Review Reports

Round 1

Reviewer 1 Report

This paper presents the first e-DNA survey in the area and represents and important evaluation toward freshwater biodiversity conservation. 

Authors must consider reviewing figure 4 A, as species names are not legible.  Table 2 should be organized in order to function also as a taxonomic table, with families names and species in first column, and in subsequent columns the information on groups. 

I take this opportunity to congratulate the authors for their complete study. I am sure that it will inspirate conservation actions with less impact on live stocks.

Author Response

R1: Authors must consider reviewing figure 4 A, as species names are not legible. 

Our answer: We revised the figure 4 as suggested by Reviewer 1 

Table 2 should be organized in order to function also as a taxonomic table, with families names and species in first column, and in subsequent columns the information on groups. 

Our answer: we re-organized the table 2 as suggested by the Reviewer 1. 

Reviewer 2 Report

Dear Authors,

I liked your MS and find your research very interesting and prompt. 

The only thing I would expect from you is enlarged conclusion. Please develop the concept of the dam building influences on each of the three areas you highlited.

Minor notes see in the attached file. Please check lines 43,320,334,338,379,448,458,495,522,561,602 and the entire text again

Comments for author File: Comments.pdf

Author Response

R2: The only thing I would expect from you is enlarged conclusion. Please develop the concept of the dam building influences on each of the three areas you highlited.

Our answer, in the revised manuscript we added the following sentence to answer Reviewer 2

"The impact of future dam building along the Mekong will then be very different regarding these regions. First, the region including the delta and its biodiversity will be less impacted, as no dam construction is scheduled in a close future and only around 20% of fish species are amphidromous. The fish biodiversity of the second region, including the Tonle Sap and sites downstream until the delta, should have a more serious effect, even if dam building is not scheduled in this area, but because 70 to 90% of species are potamodromous. Finally, the most threaten area is located upstream, from Vientiane, in Laos and Thailand, to Kampong Cham in Cambodia, because the numerous dams will be constructed in the main Mekong River, and more than 60% of fishes are migratory, reinforcing the warning of Nuon et al. (2020)."

R2: Minor notes see in the attached file. Please check:

lines 43: we removed "of the vertebrates" and change "30000" for "36000"

lines 320: We changed "T" for "The"

lines 334 & 338, we removed "..." and added "like" before the list of species.

lines 379: we entierely re-organized the Table 2 as suggested by Reviewer 1 and correct species names as suggested by Reviewer 1

lines 448, we removed "_"

lines 458, we added a space between "on" and "the"

lines 495, we removed one "that"

lines 522, we added a space between "been" and "put"

lines 561, we specified biogeographic regions by adding this to the sentence mentioned : "from upstream to downstream : two successive Headwater Regions in China; the Upper Region corresponding to the Mekong part flowing in the Yunnan province, Middle Region encompassing the Chinese border and the North of Laos ; the Middle Region corresponding to the south of Laos, Nam Chi-Nam Mun Region; Se San-Se Kong Sre Pok Region; Tonle Sap Region; Estuary Region). "

lines 602 We changed "Environmental DNA" by "The metabarcoding of eDNA"

Reviewer 3 Report

Summary:

The authors justify the present study on the fish biodiversity in the Mekong River and Delta inferred by eDNA by the increase of environmental and anthropogenic pressures on such large system (increase in salt-water penetration and disruption of the connectivity by human construction such as dams etc…). In order to better manage such large fish biodiversity and better predict the impact of future infrastructures such as dams on the Mekong, large non-lethal approaches such as eDNA is paramount. The study aims at analyzing the fish diversity along the Mekong River and Delta using an eDNA approach and at building a complementary local fish library that may better reflect local diversity and distribution (particularly for cryptic species or mislabeled species) compared to larger worldwide library such as GenBank.

Water samples were collected at several sites along the Mekong River and Delta, eDNA contained in the water was extracted, sequences and compared to existing database in order to identify which species are present in those waters and estimate how abundant they are. Classical diversity indices (H’, the Shannon diversity index and J, Pielou’s evenness index) were calculated, and clustering and multivariate analyses were performed in order to understand what factors structure the Mekong fish community.

This study recorded a total of 287 OTUs corresponding to putative fish species among which 150 were assigned to already identified species. The fish community seems to be divided in three distinct groups of sites along the Mekong, with no spatial overlap between the groups. Salinity seems to play an important role in structuring the community while the present of the major falls (Khone falls) did not seem to act as a barrier to fish connectivity.

The authors then conclude that their results clearly demonstrated the ability of the eDNA approach to investigate the structure of a fish community in a large system and to identify the potential cause for such structure. They also conclude for the need of more traditional local diversity assessment in order to increase the reliability and the intercalibration of the different database systems.

 

General comments:

The manuscript is well-written and very pleasant to read. The aim of the study is pertinent. The results are interesting for a preliminary approach (but already substantial), the material and methods and discussion are deep with sufficient references. The preparation of the manuscript for publication would not require important amendments to improve the manuscript (see Specific comments below). I therefore recommend the manuscript to be published.

 

Specific comments:

Minor issue:

You took the time to describe many limitations to the use of eDNA particularly with limited financial support to conduct more taxonomical expeditions or to build barcoding library. However, you did not explain the limitations of using a straightforward correlation between number of reads and fish abundance, which may vary between species. I personally use presence / absence data to perform such analyses, but I do understand the need of abundance data for some diversity indices that bring additional information which are useful but may be misleading if the relation is different between species by giving more importance to some species compare to other in PCA analysis for example.

I would like the reader to be informed of such limitations (few lines would be sufficient), otherwise, I have no reserve for this nice piece of work to be accepted for publication in the journal Diversity.

 

Other minor issues

Line 170: The symbol µ is missing in the sentence “…0.22 µm Sterivex…”

Line 172: I think you forget one word; I suppose you meant "...filled with RNAlater".

Line 540: The capital letter is missing at the beginning of the sentence. “Based on…” instead of “based on…"

 

Author Response

Comment: You did not explain the limitations of using a straightforward correlation between number of reads and fish abundance, which may vary between species. I would like the reader to be informed of such limitations (few lines would be sufficient), otherwise, I have no reserve for this nice piece of work to be accepted for publication in the journal Diversity.

Our answer: We definitively agree with this comment and added in the revised version of our manuscript some sentences in the M&M that point this issue and others that justify the use of the eDNA concentration as proxy of species abundance :

"Utility of eDNA for quantifying the species abundance and/or biomass is largely debated since it has been demonstrated that a number of species-specific biotic factor or abiotic may impact its concentration and biased abundance/biomass estimates (for a review see Rourke et al. 2022). However, Rourke et al. (2022) by reviewing 63 published studies that investigated relationships between eDNA and fish abundance found 90% of positive relationship. Similarly, a mixed-effects meta-analysis of data from studies that examined in laboratory or in natural environments same relationship between eDNA concentration and species abundance also concluded that even if weaker in natural environments eDNA concentration often still explained substantial variation in abundance (Yates et al. 2019). "

References has been added to the references section

Other minor issues

Line 170: The symbol µ is missing in the sentence “…0.22 µm Sterivex…”

Our answer: This has been corrected in the revised version

Line 172: I think you forget one word; I suppose you meant "...filled with RNAlater".

Our answer: That's right we added "later" after "RNA"

Line 540: The capital letter is missing at the beginning of the sentence. “Based on…” instead of “based on…"

Our answer: We changed “based on…" for “Based on…”