested line 263

260 growth -> growing

Changed as suggested line 266

261 A. pseudotsugae L. calyciformis -> A. pseudotsugae, L. calyciformis

Changed to full genus names.267-274

261 Phaeomoniella sp. - > Phaeomoniella sp.

Changed as suggested , line 267

262 M. lighni -> M. ligni

Changed to full genus names.267-274

262 Comment: Collophorina species - You have only one member of the genus Collophorina in table 3., C. euphorbiae. You have Collophora capensis but not Collophorina.

Comment is given above (names in table 3).

263 growth -> growing

Changed as suggested , line 270

264 C. niveum, , P. punctiformis -> C. niveum, P. punctiformis

Changed to full genus names.271-274

266 Uncultured sp. 2814_10 -> Uncultured sp. 2814_10

Changed as suggested , line 271 (Unidentified sp. 2814_10

266 Chaetothyriales 2814_18, and -> Chaetothyriales 2814_18, and

Changed as suggested , line 274

288 teada -> taeda

Changed as suggested , line 300

289 the diversity of fungal -> the species diversity of fungal

Changed as suggested , line 301

303 revieled -> revealed

Changed as suggested, line 316

305 growth -> growing

Changed as suggested, line 319

306 growth -> growing

Changed as suggested , line 320

307 (ORJ; KMT) -> (ORJ, KMT)

Changed as suggested, line 320

308 mainy -> mainly

Changed as suggested , line 322

316 dominat -> dominant

Changed as suggested , line 329

317 Tsuga , -> Tsuga,

Changed as suggested , line 330

319 damged -> damaged

Changed as suggested, line 332

328 Behnke-Borowxzyk -> Behnke-Borowczyk

Changed as suggested , line 341

330 dominat -> dominant

Changed as suggested , line 343

341 Cyclaeusma -> Cyclaneusma

Changed as suggested , line 354

343 compated -> compared

Changed as suggested, line356

344 Phomopsis pseudotsugae ) -> Phomopsis pseudotsugae)

Changed as suggested line 357

349 heldreichii grown under harsh growth conditions -> heldreichii growing under harsh conditions

Changed as suggested line 362

352 cyclaneusma -> Cyclaneusma

Changed as suggested line 364

357 suspectile -> susceptible

Changed as suggested , line 372

367 to needle decomposition -> to a needle decomposition

Changed as suggested , line 382

369 colloniser -> coloniser

Suggestion is accepted, line 384

370 comparisin -> comparison

Changed as suggested , line 385

378 oliveacea -> olivacea

Changed as suggested , line393

378 Coniothyrium olivaceaum  -> Coniothyrium olivaceum

Changed as suggested , line393

380 oliveacea -> olivacea

Changed as suggested , line 395

390 Phaemoniella -> Phaeomoniella

Changed as suggested , line 401

392 Caluna -> Calluna

Changed as suggested , line 405

395 Phaemoniella -> Phaeomoniella

Changed as suggested , line 407.

400 largely depend -> largely dependent

Changed as suggested, line 436.

412 All -> Both

Changed as suggested line 448

458 Low -> Law

Changed as suggested , line 664.

495 phisiological -> physiological

Changed as suggested , line 504

In many cases, authors shorten fungal genus (e.g. N. instead Neocatenulostroma etc.) when they mention taxa names in the text. It is sometimes hard to read the text because of that. So, I propose that they use full names of genera as musch as possible throughout the text.

Changed as suggested . Full names of genera were used throughout the text.

Reviewer 3 Report

The aim of the manuscript «Fungal diversity in the phyllosphere of Pinus heldreichii H. Christ - an endemic and high-altitude pine of the Mediterranean Region” was to investigate diversity and composition of fungal communities in living needles of P. heldreichii.  As Pinus heldreichii is endemic species, knowledge about fungi associated with it needles, as well as assessment of incidence of pine needle pathogens is important. In this manuscript, modern study methods (high-throughput sequencing) were performed for determination of fungal biodiversity in pine needles. As this topic is actual, the current manuscript is well suited to the journal in question, however, some improvements could be performed.

Specific comments:

Line 144.  Better use Tomicus sp. bark beetles instead of insects.

Lines159-160.  Was healthy looking and symptomatic needles mixed together? How you chose the needles to be subjected for DNA extraction?

Lines 166-167.  Why surface sterilization of pine needles was not performed?

Lines 235-236; 296 – 298. You haven’t done surface sterilization of sampled needles. Is it possible, that this fungi was an occasional contamination of needle surface?

Lines 293-294. Is there any explanation, why L. pinastri dominated at this site?

Lines 307 – 309.  Were more pathogenic or saprophytic species found at site attacked by bark beetles?

Figure. 4. This figure is not informative enough, and some colors were hard to distinguish. Do you really need this figure?

Concluding remark

The manuscript provides novel and valuable information about diversity of fungal pathogens and endophytes in needles of Pinus heldreichii. The manuscript is quite fluently written and easy to understand, however it could be improved, if revised by native English speaker.

Author Response

Please see the atacment 

GENERAL (additional explanation of study design) (For all reviewers)

The aim of our work was to characterize fungal community associated with needles of P. heldreichii:

• to make an inventory of species associated with this tree species
• to evaluate abundance and composition of fungal communities
• to compare fungal communities among different sampling sites

We were particularly interested in fungal pathogens, which can potentially affect growth and survival of the host tree. Until our study, there was no information about fungal communities associated with needles of this pine species.

In order to gather potentially broader information on fungal community, we selected different sampling sites (6 sites). They were:

• in different geographical areas i.e. covering different regions of Montenegro
• situated under different environmental conditions; we are later referring to these as different growing condition (as harsh or moderate)
• at one locality trees were additionally stressed by presence of biotic enemies/insects..

This approach enabled comparison of fungal communities developed on the same host tree under different environmental pressures. 

• In order to gather potentially higher fungal diversity (including different functional groups of fungi), for the lab work we used randomly selected parts of both symptomatic and asymptomatic f needles, which were used without surface sterilization.

Regarding the experimental design, fungal community was assessed per each site. In order to get a representative information on fungal communities at each site, multiple samples were taken: 5 needle samples from 5 twigs that were collected from 5 different trees, resulting in 25 needle samples per site. All samples per each site were amplified using primers with the same barcode i.e. 25 needles samples x 1 barcode per site. For the whole study, 150 needles samples x 6 barcodes. This resulted that the variation in fungal communities was not assessed within each site, but only among different sites.

About geographical/species range of P. heldreichii in Montenegro and in our sampling:

Different sampling sites were selected in geographically different regions of Mne. They represent characteristic and “representative” sites of species distribution on western, central and eastern areas of Mne with a geographical distance of ca 120 km among most distant sites. The sites were also at the different distance from the Adriatic sea, which is important “factor” influencing P. heldreichii distribution. Sampling was done on localities were host populations/ forests are relatively large, “important”and “representative”. It was not intention or idea to sample on all possible sites of P. heldriechii distribution in Montenegro.

It is also important to mention that the chosen localities, were monitored for tree health and general crown conditions for a long period of time i.e. more than 10 years. As it is written in the beginning of M&M section, our forest stands were generally (not ideally, but) healthy-looking, thereby representing in most cases “typical” host trees and environmental condition for each site.

Moreover, regarding the comment by R2, localities, which are mentioned in the report as Tara canyon, Durmitor NP and Hajla (Rožaje), were not  representative for P. heldreichii (although some group of trees can exist on Hajla, that it is a typical spruce habtat; in NP Durmitor P. nigra, not P. heldreichii is present). Discussion about range / distribution of P. heldreichii in Mne is not under the scope of this paper, but we acknowledge the comment about wording “whole” range, which was changed in the revised version of the manuscript. Please observe, the data presented on a P. heldreichii distribution map is not completely accurate, and thus, we added two larger areas, which previously been missing.

Further, we have a localities under different environmental pressures (referred as “harsh” and “moderate”).

About climate data and soil:

General characteristics of the climate and soil of (mountain) P. heldreichii sites are given in the Methodology section. In the Table 1, the climate type on different sampling sites according to Koppen climate classification [23] is given. In the text (lines 107-114) the data is used from climate Atlas of Montenegro [22]. The values were calculated based on 30 year series of observations and measurements (officially available data).

In Montenegro, measurements of climatic parameters are provided only from the limited number of meteorological stations, usually from lower altitudes. We do not have precise measurements of climatic parameters for sampling sites, and these were not measured by ourselves.

We are using those parameters to characterize overall climate and growing conditions for P. heldreichii trees (and associated biodiversity) at the sites, and not to present the situation at each locality during the sampling season. The same is for soil, a short general description of soil types and processes were provided.

Work with needles and needle fungal communities:

All samples collected within each site were used as pseudo-replicates. We analysed fungal community “as a whole” on each site, and compared it with fungal communities present on other sites.  

The fungal community at each site was represented by 25 needle/DNA samples (please also see above the description of experimental design). We have selected up to 5 (representative) needles per twig, with or without disease symptoms, and cut into 0,5-1 cm long segments, which were randomly selected for DNA extraction (in amounts that can fit into a 2 ml tube for grinding and extraction).

Needles were from the part of the twig which was 2-4 year old. Current /one year old needle are mainly green and rarely with symptoms, and thus, in order to get a good representation of both pathogens and other fungal species, these needles were excluded. It is not difficult to distinguish the age of needles for the last few seasons in pines. It is even more straightforward in the case of P. heldreichii as needles are grouped for each year, likely due to a short growing season. On figure 2, please see difference between 1 and 2 years old needles, and on the bottom, some fragments of 3 years old needles.

According to Editor recommendation, changes in Bioinformatic section to avoid duplications.

A map in Figure 2 with P. heldreichii distribution range has been changed because of copyrights but includes similar content. 

Specific comments:

Line 144.  Better use Tomicus sp. bark beetles instead of insects.

This suggestion is accepted. Tomicus sp. bark beetles is used instead insects (Now , line126).

Lines159-160.  Was healthy looking and symptomatic needles mixed together? How you chose the needles to be subjected for DNA extraction?

In order to gather more fungal diversity, both pathogens and endophytes, but also epiphytes fungi (whole community), for lab work we used both symptomatic and asymptomatic parts of needles, without surface sterilization.

We choose up to 5 needles per twig, containing more or less expressed symptoms ( average for the twig). 25 samples/extractions, later treated as “one group” will potentially will mediate mistakes, or subjectivity in this phase. Usually, appearance of disease symptoms along one separate needles were limited (except, where the needles were discolorated and necrotic, as consequence of Tomicus attack.) Needles were cut on segments 0,5-1 cm long parts. Randomly selected parts of needles were taken (both symptomatic and asymptomatic) for DNA extraction (in amount which can fit to 2 ml tube for grinding and extraction). This was important for appropriate assessment of abundance of species in community, also

Lines 166-167.  Why surface sterilization of pine needles was not performed?

Lines 235-236; 296 – 298. You haven’t done surface sterilization of sampled needles. Is it possible, that this fungi was an occasional contamination of needle surface?

In some other cases, especially if we are dealing with pathogens and problems they are causing, we would have done the surface sterilization to skip the species present on the surface. In some trials we in parallel tested fungal communities on needles (from the same trees, of same ages and simptoms) using surface sterilized needles or without it.

Lines 293-294. Is there any explanation, why L. pinastri dominated at this site?

Not really.

Lines 307 – 309.  Were more pathogenic or saprophytic species found at site attacked by bark beetles?

It seems that pathogens of were prevailing. It is clear that Sydowia polyspora (known as pathogen) dominated in this community with 32,2 %. Then comes  Neocatelunostroma germanicum, a  new for us, but confirmed as a pathogen on P. sylvestris (7,9). Microsphaeropsis olivaceae, which can provide some “defence “ mechanisms occurred at 16,8 %. Unidentified species were also present, as well as Geastrumia, known as a  pathogen of apple trees/fruits (2,3 %).

Figure. 4. This figure is not informative enough, and some colors were hard to distinguish. Do you really need this figure?

This figure provides more general overview about relative abundance of different fungal classes in needles of P. heldreichii. Functional significance of all fungal taxa is not known ( at this moment) but figure also shows differences /similarities among different sites, and provide  some information on the field which was not known before. The similar type of “information” was provided for example in publication [8], and sometimes is valuable, especially when many species are unidentified to species level, and when „better“ identification, and hence interpretation of results is not possible.

Concluding remark

The manuscript provides novel and valuable information about diversity of fungal pathogens and endophytes in needles of Pinus heldreichii. The manuscript is quite fluently written and easy to understand, however it could be improved, if revised by native English speaker

The language changes suggested by all Reviewers were followed, which we believe improved the manuscript.  

Round 2

Reviewer 1 Report

Fungal Diversity in the Phyllosphere of Pinus heldreichii H. Christ - an endemic and high-altitude Pine of the Mediterranean Region.

This revision has a better overall structure but there are still issues with some major issues with the conclusions based on the design of the experiment.

Previous concerns:

1) There is no control. The authors responded: "There was no DNA extraction control, but we used the best practices of DNA isolation. This does not supplement the control, but according to Lindahl et al., 2013, extraction control is not necessarily required. To avoid possible cross-contaminations, DNA extractions were done per each site separately, in sterile UV hoods, using pipette tips with filters and new sets of chemicals."

If the authors believe that a control is not necessarily required according to Lindahl et al. 2013, then they need to add this into the methods to justify why they did not have an extraction control.

2) Removal of singletons. The author's responded: "According to Lindahl et al., 2013, singletons should be removed." and "It is true that many singletons may represent rare fungal taxa, but also, they can be a consequence of sequencing biases, and can be unreliable."

Different papers can be cited for the case of keeping and removing singletons (DOI 10.1186/s40168-017-0237-y). However, if the manuscript is an inventory paper designed to identify as many fungal species/ community members as possible than why not blast the singletons for completeness? Micro-fungi can be important. It is easy to blast against UNITE for a quick determination of the reliability. No unreliable sequence will provide >80% query coverage, 94-97% similarity to genus and > 98% similarity to species.

3) NCBI is not a curated database: The authors responded: " That is true, NCBI is not a curated database and contains misidentified sequences. For species identification we did not obligatory use sequences with highest possible matches but those (still with highest, or almost highest rang (>98 %)), which are based on type material or culture collections by CBS, where it was possible. We have also checked the publication status where sequence belongs. Environmental sequences were avoided, when it was possible. 25 most common species, presented in Table 1 mainly have good  and recently published sequences, provided by eminent experts, and not rarely in publications where taxonomical position of species were discussed. UNITE is a database initially established for ectomycorrhizal fungi, and later expanded with other fungal species. We tested both UNITE and RDP, but in our case there was much lower sequence coverage, mainly because out fungal community is mainly composed of non-mycorrhizal Ascomycetes."

 

This response is wonderful but I wonder why this information was not added to the manuscript as it provides support for the taxonomic determination and describes in more detail how taxonomic determination was completed.

 

Main concern not addressed:

Justification for most of the rationale for differences based on relative abundance. Having no site replication (condensing all site data into one unit) makes it hard to determine if patterns are typical of each site. More thorough explanations is below in several places.

Analyses of the various conditions from Table 1 (Soil type, Climate, Health) using Table S1 relative abundance (NMDS (bray, k=2) or Table S1 as presence absence (PCoA, hellinger) demonstrated no statistical significance for envfit or adonis. Therefore, the conclusion that the composition was largely dependent on environmental conditions and/or health status of host trees is not supported. In addition, pheatmap of Table S1 relative abundance indicates that individual sampling site variation is likely the cause of the difference between samples (see attached pheatmap of Table S1 relative abundance). Therefore, unless there the authors can show statistical significance for this conclusion, it needs to be removed from all locations. If the authors have statistical significance, then the statistical analyses need to be included in section 2.5 statistical analyses. If the authors need assistance, I can provide R-code.

 

Line items:

108: characterissed is should be changed to characterized

 

119: Sampling was carried out in May.....

This sentence would work well at the beginning of line 115 and make the paragraph 3 sentence.

 

194: Non-fungal taxa and singletons were excluded.

This should be in the methods section 2.4.

 

198-200: The chi-square test showed that the largest difference in richness of fungal taxa was between KKO and the remaining sites (Table 2).

Fungal richness is the total number of species. KKO has 167 species which is not the largest difference for fungal richness. My understanding is that the authors are basing this on number of fungal taxa from the original number of fungal sequences per site but this has to do with one site having a great sequencing depth, not species richness. This statement cannot be supported as is because it has the same diversity index as KMT.

 

292-296: These community conclusions are based on visual ordination and not on stats. See pheatmap above. The pattern is more in line sample site variation and envfit and adonis analysis does not support moderate and harsh significance ( p =0.13 and p =0.07 respectively).

 

314-317: Not sure how this information is demonstrated in Table 2? Table does not reference species.

 

330-332: In this study, it was present in needles........with considerably higher relative abundance on symptomatic needles.....

 

If all samples contained a "random mixture of both asymptomatic and symptomatic parts of needles (line 136) how was this concluded from the samples?

 

363-366: In the present study, ...........

The relative abundance has issues when comparing to this site because all samples contained a "random mixture of both asymptomatic and symptomatic parts of needles (line 136). If more symptomatic tissue was included, then it stands to reason the abundance will be higher for some organisms and lower for others. In other terms, an alternative explanation for this result could be due to one of more samples from the site containing more symptomatic tissues. Because all the samples per site were combined, it cannot be determined if higher abundance is representative of all 5 trees or if one tree or one sample is driving the abundance pattern. Without the in-site replication data, the conclusions should suspect or alternative conclusion provided.

 

375: On P. heldreichii, N. germanicum was absent in case if M. ligni was present

suggest changing absent to not detected

 

391-393 and 410-412: No support for dependent on environmental conditions and or health status of the tree. See previous comments related to this.

Table S1: There is an A in the relative abundance table for KJ827341.

 

 

 

 

Comments for author File: Comments.pdf