Systematics of the Arboreal Neotropical ‘thorellii’ Clade of Centruroides Bark Scorpions (Buthidae) and the Efficacy of Mini-Barcodes for Museum Specimens

Round 1

Reviewer 1 Report

Basic reporting

It is an interesting and well written manuscript, meeting the high standards of the journal Diversity. The authors are specialists on the taxonomic group in question and their conclusions appear as well-supported, although the Discussion should be expanded to include more arguments in support of the efficacy of mini-barcodes (see below). The methods used are sufficiently described, including both the molecular and morphological analyses. Most of the nodes in the phylogenetic tree are well-supported.  All the relevant sampling data are provided for the specimens under study.

Minor issues

I do not think it is necessary to provide a name of the author of the family group name in the title of the paper. The title thus should be shortened appropriately. On the other hand, complete taxonomic names of the species examined, including the authors and years of the descriptions, are not included in the tables. They are possibly hidden somewhere in the text but it might be better to have them all together in a table.

The authors have decided to use a very short 125 bp region of COI. I suggest consulting a recent manuscript by Yeo et al ( https://www.biorxiv.org/content/10.1101/594952v4.full ) and incorporate in the Discussion some comments related to the finding of Yeo et al. that “mini-barcodes <200-bp perform significantly worse than full-length barcodes”.

In Table 1, mini-barcodes are not included in the dataset for three species (C. berstoni, C. catemacoensis and C. chanae). Please specify why, somewhere in the text, or is it just an omission?

In the Methods chapter, the authors mention “molecular dataset using maximum likelihood (ML) in RAxML”. However, such a tree is missing in the manuscript. Please clarify this issue.

The authors say in the Abstract that “Results … highlight the efficacy of mini-barcodes for identifying morphologically similar, cryptic species“ but this issue is not much covered in the Discussion. Please expand the Discussion and include some arguments and explanation in support of this statement. Was any analysis based just on mini-barcodes performed? What would be the difference between such a tree and a tree based on longer markers?

Author Response

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Reviewer 2 Report

The manuscript sent for review touches on an interesting topic of the use of museum specimens in species identification and the reconstruction of phylogenetic relationships. However, I have a few comments regarding the research methodology.
- The title of the work itself suggests that the age and the method of conservation of the samples are an important aspect of the study. However, the Authors did not address these features of the studied specimens. Please complete this information in the descriptions of museum specimens and refer to them on the basis of relevant references in the introduction and discussion.
- On the basis of own research, are the Authors able to state that the age and conservation method affect the quality of the sequences obtained and their usefulness in this type of analysis? This is a very interesting and important aspect of this research.
- As for the laboratory part itself, due to the degradation of genetic material in museum specimens and potential sequencing reading errors, preferably 3 repetitions for each sample should be made to confirm the result. Authors should also perform negative controls and physically separate workstations with museum and contemporary specimens to exclude contamination of the samples. However, I did not find such information in the methodology.
- Phylogenetic analysis is described in an unclear manner - how did the Authors deal with the combination within one analysis of sequences that differ significantly in length? Does it have an influence on the obtained results? Please give clear indication in the work which sequences were obtained from museum specimens.
- Have the new sequences obtained been deposited in the public database?
The discussion of the results is too focused on the phylogeographic aspects. Please refer to the references concerning the aspects suggested in the title of the manuscript.

I hope that my suggestions will help the Authors to improve their manuscript.

Best regards

Comments for author File: Comments.pdf

Author Response

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Reviewer 3 Report

The paper is clear and has shown interesting results. I suggest the authors to specify, between lines 353-364, the limits of COI in phylogenetic analyzes. In fact, it should be remembered that the mitochondrial marker used must always be validated by at least one nuclear marker. There are many papers that highlight such limitations and it would be interesting for the authors to highlight this limitation

Author Response

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Reviewer 4 Report

The present manuscript deals with the utility of mini-barcodes (fragments of 200–300 bp) of barcoding genes in the specific identification of bark scorpions. This is the first attempt to do use them in arachnids. This method would be very useful for identification of preserved museum specimens, which commonly bear fragmented and degraded DNA.

The manuscript is interesting, very well conducted and the main conclusions are supported by the presented data. I really enjoyed when reading the ms. It is a great piece of work and surely it would inspire others to test this identification tool to other arachnid groups. I only have some minor comments/suggestions:

• Some keywords are also in the title, so it is preferably to change them by different ones.
• Table 2 fits better in the Appendixes rather than in the Material and Methods section.
• I strongly recommend the authors to add some photos of the studied back scorpions. They would be highly appreciated by the non-specialist reader.
• Finally, I recommend to author to have a though about the major implications of their study. Please ensure that your work is placed in as wide a geographical context as possible and that you compare and contrast your findings with elsewhere, even spreading this methodology to other arachnids or other invertebrate groups. Perhaps the inclusion of a new heading, i.e. ‘Conclusions’, may be positive for highlighting them and also include future recommendations/ next research steps.

Author Response

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Round 2

Reviewer 2 Report

Author did not refer to my comments that were placed directly in the pdf file. 

I also think that negative controls, replications etc. are important part of laboratory procedures concerning museum specimens regardless the aim of the research - it is a good practice while working with museal samples. However, I understand that  Authors are not able to supplement these analyzes.

Author Response

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Author Response File: Author Response.docx