Downloaded from http://rsos.royalsocietypublishing.org/ on June 18, 2017
DNA barcoding uncovers cryptic diversity in 50% of deepsea Antarctic polychaetes
Madeleine J. Brasier, Helena Wiklund, Lenka Neal, Rachel Jeffreys, Katrin Linse, Henry
Ruhl and Adrian G. Glover
Article citation details
R. Soc. open sci. 3: 160432.
http://dx.doi.org/10.1098/rsos.160432
Review timeline
Original submission:
Revised submission:
Final acceptance:
18 June 2016
30 September 2016
30 September 2016
Note: Reports are unedited and appear as
submitted by the referee. The review history
appears in chronological order.
Review History
RSOS-160432.R0 (Original submission)
Review form: Reviewer 1 (Brigitte Ebbe)
Is the manuscript scientifically sound in its present form?
Yes
Are the interpretations and conclusions justified by the results?
Yes
Is the language acceptable?
Yes
Is it clear how to access all supporting data?
I did not find any supplementary material.
Do you have any ethical concerns with this paper?
No
© 2016 The Authors. Published by the Royal Society under the terms of the Creative Commons
Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use,
provided the original author and source are credited
Downloaded from http://rsos.royalsocietypublishing.org/ on June 18, 2017
2
Have you any concerns about statistical analyses in this paper?
I do not feel qualified to assess the statistics
Recommendation?
Accept with minor revision (please list in comments)
Comments to the Author(s)
My only comment to this paper is that I do not see how detection of cryptic species does help
with environmental management, aside from the fact that one may consider diversity a value in
itself (which I personally do, but decision makers may not). From my own experience with
governmental agencies I tghink it may be very difficult to get across the value of species one can
not even tell apart by looking at them. From a different angle: does the presence of many cryptic
species indicate a higfher resilience of a community? I think you might want to get a little bit
deeper into this if you want to sell your methodology to environmental and other agencies.
Review form: Reviewer 2
Is the manuscript scientifically sound in its present form?
Yes
Are the interpretations and conclusions justified by the results?
Yes
Is the language acceptable?
Yes
Is it clear how to access all supporting data?
Supporting data access is clear
Do you have any ethical concerns with this paper?
No
Have you any concerns about statistical analyses in this paper?
No
Recommendation?
Accept with minor revision (please list in comments)
Comments to the Author(s)
DNA barcoding uncovers cryptic diversity in 50% of deep-sea Antarctic polychaetes
Reviewer’s comments
A/ General appraisal:
The manuscript contains valuable information on the discrepancy between morphological and
molecular species determination and related issues with diversity estimates and cryptic diversity.
As for several other benthic invertebrates, cryptic speciation in polychaetes seems to be a frequent
phenomenon and hampers our correct estimation of faunal distribution patterns. The manuscript
is well written, relevant and contains crucial information if we are to fully understand Antarctic
Downloaded from http://rsos.royalsocietypublishing.org/ on June 18, 2017
3
deep-sea diversity. I have only small comments on a few details, and therefore suggest minor
revision. Below I have listed my recommendations per section, page and line number. As a more
general comment, I was wondering why no other markers were used to confirm/refute the
results obtained here from mitochondrial DNA (e.g., nuclear 18S or ITS sequences). I am aware of
the fact that these are generally less variable (although ITS can be almost as variable as COI in
some benthic taxa), but they can provide additional clues to speciation patterns from unlinked
loci.
B/ Specific comments:
Abstract
P.2, line 28: “normalisation to on expected...” – delete one of the prepositions
P.2, line 31: “its role of in” – delete ‘of’
Introduction
P.2, line 40: “... if species are unable adapt to...” – add ‘to’ between unable and adapt
P.2, line 43: “... accurately document and assess our understanding of current species diversity...”
– I would delete ‘our understanding of’.
P.4, line 88: the reference (Thornhill et al., 2008) for nematodes is actually dealing with Nemertea,
not Nematoda. I suggest changing this. To my knowledge, there are no published records of
cryptic nematode species in Antarctic waters, although there are several reports from other
habitats (mainly shallow and intertidal – publications by e.g., Dr. Sofie Derycke).
Methods
P.5, line 129: “comprising of” – I always thought it is either ‘being comprised of’ or ‘comprising’
without the preposition, but maybe I am mistaken.
P.6, lines 147-149: I would split this sentence into two, because now it does not read very fluently.
P.6, line 150: I suggest a comma between ‘individuals’ and ‘target’ to improve the flow of the
sentence.
P.7, line 179: I don’t understand why Table 2 is mentioned in this sentence – delete.
P.7, line 201: misspelling of ‘nucleotide’
replace by its corresponding number.
P.8, line 213: cite Mesquite and add to references? “Maddison, W. P. and D.R. Maddison. 2016.
Mesquite: a modular system for evolutionary analysis. Version 3.10 http://mesquiteproject.org”
P.8, line 219: for consistency reasons (e.g., line 111 – re-evaluate) I would use a hyphen for
reexamined.
Results
P.9, lines 254-255: Why do you mention Aglaophamus trissophyllus and Hesionidae sp. A
already here? According to the previous paragraph, they belong to scenario 2 (evidence only in
COI phylogeny).
P.9, line 256 and 261: place Table and Figure references in brackets?
P.9, line 266: dot missing in species name E. cirratoformis.
P.10, line 277: table reference in brackets.
P.10, lines 277-281: it would be nice to have an overview of the K2P pairwise distances between
species, and between species clades (e.g., table in supplementary information?). Like this, it will
be possible for the reader to determine to what extent clades differ from one another. Now there
is only Table 2 which lists whether cryptic speciation is evident or not and how many sequences
were obtained per species/species clade.
P.10, line 280-281: “... ranged from 10-15%, these were considered...”. Something is missing here.
Either you start the sentence by saying “Because interspecific differences were 10-15%, these were
Downloaded from http://rsos.royalsocietypublishing.org/ on June 18, 2017
4
considered cryptic species.” Or you leave the first part untouched and then rephrase the second
part to “ranged from 10-15%, so these were considered...”.
P.10, lines 281-284: you never mention the third 16S clade (Aglaophamus sp. (MB4) in Table 2).
What about that one? Also, line 284, Figure 6 between brackets?
P.10, lines 289 and 292: in your figure legends you use “labelled” while here “labeled”. Which one
is it?
P.10, line 292: “... and they are labelled Hesionidae sp. (MB).”
P.10, line 299-300: see previous comments about table and figure references.
P.11, line 303-305: I would use ‘consider them as cryptic species’ since you’ve used ‘deem’ twice
in this sentence.
P.11, line 313: again, it would be nice to have an overview of these K2P distances.
P.11, lines 314-317: split this sentence, or change the punctuation marks
P.11, line 318rate of COI, it is not beyond possibility that if this gene was obtained, greater variation would
have been recorded.”
P.11, line 323: see previous comment on publically vs. publicly.
P.11, line 327: “It confirmed the presence of Harmothoe fuligineum and Aglaophamus
trissophyllus.” What do you mean exactly? Did the comparison with GenBank confirm this?
Presence where? In the Antarctic? Please clarify this sentence a bit.
P.12, lines 334-336: split into two sentences: “This questions the usefulness of its subspecies
status. If the subspecies are...distinct to and located... from its parent species, we query
whether...”
Discussion
P.13, line 368: “These results indicate/show...” rather than “highlight”.
P.13, line 373: “... f
– omit ‘enough’
P.13, line 377: “to conserve the Southern Ocean as a fragile marine environment.”
P.13, line 378: “growing number of genetic datasets”?
P.13, lines 379-381: “As previously stated, cryptic diversity has now been documented [...],
however [...] investigate a single genus or species.”
P.13, lines 381-384: split into two sentences (after holothurian morphospecies). Suggestion for
second sentence (to improve flow): “Within this study, an average of three divergent lineages was
uncovered in 17 of the 28 targeted morphospecies, thereby significantly increasing species
richness.”
P.13, line 387: remove “also”
P.13, line 397: “With the exception of a few chemosynthetic species...”.
P.14, line 400-401: place brackets around “e.g. whether species are brooders of spawners, if they
have larval stages and whether these are feeding or non-feeding”.
P.14, line 402: “studies based on shallow water species.”?
P.14, lines 416-418: split into two sentences; e.g. “Genetic evidence for cosmopolitan polychaete
species does exist. For example, the tube worm...”
P.14, line 424: add comma after ‘studies’ to improve sentence clarity
P.14, lines 426-441: I do not really see the link between the first sentence and the rest of this
paragraph. The transition between them is not very logical to me. Agreed, we should increase our
knowledge on why cryptic species are important and how they differ in ecological preference or
tolerance. But in what way do your results say something about this? The experimental examples
that you refer to are interesting, but can be shortened. Can you rephrase this paragraph to make it
a bit more to the point?
Downloaded from http://rsos.royalsocietypublishing.org/ on June 18, 2017
5
P.15, lines 448-453: split sentences and rephrase. E.g.: “For example, the soft bodies of polychaetes
can be easily damaged during sample processing. The resulting presence of incomplete
specimens, especially those that have lost critical identifying features, reduces the accuracy of
species identification [65]. An example of the latter in this study was found in Aricidea where the
median antenna present on Aricidea simplex (distinguishing it from A. belgicae) was detached in
some cases leading to false identification prior to DNA sequencing.”
P.15, line 458: “the EBS used to collect specimens targets smaller individuals”
P.15, line 459: misspelling of ‘morphospecies’
P.15, line 464: simplify sentence “DNA barcoding should be considered a complimentary method
of species identification”
P.15-16, lines 465-468: this sentence does not read fluently to me. Rephrase if possible.
P.16, line 470: ‘complimentary’ is not the right adjective here. I would use ‘complementary’ or
‘congruent’. Also, better to write “between the two mitochondrial genes” since no reference was
made in the previous paragraph to the genes used.
P.16, lines 475-476: rephrase “Furthermore, within family-level polychaete studies, the retrieval
of...”
P.16, lines 488P.16, line 493: move comma to after references
P.16, line 494: start new sentence at “However, other factors remain to be investigated, [...]”
P.16, line 498: I believe the correct word here is “utmost”
Table and Figure captions
P.26, line 756: “NB”? what does this mean?
P.26, line 767: dot missing in Macellicephaloides sp. B
P.26, line 771: dot missing in Scalibregma sp.
P.26, line 778: remove “are labelled” (redundant)
P.27, line 790: dot missing in Hesionidae sp. A
P.27, line 791: Figure 4 in brackets
Decision letter (RSOS-160432)
07-Sep-2016
Dear Miss Brasier
On behalf of the Editors, I am pleased to inform you that your Manuscript RSOS-160432 entitled
"DNA barcoding uncovers cryptic diversity in 50% of deep-sea Antarctic polychaetes" has been
accepted for publication in Royal Society Open Science subject to minor revision in accordance
with the referee suggestions. Please find the referees' comments at the end of this email.
The reviewers and handling editors have recommended publication, but also suggest some minor
revisions to your manuscript. Therefore, I invite you to respond to the comments and revise your
manuscript.
• Ethics statement
If your study uses humans or animals please include details of the ethical approval received,
including the name of the committee that granted approval. For human studies please also detail
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6
whether informed consent was obtained. For field studies on animals please include details of all
permissions, licences and/or approvals granted to carry out the fieldwork.
• Data accessibility
It is a condition of publication that all supporting data are made available either as
supplementary information or preferably in a suitable permanent repository. The data
accessibility section should state where the article's supporting data can be accessed. This section
should also include details, where possible of where to access other relevant research materials
such as statistical tools, protocols, software etc can be accessed. If the data has been deposited in
an external repository this section should list the database, accession number and link to the DOI
for all data from the article that has been made publicly available. Data sets that have been
deposited in an external repository and have a DOI should also be appropriately cited in the
manuscript and included in the reference list.
If you wish to submit your supporting data or code to Dryad (http://datadryad.org/), or modify
your current submission to dryad, please use the following link:
http://datadryad.org/submit?journalID=RSOS&manu=RSOS-160432
• Competing interests
Please declare any financial or non-financial competing interests, or state that you have no
competing interests.
• Authors’ contributions
All submissions, other than those with a single author, must include an Authors’ Contributions
section which individually lists the specific contribution of each author. The list of Authors
should meet all of the following criteria; 1) substantial contributions to conception and design, or
acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it
critically for important intellectual content; and 3) final approval of the version to be published.
All contributors who do not meet all of these criteria should be included in the
acknowledgements.
We suggest the following format:
AB carried out the molecular lab work, participated in data analysis, carried out sequence
alignments, participated in the design of the study and drafted the manuscript; CD carried out
the statistical analyses; EF collected field data; GH conceived of the study, designed the study,
coordinated the study and helped draft the manuscript. All authors gave final approval for
publication.
• Acknowledgements
Please acknowledge anyone who contributed to the study but did not meet the authorship
criteria.
• Funding statement
Please list the source of funding for each author.
Because the schedule for publication is very tight, it is a condition of publication that you submit
the revised version of your manuscript within 7 days (i.e. by the 16-Sep-2016). If you do not think
you will be able to meet this date please let me know immediately.
To revise your manuscript, log into https://mc.manuscriptcentral.com/rsos and enter your
Author Centre, where you will find your manuscript title listed under "Manuscripts with
Decisions". Under "Actions," click on "Create a Revision." You will be unable to make your
Downloaded from http://rsos.royalsocietypublishing.org/ on June 18, 2017
7
revisions on the originally submitted version of the manuscript. Instead, revise your manuscript
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When submitting your revised manuscript, you will be able to respond to the comments made by
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When uploading your revised files please make sure that you have:
1) A text file of the manuscript (tex, txt, rtf, docx or doc), references, tables (including captions)
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2) A separate electronic file of each figure (EPS or print-quality PDF preferred (either format
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[email protected]
on behalf of Kevin Padian
Subject Editor, Royal Society Open Science
[email protected]
Associate Editor Comments to Author:
Associate Editor: 1
Comments to the Author:
Your paper has been seen by two reviewers. They both think it merits publication, and I agree.
However there are some suggested changes in content and emphasis in places that I would like
you to consider. Hopefully your revision will not require re-review.
Reviewer comments to Author:
Reviewer: 1
Comments to the Author(s)
My only comment to this paper is that I do not see how detection of cryptic species does help
with environmental management, aside from the fact that one may consider diversity a value in
Downloaded from http://rsos.royalsocietypublishing.org/ on June 18, 2017
8
itself (which I personally do, but decision makers may not). From my own experience with
governmental agencies I tghink it may be very difficult to get across the value of species one can
not even tell apart by looking at them. From a different angle: does the presence of many cryptic
species indicate a higfher resilience of a community? I think you might want to get a little bit
deeper into this if you want to sell your methodology to environmental and other agencies.
Reviewer: 2
Comments to the Author(s)
DNA barcoding uncovers cryptic diversity in 50% of deep-sea Antarctic polychaetes
Reviewer’s comments
A/ General appraisal:
The manuscript contains valuable information on the discrepancy between morphological and
molecular species determination and related issues with diversity estimates and cryptic diversity.
As for several other benthic invertebrates, cryptic speciation in polychaetes seems to be a frequent
phenomenon and hampers our correct estimation of faunal distribution patterns. The manuscript
is well written, relevant and contains crucial information if we are to fully understand Antarctic
deep-sea diversity. I have only small comments on a few details, and therefore suggest minor
revision. Below I have listed my recommendations per section, page and line number. As a more
general comment, I was wondering why no other markers were used to confirm/refute the
results obtained here from mitochondrial DNA (e.g., nuclear 18S or ITS sequences). I am aware of
the fact that these are generally less variable (although ITS can be almost as variable as COI in
some benthic taxa), but they can provide additional clues to speciation patterns from unlinked
loci.
B/ Specific comments:
Abstract
P.2, line 28: “normalisation to on expected...” – delete one of the prepositions
P.2, line 31: “its role of in” – delete ‘of’
Introduction
P.2, line 40: “... if species are unable adapt to...” – add ‘to’ between unable and adapt
P.2, line 43: “... accurately document and assess our understanding of current species diversity...”
– I would delete ‘our understanding of’.
P.4, line 88: the reference (Thornhill et al., 2008) for nematodes is actually dealing with Nemertea,
not Nematoda. I suggest changing this. To my knowledge, there are no published records of
cryptic nematode species in Antarctic waters, although there are several reports from other
habitats (mainly shallow and intertidal – publications by e.g., Dr. Sofie Derycke).
Methods
P.5, line 129: “comprising of” – I always thought it is either ‘being comprised of’ or ‘comprising’
without the preposition, but maybe I am mistaken.
P.6, lines 147-149: I would split this sentence into two, because now it does not read very fluently.
P.6, line 150: I suggest a comma between ‘individuals’ and ‘target’ to improve the flow of the
sentence.
P.7, line 179: I don’t understand why Table 2 is mentioned in this sentence – delete.
P.7, line 201: misspelling of ‘nucleotide’
replace by its corresponding number.
Downloaded from http://rsos.royalsocietypublishing.org/ on June 18, 2017
9
P.8, line 213: cite Mesquite and add to references? “Maddison, W. P. and D.R. Maddison. 2016.
Mesquite: a modular system for evolutionary analysis. Version 3.10 http://mesquiteproject.org”
P.8, line 219: for consistency reasons (e.g., line 111 – re-evaluate) I would use a hyphen for
reexamined.
Results
P.9, lines 254-255: Why do you mention Aglaophamus trissophyllus and Hesionidae sp. A
already here? According to the previous paragraph, they belong to scenario 2 (evidence only in
COI phylogeny).
P.9, line 256 and 261: place Table and Figure references in brackets?
P.9, line 266: dot missing in species name E. cirratoformis.
P.10, line 277: table reference in brackets.
P.10, lines 277-281: it would be nice to have an overview of the K2P pairwise distances between
species, and between species clades (e.g., table in supplementary information?). Like this, it will
be possible for the reader to determine to what extent clades differ from one another. Now there
is only Table 2 which lists whether cryptic speciation is evident or not and how many sequences
were obtained per species/species clade.
P.10, line 280-281: “... ranged from 10-15%, these were considered...”. Something is missing here.
Either you start the sentence by saying “Because interspecific differences were 10-15%, these were
considered cryptic species.” Or you leave the first part untouched and then rephrase the second
part to “ranged from 10-15%, so these were considered...”.
P.10, lines 281-284: you never mention the third 16S clade (Aglaophamus sp. (MB4) in Table 2).
What about that one? Also, line 284, Figure 6 between brackets?
P.10, lines 289 and 292: in your figure legends you use “labelled” while here “labeled”. Which one
is it?
P.10, line 292: “... and they are labelled Hesionidae sp. (MB).”
P.10, line 299-300: see previous comments about table and figure references.
P.11, line 303-305: I would use ‘consider them as cryptic species’ since you’ve used ‘deem’ twice
in this sentence.
P.11, line 313: again, it would be nice to have an overview of these K2P distances.
P.11, lines 314-317: split this sentence, or change the punctuation marks
P.11, line 318rate of COI, it is not beyond possibility that if this gene was obtained, greater variation would
have been recorded.”
P.11, line 323: see previous comment on publically vs. publicly.
P.11, line 327: “It confirmed the presence of Harmothoe fuligineum and Aglaophamus
trissophyllus.” What do you mean exactly? Did the comparison with GenBank confirm this?
Presence where? In the Antarctic? Please clarify this sentence a bit.
P.12, lines 334-336: split into two sentences: “This questions the usefulness of its subspecies
status. If the subspecies are...distinct to and located... from its parent species, we query
whether...”
Discussion
P.13, line 368: “These results indicate/show...” rather than “highlight”.
P.13,
– omit ‘enough’
P.13, line 377: “to conserve the Southern Ocean as a fragile marine environment.”
P.13, line 378: “growing number of genetic datasets”?
P.13, lines 379-381: “As previously stated, cryptic diversity has now been documented [...],
however [...] investigate a single genus or species.”
Downloaded from http://rsos.royalsocietypublishing.org/ on June 18, 2017
10
P.13, lines 381-384: split into two sentences (after holothurian morphospecies). Suggestion for
second sentence (to improve flow): “Within this study, an average of three divergent lineages was
uncovered in 17 of the 28 targeted morphospecies, thereby significantly increasing species
richness.”
P.13, line 386: “The presence/occurrence of
P.13, line 387: remove “also”
P.13, line 397: “With the exception of a few chemosynthetic species...”.
P.14, line 400-401: place brackets around “e.g. whether species are brooders of spawners, if they
have larval stages and whether these are feeding or non-feeding”.
P.14, line 402: “studies based on shallow water species.”?
P.14, lines 416-418: split into two sentences; e.g. “Genetic evidence for cosmopolitan polychaete
species does exist. For example, the tube worm...”
P.14, line 424: add comma after ‘studies’ to improve sentence clarity
P.14, lines 426-441: I do not really see the link between the first sentence and the rest of this
paragraph. The transition between them is not very logical to me. Agreed, we should increase our
knowledge on why cryptic species are important and how they differ in ecological preference or
tolerance. But in what way do your results say something about this? The experimental examples
that you refer to are interesting, but can be shortened. Can you rephrase this paragraph to make it
a bit more to the point?
P.15, lines 448-453: split sentences and rephrase. E.g.: “For example, the soft bodies of polychaetes
can be easily damaged during sample processing. The resulting presence of incomplete
specimens, especially those that have lost critical identifying features, reduces the accuracy of
species identification [65]. An example of the latter in this study was found in Aricidea where the
median antenna present on Aricidea simplex (distinguishing it from A. belgicae) was detached in
some cases leading to false identification prior to DNA sequencing.”
P.15, line 458: “the EBS used to collect specimens targets smaller individuals”
P.15, line 459: misspelling of ‘morphospecies’
P.15, line 464: simplify sentence “DNA barcoding should be considered a complimentary method
of species identification”
P.15-16, lines 465-468: this sentence does not read fluently to me. Rephrase if possible.
P.16, line 470: ‘complimentary’ is not the right adjective here. I would use ‘complementary’ or
‘congruent’. Also, better to write “between the two mitochondrial genes” since no reference was
made in the previous paragraph to the genes used.
P.16, lines 475-476: rephrase “Furthermore, within family-level polychaete studies, the retrieval
of...”
P.16, lines 488P.16, line 493: move comma to after references
P.16, line 494: start new sentence at “However, other factors remain to be investigated, [...]”
P.16, line 498: I believe the correct word here is “utmost”
Table and Figure captions
P.26, line 756: “NB”? what does this mean?
P.26, line 767: dot missing in Macellicephaloides sp. B
P.26, line 771: dot missing in Scalibregma sp.
P.26, line 778: remove “are labelled” (redundant)
P.27, line 790: dot missing in Hesionidae sp. A
P.27, line 791: Figure 4 in brackets
Downloaded from http://rsos.royalsocietypublishing.org/ on June 18, 2017
11
Author's Response to Decision Letter for (RSOS-160432)
See Appendix A.
AppendixDownloaded
A
from http://rsos.royalsocietypublishing.org/ on June 18, 2017
University of Liverpool
Nicholson Building
4 Brownlow Street
Liverpool
L69 3GP
29th September 2016
Editor, Royal Society Open Sciences
Dear Editor
On behalf of all the authors of ‘DNA barcoding uncovers cryptic diversity in 50% of deep-sea
Antarctic polychaetes’ I would like to thank you for accepting our manuscript for publication with
minor corrections.
All grammatical and structural corrections suggested by each reviewer have been edited accordingly.
Additional references have also be cited where appropriate. Specific responses to reviewers are
included below. The more major corrections and comments that have been altered include:
•
•
A table in the supplementary information of the K2P distances between morphospecies,
cryptic species and clades. This will allow a reader to determine to what extent clades differ
from one another in more detail than the current figures allow.
We have re-emphasised the link between the importance of our results and future
investigations into the functionality of cryptic species as well as the importance of accurate
biodiversity estimates for marine management.
Reponses to each of the reviewer’s comments.
Reviewer 1
“My only comment to this paper is that I do not see how detection of cryptic species does help with
environmental management, aside from the fact that one may consider diversity a value in itself
(which I personally do, but decision makers may not).”
Sentences regarding the need for accurate biodiversity estimates for effective marine management
have been retained. We regard these data to be fundamental to understanding marine ecosystems and
should be baseline data for management practices. Their question regarding whether the presence of
cryptic species indicates higher resilience of a community or not is something we will be addressing
in our future work investigating the functional traits of cryptic species. This is why we introduce
functional studies and potential impacts on ecosystem function within our discussion.
Most of the minor grammatical comments were consistent with those of reviewer 2 and have been
edited.
Reviewer 2
“I was wondering why no other markers were used to confirm/refute the results obtained here from
mitochondrial DNA (e.g., nuclear 18S or ITS sequences). I am aware of the fact that these are
Downloaded from http://rsos.royalsocietypublishing.org/ on June 18, 2017
generally less variable (although ITS can be almost as variable as COI in some benthic taxa), but
they can provide additional clues to speciation patterns from unlinked loci.”
In response to why we did not use other genetic markers, we did sequence nuclear markers, including
18S and 28S of some individuals; however these did not assist in identifying the presence of cryptic
species. ITS was not used as a genetic marker because, although we agree it could have assisted in
the identification of some cryptic and non-cryptic species, for the majority of families and genera
investigated publically available sequences for comparison were more limited and our identification
was based on between species comparisons. Furthermore the funding did not all for additional PCR
or sequencing costs.
“It would be nice to have an overview of the K2P pairwise distances between species, and between
species clades (e.g., table in supplementary information?)”
A table of all K2P distances between morphospecies, cryptic species and clades within cryptic
species was added to the supplementary information. This will allow the reader to determine to, what
extent clades differ from one another in more detail than the current figures allow.
We thank the reviewer for their extensive help with our manuscript. All the specific grammar and
editorial comments have been fixed. See below:
Abstract
P.2, line 28: “normalisation to on expected...” – delete one of the prepositions
Fixed
P.2, line 31: “its role of in” – delete ‘of’
Fixed
Introduction
P.2, line 40: “... if species are unable adapt to...” – add ‘to’ between unable and adapt
Fixed
P.2, line 43: “... accurately document and assess our understanding of current species diversity...” – I
would delete ‘our understanding of’.
Fixed
P.4, line 88: the reference (Thornhill et al., 2008) for nematodes is actually dealing with Nemertea,
not Nematoda. I suggest changing this. To my knowledge, there are no published records of cryptic
nematode species in Antarctic waters, although there are several reports from other habitats (mainly
shallow and intertidal – publications by e.g., Dr. Sofie Derycke). Fixed
Methods
P.5, line 129: “comprising of” – I always thought it is either ‘being comprised of’ or ‘comprising’
without the preposition, but maybe I am mistaken. Fixed
P.6, lines 147-149: I would split this sentence into two, because now it does not read very fluently
Fixed
P.6, line 150: I suggest a comma between ‘individuals’ and ‘target’ to improve the flow of the
sentence.
Fixed
P.7, line 179: I don’t understand why Table 2 is mentioned in this sentence – delete.
Fixed
P.7, line 201: misspelling of ‘nucleotide’
Fixed
P.7, line 205: why is the reference here written in full? _ add reference to reference list and replace
by its corresponding number.
Fixed
P.7, line 208: ‘publically’ _ ‘publicly’?
Fixed
P.8, line 213: cite Mesquite and add to references? “Maddison, W. P. and D.R. Maddison. 2016.
Mesquite: a modular system for evolutionary analysis. Version 3.10 http://mesquiteproject.org”
Fixed
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P.8, line 219: for consistency reasons (e.g., line 111 – re-evaluate) I would use a hyphen for
reexamined. Fixed
Results
P.9, lines 254-255: Why do you mention Aglaophamus trissophyllus and Hesionidae sp. A already
here? According to the previous paragraph, they belong to scenario 2 (evidence only in COI
phylogeny). Fixed
P.9, line 256 and 261: place Table and Figure references in brackets?
Fixed
P.9, line 266: dot missing in species name E. cirratoformis.
Fixed
P.10, line 277: table reference in brackets. Fixed
P.10, lines 277-281: it would be nice to have an overview of the K2P pairwise distances between
species, and between species clades (e.g., table in supplementary information?). Like this, it will be
possible for the reader to determine to what extent clades differ from one another. Now there is
only Table 2 which lists whether cryptic speciation is evident or not and how many sequences were
obtained per species/species clade. Fixed
P.10, line 280-281: “... ranged from 10-15%, these were considered...”. Something is missing here.
Either you start the sentence by saying “Because interspecific differences were 10-15%, these were
considered cryptic species.” Or you leave the first part untouched and then rephrase the second part
to “ranged from 10-15%, so these were considered...”.
Fixed
P.10, lines 281-284: you never mention the third 16S clade (Aglaophamus sp. (MB4) in Table 2).
What about that one? Mentioned in latter section. Also, line 284, Figure 6 between brackets? Fixed
P.10, lines 289 and 292: in your figure legends you use “labelled” while here “labeled”. Which one is
it?
Fixed
P.10, line 292: “... and they are labelled Hesionidae sp. (MB).”
Fixed
P.10, line 299-300: see previous comments about table and figure references.
Fixed
P.11, line 303-305: I would use ‘consider them as cryptic species’ since you’ve used ‘deem’ twice in
this sentence. Fixed
P.11, line 313: again, it would be nice to have an overview of these K2P distances.
Fixed
P.11, lines 314-317: split this sentence, or change the punctuation marks Fixed
P.11, line 318: polynoidae _ Polynoidae
Fixed
P.11, line 318-320: a few changes in this sentence to improve flow _ “Given the faster mutation
rate of COI, it is not beyond possibility that if this gene was obtained, greater variation would have
been recorded.”
Fixed
P.11, line 323: see previous comment on publically vs. publicly. Fixed
P.11, line 327: “It confirmed the presence of Harmothoe fuligineum and Aglaophamus
trissophyllus.”
What do you mean exactly? Did the comparison with GenBank confirm this? Presence where? In the
Antarctic? Please clarify this sentence a bit. Fixed
P.12, lines 334-336: split into two sentences: “This questions the usefulness of its subspecies status.
If the subspecies are...distinct to and located... from its parent species, we query whether...” Fixed
Discussion
P.12, line 351: add comma _ “Using DNA barcoding, the number of species...” Fixed
P.13, line 368: “These results indicate/show...” rather than “highlight”.
Fixed
P.13, line 369: split sentence _ “Where possible, a number of genes and ...”
Fixed
P.13, line 373: “... far more species rich that previously thought.” _ ‘than’ Fixed
P.13, line 376: “sufficient enough” _ redundancy in words – omit ‘enough’
Fixed
P.13, line 377: “to conserve the Southern Ocean as a fragile marine environment.”
Fixed
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P.13, line 378: “growing number of genetic datasets”?
Fixed
P.13, lines 379-381: “As previously stated, cryptic diversity has now been documented [...], however
[...] investigate a single genus or species.” Fixed
P.13, lines 381-384: split into two sentences (after holothurian morphospecies). Suggestion for
second sentence (to improve flow): “Within this study, an average of three divergent lineages was
uncovered in 17 of the 28 targeted morphospecies, thereby significantly increasing species richness.”
Fixed
P.13, line 386: “The presence/occurrence of...”? _ verb in third person (“suggests”)
Fixed
P.13, line 387: remove “also” Fixed
P.13, line 397: “With the exception of a few chemosynthetic species...”. Fixed
P.14, line 400-401: place brackets around “e.g. whether species are brooders of spawners, if they
have larval stages and whether these are feeding or non-feeding”. Fixed
P.14, line 402: “studies based on shallow water species.”? Fixed
P.14, lines 416-418: split into two sentences; e.g. “Genetic evidence for cosmopolitan polychaete
species does exist. For example, the tube worm...” Fixed
P.14, line 424: add comma after ‘studies’ to improve sentence clarity
Fixed
P.14, lines 426-441: I do not really see the link between the first sentence and the rest of this
paragraph. The transition between them is not very logical to me. Agreed, we should increase our
knowledge on why cryptic species are important and how they differ in ecological preference or
tolerance. But in what way do your results say something about this? The experimental examples
that you refer to are interesting, but can be shortened. Can you rephrase this paragraph to make it a
bit more to the point?
Fixed
P.15, lines 448-453: split sentences and rephrase. E.g.: “For example, the soft bodies of polychaetes
can be easily damaged during sample processing. The resulting presence of incomplete specimens,
especially those that have lost critical identifying features, reduces the accuracy of species
identification [65]. An example of the latter in this study was found in Aricidea where the median
antenna present on Aricidea simplex (distinguishing it from A. belgicae) was detached in some cases
leading to false identification prior to DNA sequencing.” Fixed
P.15, line 458: “the EBS used to collect specimens targets smaller individuals” Fixed
P.15, line 459: misspelling of ‘morphospecies’
Fixed
P.15, line 464: simplify sentence “DNA barcoding should be considered a complimentary method of
species identification”
Fixed
P.15-16, lines 465-468: this sentence does not read fluently to me. Rephrase if possible. Fixed
P.16, line 470: ‘complimentary’ is not the right adjective here. I would use ‘complementary’ or
‘congruent’. Also, better to write “between the two mitochondrial genes” since no reference was
made in the previous paragraph to the genes used. Fixed
P.16, lines 475-476: rephrase “Furthermore, within family-level polychaete studies, the retrieval
of...” Fixed
P.16, lines 488-492: very long sentence _ rephrase or shorten!
Fixed
P.16, line 493: move comma to after references
Fixed
P.16, line 494: start new sentence at “However, other factors remain to be investigated, [...]” Fixed
P.16, line 498: I believe the correct word here is “utmost” Fixed
Table and Figure captions
P.26, line 756: “NB”? what does this mean? Fixed
P.26, line 758: “epibenthic sled” _ sledge? Fixed
P.26, line 767: dot missing in Macellicephaloides sp. B
Fixed
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P.26, line 771: dot missing in Scalibregma sp.
Fixed
P.26, line 778: remove “are labelled” (redundant) Fixed
P.26, line 779: “labeled” _ labelled Fixed
P.27, line 783: “... results ‘scenario 3, ...” _ ‘scenario 3’
Fixed
P.27, line 790: dot missing in Hesionidae sp. A
Fixed
P.27, line 791: Figure 4 in brackets Fixed
Reviewer 3
We thank the reviewer for the additional comments received after manuscript acceptance. Most of
the grammatical changes and structural changes were amended in the above corrections suggested by
reviewer 2. Additional citations were added where suggested, including Kvist (2014), Puillandre et
al. (2012) and Riesgo et al. (2015) and Siciński (2011). Further comments are discussed below.
Reviewer suggested that Figure 2 should be moved to the supplementary material.
Figure 2 has not been moved to the supplementary material as the morphological characters shown in
the specimen photos are of relevance when discussing species identification. To avoid confusion we
have not altered the figure legend, at the figures point of reference in the manuscript the cryptic
species are yet to be identified and the purpose of the figure is to present the targeted morphospecies
selected.
Reviewer questions why we did not use the Automatic Barcode Gap Discovery (ABGD) to identify
cryptic species.
We did not use the Automatic Barcode Gap Discovery (ABGD) as we think that analysing both the
K2P comparisons and phylogenetic trees of our sequenced specimens and others within the same
family provides equal if not more information for species discrimination. If we had used the ABGD
model we would have still have had to make the same decisions as to what level of intraspecific and
interspecific variation is used to determine species for which, as documented, there is no universal
cut-off.
Reviewer asks why the biogeography of each cryptic species is not discussed.
The biogeography of the cryptic species was not included in this manuscript as it is not within the
scope of our current study. The primary aim was to investigate the level of cryptic diversity in
Antarctic polychaetes across multiple families, which is yet to be documented within the literature.
We feel that the increase in biodiversity by 233% resulting from DNA barcoding uncovering cryptic
species and additional morphospecies is significant enough to publish prior to our future work. These
projects will use our DNA data as a foundation to investigate the biogeographic patterns of cryptic
species combining genetic and model data as well as functional analysis.
Reviewer requested that all phylogenetic trees to be included in the supplementary material and that
more detailed phylogenetic descriptions were provided for each species within the results section.
Not all phylogenetic trees were submitted as this would have amounted to an additional 16 figures
and each would have followed one of the three scenarios described. We also hope that the K2P
values displayed in Figure and Tables 2 and S1 would emphasize this.
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Within our results section we did not provide as detailed phylogenetic descriptions as requested, as to
do this for each family would greatly change the length and focus of the manuscript. Although we
use phylogenies to determine species numbers, we want to maintain focus on the overall outcome
rather than family specific phylogenetic patterns. We have also not discussed the presence of cryptic
species within GenBank sequences e.g. Scalibregma inflatum as we do not have access to these
specimens and thus cannot re-examine the morphology nor is it within the scope of the study.
Reviewer asked for GenBank comparisons to be discussed as each species is discussed within its
given scenario.
We have kept the results sectioning the same, we did not disperse the GenBank comparisons by
species as we feel this disrupts the flow of these results and removes focus from explaining the three
scenarios.
Reviewer asks whether any morphological differences between cryptic species were found.
We did not find any morphological differences between the specimens defined as cryptic species
using our methods, however there may be differences in internal characteristics, which were not
analysed. Sentences regarding morphological taxonomy, including re-examination and the inherent
problems of identifying large numbers of specimens such as this, have not been removed. Traditional
taxonomic methods are becoming increasingly overlooked with increased DNA taxonomy and lack
of expertise and we wish to express that this is still an important part of biodiversity work. Finally,
we wish to re-emphasize that the increase in species number by 233% was a result of both cryptic
species and additional morphological species, thus the total increase is not solely a result of cryptic
diversity. We have not estimated total species diversity within the original dataset containing 20,000
specimens, as the presence of cryptic species was not consistent across families.
Yours sincerely,
Madeleine Brasier
rsos.royalsocietypublishing.org
Correction
Cite this article: Madeleine J. Brasier et al.
2017 Correction to ‘DNA barcoding uncovers
cryptic diversity in 50% of deep-sea Antarctic
polychaetes’. R. Soc. open sci. 4: 160937.
http://dx.doi.org/10.1098/rsos.160937
Correction to ‘DNA
barcoding uncovers cryptic
diversity in 50% of deep-sea
Antarctic polychaetes’
Madeleine J. Brasier, Helena Wiklund, Lenka Neal,
Rachel Jeffreys, Katrin Linse, Henry Ruhl and
Adrian G. Glover
R. Soc. Open Sci. 3, 160432. (Published online 2 November 2016)
(doi:10.1098/rsos.160432)
MJB, 0000-0003-2844-655X
The submitted correction is an updated version of figure 1. In the
correct figure, shown below, the following has been changed from
the figure in the publication:
In the COI tree (figure 1a), the sequence in purple has been
renamed Scalibregma sp. (MB2) (from (MB1) in blue).
In the 16S tree (figure 1b), the sequences labelled Scalibregma sp.
(MB3) have been recoloured to orange (from black).
2017 The Authors. Published by the Royal Society under the terms of the Creative Commons
Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted
use, provided the original author and source are credited.
0.2 substitutions per site
*
*
0.2 substitutions per site
rsos.royalsocietypublishing.org R. Soc. open sci. 4: 160937
Figure 1. Phylogenetic tree of Scalibregmatidae from Bayesian analysis using COI (a) and 16S (b). An example of results ‘scenario 1’, evidence for cryptic diversity in COI and 16S genes, cryptic species Scalibregma sp. (MB1), (MB2)
and (MB3). Outgroup: Leitoscoloplus fragilis and L. robustus (Orbiniidae), asterisk indicates significant node values (more than 95%) for Bayesian posterior probabilities. BOLD* indicates sequences obtained from a private BOLD
database.
*
(b)
Leitoscoloplos fragilis AY532341
Leitoscoloplos robustus FJ612455
Armandia intermedia KP096410
Arenicola marina AY340446
*
Ophelia limacina CMC02 HQ938304
Clymenura clypeata AY340449
Ophelia sp. CMC01 HM375493
Scalibregma sp. (MB3)
*
Ophelia limacina CMC01 HQ023646
Scalibregma sp. (MB3)
Thoracophelia mucronata KC164688
Scalibregma sp. (MB3)
*
Thoracophelia dillonensis KC164882
Scalibregma sp. (MB3)
*
Thoracophelia williamsi KC164691
Scalibregma sp. (MB2)
*
Arenicola marina HQ691225
Scalibregma sp. (MB1)
*
Clymenura clypeata KJ183005
Scalibregma sp. (MB1)
Ophelina sp. 1 MN 2008 EU752494
Scalibregma sp. (MB1)
Ophelina acuminata CMC01 HQ023898
Scalibregma sp. (MB1)
* Ophelina acuminata CMC02 HQ024166
Scalibregma sp. (MB1)
Ophelina acuminata KC164693
Scalibregma sp. (MB1)
Speleobregma lanzaroteum KC748528
Scalibregma sp. (MB1)
*
Axiokebuita millsi JN256052
*
*
Scalibregma sp. (MB1)
*
Axiokebuita sp. 1 KC748527
Scalibregma sp. (MB1)
*
Scalibregma inflatum Tsastk13 BOLD*
Scalibregma sp. (MB1)
Scalibregma sp. (MB1)
Scalibregma sp. (MB1)
Scalibregma sp. (MB1)
*
Scalibregma sp. (MB1)
Scalibregma sp. (MB1)
*
Scalibregma sp. (MB1)
Scalibregma sp. (MB1)
*
Scalibregma sp. (MB1)
Scalibregma
sp.
(MB1)
*
Scalibregma inflatum AY532331
Scalibregma sp. (MB1)
* Scalibregma inflatum SIO BIC KF511816
Scalibregma sp. (MB2)
*
Scalibregma sp. (MB3)
* Polyphysia crassa AY340471
Polyphysia crassa HM746719
Scalibregma sp. (MB3)
*
* Scalibregma sp. (MB3)
* Ophelina acuminata AY340471
Ophelina acuminata SIO BIC KF511812
* Pseudoscalibregma parvum HQ023780
Ophelina sp. B2SC00P1 KF511807
Pseudoscalibregma parvum HQ023906
Ophelina cylindricaudata HM746717
Scalibregma
inflatum
CMC01
GU672395
*
*
*
Ophelina sp. SIO BIC KF511814
Scalibregma inflatum CMC01 HQ023909
Hyboscolex pacificus HM746712
Scalibregma inflatum Ann150 BOLD*
*
*
Axiokebuita sp. KC74523
* Scalibregma inflatum TB530 BOLD*
Ophelia neglecta HM746718
Scalibregma inflatum TB549 BOLD*
Armandia bilobata DQ779604
Scalibregma inflatum CMCO2 HM473801
*
Armandia brevis KF511804
Scalibregma inflatum CMCO3 GU672496
*
*Scalibregma inflatum CMCO3 GU672569
*
Polyophthalmus sp. SIO BIC KF511805
Scalibregma inflatum CMCO3 GU672593
Armandia sp. SIO BIC KF511806
Leitoscoloplos fragilis FJ612498
Leitoscoloplos robustus FJ612499
................................................
(a)
2