Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study examines the relationship between the structure of metazoan and fish communities in a single river using eDNA metabarcoding. This is an interesting approach to understanding the biodiversity of aquatic communities and the mechanisms that shape biodiversity patterns. Although the presented idea is interesting, several significant issues compromise the quality and interpretation of the results, as well as the potential for further discussion.

The primary concern is how the authors define the term "metazoan". According to the definition I use, metazoans are multicellular, heterotrophic organisms that range from Porifera to Chordata. Thus, the L55-56 stating that "…metazoan assemblages are key components of river ecosystems, contributing to nutrient cycling, secondary production, sediment transport, and organic matter mineralization…" makes no sense. Additionally, the title is not suitable since the work examines only a small fraction of the metazoan community.

Another important concern is why the PR2 database was used for metazoan since this database is for protists. I cannot find any logical explanation for this; if one existed, it should be clearly justified in the manuscript. On the other hand, the name of the database is PR2, not RP2, as mentioned in the manuscript.

L260-264: Does the used database cover all the fish species potentially present in this river? Current studies show that there are more than 100 fish species in this river. It appears that the eDNA metabarcoding strategy employed in this study failed to detect all the species present. An appropriate strategy would have been to use multiple markers, such as the MyFish or Teleo1 primers.

A description of the Yujiang River is missing, particularly regarding features necessary to understand the degree of resolution of eDNA, such as flow and the hydrologic cycle.

It is not specified at what time of year the water samples were taken or whether they were all collected simultaneously.

It is not mentioned what equipment was used for water filtration.

L113-115 mentions that "A further 1 L of water samples were stored temporarily at 4°C for the purpose of conducting a series of environmental variable analyses." What does temporarily mean? One day or one year? Furthermore, which environmental variables were measured? Section 2.2 says that the variables were recorded in situ.

A table should be included with all the ASVs obtained. It is strange that no NNs or multiple hits were found. What criteria were used to define a species?

Caution must be taken when discussing abundance. It is incorrect to speak of the abundance of individuals since eDNA only determines the abundance of reads, which may or may not correlate with the abundance of individuals.

L171: This is true only if the database used covers all diversity.

L173-173: The number of species and genera mentioned in the text and in Figure 2 does not match Figure 3.

L176: This is true only for Chao1

Figure 3: The heatmap is not about abundance; it is about reads. It does not make sense for the log of reads to be negative. This means that less than one read was detected.

L225-227. Temperature and transparency are factors that change very rapidly over time. Since the environmental variables were measured only once at each site and were likely recorded at different times of day, it is not appropriate to use these variables to explain variations in diversity.

L236. Where do these 30 ASVs come from? What are they? Why aren't they all mentioned in Figure 6 or Figure 3?

Many species are not written in italics.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Overall, the information presented in the manuscript is sufficient to understand the idea that the authors sought to convey to the reader. However, there are a few comments.

The Results of Measurements of environmental factors are not presented. The authors could present this information in detail in an appendix or briefly in the text of the manuscript. This is especially important for such parameters as transparency and temperature, which are discussed further.

The authors filtered 1 liter of water through 0.22 micron polycarbonate filters without pre-filtration (they did not use a filter with a larger pore size). Polycarbonate filters can clog quickly. Then they perform the Mantel test (this is a statistical method used to assess the correlation between two distance or similarity matrices) and show the correlation between the composition of fish communities and water transparency. But the degree of water transparency can directly affect the filtration process (turbid water will quickly clog filters and vice versa). What filter diameter was used in the methods is also not specified. This can lead to errors in statistical analysis and give a false picture of the correlation. Nothing is written about this, but it should be noted.

It is not clear why Protist Ribosomal Reference Database (PR2) was chosen for metabarcoding? It was created (initially) for protists and only later some other metazoans were added to it. So it seems to me that for metazoans, fungi and plants it will be less effective due to limited coverage than, for example, SILVA.

18S is a rather conservative marker and may not always allow precise taxonomic typing to the species level. For example, the authors found Walkeria uva in their samples. According to literature, this is a marine species - occurs on either side of the Atlantic Ocean, in the Baltic Sea, the Mediterranean Sea, the Indian Ocean and the South China Sea. From this, we can conclude that either contamination occurred or the database is incorrect (and PR2 seems to be more focused on marine representatives of Metazoa). The authors should probably check this information for other species as well.

The article examines the correlation between environmental factors and the diversity of fish and metazoan communities. For metazoans, a correlation was found between temperature and diversity. The temperature range is not given. Meanwhile, it is known that the rate of degradation of ecoDNA in the aquatic environment, in addition to other factors, depends on temperature. It is clear that the wider the range of change, the greater the difference in the rate of degradation. This can lead to erroneous results. Therefore, the range of temperature change should be specified and this issue should be discussed.

In many places in the manuscript the appropriate font for the Latin names of species is not observed.

 

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have responded to all comments satisfactorily, and I consider the manuscript to be significantly improved.

Author Response

Comment：The authors have responded to all comments satisfactorily, and I consider the manuscript to be significantly improved.

Response： We sincerely appreciate your positive evaluation and thoughtful comments, which have helped improve the quality of our manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

In my opinion, the authors gave adequate answers within the concept of the manuscript. However, the text of the manuscript still contains typos and minor flaws (сomments are provided throughout the manuscript, please see the attached file).

I cannot judge the quality of the English language, since English is not my native language, but the manuscript definitely needs to be checked for spelling.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

In my opinion, the authors gave adequate answers within the concept of the manuscript. However, the text of the manuscript still contains typos and minor flaws (сomments are provided throughout the manuscript, please see the attached file).

I cannot judge the quality of the English language, since English is not my native language, but the manuscript definitely needs to be checked for spelling.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf