Complete Mitochondrial Genome of Decametra tigrina (A.H. Clark, 1907) (Crinoidea, Comatulida, Colobometridae) and Phylogenetic Analysis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Kim et al present a nicely laid out, thorough treatment of their generation of a new complete mitogenome for Decametra tigrina (A.H. Clark, 1907). As the first mitogenome for this genus, and one of only a handful yet generated for the family Colobometridae, this is an important addition to the available genetic resources for the Crinoidea. The data is well presented and the figures, for the most part, are particularly clean, attractive and convey their respective findings well. The sequences used for phylogenetic analysis are clearly identified by GenBank and published references, and the overall traceability of the work is clear. I have no particular concerns over the mitogenome sequencing part of this study and recommend no major or minor changes to this section. 

I have some concerns as to the clarity and interpretation of the phylogenetic analysis. The authors find a very tight relationship between three mitogenome sequences - Florometra sp, MT302206; O. serripinna (MW405444) and their D. tigrina sequence. These three form a clade within which there appears to be effectively no sequence divergence. This is noted by the authors, but in their results they further state (Line 144) that "their grouping implies that the boundaries between Mariametridae and Colobometridae may not fully reflect evolutionary relationships." I do not understand why this result implies this - none of the putative species to which the three mitogenomes in this clade have been assigned belong to the Mariametridae, and the phylogeny presented shows the only member of the Mariametridae included (Stephanometra indica) to form a distinct clade outside of the Colobometridae. 

To me, and as indicated by the authors, this result is most likely to indicate one or both or two possibilities: 1) that the one of more of the species involved have been misidentified or 2) that Oligometra serripinna and Decametra tigrina are synonyms. As the authors state, this cannot be determined without access to the specimens (and certainly the voucher for MT302206 appears inaccessible or lost) or inclusion of nuclear markers since the effective single locus of the mitochondrial genome could confuse matters. I am unclear, however as to what is meant by Line 180, "We re-examined the voucher for MW405444 and confirmed D. tigrina using a circular to pentagonal centrodorsal, relatively long cirri with about 28 segments, and sharply pointed terminal claws." Do the authors mean that they re-identified MW405444 as D. tigrina? Or just that they checked both specimens and confirmed their identifications? If the former, the phylogeny and discussion should be updated to remove reference to Oligometra altogether since it is not actually included in the phylogeny; if the latter, please re-write this section to make this clearer. 

The remaining tree topology is also interesting. As has been shown by several studies, the authors find a paraphyletic Antedonidae and Tropiometridae. As the authors note, mitogenomes are effectively single loci for phylogenetic purposes, so it would be useful to cite the two modern phylogentic works which also include nuclear genes (Rouse et al., 2013 and Hemery et al., 2013) and find the same paraphyly, not just another mitogenome study. It would also be interesting to include some discussion as to why the current authors find a completely different topology for the placement of Florometra sp MT302206 relative to Florometra serratissima AF049132 as Nam et al., 2011 who generated this sequence. 

Minor comments: 

Line 31, the correct citation for reference 1 is: Messing, C.; Gondim, A.I.; Markello, K.; Poatskievick Pierezan, B.; Taylor, K.; Eléaume, M. (2025). World List of Crinoidea. Decametra AH Clark, 1911. Accessed through: World Register of Marine Species at: https://www.marinespecies.org/aphia.php?p=taxdetails&id=204374

Line 202: Unless I have missed something, why do you discuss gene order here? 

Author Response

We have revised the manuscript throughout in accordance with all of your comments.

Please find attached our point-by-point responses.

Thank you very much for your time and valuable suggestions.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is well structured and written in a concise and clear way. It does suffer from a massive problem though: the „novel“ mitogenome of what here is identified as Decamtera tigrina is almost identical (>99.7 % sequence similarity) to two mitogenomes already present in GenBank – that of „Florometra sp. BMK-2020“ and the one of „Oligometra serripinna“. While the authors briefly discuss this issue in the manuscript, they neither come up with a clear resolution nor fully address the consequences. Several possible explanations exist:

• Two of the tree specimens that the sequences derive from are misidentified
• Oligometra serripinna and Decametra tigrina are synonyms (which seems unlikely in my opinion)
• Some kind of mix-up has happened between specimens and sequences
• There might be hybridization and mitochondrial introgression between these taxa

 

If the COI barcoding region of the novel sequence is checked in Bold Systems for species identification, the most likely identification given is „Oligometra sp.“, based on numerous barcodes. And it is near identical with another South Korean O. serripinna barcode. The resulting tree (see attached file) clearly shows that there is some internal structure within the “Oligometra” clade and that “Decametra” appears in two different clades within the tree (obviously this all has to be taken with caution, but it indicates that a second look is necessary).

What needs to be done:

• Provide a more in-depth discussion of the problem
• Provide better evidence that the identification in the present paper is correct, including documentation of all necessary morphological details that would allow others to verify or reject the identification. Additional images of key morphological details will be needed.

 

Further issues that need to be fixed:

Line 77: provide details on insert size targeted and read length

Line 78: deposit raw read in NCBI SRA and mention accession number of Short Read Archive here

Line 83: provide information on the coverage of the mitogenome, ideally if form of a graph

Line 113: statement "there were no detected gene rearrangements, …” in comparison to which taxon? The statement does not make sense without a reference taxon or condition that the observed state is compared to

Line 122: space missing btw. “67” and “bp”

Phlogenetic analysis: did you consider also calculating a tree based on AA alignments? These typically are more conserved and might be better suited to analyze taxa that likely have split many millions of years ago

Lines 142-145: “implies that the boundaries between Mariametridae and Colobometridae may not fully reflect evolutionary relationships” – this is a bold statement based on the current data – unless human error (e.g. misidentification) and natural phenomena (e.g. introgression) are ruled out such a conclusion is not valid in my opinion

Lies 152-158: here again evidence is missing – the text as currently phrased assumes that all these specimens from which the sequences derive have been correctly identified, which in comatulids, which can be difficult to identify is quite a bold assumption, I think

Lines 180-181: you state that the voucher for sequence MW405444 was re-examined, but it is not very clear what the result of the re-examination was – please rephrase and provide more details, as well as evidence (including images of that voucher specimen and key morphological details)

Line 184: provide more information on the steps taken to locate the voucher of MN883538 and (if applicable) the response of the original authors / hosting institution etc. – what was the reason that that specimen is not available – is it lost, misplaced, not deposited at an institution,…?

Line 193, first word: replace “result” by “tree”

Table 1: add localities and voucher specimen repository numbers / institutional codes

 

Summing up I recommend major revision and re-review.

Kind regards

 

 

Comments for author File: Comments.pdf

Author Response

We have revised the manuscript throughout in accordance with all of your comments.

Please find attached our point-by-point responses.

Thank you very much for your time and valuable suggestions.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The revised manuscript addresses most of the concerns raised in the review of the earlier version. While I personally would be unhappy to not be able to resolve the remaining issues (the identity of the “Florometra sp.” voucher), it is clear from the responses, that the authors do not deem this necessary. Apart from some minor issues, pointed out below, I do not see any further need for corrections.

• Line 16: replace “bella and the MW405444” by “bella and that of accession no. MW405444”
• Line 77: some details were added, but insert size used in library preparation is still not mentioned
• Line 103 ff.: much clearer now – thank you
• Line 236: information where “specimen ID 201904-Crinoid014” is deposited is missing. (it is located at the Research Institute of Basic Sciences of Incheon National University – this should be added). Did the authors get in touch with the curator of that collection and at least request photographs?
• Supplementary Fig. 1: thanks for adding the coverage graph

Kind regards

Author Response

Dear Reviewer,

Thank you very much for your additional comments on our revised manuscript.

We have carefully addressed each point and revised the manuscript accordingly.

Our point-by-point responses are provided below.

 

[Comment 1 (Line 16)] Reviewer comment: replace “bella and the MW405444” by “bella and that of accession no. MW405444”

Response: We intended to indicate a comparison between Cenometra bella and the GenBank record MW405444 (submitted as Oligometra serripinna), i.e., two separate records.

In the revised manuscript, this is written in the Abstract as: “The arrangement of genes remains conserved, matching those of Cenometra bella and the MW405444 (submitted to GenBank as Oligometra serripinna).”

 

[Comment 2 (Line 77)] Reviewer comment: some details were added, but insert size used in library preparation is still not mentioned

Response: We added the average insert size used for library construction in Materials and Methods (Section 2.2) as follows: “A genomic library with an average insert size of 300 bp constructed with the KAPA Hyper Prep Kit …”

 

[Comment 3 (Line 103 ff.)] Reviewer comment: much clearer now – thank you

Response: Thank you for your positive assessment. No further changes were required for this point.

 

[Comment 4 (Line 236)] Reviewer comment: information where “specimen ID 201904-Crinoid014” is deposited is missing… Did the authors get in touch with the curator of that collection and at least request photographs?

Response: We added the repository information for specimen 201904-Crinoid014 and clarified the limitation regarding access and curator-provided photographs in Results (Section 3.3) as follows: “The voucher for MN883538 (specimen ID 201904-Crinoid014) is reported to be deposited at the Research Institute of Basic Sciences, Incheon National University (Incheon, Republic of Korea) [18]. However, we were unable to access the specimen and obtain curator-provided photographs for re-examination in this study.”

 

[Comment 5 (Supplementary Fig. 1)] Reviewer comment: thanks for adding the coverage graph

Response: Thank you. No further changes were needed.