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Thanks for the quick turnaround with your minor revisions. I agree with your changes and the paper is now ready to go to production.
# PeerJ Staff Note - this decision was reviewed and approved by a PeerJ Section Editor covering this Section #
The more critical reviewer and I are satisfied with your revision but the reviewer noted a few things that you need to address in a very minor revision. Please proof your paper carefully to speed the production process.
I greatly appreciate the authors revision in taking into consideration my major comments and addressing each of them both in the introduction and in the discussion, as well as answering my questions. I feel the authors have done a thorough job in motivating the need for their study, including the major knowledge gaps and how the results of their current study will spur future research. I found a number of small typos that I detail under general comments. Well done, overall.
Ln 107: "Since a few years", suggest something like, "In the last few years,"
Ln 107: change "approaches become" to "approaches have become"
Ln 108: I think you mean something like "overcome possible drawbacks of traditional ways of assessment". What do you mean by the traditional way of assessment? Taxanomic?
Ln 119: Switch from "highly relies" to "relies highly"
Ln 123: Remove the extra period
Ln 126: Needs an "a" in front of "unique"
Ln 128: Needs a "the" in front of "global"
Ln 155: There is an extra period after "Cameroon"
Ln 393: "expect" should be "except" I think
Ln 475: change from "got lost" to "were lost"
Ln 477: there is a typo in "tothe"
Ln 483: Looks like a typo, not sure what word is missing here "Unique BINs were revealed for 55 species determined species", also further in the sentence, you can drop the "of" from "representing of"
Understanding molecular diversity of insect orders is important but simply presenting bar codes seems to me rather insignificant for a major research journal. Hence, I agree with the comments of reviewer 2, especially this statement: the greatest weakness was there was not a clear description of previous studies and findings. The work was also purely a description based study using DNA barcode information. Many of the most intriguing findings were suggested for future work including hypothesis driven questions. Without some description of how these results extend or improve upon previous work, it is hard to assess if this manuscript is answering a specific knowledge gap. You will need to respond decisively on that count and also consider and respond to all the points made by the three reviewers.
Clear English is used throughout, except for some minor grammatical mistakes. I have made suggestions to improve the flow of the introduction.
Literature references and sufficient field background/context is provided.
The article structure and figures look professional. The table could use some work. The Raw data is shared as a text file, but the text (lines 191-196) is missing the Dataset ID and GenBank accession numbers. Custom checks for this manuscript require me to check DNA data availability but I cannot access the deposited data online and the deposition information is not in the manuscript.
First paragraph: I would rewrite/reorder this paragraph to make it more apparent why we should care about Heteroptera and give more background on the group. 1) Aquatic insects are the dominant invertebrate taxa. 2) Heteroptera make up __ proportion of aquatic insects and are unique as a group because they contain both aquatic and terrestrial species (compared to mayflies, etc). 3) Different groups of Heteroptera you focus on and their broad distribution. 4) Palearctic region and area of focus for the paper.
The second paragraph needs work. It starts off with a topic sentence about morphological adaptations but covers much more including loss of wings, having wings and migrating, being important components of aquatic ecosystems (but as it’s written it sounds like only the winged taxa are important…), what they consume and their role as prey.
Line 62 – This topic sentence should better fit the second paragraph, perhaps add something about their ecology instead of just having it be about morphological adaptations.
Line 68-70 – different font size?
Line 67 – replace: Furthermore, we observe…, with: We also observe
Line 70 – Replace: Nevertheless, many winged… with: Other winged…
Line 94 – spell out app.
Line 99 – As a consequence, not, As consequence
Line 136 – why are you reporting a result (the number of new barcodes) in the methods section?
Line 136/137 – what are you comparing by using the published barcodes from a previous study? The aim of your study (line 118) should be modified to include all of the goals of your study, including this comparison. Or please explain more clearly how you are using the barcodes from the previous study.
Line 138-140 – Were the specimens from other countries samples that you collected (or got from others) and analyzed for this study, or were these just barcodes that already had been done?
Line 239 – Figure S2 is very hard to read because it spans so many pages, but I’m not sure what the solution is if you include every single individual.
The research is of high quality and the question is relevant and meaningful. Many aquatic insect taxa are difficult or impossible to identify to species in their larval stages (and even as female adults, as described by the authors). The use of barcoding and other techniques to identify species will allow important ecological questions to be addressed that cannot with taxonomic methods alone. Also, the authors even found species in Germany that had not been described before. Perhaps this result should be highlighted a bit more?
The research question is well defined, relevant and meaningful. The authors state how their research fills an identified knowledge gap in the identification of Heteroptera.
The DNA barcoding and analysis sections had good detail. I had a few questions regarding the species collection and identification section (listed above). The methods are described in sufficient detail to replicate.
The data are robust. I do wonder if 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).
The conclusions are well stated and new DNA barcode and relationships between different species are presented. A thorough discussion of species with high intraspecific and low interspecific differentiation is also provided.
This paper provides a nice dataset on the molecular diversity of aquatic Heteroptera. It would be interesting to provide some information or discussion on the additional information that could be gained in a typical study of Heteroptera in Germany by using the DNA barcodes instead of just using traditional taxonomy. Are a large proportion of the individuals captured impossible to identify because they are immature or females. Are certain times of year more difficult to study because of the life stage of the organisms? Perhaps a little more discussion of how DNA barcodes might change our conclusions in ecological and conservation studies? This would be useful additional information in the introduction. Improving the introduction in general (and using the comments from the Basic reporting section) would make this a stronger paper.
In this study Havemann et al. work to create a DNA barcode library for 67 species of Heteroptera or true bugs found in Germany. Overall, the manuscript was well written and very clear, though I did find a fair number of small typos, many of the detailed in my general notes. The figures were also very nicely done.
I thought the analysis and results were interesting and likely helpful for other researchers in the field, but I felt the greatest weakness was there was not a clear description of previous studies and findings. The work was also purely a description based study using DNA barcode information. Many of the most intriguing findings were suggested for future work including hypothesis driven questions. Without some description of how these results extend or improve upon previous work, it is hard to assess if this manuscript is answering a specific knowledge gap. I think it could, but it would greatly aid if the authors provided more detail on this gap is and how they are helping to fill it. There are hints of it, but nothing that seems very substantial or convincing, likely because of a lack of detail of how these new information fit into the context of what was known before.
1. Ln 112-115 It was odd to me that the authors state there have been "few" studies, but then they are able to list 11 other articles including 2 that focused on aquatic true bugs (Castanhole et al., 2013; Ebong et al., 2016). Perhaps the main weakness of the introduction is that it wasn't clear to me how they are building on any of this previous work to help bring new understanding and filling important knowledge gaps.
2. Ln 406-409 You use the term "exclusively morphologically-based taxonomy" within "some" species. But this is very confusing because there is no description of what has been done before? Even though there were 11 other studies that have "analyzed the usefulness of DNA barcodes to discriminate species of the Heteroptera" (lns 112-113).
Overall, there were a few cases where very few samples existed, this seems fine for the goal of this manuscript, however I did have one major concern to do with the species where the vouchers were lost (Ln 397). I was not completely convinced that these results should be included given there were only 2 samples to begin with and now no way of further identification or comparison.
1. In multiple places (e.g., lns, 317-319, 327-328, 338-339, 353-355) there was a statement similar to the following: "Similar to other previously discussed species pairs with low interspecific distances, more specimens have to be analyzed to validate the species status within this subgenus more in detail."
My main concern with this statement and the fact it is repeated multiple times is if this work is publishable on its own as there seems to be a lot of missing analysis and areas where additional samples are needed to draw a more clear conclusion or answer questions that are hypothesis based and not a purely descriptive study.
2. Lns 403-404 "In this study we lay the foundations for a comprehensive DNA barcode library for the aquatic Heteroptera in Central Europe and adjacent regions." This point seems debatable given my comment above. It seems a stretch to say this is a comprehensive DNA barcode library when you are also suggesting a lot of additional work needs to be done to understand fully the relationship between multiple of your species pairs? Can you justify this statement or perhaps remove/change it?
Ln 38-40 This sentence is confusing as worded and also doesn't appear to grammatically correct. Can you rearrange to help clarify? And/or split these 2 ideas up and add a bit more detail for each?
Ln 80 remove one of the "e.g."
Ln 81 "the determination by", I first assumed you meant phenological placement? But the next sentence made me realize you just mean species determination. It might help to clarify this sentence to say exactly what you mean.
Ln 92 "During the last years" doesn't sound correct. Phrased this way, it seems to say the end of something rather than the last few years.
Ln 94 "app." Is this an abbreviation? This should be spelled out, no reason to have an abbreviation here.
Ln 99-100 should be… As "a" consequence and later in the sentence by "a" unique
Ln 155 - There seems to be an inconsistent use of parentheses when using e.g. as other times they were previously not included, but are included in this sentence.
L209 - missing a "the" I think before "model"
Ln 220 - I think the "of" needs to be removed
Ln 222 - need a "an" after "and"
Ln 225 Change to "In the case"
Ln 298 - I think it should be clusters and not "cluster"?
Ln 333-335 "While this character is fairly good for most typical specimens"… fairly good for what? Identification? Also, the word character is used twice in the sentence, but I think you mean characteristic.
Ln 335-336 Not sure the purpose of this sentence, seems a bit anecdotal. Perhaps add more detail relevant to your discussion or remove it?
Ln 338 "only one (female) specimen… analyzed so far", I would suggest perhaps changing this sentence as it sounds like you have additional samples that need to be analyzed. If so, why are they not included in the current manuscript?
Ln 381 "is not scope" should be "is not within the scope"
Ln 403 "for 55 species determined species"? Not sure how this is supposed to read?
The written text is clear: the story flows, the context and aims are clearly introduced and indicated in the first section, material/methods and results are well structured and unambiguous, and the discussion of the results is not over-extrapolated and opens the door to future research. The literature provided is also context-related. The figures and associated legends are understandable without going back to the main text.
The research question is relevant and well explained: they aim to fill the barcode reference library gap for the two target groups, with a lot of the species’ being bioindicators of water quality. Filling these gaps opens the door to future eDNA studies, like rapid biodiversity inventories, temporal/spatial biodiversity investigations etc. The sampling is large in number and covers the country’s geographical range. A large proportion of the species of the two target groups presently recorded in Germany are represented, most of the time by more than one sample. The methodology is nicely described.
Conclusions are well stated, linked to original research question and limited to supporting results. The study provides reference material for future eDNA studies and underlines some limits of the DNA barcoding technic for some closely related species. Data and analyses are robust.
- line 130: were all the samples collected at adult satge? Did the authors only used the male specimens for DNA barcoding when species identification only relies on male characteristics?
-line 135-136: the sentence should go in the result section
-line 144: not recorded but I assume that they were collected in Germany. If so, please specify in the sentence.
Same line: for how many species exactly did you only had one barcode? were these samples included in the further analyses?
-line 268: h8, could you specify where is this sampling coming from? Was it collected out of Germany?
In this paragraph it might be nice to give some indications about the geographical coverage of each species sampling because the larger it is, the higher the chances to have more haplotypes recorded.
-overall, check for brackets after species name. Sometimes they are present, sometimes not.
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