The Biodiversity of Calcaxonian Octocorals from the Irish Continental Slope Inferred from Multilocus Mitochondrial Barcoding

Round 1

Reviewer 1 Report

This manuscript is very well written and compiled. I cannot comment on the molecular genetic methods but the morphology is well done. Some additions or changes might be needed before the species are described but the supplementary material is fine for the purposes of this paper.

See a few comments in the attached file. 

Les Watling

Comments for author File: Comments.pdf

Author Response

We would like to say thank you for your time reviewing our manuscript. Your thoughts and suggestions were both insightful and helpful and we hope that you find the revisions favourable and the overall manuscript improved

Reviewer: Line 88: No, it is due to the fact that the person working on the group was at a museum and did not have to do many things other than taxonomy. In contrast, many taxonomists are at universities where they have many diverse duties which occupy a lot of their time.

Response: Thank you for your comment. We have changed out statement to include this. The text now reads

“There are fewer taxonomic problems within Primnoidae due to the work of dedicated museum taxonomists, unburned by academic duties that university-based taxonomists face, and perhaps due to the frequent incorporation of genetics into their systematics which helps test and reinforce the usefulness of certain taxonomic characters (e.g., [19–21]).”

 

 

Reviewer: Line 288? Guess: Yikes

Response: Yes, we agree.  But really this just shows that substitution-based methods of species delimitation are probably not useful in this group, as we discuss later in the manuscript.

 

Reviewer: Line 455: It still looks like a bush. We have seen colonies like this at Corner Rise. I think they are all A. arbuscula.

Response: Thanks for the comment.  Interesting to hear your thoughts.  We have modified the sentence slightly, to accommodate this possibility.  It now reads:

“The single specimen sequenced as Haplotype 22 has a flattened more planar colony form (M27, Figure 4a) that does not match the description of any accepted species of Acanella known from the North Atlantic [72]. However, it is possible that this specimen is also A. arbuscula and that the full range of intraspecific variation of this species has yet to be described. Due to the current taxonomic uncertainty, the assignment was left at genus rank.”

 

Reviewer: Line 490: there is no genetic data for P. resedaeformis?? If so, how different is your haplotype from that?

Response: Thank you for pointing out our omission. When the Primnoa haplotypes (33, 34,35, and 36) are compared to the genetic data hosted on GenBank Haplotype 34 shares an identical mtMutS sequence with P. notialis (MG986917.1), P. pacifica (MF319956.1) and P. resedaeformis (AY968599.1 and MG986945.1). Haplotypes 33,35,36 are different by a single mutation.  

We have added this information to the main text so that it reads

“Finally, Haplotypes 33, 34, 35, and 36 are all diagnosed as Primnoa sp. and all share the same gross colony morphology, M31, which suggests they are all the same species and that there is just high haplotypic diversity within this species using the selected genetic markers. Genetically, the mtMutS sequence of Haplotype 34 is identical to Primnoa notialis Cairns & Bayer, 2005 (GenBank Accession Number MG986917.1 [20]), Primnoa pacifica Kinoshita, 1907 (GenBank Accession number MF319956.1 [82]), and P. resedaeformis (GenBank Accession Number MG986945.1 [20]) while Haplotypes 35, 36, and 37 have a single mutation that differentiates them from these sequences (99.87% similarity). This highlights the need for better markers to delimit between species in this genus.”

 

Reviewer: Line 551: his statement is not exactly true. Lepidisis is a useful name for particular specimens, but cannot be used indiscriminately for an unbranched whip-like octocoral colony.

Response: Thank you for your comment. We have incorporated into the main manuscript which now reads

“Lepidisis is polyphyletic [74] as lack of branching is the diagnostic character for this genus, and this trait is now recognised as labile [14,15,75]. Thus, Lepidisis cannot be used to broadly diagnose unbranched bamboo corals, and the true generic affinity of nine of the 11 presently accepted species is uncertain (see [74]). Only Lepidisis caryophyllia Verrill, 1883 (the type species) and Lepidisis cyanae Grasshoff, 1986 are considered to be representatives of the genus.”

 

 

Reviewer: Line 554, And it is possible for you to have L. cyanae based on its known distribution.

Response:  Thank you for your comment. We could not confidently diagnose any specimen of ours to L. cyanae. However, we have now explicitly mentioned this in the text

“None of our specimens matched the type description of L. cyanae (the polyp morphology and sclerite composition is best observed in fig 8 and 9 in Grasshoff [76]) which is known to occur in the Northeast Atlantic.”

 

 

Reviewer: Line585: but neither of these have been subject to RADseq?

Response: Thank you. This is a good point. We have modified the sentence slightly to make it clear that we don’t know where the true species boundaries are.  It now reads:

“Multiple putative species have also been observed to share an identical mtMutS haplotype: for example, three putative species within the genus Chrysogorgia shared a mtMutS haplotype [24], and two Narella species, N. hawaiiensis Cairns and Bayer, 2008 and N. dichotoma (Versluys, 1906) shared a single mtMutS haplotype [37]. Further genetic work with more variable markers on a wide range of specimens will help untangle what is intraspecific variation and where the species boundaries are.”

 

 

Reviewer:  Line 601: This is a problem. We don't know whether the difference you mention is just intra-specific variation. I suspect it is... but without more detailed genetic work who knows.

Response: We completely agree with your comment and thank you for spotting our inappropriate language. We have modified the sentence and it now reads

“From our data, there is no consistency in the potential species assignments among genetic delimitation methods, and without additional genome-wide data it is impossible to accurately define species boundaries, however, it is unlikely that every haplotype or every morphotype represents a distinct species.”

 

 

Reviewer: Line 627: using exclusively mitochondrial genetic methods.... the nuclear genome may be changing at a different pace, etc., and so would add something to the story.

Response:  Thank you for pointing this out. We agree that A nuclear marker may add extra detail to this delimitation and have modified our text to reflect this and show where nuclear markers can both help and hinder delimitation. The text now reads

“Due to the lack of variation in octocoral mitochondrial genes, individuals of different species might have identical DNA sequences at these markers making it likely that multiple species are represented by a single terminus. Since multiple species potentially share a terminus, genetic species delimitation methods could never delimit the species successfully, and additional avenues of investigation such as morphology are required. The added variability associated with using additional nuclear markers such as 28S may help increase the delimiting power of our concatenated alignment, as seen in the well-studied shallow-water genera Sinularia [34,83] and Ovubunda [84], and should be included in future barcoding studies. However, some nuclear markers such as the multicopy internal transcribed spacers (ITS 1 and ITS 2) may not have a shared evolution due to the heterogeneity in copies of ITS in the genome between species [85] limiting their potential usefulness as barcodes.”

 

 

Reviewer: Line 637 well, maybe….

Response: Thank you for your comment. We have changed our sentence to include a caveat that we still know very little about the intraspecific variation in deep-sea corals.  The text now reads

“bGMYC is believed to have performed the best as the differences in morphology between individuals both within and between keratoisidid haplotypes suggest that there are more putative species in our samples than recovered by any genetic delimitation and bGMYC recovered the most species. However, It is accepted that our current knowledge of intraspecific variation in octocorals is limited, and that some species, such as A. arbuscula, exhibit a wide range of distinctive morphologies, which must be considered as potential intraspecific variation, when we interpret the number of species present.”

Reviewer 2 Report

This manuscript has the potential to have substantial impact and expand on our knowledge of octocorals in the region and contribute to our understanding of octocoral taxonomy/systematics. However, the presentation and focus need major revisions to have this impact and improve clarity. Substantial amount of work and effort clearly went into this study such that you could almost have two publications resulting from the data collected and presented. I look forward to seeing a revised manuscript that is more focused and has the additional detail needed to understand the findings more fully. I make substantial suggestions for restructuring presentation; however, other restructuring option would be acceptable.  The restructuring largely depends on the goal of the study and emphasis the authors want to make.  Grammatical structure is sound.

See more specific comments below.

Abstract:

I was left wondering what the goal or hypothesis was. I think you should clarify the study goal and state it in the abstract.

What was the goal of the study?  To test methods or to evaluate diversity using different methods and secondarily to compare diversity results in light of method selection?

How confident are you in the “known species” assignments/identities?

 

Introduction

Overall impression – The flow is a bit jumpy between general deep-sea information, corals, Continental Slope features, Ireland continental slope, Whittard Canyon biodiversity, and coral taxonomy and species assignment being problematic.  I found myself wondering what the goal was and the focus was until I arrived at the “aim” statement.  With restructuring, I think you can improve flow and clarity.  I may still not know the aim until it is stated, but I will not be wondering where the information I just read fits into the next section or whether you mean general deep-sea, continental slopes, canyons, Irish slop/canyon, etc. when you make more general statements. I also will not wonder so much whether your study is a review of the three families, with an emphasis on taxa found on Irish continental slope instead of just those samples you collected from a couple cruises. I also had the impression you were focusing on Whittard canyon until I arrived at the methods and saw from where all you had samples.

 

I recommend reassessing the order of presentation. I think the following order may improve flow and clarity of the study you are presenting. Try to remove excess detail on the group-by-group morphological issues; instead group them as examples by the problem or diagnostic character or greater confidence because of morphological and genetic approaches being combined.

o   A general intro into the deep sea.

o   That deep-sea features, including those along the slopes, harbor high biodiversity, including of Octocorals, which are important and vulnerable.

o   That certain Alcyonacea are prevalent in the deep sea, but that their assignment and taxonomy is problematic. Examples of morphological and genetic issues, in particular for for the taxa of interest in this study. Good to keep in the issue of why barcoding is commonly used and that multi-locus approaches good and cost effective.

o   In part because of these species’ assignment issues, few species-level characterizations of the widely distributed coral gardens on the Irish continental slope have been conducted/completed… Examples

o   Therefore, our aim was to do X

 

I think you can introduce the different species delimitation models in the materials and methods instead of the introduction because it isn’t your goal to test those methods.  If you want it in the intro, add a second goal to compare those methods

 

Early comments before I realized the Intro should probably be restructured –

Line 33:

…landmass, that encompasses (otherwise, it modifies landmass and not the marine territory)

 

Line 37: Please rephrase this sentence “The complex topographies….” It is complex and confusing as written, so it is difficult to follow the transition between the information preceding and following this sentence. I suggest a restructuring of this paragraph as follows; however the “complex topographies” sentence still interrupts the flow a bit because Whittard Canyon is already introduced.

The deep sea is the largest and least explored ecosystem on the planet [1]. Geomorphic features, including those found on the continental slopes, have been discovered to harbour increased biodiversity, for example, submarine canyons [2], seamounts [3], and cold-water coral reefs [4]. Situated in the Northeast Atlantic at the Northwestern edge of Europe, Ireland has a marine territory of ~880, 000 km2, ten times that of its landmass, that encompasses an extensive continental slope that is heavily incised with canyons and gullies, the most notable of which is the Whittard Canyon [5]. These complex topographies at the shelf edge and slope interact with the overlying water, affecting local oceanographic and hydrodynamic conditions that promote biodiversity [6]. Such enhanced biodiversity is evident for example in Ireland’s carbonate mound provinces where residual bottom currents are increased [7], in a unique biotope comprising the large bivalves Acesta excavata and Neopycnodonte zibrowii at 600-800 m in Whittard Canyon where internal waves resuspend particulate matter [8], and through….

 

Methods –

Line 140-144 – combine these sentences. This section is missing a summary of the number of stations and where these stations were in terms of depth ranges and being on slopes, canyons, and/or other features.

Line 145 – provide more detail about subsamples (e.g., approximate size and whether there were two separate subsamples or one used for the genetic and morphological analyses)

Line 147 – in situ

Line 151 – how small is small?

Lines 160-163: report PCR reaction in concentration and not volumes

Line 165 – sequenced with amplification primers?

Line 169 – citation needed for Geneious. Which feature in Geneious did you use to assemble and trim sequences? Did you trim primers manually or using a feature in Geneious before alignment? 

Line 170 – Which MUSCLE parameters were used?

Line 172 – settings/parameters used?

Line 173 – need to report the alignment algorithms used above.

Line 175 - 3390 bp alignment

Line 182 – [55], which uses

Line 195-196 – were there specific parameter values for the models that were used for reconstructions?

Line 197 – What settings were used for bootstrap, reconstruction, and TreeCollapse? You can include all of the settings (here and for those requested above and below) in Supplemental material or in the main text. From here one, please include details of settings selected for the reconstructions, delimitations, etc.

Line 224 – Do you have a citation or other justification for this value of 0.9?

Line 231 – What characters were examined or included as part of the gross colony morphology to recognize unique morphologies? Please provide additional detail here to orient the reader to your methods so that they can be reproduced.

Lines 236-242 – Which characters were analyzed and how so? You only discuss visualization here and not the analysis or identification of characters. Some detail could be added in Supplemental material, but you should at least briefly summarize the list of characters assessed and the most common states. How many polyps were analyzed per colony and how many colonies per haplotypes?  I think you have some of this requested info in the Intro, but it belongs here.

Lines 252-254 – What were the coating settings (time or coat thickness, for example)? What were the SEM settings or setting ranges used (kV range, for example)?

Lines 255-258 – Did you first dissect the polyps  to examine sclerites from the different parts of the colonies or were they released together?  What were the characters and character states assessed? How were they assessed? Was that placement final or preliminary? If the final step to finalize assignment to lowest taxonomic unit was line 261-263, then move it to end of this section.

Lines 260-263 – What match threshold was used? Does the latter line refer only to morphology-based assignment?  Which characters were used?

Lines 264-274 – so was just that boundary used or is this the same region used as described in Line 260?  Please clarify.

Line 272 – “characters, by co-authour CBU to the…”

At the end of the methods, I am now curious about what the final methods are for species assignment.  It is not clear how the genetic and morphological data are being used together and whether comparisons of the methods or congruence of methods is being used.  For example, which method(s) for species delimitation based on genetic data are used.  What happens when the data are not in agreement? Please clarify.

 

Results –

Line 279 – otherwise stated where?

Line 282 – you can delete Family in all cases here and below.  The ending of the family name indicates they are family-level names.

Line 290 – How did that method compare to the others? In the Figure and text, I am confused about what the final species assignments are.  You use subclade/species IDs in S3 and concatenated haplotype #s and delimitation methods (morph and genetic), but I cannot tell which species are which and why they were assigned to those species.  For example, in Fig 3, is D2 one species or two..or part of one species with D1, B1, and F1? That two are labeled Eknomisis sp. is confusing.  I would use the final species assignments as labels.

Table 2 – Some of this info is essential to interpret results.  Accession numbers could be in supplemental, but a table of final assignments would be good.

Fig. 6 – Orient reader to all acronyms and coding.  D1, D2, for example, are not defined.

Lines 291-312 and onward – Consider presenting this information in a table for easier comparison of morphological features. You presented in the Intro that branching pattern is labile with other characters considered diagnostic, so I would not focus on a character not indicative of species or genus. This section needs context for comparisons.  What is large, far apart, more planar, branches quickly, etc.  What are the differences between morphotypes for M2? I fear I could not come to the same conclusions in species assignment based on morphology because it is not clear here or above which characters were used.

Fig 2-4.  I would consider replacing these with character-state presentations with select representatives for main characters used to assign to morphotype.  For example, Fig. 3 g and h and Fig3 m-o appear to have similar morphologies, so provide one image with a description of the character state assigned. The reader can go to Supplemental to see the images for each specimen, but I think the reader needs a better understanding of the characters and states used to understand species assignments in context of the final assignments.

Lines 313-544 – See comments above to clarify presentation.  I skimmed through them because I had a difficult time following the presentation of the results and who was who and why they were assigned to a taxon.  Consider presenting as follows:

Gross morphology identified X morphotypes of Family 1.  These morphotypes are distinguishable by XYZ.  Concatenated genetic data did/did not support these assignments, with X method being more consistent with morphology. Caveats and justifications or criteria used for settling on final assignment.

 

Discussion

I think a good portion of the material should be in results.  For example, the initial IDs and congruence of genetic and morphological data. Try to reduce to focusing on how the diversity and congruence of morphological data compare to the literature in this section. The paragraph starting with Line 642, for example, is mostly result that could be summarized in the discussion in a single sentence before comparing to the New England and Corner Rise study.

I would consider restructuring by finding.  For example, a section or paragraph on first reports, another or diversity comparisons, another on congruence of data and how that compares to other such studies, etc. (Not necessarily in that order). I am still unclear about the goal of the study and what you found in relation to that goal. That part of the discussion should be a major point of discussion. If it is biodiversity, how does your species richness compare to what is known from the region.  What advances did you make to understanding the biodiversity?  What is the broader impact?

Conclusion

Include an impact conclusion in this paragraph.

Author Response

We would like to say thank you for your time reviewing our manuscript. Your thoughts and suggestions were both insightful and helpful and we hope that you find the revisions favourable and the overall manuscript improved

 

Comments and Suggestions for Authors

This manuscript has the potential to have substantial impact and expand on our knowledge of octocorals in the region and contribute to our understanding of octocoral taxonomy/systematics. However, the presentation and focus need major revisions to have this impact and improve clarity. Substantial amount of work and effort clearly went into this study such that you could almost have two publications resulting from the data collected and presented. I look forward to seeing a revised manuscript that is more focused and has the additional detail needed to understand the findings more fully. I make substantial suggestions for restructuring presentation; however, other restructuring option would be acceptable.  The restructuring largely depends on the goal of the study and emphasis the authors want to make.  Grammatical structure is sound.

 

See more specific comments below.

 

Abstract:

Reviewer: I was left wondering what the goal or hypothesis was. I think you should clarify the study goal and state it in the abstract.

Response: Thanks for the comment. We knew that we would not be able to put an absolute species number on the diversity that we were observing so instead we used multiple lines of evidence from morphological diversity, genetic diversity, and genetic species delimitation methods to help us come up with an estimation. We have amended our abstract to include an explicit sentence on the aims and state immediately that we couldn’t arrive at a definitive number. The idea of the paper was to make progress in understanding the observed diversity which is why we provide a lot of gross morphology imagery, taxonomic information in the form of sclerite plates in the supplementary, and we uploaded all of our genetic data to NCBI GenBank. These resources are the first steps towards resolving the species boundaries.  

 

Reviewer: What was the goal of the study?  To test methods or to evaluate diversity using different methods and secondarily to compare diversity results in light of method selection?

Response: See above.  Relevant line from the new abstract: "Herein we investigated the utility of a superextended (>3000 bp) barcode and explored the effectiveness of various molecular species delimitation techniques with an aim to put upper and lower bounds on the estimated number of calcaxonian species in Irish waters"

 

Reviewer: How confident are you in the “known species” assignments/identities?

Response:

Good question.  Where species names are assigned, we are confident that they are correct. We have used both the available genetic data and relevant literature to arrive at those designations. Where we are unsure we have left it to the lowest rank where we are confident. e.g. Eknomisis sp. and Primnoa sp. I would like to guide the reviewer to section 2.3 where the text now reads

“We were conservative in our identifications and only identified individuals to genus and/or species when we were confident that characters matched the relevant taxonomic literature [65–73]. Where there was doubt, we assigned individuals to a higher taxonomic rank in which we had confidence.”

 

Introduction

Reviewer: Overall impression – The flow is a bit jumpy between general deep-sea information, corals, Continental Slope features, Ireland continental slope, Whittard Canyon biodiversity, and coral taxonomy and species assignment being problematic.  I found myself wondering what the goal was and the focus was until I arrived at the “aim” statement.  With restructuring, I think you can improve flow and clarity.  I may still not know the aim until it is stated, but I will not be wondering where the information I just read fits into the next section or whether you mean general deep-sea, continental slopes, canyons, Irish slop/canyon, etc. when you make more general statements. I also will not wonder so much whether your study is a review of the three families, with an emphasis on taxa found on Irish continental slope instead of just those samples you collected from a couple cruises. I also had the impression you were focusing on Whittard canyon until I arrived at the methods and saw from where all you had samples.

I recommend reassessing the order of presentation. I think the following order may improve flow and clarity of the study you are presenting. Try to remove excess detail on the group-by-group morphological issues; instead group them as examples by the problem or diagnostic character or greater confidence because of morphological and genetic approaches being combined.

o   A general intro into the deep sea.

o   That deep-sea features, including those along the slopes, harbor high biodiversity, including of Octocorals, which are important and vulnerable.

o   That certain Alcyonacea are prevalent in the deep sea, but that their assignment and taxonomy is problematic. Examples of morphological and genetic issues, in particular for for the taxa of interest in this study. Good to keep in the issue of why barcoding is commonly used and that multi-locus approaches good and cost effective.

o   In part because of these species’ assignment issues, few species-level characterizations of the widely distributed coral gardens on the Irish continental slope have been conducted/completed… Examples

o   Therefore, our aim was to do X

Response: Thank you for this feedback. We agree that a restructure was necessary. We have rewritten the introduction to reflect your feedback

 

Reviewer: I think you can introduce the different species delimitation models in the materials and methods instead of the introduction because it isn’t your goal to test those methods.  If you want it in the intro, add a second goal to compare those methods

Response: Thank you, we have moved the relevant section to materials and methods.

 Reviewer: Early comments before I realized the Intro should probably be restructured –

Response: We appreciate the reviewer's detailed treatment of the introduction. While they have suggested a restructure, we would like to acknowledge the comments made below and assure the reviewer we have also taken all of them onboard. 

 

 

Reviewer: Line 33:

…landmass, that encompasses (otherwise, it modifies landmass and not the marine territory)

Response: Thank you. We have reworded this to incorporate this change.

 

Reviewer: Line 37: Please rephrase this sentence “The complex topographies….” It is complex and confusing as written, so it is difficult to follow the transition between the information preceding and following this sentence. I suggest a restructuring of this paragraph as follows; however the “complex topographies” sentence still interrupts the flow a bit because Whittard Canyon is already introduced.

Response: Thank you. We have reworded this

 

Reviewer: The deep sea is the largest and least explored ecosystem on the planet [1]. Geomorphic features, including those found on the continental slopes, have been discovered to harbour increased biodiversity, for example, submarine canyons [2], seamounts [3], and cold-water coral reefs [4]. Situated in the Northeast Atlantic at the Northwestern edge of Europe, Ireland has a marine territory of ~880, 000 km2, ten times that of its landmass, that encompasses an extensive continental slope that is heavily incised with canyons and gullies, the most notable of which is the Whittard Canyon [5]. These complex topographies at the shelf edge and slope interact with the overlying water, affecting local oceanographic and hydrodynamic conditions that promote biodiversity [6]. Such enhanced biodiversity is evident for example in Ireland’s carbonate mound provinces where residual bottom currents are increased [7], in a unique biotope comprising the large bivalves Acesta excavata and Neopycnodonte zibrowii at 600-800 m in Whittard Canyon where internal waves resuspend particulate matter [8], and through….

Response: Thank you for this. We have amended this statement.

 

Methods –

Reviewer: Line 140-144 – combine these sentences. This section is missing a summary of the number of stations and where these stations were in terms of depth ranges and being on slopes, canyons, and/or other features.

Response: Thanks. We have incorporated this into the text and it now reads

“During two research surveys to the Irish Margin between 24^th May – 5^th June 2017 and 11^th – 23^rd August 2018 aboard RV Celtic Explorer, targeted collections of octocorals and sponges for biodiscovery gathered 70 Keratoisididae, 22 Primnoidae, 20 Chrysogorgiidae, and one Chelidonisididae (Table S1) using the robotic capabilities of ROV Holland I. All stations from 2017 were within the Whittard Canyon system between 1275 – 1988 m, except two located outside the Hovland Mound and Belgica Mound SACs at ~880 m and stations from 2018 were within small canyons and gullies along the North Porcupine Bank between 821 – 2308 m.”

 

Reviewer: Line 145 – provide more detail about subsamples (e.g., approximate size and whether there were two separate subsamples or one used for the genetic and morphological analyses)

Response: Thanks for pointing out the omission in our methods. We have amended the text to include the required information and it now reads as

“Genetic and morphological subsamples were taken and stored in 96% ethanol; remaining parts of specimens were stored dry at -80 °C. Genetic subsamples comprised a few polyps of a coral and were stored in a 1.5 ml microcentrifuge tube. Morphological subsamples were approximately 4 cm in size and stored in glass vials.”

 

Reviewer: Line 147 – in situ

Response: Thanks for pointing out this error. We have removed italics from all Greek and Latin terms such as the one mentioned as per the MDPI author style guide.

 

Reviewer: Line 151 – how small is small?

Response: Thanks for highlighting this. We have added this information into the methods. The text now reads

“Genetic and morphological subsamples were taken and stored in 96% ethanol; remaining parts of specimens were stored dry at -80 °C. Genetic subsamples comprised a few polyps of a coral and were stored in a 1.5 ml microcentrifuge tube. Morphological subsamples were approximately 4 cm in size and stored in glass vials.”

 

Reviewer: Lines 160-163: report PCR reaction in concentration and not volumes

Response: Thank you for your suggestion. We have added the final concentrations to the manuscript the text now reads

“Each 25 µl PCR reaction contained 12.5 µl of 1 x GoTaq G2 Green Mastermix (Promega), 0.5 µl of 10 µM forward and reverse primers (final concentration 0.2 µM), 9 µl of nuclease free water, and 2.5 µl of DNA template (< 250 ng as per GoTaq G2 Green Mastermix protocol recommendations).”

 

Reviewer: Line 165 – sequenced with amplification primers?

Response: Yes, it was with sequenced with amplification primers. We have amended the text to include this information.

 

Reviewer: Line 169 – citation needed for Geneious. Which feature in Geneious did you use to assemble and trim sequences? Did you trim primers manually or using a feature in Geneious before alignment?

Response: Thank you. We have provided the citation for Geneious and provided the information that we manually trimmed the primers.  The text now reads

“DNA sequences were assembled and amplification primers and poor quality bases were trimmed manually by examining chromatograms in Geneious Prime v. 2020.1.2 [40] and aligning the forward and reverse sequences using the pairwise alignment function to create a consensus sequence.”

 

Reviewer: Line 170 – Which MUSCLE parameters were used?

Response: We used default parameters Gap opening penalty of -400, and no gap extension penalty which we have now included in text

 

Reviewer: Line 172 – settings/parameters used?

Response: We used default parameters Gap opening penalty of 1.5, and an offset value of 0.14 which we have now included in text

 

Reviewer: Line 173 – need to report the alignment algorithms used above.

Response: The alignment algorithms used in both MUSCLE and L-INS-I are iterative refinement algorithms. We have included this information in the text which now reads

“Each genetic marker was aligned separately using the iterative refinement algorithm of MUSCLE [41] implemented in MEGA X [42] with default settings (Gap opening penalty of -400, and no gap extension penalty). The intergenic spacer igr4 and COI + igr1 were also aligned using the L-INS-I iterative refinement alignment in MAFFT v. 7 [43] with default settings (Gap opening penalty of 1.5 and an offset value of 0.14) to account for the different indel placement by different alignment algorithms.”

 

Reviewer: Line 175 - 3390 bp alignment

Response: Thank you for spotting this error. We have corrected it.

 

Reviewer: Line 182 – [55], which uses

Response: Thanks. We have corrected it.

 

Reviewer: Line 195-196 – were there specific parameter values for the models that were used for reconstructions?

Response: We have included information about the parameters for the model. The text now reads

“This heuristic algorithm merges partitions until the model fit does not increase further. IQTree retained three partitions (Table S2) and applied the TVM+F+R2 substitution model (transversion model where the A-G substitution rate equals the C-T substitution rate, with unequal base frequencies equivalent to the empirical base frequencies, and a FreeRate model with two rate categories) to the first partition and HKY+F+I (unequal transition/transversion rates and unequal base frequencies equivalent to the empirical base frequencies and a proportion of invariant sites) to the other two.”

Reviewer: Line 197 – What settings were used for bootstrap, reconstruction, and TreeCollapse? You can include all of the settings (here and for those requested above and below) in Supplemental material or in the main text. From here one, please include details of settings selected for the reconstructions, delimitations, etc.

Response: The bootrap method was the Felenstein 1985 method. We have correctly cited this in text now. The only parameter required for TreeCollapse is the specified bootstrap threshold to collapse at. For his, we chose < 70. The relevant text reads

“Nodes with support values lower than 70 were collapsed using TreeCollapseCL 4 (available at http://hiv.bio.ed.ac.uk).”

 

Reviewer: Line 224 – Do you have a citation or other justification for this value of 0.9?

Response: This was an omission on our part.  We accept that it would be normal to use 0.95 (i.e., P (null hypothesis of conspecifity is rejected) <0.05, but using this threshold adds little to the analysis since it returns almost every terminus as a unique species.  One of the advantages of bGMYC is that it produces a heatmap of probabilities such that different scenarios can be evaluated.  We have amended the text to explain our reasoning, and have also included the heatmap in the Supplementary Information.  We further refer to the heatmap in the discussion.  The section in the methods now reads.

“We present the results for P (conspecificity among sequences) > 0.9 since P > 0.95 returns almost every terminus as a unique species, and we include results from different probability thresholds as a heatmap in supplementary information (Figure S50).”

 

Reviewer: Line 231 – What characters were examined or included as part of the gross colony morphology to recognize unique morphologies? Please provide additional detail here to orient the reader to your methods so that they can be reproduced.

Response: Thanks for this suggestion. We have provided extra information on what we termed gross morphology in the manuscript and the text now reads

“We examined the gross colony morphology from both the in-situ and ex-situ imagery (colony structure, branching frequency, polyp density, overall colony appearance) of every sample and assigned morphotype numbers to each unique morphology.”

 

 

Reviewer: Lines 236-242 – Which characters were analyzed and how so? You only discuss visualization here and not the analysis or identification of characters. Some detail could be added in Supplemental material, but you should at least briefly summarize the list of characters assessed and the most common states. How many polyps were analyzed per colony and how many colonies per haplotypes?  I think you have some of this requested info in the Intro, but it belongs here.

Response: I have changed the text to reflect the inclusion of this information. It now reads

“Due to the limited tissue available for morphological analysis, we used a single polyp for SEM imaging, and a single polyp for in-situ visualisation of sclerites. Characters such as polyp shape, position of the tentacles, and the composition, density, and orientation of sclerites on the polyp body were examined.”

 

 

Reviewer: Lines 252-254 – What were the coating settings (time or coat thickness, for example)? What were the SEM settings or setting ranges used (kV range, for example)?

Response: Thank you for spotting our omission. We have now modified the text to include the relevant details you asked about. The text now reads

“Sclerites were gold coated for 120 seconds using a Quorum Q150R ESplus at a sputter current of 25 mA resulting in a coat thickness of approximately 10 nm. If sclerites appeared charged during SEM imagery, an additional sputter coat using the same settings was applied and the sclerites reimaged. Sclerites were imaged at 15 kV using a Hitachi S-2600 at the Centre for Microscopy and Imaging, Anatomy, School of Medicine, National University of Ireland Galway.”

 

Reviewer: Lines 255-258 – Did you first dissect the polyps to examine sclerites from the different parts of the colonies or were they released together?  What were the characters and character states assessed? How were they assessed? Was that placement final or preliminary? If the final step to finalize assignment to lowest taxonomic unit was line 261-263, then move it to end of this section.

Response: We have amended the text to clarify our methods. This information can now be found in Sections 2.2 and 2.2.2. We have explicitly stated that we compared gross morphology and the SEM findings to the relevant taxonomic literature to arrive at the genus/species designations. Where we cannot assign genus or species names we leave the assignment to family. The text now reads

2.2. Morphological investigation

“We compared the findings of the gross morphology and SEM imaging were compared to the relevant taxonomic literature to diagnose individuals to genus or species rank [62–70].”

And

2.2.2. Scanning Electron Microscopy

“Coral polyps were dissected to examine the different sclerite composition among the different anatomical parts of the coral (keratoisidids: polyp body, coenenchyme, tentacles, and pharynx, chrysogorgiids: polyp body, tentacles, and coenenchyme, primnoids: not dissected). The tissue from each part of the polyp was submerged separately in household bleach (5% sodium hypochlorite) for up to 1 hr to release sclerites which were subsequently washed with distilled water (dH₂O) followed by 300 µl of 6% hydrogen peroxide. For the Primnoidae, the polyp was submerged whole in bleach under a microscope and sclerites were sorted (marginal, medial, basal, opercular, and tentacular sclerites). The reaction between sodium hypochlorite and hydrogen peroxide causes an effervescence that removes any remaining organic residues from the surface of the sclerites. A series of ten washes with dH₂O, three washes with 70% ethanol, and three washes in 100% ethanol ensured that sclerites were clean of organic debris and residual salts from the reaction between sodium hypochlorite and hydrogen peroxide. Using a 5x0 detail paintbrush/eyelash, sclerites were mounted onto a double-sided carbon adhesive fixed to a metal stub. Sclerites were gold coated for 120 seconds using a Quorum Q150R ESplus at a sputter current of 25 mA resulting in a coat thickness of approximately 10 nm. If sclerites appeared charged during SEM imagery, an additional sputter coat using the same settings was applied and the sclerites reimaged. Sclerites were imaged at 15 kV using a Hitachi S-2600 at the Centre for Microscopy and Imaging, Anatomy, School of Medicine, National University of Ireland Galway. Qualitative descriptions of the sclerite shape and texture were noted and named according to the nomenclature established by Bayer et al. [72].”

 

 

Reviewer: Lines 260-263 – What match threshold was used? Does the latter line refer only to morphology-based assignment?  Which characters were used?

Response: Thank you. We have modified the text to include this detail. The text now reads

“For Keratoisididae, we labelled our tree termini with the subclade nomenclature established by France [14] and expanded by Watling et al. [16]. We assigned the nomenclature based on mtMutS sequences generated in this study that were identical or extremely similar (> 99.5%) to keratoisidid mtMutS sequences used in Watling et al. [16].”

 

 

Reviewer: Lines 264-274 – so was just that boundary used or is this the same region used as described in Line 260?  Please clarify.

Response: We apologise to the reviewer for the confusion.  This paragraph was in the wrong order. We have now rewritten this to be more coherent, it now reads

“Specimens were initially identified as bamboo corals based on the occurrence of proteinaceous nodes in an otherwise calcareous skeleton, and their placement in the Keratoisididae was confirmed by amplifying across the CO3 – mtMutS gene boundary, an arrangement only found in Keratoisididae and some species of Anthoptilum sea pens [64]. For Keratoisididae, we labelled our tree termini with the subclade nomenclature established by France [14] and expanded by Watling et al. [16]. We assigned the nomenclature based on mtMutS sequences generated in this study that were identical or extremely similar (> 99.5%) to keratoisidid mtMutS sequences used in Watling et al. [16].”

 

Reviewer: Line 272 – “characters, by co-authour CBU to the…”

Response: Thank you for this correction. The text has been amended.

 

At the end of the methods, I am now curious about what the final methods are for species assignment.  It is not clear how the genetic and morphological data are being used together and whether comparisons of the methods or congruence of methods is being used.  For example, which method(s) for species delimitation based on genetic data are used.  What happens when the data are not in agreement? Please clarify

Response: We hope that much of this confusion has been resolved by previous comments. Our aim was to explore the observed diversity of calcaxonians in Irish waters using a combination of methods: barcoding, genetic delimitation, and morphology and use these as tools to help estimate the number of species present. We believe that without additional specimens and genome-wide date it is impossible to reach a definitive answer as the species boundaries within these groups are not well defined.

 

Results –

Reviewer: Line 279 – otherwise stated where?

Response: Good point. Upon reflection, we think that the sentence was not needed as every unique morphotype is given a number and so it is clear when morphotypes are shared across termini.

 

Reviewer: Line 282 – you can delete Family in all cases here and below.  The ending of the family name indicates they are family-level names.

Response: Thank you for your suggestion. We have changed the text throughout as per your feedback.  

 

 

Reviewer: Line 290 – How did that method compare to the others? In the Figure and text, I am confused about what the final species assignments are.  You use subclade/species IDs in S3 and concatenated haplotype #s and delimitation methods (morph and genetic), but I cannot tell which species are which and why they were assigned to those species.  For example, in Fig 3, is D2 one species or two or part of one species with D1, B1, and F1? That two are labelled Eknomisis sp. is confusing.  I would use the final species assignments as labels.

Response:  We are using different genetic methods to show the uncertainty in species assignments. As, evident from the tree in Fig 6, we have left most termini labelled as Keratoisididae sp. to reflect the uncertainty in species assignments. This is the final assignment.  The aim of this study was to report on the observed genetic and morphological diversity, which the different genetic delimitation methods help explain, and not to define species boundaries.

 

Reviewer: Table 2 – Some of this info is essential to interpret results.  Accession numbers could be in supplemental, but a table of final assignments would be good.

Response: As I hope we have previously clarified, the aim was to provide an upper and lower estimate of species numbers in our dataset, which we have done.

 

Reviewer: Fig. 6 – Orient reader to all acronyms and coding.  D1, D2, for example, are not defined.

Response: Thanks for pointing this out: we have reworded the figure legend in question. The text now reads

“Maximum likelihood tree of all recovered haplotypes (H) and corresponding morphotypes (M) from the 3390 base pair alignment and the corresponding species delimitation results from GMYC and mPTP methods (ST = single threshold and MT = multiple threshold). Each box within each column refers to a unique species recovered by that method. 1. Family Keratoisididae. A1, B1, C1 , D1, D2 , F1, I1, and J3 refer the keratoisidid subclades defined by Watling et al. [16], 2. Family Primnoidae, and 3. Family Chrysogorgiidae. * represent bootstrap values 90 – 99. ● represents bootstrap values between 71 – 89. Nodes with support lower than 70 have been collapsed and bootstrap values not displayed.”

 

Reviewer: Lines 291-312 and onward – Consider presenting this information in a table for easier comparison of morphological features. You presented in the Intro that branching pattern is labile with other characters considered diagnostic, so I would not focus on a character not indicative of species or genus. This section needs context for comparisons.  What is large, far apart, more planar, branches quickly, etc.  What are the differences between morphotypes for M2? I fear I could not come to the same conclusions in species assignment based on morphology because it is not clear here or above which characters were used.

Response: We thank the reviewer for this helpful suggestion. We have made a table of the important morphological characters used to distinguish morphotypes of Keratoisididae (Table 3). As all Chrysogorgiidae and Primnoidae haplotypes were assigned to accepted genera or species rank, we did not include them in the table. With this table, and the corresponding images in Fig 2 -5, we believe that the differences in morphotypes are much clearer than it was before.

 

Reviewer: Fig 2-4.  I would consider replacing these with character-state presentations with select representatives for main characters used to assign to morphotype.  For example, Fig. 3 g and h and Fig3 m-o appear to have similar morphologies, so provide one image with a description of the character state assigned. The reader can go to Supplemental to see the images for each specimen, but I think the reader needs a better understanding of the characters and states used to understand species assignments in context of the final assignments.

Response: We thank the reviewer for the suggestion. However, we believe that the images showing all the morphotypes within each haplotype integral to the overall paper so we have kept them.  

 

Reviewer: Lines 313-544 – See comments above to clarify presentation.  I skimmed through them because I had a difficult time following the presentation of the results and who was who and why they were assigned to a taxon.  Consider presenting as follows:

Gross morphology identified X morphotypes of Family 1.  These morphotypes are distinguishable by XYZ.  Concatenated genetic data did/did not support these assignments, with X method being more consistent with morphology. Caveats and justifications or criteria used for settling on final assignment.

 

Response: We thank the reviewer for this suggestion, we have incorporated this into the text on Keratoisididae where there is the largest number of morphotypes assigned to only Family rank. For Primnoidae and Chrysogorgiidae we believe that since all individuals were assigned to known genus or species rank, it would have been superfluous to include them in this table 

 

Discussion

Reviewer: I think a good portion of the material should be in results.  For example, the initial IDs and congruence of genetic and morphological data. Try to reduce to focusing on how the diversity and congruence of morphological data compare to the literature in this section. The paragraph starting with Line 642, for example, is mostly result that could be summarized in the discussion in a single sentence before comparing to the New England and Corner Rise study.

Response: We have moved the mentioned paragraph into the results. It can be found in section 3.5.

 

Reviewer: I would consider restructuring by finding.  For example, a section or paragraph on first reports, another or diversity comparisons, another on congruence of data and how that compares to other such studies, etc. (Not necessarily in that order). I am still unclear about the goal of the study and what you found in relation to that goal. That part of the discussion should be a major point of discussion. If it is biodiversity, how does your species richness compare to what is known from the region.  What advances did you make to understanding the biodiversity?  What is the broader impact?

Response: Thanks for this suggestion.  We embraced it and reordered the discussion as suggested, but we could not make it flow well and reverted to the original. When we took into account your other suggestions for the discussion, such as the above comment and the addition of sub headers, we found that the original structure flowed much better. Thank you.

Regarding comparing the biodiversity and species richness to other regions, this is quite difficult. First we did not aim to put an absolute count on the number of species we found, just an estimate. The species boundaries are poorly understood in this group making any estimate of species richness impossible. Furthermore, most studies that investigate biodiversity of richness, do not just focus on a suborder like we have. These make comparisons impossible. Instead, we have tried to qualitatively compare our study with keratoisidids found elsewhere.

 

Conclusion

Reviewer: Include an impact conclusion in this paragraph.

Response: We have rewritten the conclusion to highlight the impact of the research. Thank you.

Reviewer 3 Report

This manuscript is the first to explore the biodiversity of deep sea octocorals from the canyons surrounding the Irish continental shelf. Appropriately, an integrated approach to species delimitation is applied which combines a multilocus molecular barcode with observations of the morphological characters that are most often used to define species in these groups. This is a challenging group of organisms to work with because of the lack of variability in mitochondrial barcode markers and our still-evolving understanding of the species-level taxonomy of two of the major groups of calcaxonians, the family Keratoisididae and the genus Chrysogorgia.  Recent publications based on molecular phylogenetic and phylogenomic data have demonstrated that the traditional genus-level assignments within these groups need major revision, but formal changes have not yet been made. As a result, there are few reliable names available to put on species in these groups, and simply enumerating them using alphanumeric identifiers as has been done here is the only feasible approach. The results of such species delimitation are nonetheless important because they emphasize the probable high and unique biodiversity of this environment and pave the way for future taxonomic revisions. That said, I believe the results of this particular study could be presented in a way that would be more easily digestible and better highlight the most important, general conclusions.

First, let me emphasize that this study will be of no value to anyone in the future if the voucher material is not made available in a public, curated repository. Without access to the specimen vouchers it will never be possible either to replicate/verify the results of this study or to move beyond the alphanumeric species designations to actual taxonomic revisions. If the authors cannot provide information about the permanent whereabouts of the material they have collected, I cannot endorse publication of this work. Period. If they are able to provide that information, I suggest the following changes to the organization of the manuscript will greatly improve it.

The Results section is extremely dense and detail-oriented to the point of being very difficult to read. Much of the information (which M# belongs to which H#, which methods delimited which tips as different species) is already shown in Fig. 6 and is somewhat easier to digest in that graphical format. I suggest a Table might be a more effective way to summarize the morphological distinctions between morphotypes that had the same or similar haplotypes.  For each haplotype-morphotype combination (row in table) briefly describe the major morphological features.  Or, alternatively, include columns for major morphological characters and indicate in that way what branching pattern, polyp morphology, sclerite form, etc. defines each morphotype. To me, an organization such as that (almost like a key) would be easier to interpret, especially if in the future I was trying to match something I had found with one of your morphotypes.  If the morphological details are removed from the text of the results, the section can focus more effectively on the bigger picture results, i.e. how many different species were suggested by the analyses, and the overall diversity of the major clades.

The Discussion could likewise benefit from some major reorganization to better convey the most important points. Rather than starting with details about name assignment within the specific clades, I suggest you start with an overview of the diversity you found (material that currently starts around line 605) and reasons for the uncertainty in number of taxa. After that you can deal with the more specific problems of trying to assign binomials in the different groups; I’d suggest using section headers to separate the material.

The Introduction could also be more effectively organized. Currently it starts with general information about the habitat, introduces the specific groups of octocorals you worked with, switches back to more habitat information and finally introduces problems with barcoding in octocorals and the use of automated species delimitation programs. The specific aims of your study get lost in the mix.  I suggest reorganizing the material to progress from the most general to most specific, starting with the introduction to the habitat followed by the problems with barcoding, species delimitation approaches, and concluding with the introduction to the specific groups you studied.

In the Introduction you mention ABGD as one of the more widely used methods for species delimitation from barcoding data. This, or similar methods that apply a genetic distance threshold (either discovered “automatically” or defined a priori), have been used in the majority of species delimitation studies of octocorals.  I was therefore surprised that ABGD (or the newer version, ASAP) was not used in this study. I would have been very interested in the comparison between the tree-based methods used here and the more widely-used threshold approaches. At the very least, having introduced ABGD it would be appropriate to explain why it was not used and why the tree-based methods were chosen instead.

Specific suggestions/corrections?

62: This should be “hollow cross-chambered core”, not “hollow-cross chambered”

103: “A lack of taxonomic expertise is the main reason octocorals are rarely identified to species rank” is too simplistic a statement. Poor understanding of species boundaries, lack of knowledge of intraspecific variation and what morphological characters are taxonomically important, and deficiencies of the older literature are the root causes (exacerbated by the lack of taxonomic experts to help fix these problems).

Fig. 6 legend: Please indicate in the legend what the different analysis acronyms mean (i.e. what are ST- and MT- ?)

503: note typo, Carins should be Cairns

672: Purtales should be Pourtales?

 

 

 

Author Response

We would like to say thank you for your time reviewing our manuscript. Your thoughts and suggestions were both insightful and helpful and we hope that you find the revisions favourable and the overall manuscript improved

Overall: This manuscript is the first to explore the biodiversity of deep sea octocorals from the canyons surrounding the Irish continental shelf. Appropriately, an integrated approach to species delimitation is applied which combines a multilocus molecular barcode with observations of the morphological characters that are most often used to define species in these groups. This is a challenging group of organisms to work with because of the lack of variability in mitochondrial barcode markers and our still-evolving understanding of the species-level taxonomy of two of the major groups of calcaxonians, the family Keratoisididae and the genus Chrysogorgia.  Recent publications based on molecular phylogenetic and phylogenomic data have demonstrated that the traditional genus-level assignments within these groups need major revision, but formal changes have not yet been made. As a result, there are few reliable names available to put on species in these groups, and simply enumerating them using alphanumeric identifiers as has been done here is the only feasible approach. The results of such species delimitation are nonetheless important because they emphasize the probable high and unique biodiversity of this environment and pave the way for future taxonomic revisions. That said, I believe the results of this particular study could be presented in a way that would be more easily digestible and better highlight the most important, general conclusions.

Reviewer: First, let me emphasize that this study will be of no value to anyone in the future if the voucher material is not made available in a public, curated repository. Without access to the specimen vouchers it will never be possible either to replicate/verify the results of this study or to move beyond the alphanumeric species designations to actual taxonomic revisions. If the authors cannot provide information about the permanent whereabouts of the material they have collected, I cannot endorse publication of this work. Period. If they are able to provide that information, I suggest the following changes to the organization of the manuscript will greatly improve it.

Response: We thank the reviewer for their comment and we absolutely agree. We are now in position to provide the museum assession numbers for all 113 specimens used in the study. This is now provided in the supplementary material.

 

Reviewer: The Results section is extremely dense and detail-oriented to the point of being very difficult to read. Much of the information (which M# belongs to which H#, which methods delimited which tips as different species) is already shown in Fig. 6 and is somewhat easier to digest in that graphical format. I suggest a Table might be a more effective way to summarize the morphological distinctions between morphotypes that had the same or similar haplotypes.  For each haplotype-morphotype combination (row in table) briefly describe the major morphological features.  Or, alternatively, include columns for major morphological characters and indicate in that way what branching pattern, polyp morphology, sclerite form, etc. defines each morphotype. To me, an organization such as that (almost like a key) would be easier to interpret, especially if in the future I was trying to match something I had found with one of your morphotypes.  If the morphological details are removed from the text of the results, the section can focus more effectively on the bigger picture results, i.e. how many different species were suggested by the analyses, and the overall diversity of the major clades.

Response: Thanks for the suggestion of a table - that has really helped. We have made a table of the major characters used in the description of our keratoisidids and removed the description of keratoisidid morphology out of the results text.

 

Reviewer: The Discussion could likewise benefit from some major reorganization to better convey the most important points. Rather than starting with details about name assignment within the specific clades, I suggest you start with an overview of the diversity you found (material that currently starts around line 605) and reasons for the uncertainty in number of taxa. After that you can deal with the more specific problems of trying to assign binomials in the different groups; I’d suggest using section headers to separate the material.

Response: Thank you for this suggestion. We have restructured our discussion and taken your advice to include subheadings. The discussion structure is now

4.1 Morphological and genetic diversity of calcaxonians

4.2 The usefulness of genetic species delimitation in octocorals

4.3 Comparisons of keratoisidid diversity with other regions

4.4 Interesting new occurrence records

 

Reviewer: The Introduction could also be more effectively organized. Currently it starts with general information about the habitat, introduces the specific groups of octocorals you worked with, switches back to more habitat information and finally introduces problems with barcoding in octocorals and the use of automated species delimitation programs. The specific aims of your study get lost in the mix.  I suggest reorganizing the material to progress from the most general to most specific, starting with the introduction to the habitat followed by the problems with barcoding, species delimitation approaches, and concluding with the introduction to the specific groups you studied.

Response:  Another reviewer suggested a similar outline and so we have rewritten the introduction taking both suggestions into account. We now have an introduction to the habitat, then octocorals. Next is the specific group the manuscript focuses on and the taxonomic difficulties in those groups. The next section is about barcoding and the potential problems it has. Finally, it finishes with the aims.

 

Reviewer: In the Introduction you mention ABGD as one of the more widely used methods for species delimitation from barcoding data. This, or similar methods that apply a genetic distance threshold (either discovered “automatically” or defined a priori), have been used in the majority of species delimitation studies of octocorals.  I was therefore surprised that ABGD (or the newer version, ASAP) was not used in this study. I would have been very interested in the comparison between the tree-based methods used here and the more widely-used threshold approaches. At the very least, having introduced ABGD it would be appropriate to explain why it was not used and why the tree-based methods were chosen instead.

Response: Interesting commment.  We actually didn't use ABGD because of the slow evolution of the octocoral mitogenome and we've now explained this. Following the advice of another reviewer, we have moved all mention of delimitation methods to the methods section. We have modified the text and it reads as follows

“The widespread use of DNA barcoding across the tree of life has led to heuristic methods being developed to aid species delimitation. One of the first, and still one of the more popular methods is the Automatic Barcode Discovery Gap [55] which uses pairwise genetic distances to identify a “barcode gap” which distinguishes intraspecific variation from species divergences. However, due to the slow mutating mitogenome of octocorals, many species share the same sequence across the genetic markers we have used, e.g. [34,35], meaning that in many cases there is no gap to discover and so this analysis was inappropriate for our data and not conducted.”

 

Specific suggestions/corrections?

Reviewer: 62: This should be “hollow cross-chambered core”, not “hollow-cross chambered”

Response: Thank you for picking up on this. We have amended the text.

 

Reviewer: 103: “A lack of taxonomic expertise is the main reason octocorals are rarely identified to species rank” is too simplistic a statement. Poor understanding of species boundaries, lack of knowledge of intraspecific variation and what morphological characters are taxonomically important, and deficiencies of the older literature are the root causes (exacerbated by the lack of taxonomic experts to help fix these problems).

Response: Thank you for this suggestion. We have amended the sentence to include your comments. The text now reads

 “A lack of knowledge on definitive species boundaries, lack of information of intraspecific variation and what morphological characters are taxonomically important, and deficiencies in species descriptions in the older literature are the root causes of octocorals being rarely identified to species, which is further exacerbated by the lack of taxonomic expertise.”

 

Reviewer: Fig. 6 legend: Please indicate in the legend what the different analysis acronyms mean (i.e. what are ST- and MT- ?)

Response: Thanks for pointing this out. We have amended the figure legend and it know reads

“Maximum likelihood tree of all recovered haplotypes (H) and corresponding morphotypes (M) from the 3390 base pair alignment and the corresponding species delimitation results from GMYC and mPTP methods (ST = single threshold and MT = multiple threshold). Each box within each column refers to a unique species recovered by that method. 1. Family Keratoisididae. A1, B1, C1 , D1, D2 , F1, I1, and J3 refer to the keratoisidid subclades defined by Watling et al. [16], 2. Family Primnoidae, and 3. Family Chrysogorgiidae. * represent bootstrap values 90 – 99. ● represents bootstrap values between 71 – 89. Nodes with support lower than 70 have been collapsed and bootstrap values not displayed.”

 

Reviewer: 503: note typo, Carins should be Cairns

Response:  Thank you we have corrected this mistake.

 

Reviewer: 672: Purtales should be Pourtales?

Response: Thank you we have corrected this mistake.

Round 2

Reviewer 2 Report

Thank you for thoroughly addressing my comments and suggestions.  The manuscript is much improved in clarity, flow, and presentation.  The added discussion also places the study in broader context and better highlights the importance and limitations of the study.