Natural history collections continue to inform ground beetle genetics.

shows an Oligocene origin and


Authors' reply, 26 June 2024
June 26, 2024 Dear recommender, Prof Felix Sperling, My coauthors and I would like to thank you and the reviewers for your constructive comments and for inviting us to resubmit a revised version of our manuscript.We have found your last comments helpful and have addressed all of them.We include point-by-point responses at the end of this letter with our replies and corresponding line numbers for the revision.We believe that our manuscript is much clearer and hope is now ready for publication in PCI Zoology.

Revision round #2
Decision for round #2 : Revision needed One more round needed.This version is better, with good responses to reviewers, but the ms still has editorial issues that necessitate another round of revisions by the authors.I attach the Word version of the main text file, which contains: 1.A few remaining glitches that I have edited in the Word file.

In the new biorxiv version the Figures have been uploaded in jpg format avoiding this issue.
2. Some suggested editorial revisions intended to make some of the phrasing more grammatically smooth or easy to understand (these should all be checked to ensure I haven't altered meaning).

All these suggestions have been integrated.
3. A couple of points in text that refer to other's work/opinions and need citations.

New citations have been included.
4. Noted missing caption for new Suppl.Fig. 5, which means adjusting numbers as well.

Corrected.
5. Noted that Fig. 2. has a number of problems with offset text and symbol blocks.

Corrected.
6.The references list needs some substantial cleaning up to ensure that format and information is consistent.Also please check that all refs are cited in text and vice versa.

Corrected.
Since the remaining items are editorial, I don't think this needs to go our to a second round of review.This version is better, with good responses to reviewers, but the ms still has editorial issues that necessitate another round of revisions by the authors.I attach the Word version of the main text file, which contains: 1.A few remaining glitches that I have edited in the Word file.
2. Some suggested editorial revisions intended to make some of the phrasing more grammatically smooth or easy to understand (these should all be checked to ensure I haven't altered meaning).
3. A couple of points in text that refer to other's work/opinions and need citations.4. Noted missing caption for new Suppl.Fig. 5, which means adjusting numbers as well.
5. Noted that Fig. 2. has a number of problems with offset text and symbol blocks.
6.The references list needs some substantial cleaning up to ensure that format and information is consistent.Also please check that all refs are cited in text and vice versa.
Since the remaining items are editorial, I don't think this needs to go our to a second round of review.This manuscript has recieved three very positive reviews.All of the reviews raise some editorial points, and two of them request more detailed information and discussion on the lab methods.I agree that it would help to flesh out the methods at least a bit more.Please revise your manuscript accordingly to take advantage of these supportive reviews, responding to (or refuting) each of the points raised.

Results
In the case of negative results, is there a statistical power analysis (or an adequate Bayesian analysis or equivalence testing)?[ ] Yes, [ ] No (please explain), [ ] I don't know, [X] N/A.

Discussion
Have the authors appropriately emphasized the strengths and limitations of their study/theory/methods/argument?

[X ] Yes, [ ] No (please explain), [ ] I don't know
Are the conclusions adequately supported by the results (without overstating the implications of the findings)?

[X ] Yes, [ ] No (please explain), [ ] I don't know
This is an impressive phylogenomic study of a complex lineage of ground beetles, while also serving as a powerful demonstration of the applicability of museomic techniques to such questions.The amount of data acquired from historic specimens was surprising to me (not having explored this area nearly as much as I would like), but the trees support that the vast majority of it is reliable and informative.
With regard to the criteria for excluding certain samples, these aren't too clear early on.Apparently there was nothing detected for the samples initially excluded, rather than simply very low concentrations.(As an aside, it would be interesting to see if any markers could be amplified from those extracts.Given the higher than expected success rates overall, it seems not out of the question that below detectable quantities may still be amplifiable.)Exclusion of some lower concentration samples that produced some data might merit a little more discussion.12 samples with <150 loci were left out.But it would be interesting to see if these could be placed on data sets limited to a smaller number of shared loci.I'm just interested to see what information can be squeezed out of these old specimens (thinking 100YO types and such) when necessary.
Otherwise, there is a wealth, perhaps an overabundance of discussion of the fine points of Carabus relationships.Only the hardcore fans will find this useful, while the general readers interested more in museomics will begin to wonder whether to continue reading.In many other groups this might be problematic.I suppose in a paper dealing with such a popular genus, the committed readership will be somewhat higher than for many other taxa.
I made a number of other minor corrections and comments in the pdf.But in general this is a very readable and clean manuscript, especially for a pre-print.The figures are attractive, clear, and sufficient to convey the results.

Reviewed by Julian Dupuis, 08 May 2024
Here, the authors use HyRAD-X on a combination of museum and contemporary specimens to generate a large phylogenomic dataset to assess diversification of a genus of ground beetles.This approach allowed them to generate quite a bit of data from a broad assemblage of diverse specimens, which they then used for a whole bunch of phylogenetic and population genetic analyses.From these, they focused discission on both taxonomic considerations within this genus as well as diversification/speciation dynamics.I am no carabid expert, so can't judge that aspect of the study, however from a very superficial review of pertinent Carabus literature, the authors have done a fine job in addressing outstanding taxonomic questions at both the subgeneric and more microevolutionary levels.
Overall, the study is well-written, most analyses are conducted and presented appropriately, and the system-specific discussion is well-presented and as approachable as such system-specific discussions can be for non-experts in that group.From a phylogenomic perspective, bioinformatic pipelines (including code, input files, etc.), analyses, and interpretations are solid.My only bigger-picture critique of the study is that I think more could be done to explore why some samples work and some don't work in this museomics context.As the authors point out, a lot of this is unknowable (specimen collection and preservation conditions), but the authors document that a FA was used on all raw DNA extracts, so I wonder if there are any trends with regard to fragment size distributions that might inform more interpretation of the results presented in Figure 1.
Might turn out to just be too variable to make any conclusions there, but I'd say worth a bit of added discussion to the first paragraph of that section of the Discussion.As someone who is exploring these methods in depth, that kind of methodologically important information is quite valuable, even if it's somewhat conjecture or the author's best guess.Specific comments provided below.I think this study will be a valuable addition to both 1) ground beetle researchers and 2) systematists exploring this new field of museomics.With museomics being such a young field, studies such as these have a great opportunity to add extremely valuable context to the discipline moving forward.2021?It is unclear from this section here, and that seems to be a pivotal step in library prep for this method.
Line 247-249.Commas should be used in the # loci here.Also maybe refer to Table 1 here, so it shows up near this part of the methods?Line 288.Should be "SNPs" Line 331.Although barplots and likelihood values are provided in the supplement, I'd also like to see the deltaK values (especially since that method was used to determine K, as in the methods) and would like to see the likelihood values plotted so they're more interpretable to a reader.Ultimately, following some of the best practices for STRUCTURE (10.1111/mec.14187),seeing the various statistics developed by Puechmaille might help in determining the best value of K, but given the relatively low (and uneven) sample sizes, I don't expect that they'd be terribly informative beyond what is presented here.But, as I note below, providing some more details about those analyses/interpretations would be a good addition here.The connection here to hybridization is nice, but from a purely species delimitation point of view, the diderences in the diderent mt genes is interesting.Or does a combined 3-gene network show an overall composite of the findings of the three genes individually?That might simplify a main-text figure to one thing, which I think would be a nice addition.Line 709.BioProject not currently available on NCBI-can't tell if just still embargoed or not.
Are the research questions/hypotheses/predictions clearly presented?[X ] Yes, [ ] No (please explain), [ ] I don't know Does the introduction build on relevant research in the field?[X ] Yes, [ ] No (please explain), [ ] I don't know Materials and methods Are the methods and analyses sufficiently detailed to allow replication by other researchers?[ X -with some commentary on exclusion criteria] Yes, [ ] No (please explain), [ ] I don't know Are the methods and statistical analyses appropriate and well described?[ X] Yes, [ ] No (please explain), [ ] I don't know Lines 79-81.Grammar a bit clunky-would rephrase.Line 92.Not sure what is meant by "inferences made in fine"?Line 116.Should the abbreviated form of the subgenus name be used here, as it is used down in the results/discussion?i.e., "C.(Chaetocearabus)"?Formatting of subgenera goes back and forth throughout the manuscript it seems.Personally, I like the abbreviated form, since they're all Carabus subgenera.Lines 119-120.Reword to "where hybrids are found, for example, at the Katara Pass. . ." Lines 193-194.Although I think I generally understand what is being said here, I would suggest rephrasing this sentence."Processed" could be used to mean several diderent things in this context.Line 205-208.Thank you for this transparency!I completely agree.I would rephrase slightly as "Early sampling erosion and discarded samples are commonly not. . .". Section at line 216.Was the same RNAseq-based probe kit used as in Toussaint et al.

Line 348 .
Rephrase to "enabled us to. . ." Lines 350-351.What elution volume was used for these extractions?Without that, the concentrations are rather meaningless.Alternatively, are 260/280 or 260/230 ratios available to contextualize the quality of DNA? Expanding a bit more broadly, I wonder if any of those considerations could help explain the large spread in data return across the various extracts.Or some other consideration from the FA runs that were done on raw extracts?I am not the most familiar with HyRAD/HyRAD-X literature, but trying to figure outwhat the best practices are with these museomic extraction techniques seems to be the biggest sticking point that may be keeping the broader community from advancing these methods to the next level.Adding a bit more detail/discussion to that logistical topic might be a nice balance to the very detailed taxonomic/biogeographic discussion.Line 352.I would also argue the same for fresh samples (concentrations not being homogenous)-it looks like almost a bimodal distribution among fresh samples, which might be worth noting in the text (or at least the large spread).

Figure 2 .
Figure2.Some mismatch between parts of this figure-I assume the result of bad pdf conversion or something like that.Also, as a color-blind person, I have trouble diderentiating the grey and red asterisks (the boxes are a bit easier), FYI.Also also, great illustration and pictures-cheers Conrad!Line 434.What value of K was determined to be best, and what did deltaK say?From figure3, it seems like K=8 was supported, but looking at the supplemental barplots, K=7 shows virtually the same overall pattern, and it's really only fine-scale diderences that are showing up relative to K=6.More detail about the interpretation of these results would be warranted.Line 445."i.e." not needed.Line 668.Personally, I think one or all of these networks should be included in the maintext.

Figure 3 .
Figure 3. Symbols are provided to contextualize specimens, as in fig 2, but aren't actually included in the figure (unless they also were subject to some pdf creation/conversion errors).Note, I'd answer yes to virtually all of the questions presented in the PCI Zool reviewer's list, and think I've adequately addressed my concerns above.