Comments on revised manuscript

The revised manuscript of Majeske et al is much improved in comparison to the initial submission. Some of the questions raised in the previous review, however, remain open and other new aspects have appeared. Open issues: 2) Please provide the repository number and institution where the voucher specimen has been deposited --> this issues has not been addressed in the revised version; it is unclear if a voucher specimen has been deposited or not, where it is stored and which inventory number it has; if the specimen has not been retained, this is unfortunate, but not a huge issue - it still needs to be clearly/openly stated 3) Did you verify the identification and made sure that this is D. antillarum rather than D. africanum (which allegedly has repopulated some D. antillarum habitats in the Caribbbean and GoM) --> this issue too has not been addressed; at the very least I would expect a statement that the authors were aware of this second Atlantic Diadema species and how they made sure they really had D. antillarum 7) Please provide a coverage graph --> the coverage graph is mentioned in the text, but not provided in the paper 9) Please explain what exactly was used for the analysis - the full nucleotid sequence including non-coding regions, just the CDS of the protein coding genes, or ? --> this is still unclearly formulated in the paper - I assume the whole mitogenome sequence was used, but the wording is very ambiguous; this needs to be very clearly stated in the material and metods section 11) Please explain the choice of the model used in the analysis - was some Modeltest run? --> this information is still lacking New issues: A) The description of the assembly process is still rather unclear - this needs to be better explained. For example, was any kind of preprocessing (read triming etc.) done? Which parameters were chosen for the various programms employed? How did the two-stage read extraction process really work - the wording in the manuscript is very unclear regarding this aspect B) The raw data need to be deposited in the GenBank Short Read Archive (SRA), in the Github repository only the extracted mitochondrial reads are available - this is insufficient to repeat the assembly process and analyses carried out in the present manuscript C) The fasta file included in the Github repository has 23 positions that are redundant (overlapping with the start of the sequence) - they need to be removed before submisson D) There is some inconsistence on the length of the mitogenome, the text says 15,708, the figure says 15,707 - the latter, judging form the files in your Github repository, is correct --> please make sure the information given is consistent E) No information is given on the reason for chosing the particular evolutionary model that has been used in the phylogenetic analysis F) The phylogenetic analysis has been done by NJ-methods, which are fast but can subject to a lot of problems, it would be better to use MAximu Likelihood (or Bayesian) methods G) The authors have made an important discovery in relation to the mitogenome deposited as "Echinothrix diadema" in GenBank. Rather than to speculate on the reasons that is the sister of D. antillarum in their analysis the authors should simply which of their hypotheses (AT-bias vs. misidentification) is correct. All the tools that are needed are already available in Genbank! There is an extensive dataset of three mitochondrial markers (12S, ATP6, ATP8; https://www.ncbi.nlm.nih.gov/popset/?term=MW329515 etc.) available for Echinothrix, which includes hundreds of sequences and encompases material from the complete geographical range of the genus (Coppard et al. 2021 https://www.nature.com/articles/s41598-021-95872-0). In addition, there are 16S sequences available for D. savignyi, the suspected candidate of the misidentification. I have downloaded these sequences and run preliminary analyses with with a subset of the sequences. These clearly show that the "E. diadema" mitogenome has nothing to do with true E. diadema and that it is a Diadema. While the data basis for Diadema is less extensive than for Echinothrix there are 16S sequences of D. savignyi (GenBank PopSet: 673458050) that are identical to part of the 16S sequence of the alledged "E. diadema" mitogenome. Thus I am convinced that the second hypothesis (misidentification) of the authors is correct. This is an important finding that should be discussed in depth in the manuscript. I am includid the alignments and trees that I made in the attachment - similar analyses and trees should be included in the manuscript. Link to download the attachments: https://we.tl/t-y7ypbnZYPQ Summing up, I recommend acceptance after major revision. Kind regards Andreas Kroh, NHM Vienna, 11/9/2022