-->PONE-D-26-08592-->-->First eDNA-Based Monitoring of Marine Vertebrates in the Urban Marine Ecosystem of Abu Dhabi: Mangrove and Seagrass beds as biodiversity reservoirs?-->-->PLOS One

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Reviewer #1: No

Reviewer #2: Yes

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-->2. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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-->5. Review Comments to the Author

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Reviewer #1: This manuscript presents an environmental DNA (eDNA) metabarcoding survey of marine vertebrate biodiversity across four urban coastal habitats in Abu Dhabi. While the study has the potential to generate interesting insights, several methodological limitations, some of which (in my opinion) may not be addressable at this stage, render it unsuitable for publication in PLOS ONE. First, it seems like field blanks were not included in the surveys, there is no mention of DNA extraction controls, or PCR negative controls. This raises concerns about how potential contamination during both field sampling and laboratory processing was assessed and mitigated. This represents a major shortcoming of the study. Second, the sampling design is insufficient to support robust ecological inference. The study is based on only four sites sampled over two days, with duplicate filtrations. The lack of temporal replication, limited spatial replication within habitat types, and the absence of a clear distinction between technical and biological replicates weaken the strength of the conclusions. Third, only approximately 25% of taxa were resolved to the species level. Although this limitation is acknowledged, it is not adequately integrated into the interpretation of the results. Fourth, statistical analyses comparing richness/diversity indices across sites should account for spatial autocorrelation, otherwise the authors need to prove with strong arguments that there is no spatial autocorrelation.

Minor comments:

Lines 37-40: Please rephrase. The words “worrying” and “with regard to” are not appropriate in scientific writing.

Lines 41-42: The word “inventory” assumes that the authors have done a very comprehensive eDNA survey. Also, doing a one-off survey does not result into a marine vertebrate inventory. I am not sure if four sites count as comprehensive. I recommend toning down this claim. Also, please include the genes targeted in this eDNA survey, as this info can provide an idea of how comprehensive were these surveys.

Lines 51-55: this paragraph should come after the introductory sentences. Also, please rephrase.

Lines 81-82: This seems repetitive, I recommend deleting.

Lines 115-116: Please tone down this claim.

Line 116: What does “Using a standardized workflow of water sampling” mean, please give a bit more detail about who standardized this method.

Lines 127-131: I suggest removing this, as it has already been explained in the Introduction.

Figure 1 can be improved. Please add a reference map and a better delineation of the sampling sites. By looking at this map, I don’t think these scattered four sites would give an “inventory” of the marine vertebrates of Abu Dhabi.

Lines 146-150: I understand that at each site, samples were collected along two transects? If this is the case, please say so and provide the GPS points and length of the transects. Also, please describe briefly the protocol cited (ref #40).

Lines 151-152: what about field blanks? This is such an important aspect in eDNA fieldwork. I am afraid that there is no way of proving that the results of this study are not result of sample contamination if there were no field blanks. Also, there is no mention of DNA extractions and extraction controls, or of PCR negative controls.

Lines 156-158: I think the right approach to detect “all” marine vertebrates would have been to sequence more than one gene. There are fish, elasmobranchs, mammals and birds’ specific universal assays available. The choice of only one gene needs to be justified.

Lines 159-161: The bioinformatics need much more detail than three lines. Include filtering parameters, and software/version used.

Lines 104-248: It is unclear how the authors can demonstrate that the detected species originate solely from the sampled environment rather than being influenced by potential contamination during field collection or laboratory processing.

Line 250: I am not sure if this is the right statistical analysis. I assume there is a strong spatial autocorrelation between sites, and this needs to be taken into account for statistical analyses. Without proving that data is not spatially autocorrelated, statements such as “Taxonomic dissimilarity was generally moderate to low across site” could be arising from incorrect analysis.

Reviewer #2: Overall, this paper presents a tidy eDNA metabarcoding exploration of four sites off the coast of Abu Dhabi. The methods need to be clarified in a few places, but the paper is an interesting inventory of species that can be used as a baseline for future research.

In addition to specific methodological details which must be included before publication (ie, identity threshold in OBItools and reference database curation methods), I recommend the authors cut down/streamline a little bit of the introduction of the region as a whole and instead focus on the actual sampling sites to introduce readers to your hypotheses. IE, how are these sites different from each other? What role does each ecosystem play in harboring diversity in UAE waters, and how does that affect your expectations of what you’re going to find? What patterns have previous, non-eDNA surveys found?

Basically, explain the biological reasoning behind why these four sites were picked, and what you expect to find. I say this because I think you have a really cool setup here and it can answer interesting questions, and I think it should be highlighted. I also think it will both contextualize and help with the flow of the discussion by giving you something to call back to.

Below are my line-specific edits.

Line specific review:

67 – Low species diversity compared to the same habitats in other, less extreme waters, right? The introduction of mangroves and coral reefs, famous for their high diversity, makes this sentence a bit hard to follow.

74 – climate projects for what region specifically? The whole paragraph varies between “UAE waters” and what I think is referring to the whole Arabian gulf. Probably an easy change to clarify.

164 – MOTUs were checked “meticulously by hand”. Checked against what? A taxonomic database (ie, BLAST, a local database, BOLD, etc.)? Or checked for a certain % identity? This is a key step for your analysis, so please be as accurate as possible. I understand you used OBITools – please be more descriptive. I assume you used ecotag? What did you set your % identity cutoff as (and why?) What did you use as your reference sequences? How did you develop that reference database? Did you use ecoPCR? If it was EMBL, which version, accessed on what date? If it was hand-curated, did you include common contaminants (ie, human, domestic animals)? Are you confident in how robust your database is?

166 – So is a “taxonomic redundancy” a case where two MOTUs were above your % identity similarity threshold, yet still assigned to the same species? We need far more detail here about how you assigned MOTUs to species.

187 – Missing word – “Unifrac also allowed [us?] to decompose…”

207-219 – This is a pretty long description of taxa that are available in a table. I think given the length of the paper, this paragraph can be removed unless any of these were new records. I do appreciate noting that one of your taxa was confirmed with visual observation, though.

238 – You vary between italics and regular face for family names. Just a formatting note.

307 – Typo: “Trough” for “through”

311- It isn’t a great message to just blame your insufficient reference database on the “current limitations of reference databases.” Doing it yourself is always an option – a curated reference database is always best, and producing the sequences for projects like these is a primary way the sequencing databases grow. Producing location-relevant sequences is something every metabarcoding project should consider, especially one developing a forward-looking protocol for monitoring a specific region. I appreciate that this project probably was not of large enough scope to produce any sequencing, but with the cheapness and availability of Sanger sequencing, a sentence about why the project did not/could not produce relevant sequences for known teleosts might be appropriate.

312 – I also want to ask – Is the failure to resolve at the species level for so many teleosts exclusively because they aren’t in the database, or is there evidence that some of them are not variable/distinct enough at these primers to be resolved? If so, please mention it, because it represents a different kind of limitation for this study into the future.

320 – Another formatting note, you vary whether you spell out or use numbers for numbers <10, in the results you used numerals, but here you spell out. The PLoS editors will have guidance for this, no biggie.

377 – Typo: “Areas” for “Area”.

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Reviewer #1: Yes:  Cecilia Villacorta-Rath

Reviewer #2: No

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First eDNA-based monitoring of marine vertebrates in the urban marine ecosystem of Abu Dhabi: mangrove and seagrass beds as biodiversity reservoirs?

PONE-D-26-08592R1

Dear Dr. Jung,

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