Changes in Soil Fungal Diversity and Composition along a Rural–Urban Gradient

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Changes in Soil Fungal Diversity and Composition along a Rural–Urban Gradient

Hans-Peter Rusterholz and Bruno Baur

Some Questions

Line 151. 2.4. Soil Sampling and Physiochemical Properties. Please give WRB (World Reference Base for Soil Resources) soils name and morphological description.

Line 419. However, no relationship was found between soil pH and soil fungal richness in our study. Maybe we need to describe that there is no or weak relationship on soil moisture

Author Response

Reviewer 1

Line 151. 2.4. Soil Sampling and Physiochemical Properties. Please give WRB (World Reference Base for Soil Resources) soils name and morphological description.

Following the suggestion of the reviewer we added the WRB soil name and a morphological description.

Line 419. However, no relationship was found between soil pH and soil fungal richness in our study. Maybe we need to describe that there is no or weak relationship on soil moisture, soil organic matter, nitrogen and plant-available phosphorus content?

The reviewer may have over-seen that soil pH, soil organic matter, total organic nitrogen content and plant-available phosphorus content are inter-correlated. Thus, we considered exclusiverly soil pH and soil moisture as co-factors in all statistical analyses. Furthermore, soil fungal richness was signifcantly affected by soil moisture. We therefore made no changes in the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The manuscript is devoted to the environmentally important topic – the variations in soil fungal communities along a rural-urban gradient. The authors measured various characteristics of the vegetation cover and soil chemistry along the gradient in order to estimate their influence on soil mycobiota. However, there are some principal aspects, which need to be clarified and improved.

First of all, the authors should properly distinguish between "community" and "OTUs". In fact, they studied fungal communities at different taxonomic levels – from species (OTUs) to phyla.  In the study, fungal ITS genes were amplified, and "taxonomy with short reads is focused on ITS1 and ITS2 regions, considered as the official barcoding markers for species-level identification in fungi" (Schoch et al., 2012, https://doi.org/10.1073/pnas.1117018109). Taxonomy assignments of fungal phylotypes were performed against the UNITE database, and "UNITE regularly clusters all ITS sequences at several sequence similarity thresholds to obtain approximate species-level OTUs referred to as species hypotheses" (Nilsson et al., 2019, doi: 10.1093/nar/gky1022). Therefore, such expressions as "diversity of OTUs" (by the Shannon index) and "evenness of OTUs" should be substituted by "diversity and evenness of fungal communities", and the titles of the corresponding subsections in Results and Discussion should be corrected.

The authors use such strange expressions as "plant and tree species" or "species composition of plants, shrubs and trees". Probably they mean "herbaceous plants" because shrubs and trees also belong to plants.

Subsection 2.4. The authors did not measure any physical soil properties (color, texture, structure, porosity, density, consistence, aggregate stability, and temperature), only chemical ones.

Subsection 2.6. The description of fungal functional guild determination is absent.

On my mind, the authors placed excessive information in the table footnotes, some of which can be definitely moved to the table captions.

Discussion mainly answers on the question "how" and much less – "why". For example, could the decrease in relative abundance of Basidiomycota (the great majority of which definitely belong to ectomycorrhizal fungi) with the increase of urbanization be associated with the decrease in tree cover? The authors wrote, "Most Chytridiomycota are saprotrophic fungi living on decaying plant material" (lines 443-444). But exactly the same is applicable to Ascomycota (this very important and extremely diverse fungal phylum even does not mention in Discussion). Many chytridiomycetous fungi are water-dependent in their life cycle – so what about the association of their abundance with soil moisture? Additionally, it is rather strange that no or very low abundance of Glomeromycota (arbuscular mycorrhizal fungi) has been detected in the studied soil, despite the abundant presence of herbaceous plants along the gradient. Is there any explanation of this fact?

All other comments and suggestions are inserted into the PDF version of manuscript, which is attached.

Author Response

Reviewer 2

The manuscript is devoted to the environmentally important topic – the variations in soil fungal communities along a rural-urban gradient. The authors measured various characteristics of the vegetation cover and soil chemistry along the gradient in order to estimate their influence on soil mycobiota. However, there are some principal aspects, which need to be clarified and improved.

First of all, the authors should properly distinguish between "community" and "OTUs". In fact, they studied fungal communities at different taxonomic levels – from species (OTUs) to phyla. In the study, fungal ITS genes were amplified, and "taxonomy with short reads is focused on ITS1 and ITS2 regions, considered as the official barcoding markers for species-level identification in fungi" (Schoch et al., 2012, https://doi.org/10.1073/pnas.1117018109). Taxonomy assignments of fungal phylotypes were performed against the UNITE database, and "UNITE regularly clusters all ITS sequences at several sequence similarity thresholds to obtain approximate species-level OTUs referred to as species hypotheses" (Nilsson et al., 2019, doi: 10.1093/nar/gky1022). Therefore, such expressions as "diversity of OTUs" (by the Shannon index) and "evenness of OTUs" should be substituted by "diversity and evenness of fungal communities", and the titles of the corresponding subsections in Results and Discussion should be corrected.

Please note that we did not use ITS1 and ITS2 markers (we used ITS3 and ITS4 markers). Nonetheless, following the suggestion of the reviewer we replaced “diversity of OTUs” and “evenness of OTUs” by “diversity and evenness of fungal communities” throughout the manuscript.

The authors use such strange expressions as "plant and tree species" or "species composition of plants, shrubs and trees". Probably they mean "herbaceous plants" because shrubs and trees also belong to plants.

We replaced “plant” by herbaceous plant” throughout the manuscript.

Subsection 2.4. The authors did not measure any physical soil properties (color, texture, structure, porosity, density, consistence, aggregate stability, and temperature), only chemical ones.

We improved the text by deleting “physiochemical” throughout the manuscript. Instead we use “soil properties”.

Subsection 2.6. The description of fungal functional guild determination is absent.

We added one sentence that describes fungal functional guild determination.

On my mind, the authors placed excessive information in the table footnotes, some of which can be definitely moved to the table captions.

Please note that we followed the guidelines of the journal. However, we made some improvements in the tables suggested by the reviewer.

Discussion mainly answers on the question "how" and much less – "why". For example, could the decrease in relative abundance of Basidiomycota (the great majority of which definitely belong to ectomycorrhizal fungi) with the increase of urbanization be associated with the decrease in tree cover? The authors wrote, "Most Chytridiomycota are saprotrophic fungi living on decaying plant material" (lines 443-444). But exactly the same is applicable to Ascomycota (this very important and extremely diverse fungal phylum even does not mention in Discussion).

Please note that most “why” answers are speculative, largely due to the lack of necessary studies. However, we improved the Discussion by mentioning that tree cover was not affected by urbanization and can therefore not be a factor influencing the relative abunfance of Basidiomycota. We also mentioned the very important and extremely diverse fungal phylum Ascomycota.

Many chytridiomycetous fungi are water-dependent in their life cycle – so what about the association of their abundance with soil moisture?

We did not find a linear relationship between relative abundance of chytridiomycetous fungi and soil moisture. We inserted this information in the Results and improved the Discussion.

Additionally, it is rather strange that no or very low abundance of Glomeromycota (arbuscular mycorrhizal fungi) has been detected in the studied soil, despite the abundant presence of herbaceous plants along the gradient. Is there any explanation of this fact?

Other studies found also a very low number of arbuscular mycorrhizal fungi (7 out of a total of 327 mycrorhizal fungi; Wubet et al., 2012) or no Glomeromycota in the beech forest soils (Gorfer et al., 2021). It could be that the primers used do not sufficiently recognize Glomeromycota or that this fungal phylum only occurs in very low abundance in the forest soil examined, below the detection limit. However, the information on the particular topic is too vague to be mentioned in the Discussion.

In addition, we improved the text and explained which percentages present the relative frequency of the OTUs (fungal species) and which percentages refer to the relative abundance of the fungal species (or groups).

All other comments and suggestions are inserted into the PDF version of manuscript, which is attached.

We considered the minor remarks and suggestions in the pdf-version of our manuscript (mainly replacements of a term by another term).

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript looks better; however, there are still some doubtful points.

Lines 8 and 39 – what does "ecological diversity" mean? Functional diversity?

Line 93: What about other functional groups of fungi - they are not sensitive to disturbance? Moreover, [31] refers to arbuscular mycorrhyzal fungi, and the study revealed AMF in very low abundance.

Subsection 2.2. – the description of soil profile should be provided with the relevant reference.

Line 97: "differences in the composition of fungal phyla" – but according to the results, only relative abundances and frequencies of occurrence of fungal phyla were studied.

Subsection 3.3. "frequency of occurrence" - it should be specified in M&M how this parameter was calculated.

As to Discussion, I am still not satisfied with it. Even if the most "why" answers are "speculative", they should be given. For example, why is – on the authors' mind – the richness of fungal communities negatively related to the degree of urbanization, while the heterogeneity (Shannon index) and evenness of the communities – positively? When I previously mentioned the possible association between the decrease in relative abundance of Basidiomycota (i.e. ectomycorrhizal fungi) along the rural-urban gradient and the decrease in tree cover, I meant not the decrease in canopy closure, but the decrease in number of trees (tree individuals) as the hosts for EM. Still, the authors did not discuss the variations in Ascomycota along the rural-urban gradient, because to write "and Ascomycota" on line 560 is definitely not enough. Yes, Ascomycota is extremely diverse phylum containing species with different morphology, mode of reproduction, and ecological preferences, but nevertheless…

All other comments and suggestions are inserted into the PDF version of manuscript, which is attached.   

Comments for author File: Comments.pdf

Comments on the Quality of English Language

 Minor editing of English language required

Author Response

Reviewer 2

The manuscript looks better; however, there are still some doubtful points.

Lines 8 and 39 – what does "ecological diversity" mean? Functional diversity?

We replaced ecological diversity by functional diversity.

Line 93: What about other functional groups of fungi - they are not sensitive to disturbance?

Please note that the hypotheses are formulated at the beginning of the research and should not be reformulated afterwards – depending on the results. However, we slightly modified the wording.

Moreover, [31] refers to arbuscular mycorrhyzal fungi, and the study revealed AMF in very low abundance.

We replaced this reference by a more appropriate one which assessed the effects of disturbance on both ectomycorrhizal and arbuscular soil fungi.

Rodrigez-Ramos, J.C., Cale, J.A.; Cahill Jr, J.F.; Simard, S.W.; Karst, J.; Erbilgin, N. Changes in soil fungal community composition depend on functional group and forest disturbance type. New Phyt. 2021, 229, 105-1117. DOI: 10.1111/nph.16749.

Subsection 2.2. – the description of soil profile should be provided with the relevant reference.

As suggested we inserted a reference.

Line 97: "differences in the composition of fungal phyla" – but according to the results, only relative abundances and frequencies of occurrence of fungal phyla were studied.

Phyla composition is by defenition the combined measure of proportion of occurrence and relative abundance of the different phyla. We presented these data in Table S2 and lines 452–455 in the Results section in the original version of the manuscript.

Subsection 3.3. "frequency of occurrence" - it should be specified in M&M how this parameter was calculated.

To simplify to presentation of the results we decided to delete the sentence on frequencies of occurrence. The focus is given on relative abundance.

As to Discussion, I am still not satisfied with it. Even if the most "why" answers are "speculative", they should be given. For example, why is – on the authors' mind – the richness of fungal communities negatively related to the degree of urbanization, while the heterogeneity (Shannon index) and evenness of the communities – positively?

We extended the Discussion to explain why the richness of fungal communities decreases with increasing urbanization, while the Shannon index and evenness of the communities increases.

When I previously mentioned the possible association between the decrease in relative abundance of Basidiomycota (i.e. ectomycorrhizal fungi) along the rural-urban gradient and the decrease in tree cover, I meant not the decrease in canopy closure, but the decrease in number of trees (tree individuals) as the hosts for EM.

The number of tree individuals did not differ among different degrees of urbanization. It should also be noted that the number of trees stems does not necessarily correspond to the number of tree individuals because an individual can have two or even more stems. We therefore no longer took the number of tree individuals into account in the analyses. For the assessement of the potential effect of the degree of urbanization, we selected areas dominated by European beech (80–90% of all tree individuals) in each of the 20 forests. Previous analyses revealed that the composition of tree species was not affected by the degree of urbanization. Based on the very small number of tree species present in the forest areas, we did not incclud this result in the manuscript. The forest areas studied were dominated by tree species which are EM hosts inculding Fagus sylvatica (80–90% of all stems), Quercus robur and Quercus petraea (5–10%) and single Carpinus betulus and Pices abies stems. The number of stems of these tree species was not affected by the degree of urbanization. Single Acer pseudoplatanus and Fraxinus excelsior trees which are host for arbusculare maycorrhiza occured in the forest areas. We add this information in the Discussion,

Still, the authors did not discuss the variations in Ascomycota along the rural-urban gradient, because to write "and Ascomycota" on line 560 is definitely not enough. Yes, Ascomycota is extremely diverse phylum containing species with different morphology, mode of reproduction, and ecological preferences, but nevertheless…

We improved the ms to explain why Ascomycota did not response to the increasing degree of urbanization.

All other comments and suggestions are inserted into the PDF version of manuscript, which is attached.

We considered the comments and suggestions in the pdf-document.