Richness of Arbuscular Mycorrhizal Fungi in a Brazilian Tropical Shallow Lake: Assessing an Unexpected Assembly in the Aquatic-Terrestrial Gradient

Round 1

Reviewer 1 Report

The authors have done a great and thorough job, but there are questions and/or inaccuracies

Abstract

Line 32: The word „restinga“ is mentioned for the first time in „Abstract“, but its meaning is not explained, and it is not at all clear what the significance of its mention in this chapter is.

Introduction

Lines 45-46: misleading citation – for example, sentence „However, studies on AMF diversity, distribution, and function were conducted mainly in terrestrial environments [17]“ – this reference 17 is about aquatic habitats (lines 491-492: [17] Queiroz, M.B.D.; Jobim, K.; Vista, X.M.; Leroy, J.A.S.; Gomes, S.R.B.S.; Goto, B.T. Occurrence of Glomeromycota species in aquatic habitats: a global overview. Mycotaxon 2020, 135, 469-469. https://doi.org/10.5248/135.469 ) and the global view is presented here.

Lines 88-90: the authors emphasize in the aim that „...The present study aimed to characterize the occurrence and structure of the AMF community in a terrestrial-aquatic transition area in an oligotrophic shallow lake impacted by anthropogenic action“, however, the authors do not provide a comparative analysis of the situation in restanga(s) where anthropogenic activities did not occur.

 Materials and Methods

Line 98 (Fig. 1 c): the picture should be corrected because it is difficult to understand – the 10 m scale (left) is significantly larger than the 30 m scale on the right side of the figure (bottom).

Table 1: it is not clear what this abbreviation means: „cmolc.dm^-3“ and „mg.dm^-3“; the lines of the table are drawn indistinctly/messily OR, if the lines thus given have meaning, the description of the table should be clearer.

Line 141: website INVAM – http://invam.caf.wvu.edu/ – this site can’t be reached.

Lines 156, 166, 207, 340-341, etc.: unchecked text, e. g. whether or not there should be spaces between letters, numbers and symbols or missing letter (“specie”)

Line 180: the sentence “The first three principal components were extracted to…” – does this refer to the PCA plot in the "Supplementary Material" that was not available? Figure 2 (in “Results”) shows only two PCA plots.

Line 190: the link to the 82nd reference does not work.

 Results

Table 2: the upper right column of Table 2 is presented unaligned (names are presented on one line, on two lines), making it very difficult to read.

Line 222, 260: the link to the “Table S1 in the supplementary material”, “Table S2 in the supplementary material“ – does not work.

Lines 223-226: Latin names of genus and/or species must be written in italics.

Line 366: there is a reference to Figure 3a in the text, but Figure 3 does not have part "a".

Lines 376-377: the same situation as above (in the line 366) – maybe the authors could clarify if the references to the pictures (such as (Figures 3a, 4) are really correct according to what is stated in this sentence (“However, the richness and number of glomerospores for the aquatic condition superior to the terrestrial condition was unexpected (Figures 3a, 4)”.

Table 3: first row of the table – the meaning of these abbreviations is not explained in the text (such as Sum Sq, Df); it is also unclear whether the same letters in the description of Table 3 and the same letters (S, H, C, J) under the table have the same meaning and this information is merely repeated or not.

Table 3 and Table 4: it is not clear why so many characters under the table (as in lines 268 and 321: "0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1 "), because other characters (except “*”) – not visible.

Lines 267 and 320: what does it mean „e“ in the sentence “Signif. codes: S- richness, H- diversity of Shannon, J- equitability of Pielou e C- dominance of...“

 Discussion

The authors emphasize the high diversity of AMF the “Results” (lines 210-211: "A total of 50 species were found in aquatic conditions, 42 and 33 in the rainy and dry seasons, respectively"), also, a complete list is provided (Table 2). However, it is not clear why the authors did not address this topic in the “Discussion” section: although the title states "...we assess a suite of unexpected richness..." and provides a long list of organisms found, only three (belonging to 2 species and 1 genus) are identified as the main ones and there has been minimal discussion of this issue, although if the authors were to compare their results with already published results, both local (Brazilian) and global, the value of this publication would be significantly higher. Therefore, although the authors emphasize diversity research in the title of the article, the results of the research represent the results of only one place, which are not analyzed even in the existing restinga(s) research conducted in Brazil or in other aquatic condition in other countries, so the work is sufficiently local. It is not clear how reliable the results are, the restinga research data only represent the area of one research site.

Line 431: the webpage www.mdpi.com/xxx/s1 – since the link does not work [“Error 404 - The webpage you are looking for could not be found. The URL may have been incorrectly typed, or the page may have been moved into another part of the mdpi.com site”], it is not possible to view the additional information, where the authors indicate Table S1 and Table S2 should be. Therefore, it is impossible to fully analyze and understand the interpretations of the information and results presented by the authors.

 Reference

Line 636: reference No. 82 – this site can’t be reached.

Author Response

Dear Doctor,

we are sending a corrected version of our paper intended for publication in Diversity. The title of the paper is “Richness of Arbuscular Mycorrhizal Fungi in a tropical shallow lake: assessing an unexpected assembly in the aquatic-terrestrial gradient”; (Ms. diversity-1968792).

Below are our responses to the remarks and suggestions of Reviewers 1. We would like to thank them for all valuable comments that helped us to improve the manuscript.

REVIEWER 1

The authors have done a great and thorough job, but there are questions and/or inaccuracies. 

Authors’ reply: Thank you for the kind message. 

 

Abstract

 

Line 32:  The word “restinga“ is mentioned for the first time in “Abstract“, but its meaning is not explained, and it is not at all clear what the significance of its mention in this chapter is.

Authors’ reply: We agree that the first mention of restinga is not clear. We replace "restinga" to "coastal sand plain vegetation".   

 

Introduction

Lines 45-46: misleading citation – for example, sentence “However, studies on AMF diversity, distribution, and function were conducted mainly in terrestrial environments [17]“ – this reference 17 is about aquatic habitats (lines 491-492: [17] Queiroz, M.B.D.; Jobim, K.; Vista, X.M.; Leroy, J.A.S.; Gomes, S.R.B.S.; Goto, B.T. Occurrence of Glomeromycota species in aquatic habitats: a global overview. Mycotaxon 2020, 135, 469-469. https://doi.org/10.5248/135.469 ) and the global view is presented here.

Authors’ reply: We change the reference. 

 

Lines 88-90: the authors emphasize in the aim that „...The present study aimed to characterize the occurrence and structure of the AMF community in a terrestrial-aquatic transition area in an oligotrophic shallow lake impacted by anthropogenic action“, however, the authors do not provide a comparative analysis of the situation in restinga(s) where anthropogenic activities did not occur. 

Authors’ reply: We did not provide a comparative analysis because it was not our goal to compare the impact of anthropogenic actions. Besides, the studied site does not provide the possibility to test the impact of anthropogenic action. Our goal was to compare the diversity and community structure of AMF between sediments collected from a shallow lake and the soil from a nearby vegetation area. However, the studied lagoon is a touristic area used to recreative activities during all year. For this reason we informe in the paper that the area is impacted by anthropogenic action.

 

Materials and Methods

Line 98 (Fig. 1 c): the picture should be corrected because it is difficult to understand – the 10 m scale (left) is significantly larger than the 30 m scale on the right side of the figure (bottom).

Authors’ reply: Specific modifications were made to figure 1c, the scale was removed mainly because it does not represent the real context and the indication of transects was added to make the image clearer. What we want to show in the image are two transects with a length of 30 m each and 10 meters apart from each other, in addition to the four plots inserted in each transect. Thus, for a better understanding of the study design represented in Figure 1c, a detailed description was made in sub-item 2.2 of the materials and methods.

 

Table 1: it is not clear what this abbreviation means: „cmolc.dm-3“ and „mg.dm-3“; the lines of the table are drawn indistinctly/messily OR, if the lines thus given have meaning, the description of the table should be clearer.

Authors’ reply: Done. 

 

Line 141: website INVAM – http://invam.caf.wvu.edu/ – this site can’t be reached.

Authors’ reply: Thanks for the observation. The correction was made - https://invam.ku.edu/species-descriptions - the collection was transferred from West Virginia to Kansas.

 

Lines 156, 166, 207, 340-341, etc.: unchecked text, e.g. whether or not there should be spaces between letters, numbers and symbols or missing letter (“specie”)

Authors’ reply: Done.

 

Line 180: the sentence “The first three principal components were extracted to…” – does this refer to the PCA plot in the "Supplementary Material" that was not available? Figure 2 (in “Results”) shows only two PCA plots.

Authors’ reply: In the supplementary material we have Table S1 showing the percent variance explained by each of the five principal components. In PCA analyses we should consider hold on to principal components that explain at least 70% of the variance cumulatively. We graphically represent only the first 3 components in figure 2 because components 4 and 5 were visually redundant. In figure 2A we have PC1(Dim1) with PC2 (Dim2) and in figure 2B we have PC1 (Dim1) with PC3 (Dim3).

 

Line 190: the link to the 82nd reference does not work.

Authors’ reply: Thanks for the observation. The correct link has been added - https://CRAN.R-project.org/package=RVAideMemoire

 

Results

Table 2: the upper right column of Table 2 is presented unaligned (names are presented on one line, on two lines), making it very difficult to read.

Authors’ reply: This misalignment in table 2 has been corrected, however it is now named table 1.

 

Line 222, 260: the link to the “Table S1 in the supplementary material”, “Table S2 in the supplementary material“ – does not work.

Authors’ reply: Thanks for the observation. Our sincere apologies for this mistake. We are submitting the corrected version including the supplementary material.

 

Lines 223-226: Latin names of genus and/or species must be written in italics.

Authors’ reply: Corrections to species names were made.

 

Line 366: there is a reference to Figure 3a in the text, but Figure 3 does not have part "a".

Authors’ reply: We fixed this error.

 

Lines 376-377: the same situation as above (in the line 366) – maybe the authors could clarify if the references to the pictures (such as (Figures 3a, 4) are really correct according to what is stated in this sentence (“However, the richness and number of glomerospores for the aquatic condition superior to the terrestrial condition was unexpected (Figures 3a, 4)”.

Authors’ reply: Thank you for all the remarks. We correct all mistakes.

 

Table 3: first row of the table – the meaning of these abbreviations is not explained in the text (such as Sum Sq, Df); it is also unclear whether the same letters in the description of Table 3 and the same letters (S, H, C, J) under the table have the same meaning and this information is merely repeated or not.

Authors’ reply: The letters in the description of table 3 and below have the same meaning. Therefore, this repetition of information has been corrected. Also, the meanings of Sum Sq and Df have been added below the table for better understanding. Also, table 3 has been renamed as table 2, where you will be able to observe these changes.

 

Table 3 and Table 4: it is not clear why so many characters under the table (as in lines 268 and 321: "0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1 "), because other characters (except “*”) – not visible.

Authors’ reply: When we perform ANOVA in R, the output tables will contain p-values for the variables along with corresponding significance codes.  They represent a measure of p-value variation. We use an alpha level of α = .05, then all values less than 0.05 are significant and have a corresponding code. In the range 0 to 0.001, the assigned code is '***'. Values between 0.001 and 0.01 have code '**', values between 0.01 and 0.05 have code  '*'. When the values range from 0.1 to 1 it has no significance code ' '.

 

Lines 267 and 320: what does it mean „e“ in the sentence “Signif. codes: S- richness, H- diversity of Shannon, J- equitability of Pielou e C- dominance of…“

Authors’ reply: This sentence refers only to table 2, as it shows these parameters explored by the linear and generalized linear models. Thus, this information was excluded from line 313, below table 3.

 

Discussion

The authors emphasize the high diversity of AMF the “Results” (lines 210-211: "A total of 50 species were found in aquatic conditions, 42 and 33 in the rainy and dry seasons, respectively"), also, a complete list is provided (Table 2). However, it is not clear why the authors did not address this topic in the “Discussion” section: although the title states "...we assess a suite of unexpected richness..." and provides a long list of organisms found, only three (belonging to 2 species and 1 genus) are identified as the main ones and there has been minimal discussion of this issue, although if the authors were to compare their results with already published results, both local (Brazilian) and global, the value of this publication would be significantly higher. Therefore, although the authors emphasize diversity research in the title of the article, the results of the research represent the results of only one place, which are not analyzed even in the existing restinga(s) research conducted in Brazil or in other aquatic condition in other countries, so the work is sufficiently local. It is not clear how reliable the results are, the resting research data only represent the area of one research site.

Line 431: the webpage www.mdpi.com/xxx/s1 – since the link does not work [“Error 404 - The webpage you are looking for could not be found. The URL may have been incorrectly typed, or the page may have been moved into another part of the mdpi.com site”], it is not possible to view the additional information, where the authors indicate Table S1 and Table S2 should be. Therefore, it is impossible to fully analyze and understand the interpretations of the information and results presented by the authors.

Authors’ reply:Thank you for your suggestions. We improve sentences in the lines 321-346 to be more clear about the importance of our finds and compare with others studies in sand dunes ecosystems.

 

Reference

Line 636: reference No. 82 – this site can’t be reached.

Authors’ reply: Thanks for the observation. The correct link has been added - https://CRAN.R-project.org/package=RVAideMemoire

Author Response File: Author Response.docx

Reviewer 2 Report

This manuscript "Arbuscular Mycorrhizal Fungi in a tropical shallow lake: assessing an unexpected richness assembly in the aquatic-terrestrial gradient" is an interesting piece of work worth to be considered but it needs some revisions. The comments and suggestions are annotated in the manuscript. Authors are advised to change the title to a more attractive one. 

Comments for author File: Comments.pdf

Author Response

Dear Doctor,

we are sending a corrected version of our paper intended for publication in Diversity. The title of the paper is “Richness of Arbuscular Mycorrhizal Fungi in a tropical shallow lake: assessing an unexpected assembly in the aquatic-terrestrial gradient”; (Ms. diversity-1968792).

Below are our responses to the remarks and suggestions of Reviewers 2. We would like to thank them for all valuable comments that helped us to improve the manuscript.

REVIEWER 2

This manuscript "Arbuscular Mycorrhizal Fungi in a tropical shallow lake: assessing an unexpected richness assembly in the aquatic-terrestrial gradient" is an interesting piece of work worth to be considered but it needs some revisions. The comments and suggestions are annotated in the manuscript. Authors are advised to change the title to a more attractive one.

Authors’ reply: We agree with the need for a more attractive title and the suggestion was considered.

 

Title: Richness of Arbuscular Mycorrhizal Fungi in a Brazilian tropical shallow lake 

Authors’ reply: As suggested, the title was changed to “Richness of Arbuscular Mycorrhizal Fungi in Brazilian tropical shallow lake: assessing an unexpected assembly in the aquatic-terrestrial gradient”.

 

Line 43: Approximately 350 AMF species have been described [3,12] and this is not completed 

Authors’ reply: Done. We modify the sentence to be more clear.

 

Line 82: 22,

Authors’ reply: The error was adjusted by adding a comma after the number 22 on line 81.

 

Line 83: community 

Authors’ reply: The word communities was replaced by community.

 

Line 90: Replace This aim to fulfill this aim 

Authors’ reply: The suggested replacement was performed on line 89.

 

Line 91: Replace communities to community

Authors’ reply: The word communities was replaced by community on line 90.

 

Line 108: Poaceae, Cyperaceae, Fabaceae, Rubiaceae and Chrysobalanaceae (arrange alphabetically)

Authors’ reply: The plant species families were ordered alphabetically in line 108 and 109.

 

Lines 334: replace biomes to biomes' 

Authors’ reply: The replacement was performed on line 346.

Line 335: Replace The present study to In the present study,

Authors’ reply: The replacement was performed on line 347.

 

Line 340: Replace specie to species

Authors’ reply: The replacement was performed on line 353.

 

Verify order of table numbers. 

Authors’ reply: The numerical order of the tables was checked and we did not find any inconsistency. However, due to the suggestion of other reviewers some tables were transferred to the supplementary material and the order of the tables was changed.

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript submitted by Gomez et al. addresses a fascinating topic related to arbuscular mycorrhizal fungi diversity in an aquatic-terrestrial gradient. However, after reading the manuscript, I feel that the authors must provide some improvements. In the Abstract, the contextualization must be reduced, and the section must focus on the main findings of this manuscript. In the introduction section, the authors must provide only one objective. The main criticisms that I have are in the materials and methods section. I feel that just subjective classifications were made to analyze and classify the mycorrhizal diversity ust be complemented with objective data (e.g., molecular tools). I think that the authors based most of the analyses on such observations. To strengthen the result presentation, the authors must provide pictures of the structures to perform the assignment (only the most representative in the manuscript and the others in the supplementary). The authors must reduce the number of figures and tables. The authors must ensure that the discussion is related to their findings. I feel that some sections are out of topic. How do the authors confirm that the presence of fungal structures is related to activity? And how do the environmental conditions modify the diversity of fungal spores? Was sampling performed precisely at the same point in the wet and dry seasons? These are questions that the authors must answer in the manuscript.

Author Response

Dear Doctor,

we are sending a corrected version of our paper intended for publication in Diversity. The title of the paper is “Richness of Arbuscular Mycorrhizal Fungi in a tropical shallow lake: assessing an unexpected assembly in the aquatic-terrestrial gradient”; (Ms. diversity-1968792).

Below are our responses to the remarks and suggestions of Reviewers 3. We would like to thank them for all valuable comments that helped us to improve the manuscript.

REVIEWER 3

The manuscript submitted by Gomez et al. addresses a fascinating topic related to arbuscular mycorrhizal fungi diversity in an aquatic-terrestrial gradient. However, after reading the manuscript, I feel that the authors must provide some improvements. In the Abstract, the contextualization must be reduced, and the section must focus on the main findings of this manuscript. In the introduction section, the authors must provide only one objective. The main criticisms that I have are in the materials and methods section. I feel that just subjective classifications were made to analyze and classify the mycorrhizal diversity ust be complemented with objective data (e.g., molecular tools). I think that the authors based most of the analyses on such observations. To strengthen the result presentation, the authors must provide pictures of the structures to perform the assignment (only the most representative in the manuscript and the others in the supplementary). The authors must reduce the number of figures and tables. The authors must ensure that the discussion is related to their findings. I feel that some sections are out of topic. How do the authors confirm that the presence of fungal structures is related to activity? And how do the environmental conditions modify the diversity of fungal spores? Was sampling performed precisely at the same point in the wet and dry seasons? These are questions that the authors must answer in the manuscript.

Authors’ reply: Thank you for general remarks. We made several improvements in the text following remarks provided for 3 reviewers. In the abstract section we reduce the contextualization to focus on aquatic ecosystems. In the end of discussion sections, following editor suggestions, we prepare considerations about limitations and future perspectives in this topic such as molecular tools methods. The molecular methods are an important tool to access the AMF diversity mostly to respond to specific questions. However, the spore based morphological approach is an efficient method to evaluate AMF diversity (and easy to compare with others studies), mainly when the research team is able to do it, which is our case. The last and senior author of this research article has been using this method for a long time and in several publications. Nevertheless, we agree high-throughput sequencing technology can be useful to improve the data set about richness and composition of a community but it is not our goal. Furthermore, our unpredictable results, even using morphological techniques, can be used to guide new ecological hypotheses that can and should use molecular methods. We do not provide pictures of fungal species in the main file because the paper already presents several figures/tables. Other reviewers suggest removing figures and tables as supplementary files. In this case, we transferred from the main text to the supplementary material only the table referring to the physical-chemical attributes and we did not exclude any other tables or figures, considering that they are relevant information for the understanding of our findings. Furthermore our paper is not taxonomic (despite using taxonomy as an ecological tool) and all information about methods used for identification was presented (in detail) in the material and methods section.

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

Dear editor. After reading the author's response, I feel (again) that the main issue related to identifying such a wide diversity of fungal diversity must be confirmed with objective data. I do not doubt the expertise of the senior researcher in the field, but objective evidence is necessary for this manuscript. The authors must provide such information by attaching pictures in the supplementary material in which there is no page limit. 

Additionally, some previous comments were not answered by the authors. How do the authors confirm that the presence of fungal structures is related to activity? And how do the environmental conditions modify the diversity of fungal spores? Was sampling performed precisely at the same point in the wet and dry seasons? 

Author Response

Dear Doctor,

We are sending a corrected version of our paper intended for publication in Diversity. The title of the paper is “Richness of Arbuscular Mycorrhizal Fungi in a tropical shallow lake: assessing an unexpected assembly in the aquatic-terrestrial gradient”; (Ms. diversity-1968792).

Below are our responses to the remarks and suggestions of three Reviewers. We would like to thank them for all valuable comments that helped us to improve the manuscript.

We are waiting for your decision and wish you all the best.

Bruno T. Goto

Reviewer 3: Dear editor. After reading the author's response, I feel (again) that the main issue related to identifying such a wide diversity of fungal diversity must be confirmed with objective data. I do not doubt the expertise of the senior researcher in the field, but objective evidence is necessary for this manuscript. The authors must provide such information by attaching pictures in the supplementary material in which there is no page limit.

Authors’ reply: We agree that good pictures in the taxonomic papers are useful to confirm spores identities but to do this properly it is necessary to present several pictures of the same species as a request to detect (and confirm) the most important diagnostic features. Meanwhile, to meet the reviewer's requirement, we present a plate with the best images obtained from the original material. Unfortunately, due the old slides prepared in 2015, we did not obtain good images for all species. During the project development we did not obtain pictures of the species identified because several papers published in this field (ecological papers using morphological approach) do not present such requirements. Unfortunately several slides was dry and impossible to be used to obtain good pictures. Nevertheless, we are convinced that our identifications are accurate, taking care to confirm the species based on well-defined criteria (see material and methods) for species definition. We hope that our explanation and plate carefully presented in the supplementary files (Figure S1) can be considered by reviewer requirements.

Reviewer 3: Additionally, some previous comments were not answered by the authors. How do the authors confirm that the presence of fungal structures is related to activity?

Authors’ reply: We are not sure about how to clarify this remark in the discussion. We are cognizant that the presence of glomerospores in the aquatic sediment does not necessarily reflect on its activity. We argue (line 418-420) that the greaterrichness in the aquatic sediment, especially in the rainy season, could be due to hydrochory (glomerospores transfer from the edge to the lake’s interior). Nonetheless, less than 50% of the species were shared between terrestrial and aquatic conditions (Table 1) and there were statistical differences between aquatic/rainy and terrestrial/rainy interactions (line 245-246). These results suggest that there is a dynamic of sporulation, and that the high diversity in sediment may not simply be due to the flow from the terrestrial environment. We increment this argument on lines 420-426).

Reviewer 3: And how do the environmental conditions modify the diversity of fungal spores?

Authors’ reply: We assess whether AMF community structure differs between aquatic and terrestrial conditions and between rainy and dry seasons. Factors such as PH, nutrient content of soil/sediments and plant hosts can affect AMF sporulation and as the ecological analyzes were based on identification of spores obtained in the field, the results related to the seasonal dynamics of the species may be related to the differences in sporulation responses by different species as presented in lines 402-405. However, it was not our goal to evaluate how environmental factors influence the AMF community in these two conditions and seasons. Therefore, our analysis does not allow us to answer this question in an objective manner. In our discussion we cite some likely environmental factors, whose variation may have caused differences in AMF parameters (lines 398-401).

Aquatic environments are historically overlooked in terms of AMF diversity and further research is still needed to understand the dynamics and function of these fungi in this system. It is not possible to answer all questions in just one article, but we consider that our article brings important contributions for understanding the AMF diversity in these systems. For instance, (i) this is only the third study carried out in Brazilian aquatic environments, despite its enormous hydrographic dimension and a significant wealth of macrophytes, and the first to investigate the influence of seasonality, (ii) unlike other studies in aquatic environments, we assess the adjacent terrestrial vegetation, (iii) we found an expressive and unexpected richness of species in the sediment, encouraging more researchers to investigate these systems, (iv) besides we have increased information on the AMF diversity that inhabits Brazilian Coastal Sandy Plain vegetation.

Reviewer 3: Was sampling performed precisely at the same point in the wet and dry seasons?

Authors’ reply: Sediment samples were carried out in the same area in October/2015 and May/2016 during the dry and rainy seasons. Collection efforts were to sample soil and sediments from the same host plant in both environments as mentioned in the lines 117-119.

 

 

Author Response File: Author Response.pdf

Round 3

Reviewer 3 Report

Although I think objective data is necessary for the manuscript, I understand the point of view of the authors and the novelty of the study