Fungal Diversity and Dynamics during Long-Term Immersion of Conventional and Biodegradable Plastics in the Marine Environment

Round 1

Reviewer 1 Report

“Fungal Diversity and Dynamics during Long-Term Immersion of Conventional and Biodegradable Plastics in the Marine Environment” by Philippe et al.

Overall, this manuscript is well-written and certainly worthy for publication, as it contains novel and significant information about fungal plastisphere in marine environments. I enjoyed reading it and there are a few concerns which need to be addressed for better publication. 

Major comments:

Firstly, I feel that the information regarding samples and methods used in the study is scarce. More detailed information is needed. How samples were deployed, as well as some information about the characteristics of locations would be useful (hanging in water column/lay on sediment, etc., sample photos in supplementally figs could also be helpful), sample types (film/pellets), how much volume of water was collected for DNA extraction, size of plastics immersed/used for DNA extraction, environmental data corresponding to each sample, etc.

My main concern about this manuscript is the sampling bias, which could mislead the results. For example, the collecting time of the plastic samples were quite biased for each sample. i.e. PVC samples were collected intensively in the early immersion period, such as 1-30 days in Toulon, but not in the middle period. In contrast, PVC samples were intensively collected in the middle period, but not in the early stage from Brest. I strongly feel that this sampling bias would influence the analysis measuring the impact of immersion duration towards fungal communities on plastic materials. Especially, since it was indicated in this study that location is the most effective factor for fungal diversity, it is better to analyze the impact of duration time individually for each location. Also, there seems to be a sampling bias for water/environmental samples as well. Missing water samples from Banyuls-sur-mer and Brest which showed the highest fungal diversity, could mislead the result of lower fungal diversity in water/environmental samples. Moreover, there seems to be sampling bias on the number of samples as well, according to Supplementary table 1. Triplicates or more were available in some conditions, while a single sample was available for others. In my understanding, increasing the number of samples will increase the total number of ASVs detection. These issues should be addressed by individual analysis on each location, etc.  

Followings are some specific comments:

L205, In our experience, the community amplified by fungal-specific primer from plastic materials often identify as non-fungi, and using UNITE database won’t provide accurate identification sometimes. I wonder similar experience was observed in this study as well.

L228-230, In relation to the previous comment, what was the percentage of reads which were assigned as fungi? Also, were these sequence data submitted to any public databases?

L255-284 and related other parts, Please refer to the major comments.

L272, et?

L308, Alternaria should be in italics.

L345, It doesn’t look like a contrasting proportion from Figure 4E, as there are not much difference in the size of the bubble plots. The presentation can be improved.

L345-353, The number of samples for water columns is much smaller than PVC samples and samples were not available from all locations. This sampling bias could affect the low relative proportion in surrounding waters. This should be confirmed by comparing with the same sampling effects. These comments also apply for the 18S analysis as well – please refer to the major comments.

Figure 4, How were these 11 genera selected? Were they the top 10 most abundant genus?

L370-375, It would be useful to know the relation of temperature and the location. Did specific locations always have a high/low temp? or did each location have a variety of temperature ranges depending on the collection timing, etc.

Supplementary figures 2C and 2D, abbreviations or explanation for tidal location need to be written in fullform somewhere.

L401-403, “a difference that may be attributed to the smaller number of total fungal ASVs obtained in the 18S…” I think that this explanation is not quite correct.

L416-419, Same comments as L345-353.

L499-503, I feel that this explanation is not quite right.

Figure 8D, It would be useful to see which plots indicate which materials are within conventional and biodegradable plastics (i.e. PEO, OXO, PCL, PHBV).

Figure 9, It was mentioned earlier in the manuscript that fungal diversity was much higher in surrounding water than on plastics. However, it looks like more diverse fungi were detected from plastics in Figure 9. May be this should be explained.

Section 4.2, Were there any trends that fungal community on conventional plastics match more with environmental (water) communities than fungal communities on biodegradable plastics? Accumulation of degrading fungi on biodegradable plastics can explain the lower fungal diversity on biodegradable than conventional plastic. I wonder if such a trend observed in the analysis actually depends on the installation period? i.e. fungal diversity on biodegradable plastics is made less with longer immersion periods?

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

General Comments

This work assesses the fungal community associated with conventional and biodegradable plastics under different conditions both in a marine natural environment and under controlled laboratory conditions. The authors used the ITS2 region, which is the most common region used in meta-barcoding analyses, and the V4 region of the 18S gene. These two regions represent a good choice for assessing biodiversity. However, the taxonomic precision of these regions at the species level is hampered by the amount of data available in public databases like UNITE and the lack of cultures. Thus, the identifications at the species level provided in the discussion should be considered with caution. Despite this, the results section represents data at the genus level and manages to describe the fungal communities recovered in each treatment. This enabled the authors to conclude that fungi form different communities on plastic substrates and the surrounding water, communities growing on plastic substrates change over time and comprise different taxa depending on the geographical location. These conclusions improve the knowledge on fungal population dynamics, a discipline still in progress.

Specific comments

- The meaning of PVC is not stated in the text. It would be interesting to include a brief description of this polymer in the introduction section, because it represents the only substrate assessed in the natural environment.

- ITS2 is a region between the 5.8S and 28S genes, but ITS2 is not a gene.

- In general, the resolution of the figures should be improved. Moreover, figure captions are missing information. Figures should be self-explanatory and provide all the information required to understand the figure without reading the text. In particular:

a) In figure 1B, 2B, 3B 4C, 4D, 5B, 6B, 7C, 7D, 8B and 9the unit of time is missing in both the graph and the caption.

b) Figure 9 is missing the letter that identifies each graphic.

c) Figure captions should mention ITS 2 instead of just ITS, because only a portion of the whole ITS region was analysed. In this sense, when required, figure captions should specify the V4 region of the 18S gene instead of just 18S dataset. Moreover, figure 2 repeats “ITS dataset” twice.

d) Figures 3, 6, 7 and 8 are too small and, in particular, the lines of the hierarchical clustering and some plot lines are too thin and subtle.

e) The location abbreviations used in figures 3D and 6D are too small and they are not explained in the text.

f) Figure 4C is missing the numbers for each relative percentage dot.

Section 3, results, contains the template text of the journal.

- Line 105, it should say targeting instead of targeted.

- Lines 618 to 620 and 627 to 629, the writing is confusing and need clarification.

- Check citation in the text since format like this “Lacerda et al. (2020) [39]” is not correct.

Author Response

Please see the attachment

Author Response File: Author Response.pdf