Expanding the Microcolonial Black Fungi Aeminiaceae Family: Saxispiralis lemnorum gen. et sp. nov. (Mycosphaerellales), Isolated from Deteriorated Limestone in the Lemos Pantheon, Portugal
, João Trovão 1, LuÃs Fernandes 1, Nuno Mesquita 1, Igor Tiago 1 and António Portugal 1,2,3,*
Round 1
Reviewer 1 Report
The paper fits with the aim of the special issue and the common knowledge could benefit from this publication, but there are many items to be addressed. The manuscript is composed of two main items the taxonomical positioning of two new black fungal strains and, as counterparts, tests performed to assess their deteriogenous potential and limit for life. This is an uncommon choice since, generally, the strain described is characterized for basic growth requirement (e.g. temperature and culture medium – namely malt extract agar, potato dextrose agar, Czapek, and oatmeal) only, while the eco-biological characterization could be a standalone item. Besides the huge work done and data achieved, it is essential that the authors accompany the readers (even the beginners in this field) in understanding, explaining why each test has been performed and what the gain achieved. In this light, the first suggestion raised from the title doesn’t reflect the dual content mentioned above. The same problem affects the introduction, which should be reworked, removing results from there (L92-102 should be dramatically reduced) and defining the other work aims that you can list, for example, as a), b), c). This could be useful for study design understanding. The materials and methods (MM) should be more strictly organised/rationalised, removing results (e.g., L163-172, and Table 1) and giving order to all tests. There were incongruences between the citation reported and the conditions applied in the experiment (e.g., temperature, salt) and is not introduced the reason why some tests such as pH, hot tolerance, freeze-thawing etc have been applied even because no similar conditions characterize the isolation source. And order is also required in figures where sub-figures are not regular, and lettering doesn’t follow the typical reading frame from left to right. The reference list is characterized mainly by old papers and should be revised consistent with the expected trend. I’m not telling that old papers should not be cited but in your manuscript there are too many…as nothing better has been done more recently- It is common that since early steps, discoveries are generally elaborated and updated step by step as the common knowledge increases. I noted that a little bit less than one-third is represented by papers published more than 20 years ago, 30% of papers published in the last ten years and the remaining is represented by papers published in the range 11-19 years ago.
Below are some additional detailed comments
Title_microcolonial black fungali Aeminiaceae
Keywords should be reworked since they are not effective and are not able to direct the readers’ search to your work.
Aeminiaceae is already indexed because part of the title and should be changed; Biodeterioration be more precise, give a context to this word since there are many types of biodet (stone, wood, paper, ceramics etc). Limestone; is too general, is you’re a peculiar type of limestone? There are thousand types. New species; too general, species of microorganisms, mammals, amphibians, fishes, plants…Phylogeny too general, of what. Taxonomy too general, taxonomy of what?
Since the connection with the biodeterioration item is strong it would be fine to better define limestone since there are thousand of them. Since the early lines, you should talk about Ançã limestone, and features should be given, such as porosity, strength, colour and so on). The advantage could be double, since you gave additional info useful from a conservative point of view, and count on a more efficient keyword (Ançã limestone).
L37 - engage in interactions sounds wordy; please change it to interact
L100 “Extremaceae and Neodevriesiaceae (now part of Mycosphaerellales, Capnodiales s. lat.)” add reference
L126 aerobically remove, it is obvious
L128-129 (the medium from which they were originally recovered). Remove the abbreviation RB is three lines above.
L129 What about the strains deposited in a culture collection? You must add this info
L131 of both isolates- remove, not necessary
L132 with several modifications – please disclose in which consist of modifications because if the reader hasn’t familiar with the method cannot discriminate between the standard and used protocol.
Since MEA has different recipes, please disclose yours.
L134 PCR-style microtube – please change to 0.2 mL PCR microtube
L133-134 please change to: About 1 mm2 of biomass was taken from colonies, placed into 0.2 mL PCR microtubes with 20 μL of extraction solution and incubated in a thermocycler using the following protocol
L139 two nuclear genes regions – two nuclear genes are mentioned, but three targets were listed. Formally ITS is not a gene; all of them are nuclear regions. Please change to three nuclear regions
L148-151 remove, not necessary
L159-160 Obtained consensus sequences were deposited in the GenBank database with the accession numbers: OR081767-OR081768 for ITS, OR081765-OR081766 for LSU, and OR074926, OR074927 rpb2. The sentence should be removed from MM because it is a result. In detail, “Obtained consensus sequences were deposited in GenBank database” should be moved to section 2.2 (L154); the rest should be cancelled because already listed in Table 1
L194-203 Table 1. the Strain n/ Strain ID (not isolate) column should be moved next to the Species name. Instead of substrate, it’s better using Source. The grey shade is not necessary since the new strains are bold.
Be consistent in reporting the collection names (e.g., there are some that use the national language and other such DMSZ and CBS that have been translated). Be consistent in identify where are the collections, namely Institution, city (state for USA), and country for all of them. 18S nrDNA is not part of ITS
L206 Disclose MEA recipe; please explain why you used so uncommon media for colony morphological characterization; please explain why so different periods of incubation (i.e., 2-6 months). What about the temperature of incubation used? Please also explain the selection of culture media for microscopic description and give info on temperature and incubation period. In my opinion, it is better to reduce conditions because it is difficult to follow. In any case, at this stage, the reader doesn’t know that your strain is halophilic.
L216… physiological characterization -the references don’t fit well with the title and conditions used. Moreover, it is necessary to disclose the reason for this kind of investigation. Are these conditions in such a way connected to conditions present in the site or possible treatments for their eradication/control?
Anyway, the thermal characterization doesn’t reflect the condition used in the cited works (Sterfl- Selbm). Temperatures tested are too limited to have a confident growth limit and optimum and too irregular to have significance. This is an essential requirement.
Similar incongruences are recorded for sodium chloride tolerance tested by Sterflinger on MEA from 0-27 with 1% increases, not DSMZ 372-Halobacteria medium from 0-30 with increases of 5. How is calculated the salt percentage in that medium? The declared concentration is indeed well above the NaCl solubility limit in water, and it could be useful from an applicative point of view.
L245-253 UV tolerance and plate vitality test. Please explain how the experiment was performed. In detail, how the mycelium has been prepared since the shadow effect can interfere with the real tolerance limit and 0.5 cm seems to be a big slice. How about the plating, counting and negative control? Please specify if the mycelium is fresh or dehydrated; similar comments are for heat and cold stress tests.
L266-278 Please explain the reason for choosing each culture medium, their recipe and what you expect from each of them. What’s the reason for modifying the B4 medium? In particular, please explain the reason for choosing creatine sucrose agar since the medium containing calcium carbonate is already a test for acid production. What’s the meaning of pH in table 2?
L294 within the Mycosphaerellales order – Since each figure or table should be considered a standalone material, this information should be reported in the figure or in the relative caption.
L312-314 Nevertheless, since Extremaceae and Neodevriesiaceae were included in Mycosphaerellales and considering their close relationship to Aeminiaceae, the latter should also be considered as part of Mycosphaerellales order – you can prove it without doubt including in the dataset some capnodilean fungi
Figure 1. DPI quality should be increased. The inclusion of the new strains into the Mycosphaerellales family is not explicit since the figure did not show orders (namely Mycosphaerellales and Cladosporiales).
L322 this family – which family?
L333-334 empty lines
L356 «Lemni» meaning «from Lemos. It seems there is an extra “n". the surname is Lemos or better de Lemos, so it should be Latinised as Lemosii as it was for Louis de Lemos whose latin translation was Ludovici Lemosii (https://www.abebooks.com/9781104255503/Ludouici-Lemosii-Medici-Physici-Tres-1104255502/plp?cm_sp=plped-_-1-_-image) and Brazilian orchid Catasetum lemosii- More important, lemnus in latin exists and refers to Greek island Lemnus/Lemno, so lemni indicates a link with the Greek island. There is also an extra 0 in coordinates
L434 -optimal growth – you cannot be sure since 15 °C has not been measured
L439-441, are the found differences significant? In figure 4, the colony figures are not significant; please remove them, giving more importance to graphs that should be improved adding SD or SE bars and asterisk when significant differences are recorded.
Table 3. The usual way to indicate positive and negative responses are using + (plus) and –(minus) , sometimes with more plus (++) to indicate also quantitative response. Please change
The data reported as appendix should be reported as supplementary material
Reference 15. Sibanda, T.; Selvarajan, R.; Tekere, M. Synthetic extreme environments: Overlooked sources of potential biotechnologically relevant microorganisms. Microb. Biotechnol. 2017, 10, 570–585. It sounds inappropriate because focuses on bacteria
the English need minor checks once major revisions are applied
Author Response
Dear reviewer,
I would like to express my gratitude for your highly insightful comments and the valuable constructive criticism provided. Enclosed within the attached document are detailed responses addressing each of the comments and questions raised.
Thank you for your valuable input!
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Diana S. Paiva et al. present a research manuscript entitled “Expanding the microcolonial black fungi Aeminiaceae family: 2 Saxumspirale lemni gen. et sp. nov. (Mycosphaerellales), isolated from deteriorated limestone in the Lemos Pantheon, Portugal“ for possible publication in JOF.
The topic of the study is of high interest for the research community working on biodeterioration and biodegradation of stone monuments since black fungi are major biodeteriogenic agents that are still largely unknown.
The main originality of the research is the description of a new genus and a new species in the Aeminiaceae family based on a multi-locus phylogeny (ITS rDNA, LSU, rpb2) and phenotypic characters (morphology, physiology, ecology).
The manuscript is well written, easy to read, but it needs improvements in text and illustrations. The strength of this study is also its weakness because few data are available on this family of fungi, which makes the classification proposal fragile.
Introduction
Page 2 Line 58. The authors must cite this recent review that is directly related to their work:“ De Leo, F.; Marchetta, A.; Urzì, C. Black Fungi on Stone-Built Heritage: Current Knowledge and Future Outlook. Appl. Sci. 2022, 12, 3969. https://doi.org/10.3390/app12083969.
Page 2 paragraph Lines 79-88. This paragraph should be reviewed because it breaks the sequence of ideas. Thus, the part on Portugal must go with the following paragraph which deals with the study site and the general information on the BFs must go with the previous paragraph.
Page 2 lines 96-97 “However, they did not cluster with any known representatives of this family“. As there is only one representative of this family, this sentence appears abusive.
Materials & Methods
Page 3 Lines 115-121. Additional information about samples L1 to L4 are needed, in particular information about differences between the 4 samples. How far between them? What macroscopic differences (colour, surface appearance, degradation, salts)? Difference in sampling technique between them (invasive, non-invasive)? etc.
In fact, since only isolates from the L4 sample seem to be included in the results of the manuscript, the authors could restrict the article to this sample.
Page 3 Line 122 and Table 1. In the M&M section, the authors speak of 16 isolates but in Table 1 There are 20 isolates. The authors must standardize this and speak in the M&M of the 20 isolates presented next.
Page 4 Line 161. Change “our sequences“ to “the sequences obtained“.
Page 4 Lines 155-172. The % of similarity and relation to published work (Vázquez-Nion et al. 2016) are results and discussion that need to move to corresponding sections.
Page 6 paragraph “2.4.4. UV tolerance”. The doses of UV radiation applied must be specified here.
Results & Discussion
Page 7 Lines 282-289. As requested before for Lines 155-172 Page 4, the % of similarity for the different sequences must be presented and discussed here. Some % are low, and have to be discussed in relation to similar published works.
Page 7 Lines 295-302 and Figure 1. With only another species and 10 sequences, the authors should be careful in their discussion and conclusions. The enrichment of databases with new sequences is necessary to confirm and clarify this classification.
Figures 2, 3, 4, and 5, a size scale bar must be systematically added to each photograph (macro and micromorphology).
The Figure 4 is very difficult to understand and essential information is missing. What isolate is tested for each parameter? The results must be shown for the two isolates. The nature of the agar medium used for each test must be indicated in the figure legend. Y axes with tick marks should be added.
Table 3. Abbreviations should be defined in the table caption (CREA, CGA, MMA, B4, MB4, HM10%). The green, red colour code is useless. Table 3 must be presented after Figure 5 and the corresponding text (Lines 493-505) must also be moved. A relationship between the phenotypes observed in vitro and the biodegradation of the stone from the sampled area has to be done.
Lines 536-546. A discussion and bibliographical references on fungi having this behaviour and capable of inducing mechanical stress on the stone could enrich this paragraph.
Minor editing of English language required
Author Response
Dear reviewer,
I would like to express my gratitude for your highly insightful comments and the valuable constructive criticism provided. Enclosed within the attached document are detailed responses addressing each of the comments and questions raised.
Thank you for your valuable input!
Author Response File: Author Response.pdf
Reviewer 3 Report
Manuscript ID jof-2492676 presents comprehensive description of a novel fungal genus and species, i.e. Saxumspirale lemni, within Aeminiaceae family of Mycosphaerellales order, isolated from deteriorated limestone monument in Portugal. Description of novel microcolonial fungus is further complemented with data regarding physiological and biodegradative properties of two isolates. The study is soundly designed and manuscript well written and of the interest to the readers of JoF, as well as anyone interested in science of CH biodeterioration and biology of RIF.
There is only one minor detail that I would like resolved before the manuscript is accepted for publication. I feel that lines 99-107 (Introduction) and line 128 (M and M) do not belong in the respective sections but Results and Discussion section as they describe part of the results.
Author Response
Dear Reviewer,
Thank you for your valuable feedback on our manuscript. We have carefully addressed the points you raised in the attached document. We sincerely appreciate your thoughtful evaluation and look forward to hearing from you soon.
Best regards!
Author Response File: Author Response.pdf
Reviewer 4 Report
I have read the paper jof-2492676 entitled “Expanding the microcolonial black fungi Aeminiaceae family: Saxispiralis lemnorum gen. et sp. nov. (Mycosphaerellales), isolated from deteriorated limestone in the Lemos Pantheon, Portugal” by Paiva et al. The study is interesting, and I recommend accepting the manuscript after the following revisions.
General comments
In the work “Expanding the microcolonial black fungi Aeminiaceae family: Saxispiralis lemnorum gen. et sp. nov. (Mycosphaerellales), isolated from deteriorated limestone in the Lemos Pantheon, Portugal” the isolation of two unknown microcolonial black fungus from a deteriorated limestone funerary art piece at the Lemos Pantheon, a national monument located in Águeda, Portugal, was carried out. These were thoroughly studied using a polyphasic approach, consisting in molecular, morphological and physiological analyses and as result, a new a new genus, Saxispiralis gen. nov., and a new species, Saxispiralis lemnorum sp. nov., were proposed. The biodeteriorative potential of the fungal isolates was further evaluated, to better understand the potential contribution to the overall stone monument biodeterioration. The topic is interesting, giving a contribution to the understanding of the fungal diversity involved in the biodeterioration of limestone heritage and to expanding the knowledge of the complex taxonomy of black fungi. The work overall is well presented, the paragraphs are well balanced, and it was easy to read, although some sentences should be written better. The bibliography is complete.
Specific comments
Line 28: Please change “it’s” with “its”.
Lines 172-181: I think this part should be placed in the Results section.
Lines 221-222: This sentence is not clear here. Is “Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures 372-Halobacteria medium” the full name of the cultural medium?
Line 227: Please add manufacturer and country for synthetic low-nutrient agar (SNA), as was done for previous media.
Lines 254-255:“The final pH of the medium was adjusted 254 to 8 for each tested NaCl concentration”. Why did you use alkaline pH for this test?
Line 272: Did the exposure was performed with open lid of the Petri dishes?
Line 300: “the occurrence of such phenomena was not observed” this sentence should be placed in the results, please.
Author Response
Dear Reviewer,
Thank you for your valuable feedback on our manuscript. We have carefully addressed the points you raised in the attached document. We believe these revisions have improved the quality of the paper and made it more suitable for publication. We sincerely appreciate your thoughtful evaluation and look forward to hearing from you soon.
Best regards!
Author Response File: Author Response.pdf
Reviewer 5 Report
The manuscript is very interesting, studying a less-known group of fungi of highly ecological and archaeological importance. However, there are some sections that require revision. Materials and methods require more information and references. Also, statistical analyses details and results are required. Comments are provided to authors below.
Abstract
L27-29: I suggest to revise the style of this sentence. Please, use "of" instead of the genitive and avoid the abbreviated form "'s" for the verb.
Keywords
There is a non English word before Limestone: is it necessary? Is there an alternative one, keeping the technical meaning? The name of the fungal order is also in the title, do you want to keep it?
Please, also check the maximum total number of the keywords allowed in the guidelines for authors.
Introduction
L81-84: I suggest to revise the sentence since it is not clear. It is not clear what you mean with "limited competitiveness" of fungi, since in the stone substrates they result to be competitive with respect to other organisms. I suggest to consider more the great specialisation of these fungi to these "extreme" microhabitats and reformulate the sentence.
L87: I suggest to change in "is not an exception".
L99-113: in this part, some results are presented. I suggest to limit this choice to results section, if it is possible, while you could better introduce the taxonomic aspects of these not-fully described group of fungi and the involved mechanisms of fungal biodeterioration, and enumerate the aims of your study.
Materials and methods
L124: please check the use of the term "pathologies" for this context.
All used components of media, chemicals, software need to be reported with commercial brands, city and country. Please revise all the text, such as for example in lines 137 and 140.
L145: generally weight is used instead of volume. You should provide the weight and cite a reference followed for this analysis.
L172-181: in this part, results are presented with some explanations. I suggest moving this part to results and discuss sections.
L220-231: please, cite some references to support this part and justify the followed methods. Why 23°C?
L234-237: this part is not clear, please revise it.
How was the inoculation carried out? Please, specify here. In lines 245-247, 251-253, 262-263, it is reported an inoculation method used, but it is not clear which instrument was used and which references were followed.
L254-255: this sentence is not clear: please check the pH value which is high and the chemical used to adjust the pH, since NaCl does not change pH, while HCl and NaOH are generally used.
L272-274: please, explain how the mycelial piece was chosen and support this method with references. Please do the same for L277-278.
To reduce repetitions and improve clearance, please, revise the entire section 2.4.
Please revise section 2.5 and table 2, since there are no references cited, inoculation is not clear, as well as media composition and require a check (e.g. table 2, 0 g of calcium acetate?). Which strain of Penicillium brevicompactum was used?
Moreover, how could authors identify the fungal secondary minerals (e.g. oxalate)? Morphological analysis is not sufficient. Please, clarify.
Did you perform any statistical analyses? How many replicates for the performed tests? Please, explain and integrate it in the text and in the results and discussion section.
Results and discussion
Figure 3: C,D, F, G and H are cut, please provide uncut figures. Moreover, please, provide scale bars for all figures and report them in the caption.
Do the same for Figure 4.
L486-491: Please integrare your results with statistics.
L492-496: please, revise this part, improving style and cleanness and supporting your explanations with references.
Figure 5: for the colony figure for pH; please consider replacing it with a figure with a single colony and check your data. More colonies in one plate can provide underestimation of fungal growth due to different interactions or competition for resources.
Please provide high resolution figures.
Other comments:
have you thought to test the fungus in liquid medium and measure pH modifications?
Have you considered studying organic acid production by this fungus?
References
Please, check again the references considering the guidelines for authors.
Only minor editing of English is required.
Author Response
Dear Reviewer,
Thank you for your valuable feedback on our manuscript. We have carefully addressed the points you raised in the attached document. We believe these revisions have improved the quality of the paper and made it more suitable for publication. We sincerely appreciate your thoughtful evaluation and look forward to hearing from you soon.
Best regards!
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
There is a profound contrast between words and deeds; the authors reiterate appreciation for the reviewer’s comments, but little or nothing has been changed/ improved compared to the previous version.
Here is a short list of the suggestions skipped and still pending.
· The title doesn’t reflect the dual content mentioned above. The same problem affects the introduction, which should be reworked, removing results from there (L92-102 should be dramatically reduced) and defining the other work aim/s. This could be useful for study design understanding.”
· Keywords. The authors should keep in mind (1) how search algorithms work. If you put in whatever search engine “new species” you can be sure that mammals, amphibians, fishes, plants, or other types of microorganisms will be in your output. (2) Words indexed in the title are not allowed. (3) keywords should improve finding your work and not the journal. So, you must use kw to characterize your manuscript, if you don’t do it, you are running 110 m hurdles and having your knees tied. (https://www.editage.com/insights/how-to-create-keywords-for-a-research-paper#:~:text=Keywords%20should%20ideally%20be%20phrases,closely%20related%20to%20your%20topic.)
· There were incongruences between the citation reported and the conditions applied in the experiment (e.g., temperature, salt) and is not introduced the reason why some tests such as pH, hot tolerance, freeze-thawing etc have been applied even though no similar conditions characterize the isolation source. Considering the explanation given about your “based on protocols ”as a free inspiration is not consistent with common sense, and the citation of Sterflinger and Selbmann as it is misleading; most important, explain why you decided to test three temperatures only to measure pH and the info about DSMZ 372 HM is very important from an applicative point of view. Share your experience with readers (I asked of salt concentration because of tricks in medium preparation).
· Order is also required in figures where sub-figures are not regular, and lettering doesn’t follow the typical reading frame from left to right- in this item, JoF guidelines are quite general because they are based on common sense. Order is needed for fast and effective reading, and you randomly placed them. Please also check the inconsistency between the figure and the captions. Sorry, last time, I missed pointing it out. (e.g. (A-C) Colony appearance on PDA before and after maturation, with melanization progressing to fully black colour. (1) it is expected to follow the growth of the same colony till the total black stage; (2) there are a full stop and semicolon; please cancel one of them
· “Since MEA has different recipes, please disclose yours.”JoF, as all MDPI journals require detailed MM to allow experiment reproducibility, the info you gave me should be part of the manuscript (suppliers and MEA recipe too).
· “L245-253 UV tolerance and plate vitality test. Please explain how the experiment was performed. In detail, how the mycelium has been prepared since the shadow effect can interfere with the real tolerance limit, and 0.5 cm seems to be a big slice. How about the plating, counting and negative control? Please specify if the mycelium is fresh or dehydrated; similar comments are for heat and cold stress tests”.If I ask you, it means that the info is missing in the text. Moreover, being micro-colonial fungi well known for their three-dimensional clumped growth is basic to know if you are testing single cells or not. The shadow I was talking about was that given by the external layer that protects the inner cells from UV damage. Please amend the text
· L266-278 Please explain the reason for choosing each culture medium, their recipe and what you expect from each. What’s the reason for modifying the B4 medium? In particular, please explain the reason for choosing creatine sucrose agar since the calcium carbonate medium is already a test for acid production. What’s the meaning of pH in Table 2? Dear authors, you don’t have to explain to me; you should add that information to the text. About pH obviously was not referred to the general meaning, but the meaning in table 2 (not explained as a note); is it referred to the final pH of the medium?
· L255 physiological solution – please change it to saline because the true physiological solution is not only isotonic but also work as a buffer solution.
· What was the concern with the Curator of Mycobank? The species epithet still creates a link with Lemnos Island, this time using the plural genitive.
· L434 -optimal growth – you cannot be sure since 15 °C has not been measured. The same in MM L234. To determine the optimal temperature growth range…Since you didn’t measure MM should be modified to something like “the growth rate was measured at 5, 20, 25…°C “…in the result section, the sentence should be something like “At the tested conditions, the best growth recorded was..” please amend.
Author Response
The authors would like to express their sincere gratitude to the reviewer for the valuable suggestions, corrections, and constructive criticisms of the manuscript. We have thoroughly reviewed and revised the original document to address pertinent concerns and have made our best efforts to provide thorough responses to the raised questions. Our revisions to the original text have been highlighted in green.
We hope that the revisions provided are sufficient to make our manuscript suitable for publication and look forward to hearing from you at your earliest convenience.
“There is a profound contrast between words and deeds; the authors reiterate appreciation for the reviewer’s comments, but little or nothing has been changed/ improved compared to the previous version.”
- The authors regret this feedback; however, we would like to reiterate that all comments have been carefully analysed and taken into consideration. Whenever they contributed positively to the article and were not solely a matter of personal preference, they were incorporated into the text. Additionally, it is important to note that besides the sections highlighted in blue, the sections marked in purple represent changes made in response to similar comments from different reviewers and other minor corrections necessary to ensure the accuracy of the provided information. Consequently, a substantial portion of the article has been restructured to accommodate the suggestions. Regardless, we sincerely appreciate the reviewer's efforts in helping to improve the quality of our manuscript.
“The title doesn’t reflect the dual content mentioned above. The same problem affects the introduction, which should be reworked, removing results from there (L92-102 should be dramatically reduced) and defining the other work aim/s. This could be useful for study design understanding.”
- The authors extend their sincere gratitude for the valuable comment provided by the reviewer. While the decision has been made to retain the original title, as it effectively encompasses the article's content, significant efforts have been dedicated to adjusting the abstract section and the concluding part of the introduction to ensure clear research objectives and better reflect the dual content, in response to the reviewer's suggestions. Additionally, we would like to clarify that the brief mention of the main results adheres to the guidelines outlined by the Journal of Fungi (JoF), which state in the instructions for the preparation of the introduction section, "Finally, briefly mention the main aim of the work and highlight the main conclusions."
“Keywords. The authors should keep in mind (1) how search algorithms work. If you put in whatever search engine “new species” you can be sure that mammals, amphibians, fishes, plants, or other types of microorganisms will be in your output. (2) Words indexed in the title are not allowed. (3) keywords should improve finding your work and not the journal. So, you must use kw to characterize your manuscript, if you don’t do it, you are running 110 m hurdles and having your knees tied. (https://www.editage.com/insights/how-to-create-keywords-for-a-research-paper#:~:text=Keywords%20should%20ideally%20be%20phrases,closely%20related%20to%20your%20topic.)”
- As suggested, the keywords have been carefully reviewed and modified accordingly.
“There were incongruences between the citation reported and the conditions applied in the experiment (e.g., temperature, salt) and is not introduced the reason why some tests such as pH, hot tolerance, freeze-thawing etc have been applied even though no similar conditions characterize the isolation source. Considering the explanation given about your “based on protocols”as a free inspiration is not consistent with common sense, and the citation of Sterflinger and Selbmann as it is misleading; most important, explain why you decided to test three temperatures only to measure pH and the info about DSMZ 372 HM is very important from an applicative point of view. Share your experience with readers (I asked of salt concentration because of tricks in medium preparation).
L434 -optimal growth – you cannot be sure since 15 °C has not been measured. The same in MM L234. To determine the optimal temperature growth range…Since you didn’t measure MM should be modified to something like “the growth rate was measured at 5, 20, 25…°C “…in the result section, the sentence should be something like “At the tested conditions, the best growth recorded was.” please amend.”
- The authors would like to once again clarify that the protocols used are adapted from the cited references, rather than being a duplication of them. It is not a matter of "free inspiration", as mentioned, but rather small adjustments tailored to the tested organism and the intended objectives. For instance, when it comes to temperature, the aim was not to identify a single preferential temperature, but rather to determine the temperature range where the organism can grow and survive, thus classifying it accordingly within this interval. Furthermore, this information is later reinforced in the high and low-temperature tolerance assay. Nevertheless, the manuscript has been further reviewed and text slightly modified to accommodate the suggestion provided. Regarding the DSMZ - HM medium, the initial isolates leading to the creation of the Aeminiaceae family were obtained using this specific medium, as mentioned in the relevant article, hence its use and relevance. Additionally, its original recipe, as verified on the DSMZ website, includes 20% NaCl, making it inherently stable at high salt concentrations. Nonetheless, we have adjusted the text and added further details to address this point.
“Order is also required in figures where sub-figures are not regular, and lettering doesn’t follow the typical reading frame from left to right- in this item, JoF guidelines are quite general because they are based on common sense. Order is needed for fast and effective reading, and you randomly placed them. Please also check the inconsistency between the figure and the captions. Sorry, last time, I missed pointing it out. (e.g. (A-C) Colony appearance on PDA before and after maturation, with melanization progressing to fully black colour. (1) it is expected to follow the growth of the same colony till the total black stage; (2) there are a full stop and semicolon; please cancel one of them”
- As suggested, the lettering of the images has been revised accordingly. Additionally, the caption of each figure has been carefully verified and accurately corresponds to the respective image. However, we would like to clarify that if "common sense" were always from top to bottom and from left to right, there would be no need for letters on the images and their respective captions, as the order would be standardized. Nonetheless, readers will always refer to the corresponding lettered caption, regardless of its positioning. Additionally, the order was chosen simply to maintain grouping of the same medium or similar structures (or related), and not randomly.
“Since MEA has different recipes, please disclose yours.”JoF, as all MDPI journals require detailed MM to allow experiment reproducibility, the info you gave me should be part of the manuscript (suppliers and MEA recipe too).”
- As recommended, the manufacturer of the respective culture media, when applicable, has been indicated the first time each medium is mentioned in the text. Otherwise, the respective recipes are provided in detail.
“L245-253 UV tolerance and plate vitality test. Please explain how the experiment was performed. In detail, how the mycelium has been prepared since the shadow effect can interfere with the real tolerance limit, and 0.5 cm seems to be a big slice. How about the plating, counting and negative control? Please specify if the mycelium is fresh or dehydrated; similar comments are for heat and cold stress tests”.If I ask you, it means that the info is missing in the text. Moreover, being micro-colonial fungi well known for their three-dimensional clumped growth is basic to know if you are testing single cells or not. The shadow I was talking about was that given by the external layer that protects the inner cells from UV damage. Please amend the text”
As indicated in the manuscript, the assay was performed on small fragments collected from a fresh and fully matured culture, not on individual cells. Also, if the mentioned shadow effect refers to the physical and structural characteristics of the fungus, such as the presence of highly melanized cells or the production of protective substances, as explained in the introduction section, these are precisely some of the intrinsic features of these fungi that enable them to survive various stress factors, including UV. Therefore, the ability of this fungus to grow without any impairment after exposure to UV is precisely due to these adaptations; it is not an additional effect but rather a structural adaptation of these fungi. We hope we have understood the question and adequately clarified the matter. Nevertheless, in order to provide further clarification on this matter, the inoculation process has been elaborated in more detail in the text.
“L266-278 Please explain the reason for choosing each culture medium, their recipe and what you expect from each. What’s the reason for modifying the B4 medium? In particular, please explain the reason for choosing creatine sucrose agar since the calcium carbonate medium is already a test for acid production. What’s the meaning of pH in Table 2? Dear authors, you don’t have to explain to me; you should add that information to the text. About pH obviously was not referred to the general meaning, but the meaning in table 2 (not explained as a note); is it referred to the final pH of the medium?”
- The authors would like to clarify that the selection of culture media and the corresponding tests was solely based on the substrate type from which the fungus was obtained, as stated at the beginning of both the Materials and Methods and Results and Discussion sections. These tests are commonly recommended in the literature for identifying fungal deteriorative characteristics in limestone. This also applies to the modified B4 medium, where the calcium source is adjusted based on the main constituent of limestone. Additionally, we would like to emphasize that the Calcium Carbonate Solubilization or Dissolution assay can be complemented by the evaluation of pH media modifications, which are complementary tests and not duplicates. For further information or clarification on this matter, we provide a recently published review on this topic: https://doi.org/10.3390/app11094196. However, in order to better accommodate the suggestion made, Table 2 has been readjusted to include information about the test, the corresponding culture medium, detailed recipe, final pH, and the expected outcome in case of a positive reaction, along with their respective references.
“L255 physiological solution – please change it to saline because the true physiological solution is not only isotonic but also work as a buffer solution.”
- As suggested, the text has been revised accordingly.
“What was the concern with the Curator of Mycobank? The species epithet still creates a link with Lemnos Island, this time using the plural genitive.”
- As part of the standard procedure for describing a new species, all proposed names for a new species are thoroughly reviewed by the MycoBank curator to ensure adherence to proper nomenclatural guidelines. Accordingly, the name will be retained as stated in the article.
Reviewer 5 Report
I have only few commets to authors:
L263:Please, correct "was" and replace it with "were" (...mycelia were incubated...)
Table 4: the media components should have the same information about manufacturers, country and town like used media or software.
L361,369: "s. lat." not in italics
Author Response
Dear Reviewer,
We sincerely appreciate your highly insightful comments and valuable criticisms of our manuscript, which have significantly improved the overall quality of the document. We have thoroughly addressed all your concerns and revised the original manuscript accordingly. We hope that the revisions provided are sufficient to make our manuscript suitable for publication, and we look forward to hearing from you at your earliest convenience.
Thank you again for your time and effort in reviewing our manuscript!
