PONE-D-26-15381

Taxonomy-free approach overcomes the gaps in ecological knowledge: the case of foraminiferal metabarcoding applied to environmental impact assessment

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Partly

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

Reviewer #1: N/A

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The manuscript addresses an important and timely topic, namely the use of a taxonomy-free approach in foraminiferal eDNA metabarcoding for the assessment of the ecological quality of benthic habitats. The central idea is interesting and potentially valuable: the authors demonstrate that a taxonomy-free framework may exploit the molecular signal more effectively than conventional morphological or taxonomically assigned molecular indices, particularly in the presence of a high proportion of monothalamous taxa whose ecology remains poorly understood. The study is based on data collected around the Armida gas platform in the northern Adriatic Sea.

It is evident, and on this point I fully agree with the authors, that relying exclusively on morphologically identified species for ecological status assessment is insufficient. In my view, such an approach often fails to make full use of the available biodiversity data, which may in turn lead to questionable interpretations of the results.

In the Methods section, I would recommend explaining in greater detail why the SWARM clustering algorithm was selected and how exactly it was parameterized, so that it is clear how the MOTUs were defined. At line 211, it would also be appropriate to describe the filtering of HTS data, as this abbreviation has not been introduced previously. I also find the wording in lines 211–212 problematic: “to improve the congruence in terms of alpha diversity the MOTUs represented by less than 1000 reads were excluded.” This justification appears rather subjective. It is not clear what the reader should understand by “improve the congruence in terms of alpha diversity,” nor is it sufficiently explained why the threshold of 1000 reads was chosen. If I understand correctly, the algorithm originally produced nearly 23,000 MOTUs, but after excluding units represented by fewer than 1000 reads, only 343 remained. This represents a very substantial reduction of the dataset, exceeding 85%, and therefore the rationale for this step should be explained much more convincingly and in greater detail.

A similarly unclear step is presented at line 216: “The MOTUs assigned to the same morphospecies have been combined, reducing the total number of assigned MOTUs to 43.” If MOTUs were generated using the SWARM algorithm, and if the text simultaneously suggests that morphospecies may not reliably reflect actual diversity, then it is not entirely clear why MOTUs assigned to the same morphological species were subsequently merged. In my opinion, this procedure deserves a much clearer justification. Overall, I believe that the section between lines 206–217 is not formulated with sufficient clarity and in places appears overly subjective. I would recommend that this section be carefully revised, clarified, and reworked, because the resulting dataset is fundamental to all subsequent analyses and interpretations. It would also be useful to provide the results of the same analyses without these subjective dataset modifications, at least in the supplementary materials, so that the reader can assess whether, and to what extent, these steps affected the results. On the one hand, the authors emphasize the strength of DNA-based data; on the other hand, they substantially reduce that signal themselves.

The reference conditions for gEQR are defined internally from the 95th percentile of the investigated stations. This may be a practical solution, but if the entire study area already includes anthropogenically affected stations, the “reference conditions” may not be truly reference conditions. As a result, the classification of gEQR may be biased. The authors should state this limitation more explicitly in both the Methods and the Discussion and explain why this procedure is acceptable in the present context. The use of the 95th percentile as the reference value appears rather risky to me, and in my opinion it would be appropriate to explain whether no alternative method of deriving reference values was available, or why such an alternative was not used.

In presenting the results, I would recommend harmonizing the orientation of the scales in Figure 5. At present, EQR and gEQR are displayed with EcoQS running from “High” at the top to “Bad” at the bottom, whereas AMBI and gAMBI are oriented in the opposite direction, i.e. from “Moderate” at the top to “High” at the bottom. I understand that this reflects the values of the indices themselves, as one increases while the other decreases, but in the figures the EcoQS categories are more important than the raw index values. The current arrangement may therefore be confusing for the reader, and a unified orientation would be preferable.

At lines 267–268, the meaning of the statement “Replicates were partitioned by identity (A, B, and C), and in each iteration” is not sufficiently clear to me. The Methods indicate that three replicates were collected from each core, but later in the DNA methodology it is stated that three replicates were generated from each sample. I therefore cannot deduce unambiguously whether three replicates per sampling site were sequenced, or whether each biological replicate was further processed into three PCR replicates, resulting in a total of nine sequenced replicates. Likewise, it is not clear what exactly the A, B, and C partitions represent. This part of the methodology should therefore be specified much more precisely. At the same time, it is not explained why the authors chose this particular partitioning scheme for the leave-one-replicate-out procedure. For example, it is unclear why the replicates were not grouped according to classes of Ba concentration, given that this variable plays a central role in the study.

I will not assess the statistical analyses in detail, as this is not among my main areas of expertise. Nevertheless, I would like to comment on the interpretation of some of the results. At line 355, a trend in Exp(H) is reported; however, in my opinion, Figure 3 does not show a clear trend either in Exp(H) or in DNA-based diversity. I do not know whether such a trend could be formally tested, but based on the figures alone I would be much more cautious in making statements about trends.

More generally, I have the impression that the results based on EQR and AMBI are at times interpreted more in line with the authors’ expectations than strictly according to how they are presented in the figures. For example, lines 399–400 state: “Conversely, EcoQS worsened with Foram-AMBI along the 0 to 2000 m transects,” yet this conclusion does not seem unambiguous to me from the corresponding graphs. In my opinion, the East transect does not show a deterioration in EcoQS, and neither does the West transect, apart from a minor deviation around 50 m. I perceive a similar mismatch between the text and the visually presented results in other parts of Figure 5 as well. I would therefore recommend reconsidering the interpretation of the results in this section and formulating it more conservatively, so that it more faithfully reflects the graphical outputs themselves.

In conclusion, I regard the taxonomy-free approach to DNA metabarcoding data as very promising and, given the current limitations of classical morphotaxonomy and the exponentially increasing volume of new data, essentially the only realistic and appropriate way forward. The manuscript has several strengths: a clearly articulated motivation, a relevant applied research question, and a convincing argument that a substantial portion of the biological signal in eDNA datasets remains unused when the analysis relies solely on taxonomically assigned MOTUs. However, the reported results and their interpretation do not yet appear fully convincing to me.

Reviewer #2: The paper's draft "Taxonomy-free approach overcomes the gaps in ecological knowledge: the case of foraminiferal metabarcoding applied to environmental impact assessment" of Bouchet and colleagues deals with a critical challenge in marine biomonitoring: the inability of traditional (morphology-based) methods to capture the full diversity of foraminifera, particularly soft-walled taxa (monothalamids), which are often dominant but difficult to identify. The document proposes and validates a taxonomy-free approach based on environmental DNA (eDNA) metabarcoding, using the "Armida" gas platform in the Adriatic Sea as a case study. Once published, this article could represent a high-value contribution that shifts the biomonitoring paradigm towards genomic ecology. It successfully demonstrates that it is not necessary to know the "name" of each organism to assess the health of an ecosystem, as long as one knows its response to environmental stress. It is a pioneering work that facilitates the adoption of eDNA in international regulations (such as the European WFD). The value of this manuscript is potentially high, but it needs some revision in this sense.

Both the title and abstract are effective, designed to maximise the visibility and key points of the contribution. I suggest removing the keywords already reported in the title and replacing them with related ones to improve the document's soundness. Probably adding some numeric key results in the abstract (eg, eDNA/morphology MOTUs) could enhance the soundness of your data.

The introduction section is well-drafted with all the necessary information given to the reader to understand the methods and the author's research question.

The sampling design looks properly set, with adequate numbers and replicas, scientifically strong, even if some confusion is generating later in their "partitioning", what do you mean with replicas partitioning?

Methods and analyses were well applied and supported by previous literature, also from the authors wich demonstrate experience in this regard between the lines. Rather, I have some doubts about the data filtration with a threshold of 1000 reads. Due to the nature of the sampled matrix and the specific biomarker, could it represent a correct choice, or was it influenced by some methodological limitations? Please argue this better.

A point to better argue in the discussion is that eDNA can persist in the sediment for a long time, even after the organisms die. Although the authors cite the preservation kit, a more in-depth discussion of the distinction between "live" DNA and "dead" DNA could add more accuracy to the study results, even to contextualise whether the indices reflect the platform's current health or an average over recent years. The authors used the 0-1 cm layer to mitigate this problem, which is the correct standard procedure, but at the same time, the influence of this problem also affects the methodology (short length MOTUs), which could be discussed and integrated. Moreover, I suggest depositing all the raw data in a public repository to give the reader access.

The limitations of the approach were adequately stated by thre authors, such as the influence of river of the study area on the chemical equilibria. The link between barium and gas platforms is supported by a solid literature, confirming that the choice of stress parameter is correct. Since the approach is "calibrated" to local Barium, the transferability of the ecological groups defined in this study to other geographical areas requires further validation. Since the Barium thresholds (Table 1) were created by the authors themselves because they did not exist in the literature, this point remain a potential limitations of this study replication by other authors, to hihglight in the conclusion section.

Although, as specified by the authors, the lack of ecological knowledge on monothalamids is a bottleneck that only the taxonomy-free approach can currently circumvent. Indeed, even if the approach of this study is taxonomy-free, improving genomic databases remains essential to better understand the functional biology of the detected taxa, which could be well-assessed in the conclusion section too, which in the present form resulted poor in this sense, focusing just on the effectivenes of the method/biomarker proposed (proper results).

Best regards

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Reviewer #1: No

Reviewer #2: No

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Dear Dr. Bouchet,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Sep 10 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Claudio D'Iglio, Ph.D.

Academic Editor

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Additional Editor Comments:

Please improve some point of the Discussion section to resolve the minor concerns that emerged from revison. Please follow the reviewer suggestions.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #2: Yes

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #2: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: Dear Authors, thanks for following my previous suggestions on your manuscript. Looking at its revised draft, I found improvements from all points of view, especially for accuracy on methods and limitations.

Regarding my comment 12, and 1 of the other reviewers, I suggest improving your comments on the manuscript also in the discussion section utilising the same explanation that you add in the report to argue your choice:

"...the aim of data filtration, which is to remove the potential noise that could represent DNA molecules of extraneous origins (e.g. transported and preserved in the sediment). This DNA generally occurs at lower abundances and the common way to remove it is to filter out the low abundance sequence variants (ASVs). This also allows to remove the copies of rare genetic variants resulting from intragenomic polymorphism common in ribosomal operons of foraminifera. The filtration threshold is arbitrary. We choose 1000 reads to ensure that the analysed data correspond to the local population actually living there. We consider this step essential for the robustness of metabarcoding data in ecological studies."

I think it sounds clearer and more accurate to introduce the discussion of your results based on the applied methodology, and it could be useful for the readers.

Same for the comment 8 response:

"...We decided to use part of the dataset for calibrating the assignment to ecological group to limit circular reasoning which could have severly biased our work. Different options could have been tested, like leave-one-station-out procedure. We considered that by working at replicate level, it allowed us to consider the full Ba gradient captured in this work, therefore providing a more robust calibration, hence assignment of MOTUs to eg for Foram-gAMBI calculation"

This could also be reported in the discussion section to clarify this aspect.

Best regards

The Reviewer

Reviewer #3: (No Response)

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Reviewer #2: Yes: Marco Albano

Reviewer #3: Yes: LAZARO LUIZ MATTOS LAUT

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Taxonomy-free approach overcomes the gaps in ecological knowledge: the case of foraminiferal metabarcoding applied to environmental impact assessment

PONE-D-26-15381R2

Dear Dr. Bouchet,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Claudio D'Iglio, Ph.D.

Academic Editor

PLOS One

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Reviewers' comments: