PONE-D-23-12738Abundant, diverse, unknown: Extreme species richness and turnover despite drastic undersampling in two closely placed tropical Malaise trapsPLOS ONE

Dear Dr. Chimeno,

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PLOS ONE

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Reviewers' comments:

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Comments to the Author

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

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

Reviewer #1: Yes

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 requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Review of ‘Abundant, diverse, unknown: Extreme 1 species richness and turnover despite

2 drastic undersampling in two closely placed tropical Malaise traps’

I can find very little to criticise in this manuscript. I suppose that none of the results are particularly surprising, as the authors acknowledge, but the empirical demonstration of massive local heterogeneity and very high species richness are very useful, and the methods will be very useful for the development of further studies at different scales. I would hope that the availability of large numbers of barcodes for ‘dark taxa’ would also promote further taxonomic work.

For those samples for which you filled 15 plates, rather than all specimens, what was the protocol for choosing individuals to sequence? Or were specimens chosen randomly until the plates were full?

What happens to the specimens? Are they available for taxonomic work?

I couldn’t find the datasets which are deposited on BOLD. Are they publicly available yet? I was hoping to scan through the areas I know best (Ichneumonoidea) and sense check the results.

Lines 46-47: I don’t think ‘dark taxa’ need be defined by a complete absence of taxonomic expertise, but clearly the expertise is very limited and completely inadequate to the job.

Change ‘an increase by…’, to ‘an increase of…’ (eg., line 205)

Reviewer #2: The manuscript by Chimeno et al. compares the insect communities collected using two closely spaced Malaise traps in Sumatra. It presents a well sampled dataset of ~70K insects and compares these two sites in terms of alpha and beta diversity. The findings of high species diversity of insects along with the distinct communities in nearby sites are of interest for wider scientific community. However, I would suggest the following be considered:

The paper shows surprisingly distinct communities across the two sites despite their proximity. But the manuscript has a very limited description of the sites being compared (other than them being forest edge sites). It would be important to describe the two sites in the study in detail and the area around in general in the methods section of the manuscript.

The manuscript furthermore characterizes the insect communities at family level but does not describe how the family level identifications were made for the insects. This should be properly documented in the methods. Similarly, standard barcode protocols of CCDB is cited at L93 but the link directs to several other links: It would be good if the major steps are described (target region, extractions, sequencing methods) given the linked protocols may change in future. Regarding data analysis, how were the sequences aligned?

While those familiar with the topic would not find the assumption that the specimens sequenced in this project (given they are from Southeast Asia and given the insect families they belong to) are from dark taxa ‘for which little taxonomic… information is available’ (L277) surprising, it is worth noting that the manuscript does not document this finding. It would be valuable to characterize how many of the OTUs sequenced in this project are (1) present in the barcode databases and (2) are identified to species level. This further links to the title of the paper abundant diversity and “unknown”.

A key issue relates to the discussion of the results. The authors find high diversity, distinct communities and that most species and specimens belong to a few families. However the discussion of the solution to these challenges is not developed; i.e. what are the recent methods that can be used to expedite the work for taxonomists and how can such large scale studies be conducted given that even 70K specimens are not sufficient for studying these sites? What is the proposal for future research, particularly in tropics?

This may be a preference, but I find that structurally the first section of the discussion “Employing DNA-based delimitation methods: working with 225 species proxies” is less interesting than several of the key findings described in the paper and it would be more interesting to highlight the key findings of the manuscript first.

Minor:

L152: The links are not working for me

L244: -21% and -14% is confusing to me. 21% of 6283 (RESL) is 1319 and -21% would imply 4960 ASAP OTUs based on table 2, but doesn’t match 5185. Perhaps it should be +21% if the reference is ASAP OTUs. But overall, I would suggest not using % here given the clusters themselves are not necessarily the same. Same for L239.

L278-282: mentions eight families but lists only seven

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Reviewer #1: Yes: Gavin Broad

Reviewer #2: No

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PONE-D-23-12738R1Abundant, diverse, unknown: Extreme species richness and turnover despite drastic undersampling in two closely placed tropical Malaise trapsPLOS ONE

Dear Dr. Chimeno,

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 04 2023 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.

Please include the following items when submitting your revised manuscript:

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• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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We look forward to receiving your revised manuscript.

Kind regards,

Pierfilippo Cerretti, Ph.D.

Academic Editor

PLOS ONE

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1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

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

Reviewer #2: Yes

**********

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The authors have addressed my comments. Upon review, and looking through the links that are now available I have a few suggestions.

The numbers in BOLD datasets doi.org/10.5883/DS-GMTINDO1 and doi.org/10.5883/DS-GMTINDO2 correspond to 52239 and 24938 specimens (and strangely enough, sequences) which is slightly different from the numbers in L254 (52362 and 25135) and the actual number of sequences is far fewer. The downloaded sheets from figshare correspond to the values in the manuscript. Based on "BIN_data" from figshare, approximately 12,000 specimens have 0bp barcodes, but the number of sequences in the BOLD page seems to be too high, though upon download the sequence count gets closer, though specimen count is still off. I am not sure what is going on, it would be good if a field of sequence is added to one of the figshare spreadsheets for straightforward access to data for future users along with a flag of which sequences were used for final analysis.

The authors have stated that BOLD taxonomy is used for family level classification. Given these were not morphologically examined/verified, it would be good to mention in the discussion this limitation/potential errors propogating in sequence based identifications.

L361: Edit to "in temperate regions", and given temperate samples are not involved in this study, this should be cited, and the similarity in pattern has been quantified in https://doi.org/10.1038/s41559-023-02066-0

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Reviewer #2: No

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Abundant, diverse, unknown: Extreme species richness and turnover despite drastic undersampling in two closely placed tropical Malaise traps

PONE-D-23-12738R2

Dear Dr. Chimeno,

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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Pierfilippo Cerretti, Ph.D.

Academic Editor

PLOS ONE

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