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Dear Dr. Mitchell and colleagues:
Thanks for revising your manuscript based on the concerns raised by the reviewers. I now believe that your manuscript is suitable for publication. Congratulations! I look forward to seeing this work in print, and I anticipate it being an important resource for groups studying flower beetle systematics, as well as cryptic speciation. Thanks again for choosing PeerJ to publish such important work.
[# PeerJ Staff Note - this decision was reviewed and approved by Vladimir Uversky, a PeerJ Section Editor covering this Section #]
Dear Dr. Mitchell and colleagues:
Thanks for submitting your manuscript to PeerJ. I have now received three independent reviews of your work, and as you will see, the reviewers raised some minor concerns about the research. Despite this, these reviewers are optimistic about your work and the potential impact it will have on research studying flower beetle systematics, as well as cryptic speciation. Thus, I encourage you to revise your manuscript, accordingly, taking into account all of the concerns raised by both reviewers.
While the concerns of the reviewers are relatively minor, this is a major revision to ensure that the original reviewers have a chance to evaluate your responses to their concerns. There are many suggestions, which I am sure will greatly improve your manuscript once addressed.
Please note that reviewer 2 has included a marked-up version of your manuscript.
Please use the comments by the reviewers to add missing information where possible. Try to restructure your manuscript for clarity, avoiding redundancy and streamlining sections for effective delivery.
Therefore, I am recommending that you revise your manuscript, accordingly, taking into account all of the issues raised by the reviewers. I do believe that your manuscript will be greatly improved once these issues are addressed.
Good luck with your revision,
The background information is mostly clear although could be improved by directly stating the number of diversity of Australian genera in the first paragraph of the introduction, or quantified following statement on 65% of genera on line 56.
The second paragraph is largely a non-sequitur and while the biology is important, this paragraph seems to largely be irrelevant as there are no-discussions or conclusions relating to biology. If the authors decide to include this information, it would be better suited towards the end of the Introduction, and prehaps relating to the utility of generating this barcode library.
Goals are stated clearly but there is room for a hypothesis that barcodes are likely to identify numerous undescribed species. This could be supported by the limited taxonomic attention in the last 65 years, high levels of endemism, cryptic species complexes already identified and estimates of undescribed proportion of Australian fauna.
English is clear. Citations are sufficient to provide context.
This study provides the first insights in to the hidden diversity of Australian cetoniines and fills an important gap in our knowledge of scarabs as well as the Australian fauna.
The design and investigation is rigorous and methods are described in detail. The authors should be commended that this first investigation provides barcodes for almost 50% of species and all common genera. It provides a clear framework for further investigation and the utility of barcodes for identification.
I'd like to see a short discussion of the utility of this barcode library beyond taxonomy, e.g. the potential agricultural importance mentioned in the introduction, or ability to match life-cycles.
I was intrigued when I read in the introduction that both larvae and adults were sampled and that one of the goals of this paper were to facilitate life-cycle matching but these larvae didn't get mentioned in the discussion or conclusion. Were they from easily identifiable species or were they matched using these barcodes?
The taxonomic interpretations were robust. I think its great that when possible, multiple samples were included per species. It would be useful to include locality information either on the trees or in a table so readers don't need to refer to BOLD to decipher locality information from this data set.
Overall, this is an important contribution to the field.
In this study Mitchell and co-authors Liu et al. analyzed about 280 specimens of Australian cetoniine flower beetles using the DNA barcode fragment. During the last years, DNA barcoding has become an important and popular tool in modern biodiversity studies as well as taxonomic research. Based on the given data and results, I think that the analysis and results are interesting and likely helpful for other researchers in this research field. In my eyes, a clear, unambiguous and professional English language has been used. The article structure and figures look professional. The literature provided is also context-related, but some important references are missing (see sticky notes in the uploaded pdf). The figures and associated legends are understandable without going back to the main text.
I feel, however, that a discussion of possible pitfalls of using mtDNA in terms of species delineation and specimen identification, e.g., incomplete lineage sorting, the presence of Wolbachia, NUMTS etc. as part of the introduction or discussion will improve the quality of the manuscript. Such effects can cause unexpected mitochondrial diversity as well.
Furthermore, it is obvious that DNA barcoding will play an essential role in modern molecular biodiversity research, e.g., the effective use of high-throughput sequencing technologies (meta-barcoding) highly relies on detailed barcode libraries. These aspects should be also discussed.
The research question is well defined, relevant and meaningful. The authors state how their research fills a serious knowledge gap in the taxonomy of Australian flower beetles. Methods are described in sufficient detail to replicate. However, the authors may consider using additional species delimitation approaches (e.g., GMYC, bPP). Do you will get similar results in comparison to the BIN/RESL approach? This would be interesting to know. For some species cluster, e.g., Chondropyga dorsalis, you may also think about using statistical parsimony networks as implemented in PopART (http://popart.otago.ac.nz/index.shtml) to visualize observed molecular patterns.
I wonder if the results would be different if more range wide samples of different species were included. In some cases the samples sizes were quite small (i.e. one individual). However, the conclusions are well stated, linked to original research question and limited to supporting results. Overall, data and analyses are robust.
Please check the uploaded pdf for more specific comments.
The manuscript is generally well written and conforms to the PeerJ standards on structure. There are only a handful of relatively small issues which should be easy to address. The introduction provides a good overview of Australian Cetoniinae and previous taxonomic work on the group. Sequences are made available through both BOLD and GenBank, and detailed specimen metadata and images are provided through BOLD. The figures are of good quality. In particular, the illustrated and color-coded supplementary trees are visually appealing, and I wonder if at least one of them (or parts of one) could be transformed into a proper figure in the manuscript instead of being “hidden” in the supplements. Some parts of the manuscript should be moved elsewhere to improve the flow of the text and avoid confusing the reader - see detailed comments below under General comments.
The need and motivation for the present study and its goals are clearly stated. The methods are sufficient to achieve the stated goal, i.e. providing an initial DNA barcode library for the focal taxon to serve as a starting point for more thorough taxonomic studies. Some basic details of PCR and sequencing protocols (primers, sequencing platform, etc) should be added in Materials and Methods instead of simply referring to the previous publication. Rooting of the trees is not justified in any way in Material and Methods, but the justification is briefly mentioned in Discussion (lines 297-299). This note should be moved to the last section of M&M where the tree inference methods are described. Apart from these minor issues, the methods are well reported, and I see no ethical or technical problems.
All primary data are made available through both BOLD and GenBank. Data and image quality on BOLD are mostly exemplary, although collection data beyond the country are not reported for many specimens. I assume that this is due to deficient label data on the sampled specimens rather than an omission on the authors’ part. Conclusions are not overarching and are supported by the results. A few more specific comments are listed below under General comments.
Line 71: 4000 is a gross underestimate. A quick search on Web of Science with the term “DNA barcod*” as topic resulted in 7262 articles and 1000 or so hits for other document types. The 2003 paper by Hebert et al. (https://doi.org/10.1098/rspb.2002.2218) has been cited more than 10000 times according to Google Scholar.
Line 76: “…aid taxonomic research in many families” - Consider citing a few studies as examples
Line 185-187: A public project on BOLD works fine for data sharing in this case, but in future work, I suggest using the dataset feature on BOLD instead. Specimen records from multiple projects can be combined into one dataset, and the same records can be added into multiple datasets, without any need to move records between projects. A dataset can also be assigned a DOI. I believe the dataset feature will be useful in your future work on Australian Cetoniinae when more sequence data are added to many of the taxa covered here.
Line 214: As mentioned above, please move the note on lines 297-299 here to justify your approach to rooting the trees.
Line 218: How were the larvae identified – by associating them with adults based on barcodes or morphologically? Were some taxa represented by larvae only? Consider adding a note on sampling larvae as well as adults in Material & Methods. If larvae were sampled in order to test identification of larvae based on barcodes, add text appropriately in M&M, Results, and Discussion.
Line 239-246: Mainly a matter of personal preference, but I think the information in this paragraph would be better presented in a table including both monophyletic and non-monophyletic genera.
Line 251-252: Representatives of two genera appearing in the same BIN is highly unusual and is almost always due to operational errors. Is this possibly a case of poorly resolved genus-level taxonomy? Can you exclude the possibility of cross-contamination or specimen mix-up?
Line 291-293: This seems to belong to the Results section rather than here, especially since there is no mention of these results in Results in the current version of the manuscript.
Line 306-313: Move this paragraph up before the previous one to make the text more coherent
Line 314-320: Join to a single paragraph
Line 339: probabilities -> probability
Fig. S1 legend: Baysian -> Bayesian
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