Adaptive phenotypic response to climate enabled by ...rsos.royalsocietypublishing.org/content/royopensci/3/10/160299...epigenetics in a K-strategy species, the fish Leucoraja ocellata

© 2016 The Authors. Published by the Royal Society under the terms of the Creative Commons

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Review History

RSOS-160299.R0 (Original submission) Review form: Reviewer 1 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? Yes. Do you have any ethical concerns with this paper? No

Adaptive phenotypic response to climate enabled by

epigenetics in a K-strategy species, the fish Leucoraja

ocellata (Rajidae)

Jackie Lighten, Danny Incarnato, Ben J. Ward, Cock van Oosterhout, Ian Bradbury, Mark

Hanson and Paul Bentzen

Article citation details R. Soc. open sci. 3: 160299. http://dx.doi.org/10.1098/rsos.160299

Review timeline

Original submission: 2 May 2016 1st revised submission: 8 September 2016 2nd revised submission: 26 September 2016 Final acceptance: 26 September 2016

Note: Reports are unedited and appear as submitted by the referee. The review history appears in chronological order.

on June 29, 2018http://rsos.royalsocietypublishing.org/Downloaded from

http://rsos.royalsocietypublishing.org/

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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) Review of: “Adaptive phenotypic reponse to climate change enabled by epigenetics in a K-strategy species, the fish Leucoraja ocellata (Rajidae).” – Lighten et al., 2016 The authors compose a cohesive and well-constructed story with their findings. They clearly show the morphological differences between two distinct populations of skate and identify epigenetic changes associated with these morphological traits, providing strong support for their hypothesis; Leucoraja ocellata have the potential to adapt to rapidly changing environments through epigenetic mechanisms. They identify many up- and down-regulated genes that directly and indirectly impact temperature stress response, morphology, and life history traits. They highlight the clear differences in the two populations: maturation time, egg case size, lifespan, body size, among other morphological characteristics. While the article has technical components in terms of the genetic tools and techniques used, the authors were able to communicate their results and the significance of their findings without difficulty. Valuable connections are drawn to link the genetic components to physiological, ecological, and behavioural mechanisms that strengthen their findings, drawing from fundamental literature to support their findings and linking it to recent literature for relevance in today’s changing climate. They provide a comprehensive list of explanations for changes in gene expression to support their findings. The authors also draw connections to social components, with the species vulnerable to fishing activities as by-catch. This article will be a novel and valuable addition to the current scientific literature. The following comments should be addressed: 1. Page 4, second paragraph. You indicate that natural epigenetic changes to climate change have not been identified in fish populations. Is there other literature on land and non-fish species that show these epigenetic changes? 2. Page 6, second paragraph. It appears that the sample size was 2. While this may be appropriate for this type of genetic study, you should address the variance and uncertainty in the results with this study design. What are the implications of pseudoreplication when sampling from the same individual? 3. Page 12-13, last paragraph. The authors indicate changes in growth between populations. Is it growth (rate) that is different between the two environments, or max size? Table 1 does not summarize difference in growth rate. 4. Page 13, second paragraph, last sentence. The authors can specify how oxidative stress affects organismal aging (increase aging). 5. Page 14, first paragraph, last sentence. The authors should provide a citation for this statement. 6. This study identifies the adaptive changes in gene expression to different climates. However, in the title and throughout the article, climate change is introduced as a component leading to epigenetic differences. Climate change is different than the environmental differences between these two populations. Instead, the authors should clearly state the implications of your



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study to climate change. The title is also misleading as it suggests that these species have already shown epigenetic responses to climate change. 7. The study supports epigenetic adaptation across relatively short time scales. The authors may want to consider the implications of their study and the fact that chondrichthyes, in general, having persisted relatively evolutionary unchanged through many eras and through vast changes in climate (e.g., ice ages). 8. It would be useful to include a table to explain the linkages of the specific epigenetic responses (up/down regulation of a certain process) mean to the adaptation to environmental changes. It is not apparent in the manuscript. As the manuscript aims to build such linkages, it would be helpful for readers if the author could include such a table e.g., the first column is the process, second column as up/down regulation or no change, and then final column as inferred links to adaptation to climate. The following minor changes to be addressed: 9. Page 5, line 121. Consider changing “K-strategy” to “K-strategist”. 10. Page 6, line 173. Grammar: “manufactures” to “manufacturer’s”. 11. Page 9, line 262. Grammar: “an 95%” to “a 95%”. 12. Page 11, line 311. Spelling: “exposer” to “exposure”. 13. Page 21, figure 1. Identify study sites on map. This is an integral component to your study, showing the geographical and environmental differences between the populations. 14. Page 22-23, figure 2. This figure is not properly labelled and referenced in text. 15. Page 24-26, figure 3-5. These figures were not labelled properly and did not correspond to labels in text (e.g., ‘a’, ‘b’).

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? Yes Do you have any ethical concerns with this paper? No Have you any concerns about statistical analyses in this paper? No



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Recommendation? Accept with minor revision (please list in comments) Comments to the Author(s) The manuscript reports results from a study comparing transcriptomes of two populations of the winter skates. The topic is timely and the protocols appear to be executed appropriately. The scope of the study is somewhat limited to two populations, and I do not quite understand why the study was not extended to investigate also other, more southern, populations. Most importantly, the authors need to clarify the collection methods, and they need to include a broader reference to the literature on the effect of temperature on skates in light of climate change beyond the fisheries studies. I recommend that these issues are addressed in a revision. Minor comments and suggestions: L.52: “factor” instead of condition L.57: some species shrunk their range, i.e. didn’t shift towards higher latitudes, but they rather lost the southern population L.60-61: I’m confused. If you’re talking about plastic physiological responses it is not “adaptation” but “acclimation”, please clarify. L.63-65: rephrase this whole sentence, it’s awkward. Line 74 and on.. : if you are talking about phenotypic plasticity, then I think you need to report also physiological studies (see general comments). L.121,280,291: “elasmobranchs” misspelled L.123 and on: perhaps because many species of skates don’t seem to travel very far? L.150: how did you collect? Could the collection methods have affected gene expression? How many skates? And at which temperature? L.287-289: rephrase, some of the wording -I think- is not so appropriate for a paper. L.302: need to explain these effects – expand this paragraph L.375 “affect” Fig.1 It would be useful to see the 2 sampling spots on the map

Review form: Reviewer 3 Is the manuscript scientifically sound in its present form? No Are the interpretations and conclusions justified by the results? Yes Is the language acceptable? No Is it clear how to access all supporting data? Data were included, but were lacking clear explanation. Do you have any ethical concerns with this paper? No Have you any concerns about statistical analyses in this paper? No



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Recommendation? Reject Comments to the Author(s) Dear authors, Although the manuscript could have potential, in its current form it is incomprehensible to the wider audience. Although it is possible that it can be understood by scientists who already have a fairly extensive knowledge on (epi)genetics, this would not be the case for a majority of readers. The Introduction and Discussion sections can be fairly well understood, but there is no good connect with the (highly technical) Methods and Results sections. The key findings cannot be grasped. The key findings seem to be mentioned in Discussion, lines 289-293 ("Our study suggests that a phenotypically plastic response through epigenetic regulation ... take place over time." However, it remains unclear how the actual results section really supports this. The Methods and Results sections require significant clarification. - The figures are also of insufficient quality (e.g., the map in Fig. 1 should have the names of places referred to in text included - the reader cannot be expected to look up in an atlas to check where Northumberland Strait is). It is, moreover, important to realise that, although during summer the shallow southern Gulf of St Lawrence is warmer than the open Bay of Fundy, the reverse is true during winter. At least this point - which might interfere with the hypothesis to be tested - needs to be acknowledged. - Although the hypothesis on epigenetics is interesting and potentially widely relevant, the paper, as is, does not allow the typical reader of Royal Society Open Science to understand whether the results presented actually do, or do not, support it. Significantly improved presentation is needed, where the authors should focus to make this article more reader-friendly. Specific points: Simplify wherever possible throughout document - e.g. 7000 years ago (instead 7ka); avoid introducing difficult terms if used once or twice only (e.g. 'epigenetic augmentation'). Line 54 simplify; warmer temperatures mean higher oxygen demand due to higher metabolism, and lower oxygen levels in water. Note that oxygen levels also tend to be lower deeper down in the ocean. Line 95. Reference (17) appears to be about mammals, not about marine fish that can very quickly switch genes on or off (?) Line 117. These references (17,18) appear incorrect. Line 151. Correct explanation DFO. Line 156. Probably some mnore information on Eurofins MWG needed (e.g. location, what sort of company it is). Lines 177-236. I could not comment on these sections owing to the technical nature, but would suggest to go through this and make this more easily accessible to wider readership. The broad readership should be able to get a feel, although not necessarily understand the detail. Line 258 the reference to table S1 seems incorrect (I could not find this table in suppl. mat.). Likewise, the figures 68.6% and 70.5% could not be found anywhere in suppl. mat. Line 252, this did not seem supported by figure S2a-b. There was no caption for figure S2a-b (or c or d). Lines 268-277: I could not understand this (and would worry that most potential readers would neither). Does this support adaptive phenotypic plasticity? Lines 272. I could not see how this statement was supported by figures 3-5 (which were lacking a caption). Lines 280-288. This is introductory text, and does not seem to fit at a start of discussion. Lines 289-293. It is not clear how this message is derived from the results. The results are stated too technically and this conclusion is not evident. Lines 297-300. please clarify. P.S. recommend to do a better spelling check; for example, this single sentence (caption of table S2) contains 4 spelling mistakes: "Statistcs of de novo transcripome assembly for the southern Gulf of St. Lawrnece (sGSL) and Scotian Shelf Winter Skate (Leucoraja ocellata) compared to



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statistics from the published transcriptome of the closely relataed Little Skate (Leucoraja erinacea)"

Decision letter (RSOS-160299) 08-Aug-2016 Dear Dr Lighten, The editors assigned to your paper ("Adaptive phenotypic response to climate change enabled by epigenetics in a K-strategy species, the fish Leucoraja ocellata (Rajidae)") has now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Subject Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance. Please submit a copy of your revised paper within three weeks (i.e. by the 31-Aug-2016). If we do not hear from you within this time then it will be assumed that the paper has been withdrawn. In exceptional circumstances, extensions may be possible if agreed with the Editorial Office in advance.We do not allow multiple rounds of revision so we urge you to make every effort to fully address all of the comments at this stage. If deemed necessary by the Editors, your manuscript will be sent back to one or more of the original reviewers for assessment. If the original reviewers are not available we may invite new reviewers. To revise your manuscript, log into http://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." Your manuscript number has been appended to denote a revision. Revise your manuscript and upload a new version through your Author Centre. When submitting your revised manuscript, you must respond to the comments made by the referees and upload a file "Response to Referees" in "Section 6 - File Upload". Please use this to document how you have responded to the comments, and the adjustments you have made. In order to expedite the processing of the revised manuscript, please be as specific as possible in your response. In addition to addressing all of the reviewers' and editor's comments please also ensure that your revised manuscript contains the following sections as appropriate before the reference list: • Ethics statement (if applicable) 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 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



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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-160299 • 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. Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Yours sincerely, Andrew Dunn Senior Publishing Editor Royal Society Open Science on behalf of Kevin Padian Subject Editor, Royal Society Open Science [email protected] Comments to Author: Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) Review of: “Adaptive phenotypic reponse to climate change enabled by epigenetics in a K-strategy species, the fish Leucoraja ocellata (Rajidae).” – Lighten et al., 2016



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The authors compose a cohesive and well-constructed story with their findings. They clearly show the morphological differences between two distinct populations of skate and identify epigenetic changes associated with these morphological traits, providing strong support for their hypothesis; Leucoraja ocellata have the potential to adapt to rapidly changing environments through epigenetic mechanisms. They identify many up- and down-regulated genes that directly and indirectly impact temperature stress response, morphology, and life history traits. They highlight the clear differences in the two populations: maturation time, egg case size, lifespan, body size, among other morphological characteristics. While the article has technical components in terms of the genetic tools and techniques used, the authors were able to communicate their results and the significance of their findings without difficulty. Valuable connections are drawn to link the genetic components to physiological, ecological, and behavioural mechanisms that strengthen their findings, drawing from fundamental literature to support their findings and linking it to recent literature for relevance in today’s changing climate. They provide a comprehensive list of explanations for changes in gene expression to support their findings. The authors also draw connections to social components, with the species vulnerable to fishing activities as by-catch. This article will be a novel and valuable addition to the current scientific literature. The following comments should be addressed: 1. Page 4, second paragraph. You indicate that natural epigenetic changes to climate change have not been identified in fish populations. Is there other literature on land and non-fish species that show these epigenetic changes? 2. Page 6, second paragraph. It appears that the sample size was 2. While this may be appropriate for this type of genetic study, you should address the variance and uncertainty in the results with this study design. What are the implications of pseudoreplication when sampling from the same individual? 3. Page 12-13, last paragraph. The authors indicate changes in growth between populations. Is it growth (rate) that is different between the two environments, or max size? Table 1 does not summarize difference in growth rate. 4. Page 13, second paragraph, last sentence. The authors can specify how oxidative stress affects organismal aging (increase aging). 5. Page 14, first paragraph, last sentence. The authors should provide a citation for this statement. 6. This study identifies the adaptive changes in gene expression to different climates. However, in the title and throughout the article, climate change is introduced as a component leading to epigenetic differences. Climate change is different than the environmental differences between these two populations. Instead, the authors should clearly state the implications of your study to climate change. The title is also misleading as it suggests that these species have already shown epigenetic responses to climate change. 7. The study supports epigenetic adaptation across relatively short time scales. The authors may want to consider the implications of their study and the fact that chondrichthyes, in general, having persisted relatively evolutionary unchanged through many eras and through vast changes in climate (e.g., ice ages). 8. It would be useful to include a table to explain the linkages of the specific epigenetic responses (up/down regulation of a certain process) mean to the adaptation to environmental changes. It is not apparent in the manuscript. As the manuscript aims to build such linkages, it



9

would be helpful for readers if the author could include such a table e.g., the first column is the process, second column as up/down regulation or no change, and then final column as inferred links to adaptation to climate. The following minor changes to be addressed: 9. Page 5, line 121. Consider changing “K-strategy” to “K-strategist”. 10. Page 6, line 173. Grammar: “manufactures” to “manufacturer’s”. 11. Page 9, line 262. Grammar: “an 95%” to “a 95%”. 12. Page 11, line 311. Spelling: “exposer” to “exposure”. 13. Page 21, figure 1. Identify study sites on map. This is an integral component to your study, showing the geographical and environmental differences between the populations. 14. Page 22-23, figure 2. This figure is not properly labelled and referenced in text. 15. Page 24-26, figure 3-5. These figures were not labelled properly and did not correspond to labels in text (e.g., ‘a’, ‘b’). Reviewer: 2 Comments to the Author(s) The manuscript reports results from a study comparing transcriptomes of two populations of the winter skates. The topic is timely and the protocols appear to be executed appropriately. The scope of the study is somewhat limited to two populations, and I do not quite understand why the study was not extended to investigate also other, more southern, populations. Most importantly, the authors need to clarify the collection methods, and they need to include a broader reference to the literature on the effect of temperature on skates in light of climate change beyond the fisheries studies. I recommend that these issues are addressed in a revision. Minor comments and suggestions: L.52: “factor” instead of condition L.57: some species shrunk their range, i.e. didn’t shift towards higher latitudes, but they rather lost the southern population L.60-61: I’m confused. If you’re talking about plastic physiological responses it is not “adaptation” but “acclimation”, please clarify. L.63-65: rephrase this whole sentence, it’s awkward. Line 74 and on.. : if you are talking about phenotypic plasticity, then I think you need to report also physiological studies (see general comments). L.121,280,291: “elasmobranchs” misspelled L.123 and on: perhaps because many species of skates don’t seem to travel very far? L.150: how did you collect? Could the collection methods have affected gene expression? How many skates? And at which temperature? L.287-289: rephrase, some of the wording -I think- is not so appropriate for a paper. L.302: need to explain these effects – expand this paragraph L.375 “affect” Fig.1 It would be useful to see the 2 sampling spots on the map



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Reviewer: 3 Comments to the Author(s) Dear authors, Although the manuscript could have potential, in its current form it is incomprehensible to the wider audience. Although it is possible that it can be understood by scientists who already have a fairly extensive knowledge on (epi)genetics, this would not be the case for a majority of readers. The Introduction and Discussion sections can be fairly well understood, but there is no good connect with the (highly technical) Methods and Results sections. The key findings cannot be grasped. The key findings seem to be mentioned in Discussion, lines 289-293 ("Our study suggests that a phenotypically plastic response through epigenetic regulation ... take place over time." However, it remains unclear how the actual results section really supports this. The Methods and Results sections require significant clarification. - The figures are also of insufficient quality (e.g., the map in Fig. 1 should have the names of places referred to in text included - the reader cannot be expected to look up in an atlas to check where Northumberland Strait is). It is, moreover, important to realise that, although during summer the shallow southern Gulf of St Lawrence is warmer than the open Bay of Fundy, the reverse is true during winter. At least this point - which might interfere with the hypothesis to be tested - needs to be acknowledged. - Although the hypothesis on epigenetics is interesting and potentially widely relevant, the paper, as is, does not allow the typical reader of Royal Society Open Science to understand whether the results presented actually do, or do not, support it. Significantly improved presentation is needed, where the authors should focus to make this article more reader-friendly. Specific points: Simplify wherever possible throughout document - e.g. 7000 years ago (instead 7ka); avoid introducing difficult terms if used once or twice only (e.g. 'epigenetic augmentation'). Line 54 simplify; warmer temperatures mean higher oxygen demand due to higher metabolism, and lower oxygen levels in water. Note that oxygen levels also tend to be lower deeper down in the ocean. Line 95. Reference (17) appears to be about mammals, not about marine fish that can very quickly switch genes on or off (?) Line 117. These references (17,18) appear incorrect. Line 151. Correct explanation DFO. Line 156. Probably some mnore information on Eurofins MWG needed (e.g. location, what sort of company it is). Lines 177-236. I could not comment on these sections owing to the technical nature, but would suggest to go through this and make this more easily accessible to wider readership. The broad readership should be able to get a feel, although not necessarily understand the detail. Line 258 the reference to table S1 seems incorrect (I could not find this table in suppl. mat.). Likewise, the figures 68.6% and 70.5% could not be found anywhere in suppl. mat. Line 252, this did not seem supported by figure S2a-b. There was no caption for figure S2a-b (or c or d). Lines 268-277: I could not understand this (and would worry that most potential readers would neither). Does this support adaptive phenotypic plasticity? Lines 272. I could not see how this statement was supported by figures 3-5 (which were lacking a caption). Lines 280-288. This is introductory text, and does not seem to fit at a start of discussion. Lines 289-293. It is not clear how this message is derived from the results. The results are stated too technically and this conclusion is not evident. Lines 297-300. please clarify. P.S. recommend to do a better spelling check; for example, this single sentence (caption of table S2) contains 4 spelling mistakes: "Statistcs of de novo transcripome assembly for the southern Gulf of St. Lawrnece (sGSL) and Scotian Shelf Winter Skate (Leucoraja ocellata) compared to statistics from the published transcriptome of the closely relataed Little Skate (Leucoraja erinacea)"



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Author's Response to Decision Letter for (RSOS-160299) See Appendix A.

RSOS-160299.R1 (Revision) Review form: Reviewer 3 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? Yes 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) See attached. See Appendix B.

Decision letter (RSOS-160299.R1) 23-Sep-2016 Dear Dr Lighten: On behalf of the Editors, I am pleased to inform you that your Manuscript RSOS-160299.R1 entitled "Adaptive phenotypic response to climate enabled by epigenetics in a K-strategy species, the fish Leucoraja ocellata (Rajidae)" 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.



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The reviewers and Subject Editor 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 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-160299.R1 • 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. Please note that we cannot publish your manuscript without these end statements included. We have included a screenshot example of the end statements for reference. If you feel that a given



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heading is not relevant to your paper, please nevertheless include the heading and explicitly state that it is not relevant to your work. 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 02-Oct-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 revisions on the originally submitted version of the manuscript. Instead, revise your manuscript and upload a new version through your Author Centre. When submitting your revised manuscript, you will be able to respond to the comments made by the referees and upload a file "Response to Referees" in "Section 6 - File Upload". You can use this to document any changes you make to the original manuscript. In order to expedite the processing of the revised manuscript, please be as specific as possible in your response to the referees. 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) and figure captions. Do not upload a PDF as your "Main Document". 2) A separate electronic file of each figure (EPS or print-quality PDF preferred (either format should be produced directly from original creation package), or original software format) 3) Included a 100 word media summary of your paper when requested at submission. Please ensure you have entered correct contact details (email, institution and telephone) in your user account 4) Included the raw data to support the claims made in your paper. You can either include your data as electronic supplementary material or upload to a repository and include the relevant doi within your manuscript 5) All supplementary materials accompanying an accepted article will be treated as in their final form. Note that the Royal Society will neither edit nor typeset supplementary material and it will be hosted as provided. Please ensure that the supplementary material includes the paper details where possible (authors, article title, journal name). Supplementary files will be published alongside the paper on the journal website and posted on the online figshare repository (https://figshare.com). The heading and legend provided for each supplementary file during the submission process will be used to create the figshare page, so please ensure these are accurate and informative so that your files can be found in searches. Files on figshare will be made available approximately one week before the accompanying article so that the supplementary material can be attributed a unique DOI. Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Best wishes Alice Power Editorial Coordinator Royal Society Open Science [email protected] on behalf of Kevin Padian Subject Editor, Royal Society Open Science [email protected]



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Comments to Author: Reviewer: 3 Comments to the Author(s) See attached.

Author's Response to Decision Letter for (RSOS-160299.R1) See Appendix C.



Supporting Data on Dryad:

Journal editors and anonymous peer reviewers may access data on Dryad using the following

temporary URL: http://datadryad.org/review?doi=doi:10.5061/dryad.6mr8n

Reviewers' Comments to Author:

Reviewer: 1

Comments to the Author(s)

Review of: “Adaptive phenotypic response to climate change enabled by epigenetics in a K-strategy

species, the fish Leucoraja ocellata (Rajidae).” – Lighten et al., 2016

The authors compose a cohesive and well-constructed story with their findings. They clearly show the

morphological differences between two distinct populations of skate and identify epigenetic changes

associated with these morphological traits, providing strong support for their hypothesis; Leucoraja

ocellata have the potential to adapt to rapidly changing environments through epigenetic mechanisms.

They identify many up- and down-regulated genes that directly and indirectly impact temperature stress

response, morphology, and life history traits. They highlight the clear differences in the two populations:

maturation time, egg case size, lifespan, body size, among other morphological characteristics.

While the article has technical components in terms of the genetic tools and techniques used, the

authors were able to communicate their results and the significance of their findings without difficulty.

Valuable connections are drawn to link the genetic components to physiological, ecological, and

behavioural mechanisms that strengthen their findings, drawing from fundamental literature to support

their findings and linking it to recent literature for relevance in today’s changing climate. They provide a

comprehensive list of explanations for changes in gene expression to support their findings. The authors

also draw connections to social components, with the species vulnerable to fishing activities as by-catch.

This article will be a novel and valuable addition to the current scientific literature.

>Thank you very much for your positive comments and interest.

The following comments should be addressed:

1. Page 4, second paragraph. You indicate that natural epigenetic changes to climate change have not

been identified in fish populations. Is there other literature on land and non-fish species that show these

epigenetic changes?

>An important point. Epigenetic studies on wild populations are very rare as generally isogenic

experimental lines are used. However we included a reference to a very recent study that showed

epigenetic mechanisms operating in wild caught mammals, albeit still though experimental

manipulation. Indeed, to the best of our knowledge, our study provided the first comprehensive analysis

of epigenetic adaptation of fish in the natural environment.

Appendix A on June 29, 2018http://rsos.royalsocietypublishing.org/Downloaded from

http://datadryad.org/review?doi=doi:10.5061/dryad.6mr8n


“. Experimental studies using wild caught guinea pigs have even shown that inheritance of epigenetic

modifications may be important in determining the survival of mammalian species during increased

environmental warming (22)”

2. Page 6, second paragraph. It appears that the sample size was 2. While this may be appropriate for

this type of genetic study, you should address the variance and uncertainty in the results with this study

design. What are the implications of pseudoreplication when sampling from the same individual?

>We agree this is an important point we initially did not clarify. We have now included a second

paragraph to the Discussion section “Regulation of gene expression”, as follows:

“Methodologically speaking, although we included four samples of Winter Skate in transcriptome

characterization, by pooling a male and a female per population we effective sequenced two biological

replicates. For such RNA-Seq experiments, random variation in gene expression patterns may be very

small, with highly correlated estimates of sequence fold-change expression between biological

replicates (e.g. >0.99 (65)). However, a recent study has shown that increasing biological replicates also

increases the power in detecting differentially expressed transcripts in RNA-Seq studies (66). Therefore,

although we are confident in our estimates of differential transcript expression, we also acknowledge

that more biological replicates across populations -and additional gene expression detection methods

e.g. bisulfite sequencing to characterize patterns of DNA methylation- would be advantageous for a

fuller description of Winter Skate epigenetic adaptation.”

3. Page 12-13, last paragraph. The authors indicate changes in growth between populations. Is it growth

(rate) that is different between the two environments, or max size? Table 1 does not summarize

difference in growth rate.

>Thank you for highlighting this potentially valuable metric. In the introduction we now include the

relevant information from an already cited study that showed no significant difference in growth rate

between the two populations of skate

Line 114. “Previous studies have shown that individuals in the southern Gulf of St. Lawrence (sGSL)

population appear to be an undescribed endemic, which are substantially divergent from the Scotian

shelf population in morphology (e.g. significantly smaller body size despite no significant difference in

growth rate)…”

4. Page 13, second paragraph, last sentence. The authors can specify how oxidative stress affects

organismal aging (increase aging).

>Now clarified “Oxidative stress is known to effect organismal aging by disrupting processes of glucose

uptake and lipid synthesis at the cellular level (57, 58)…”



5. Page 14, first paragraph, last sentence. The authors should provide a citation for this statement.

>Added

6. This study identifies the adaptive changes in gene expression to different climates. However, in the

title and throughout the article, climate change is introduced as a component leading to epigenetic

differences. Climate change is different than the environmental differences between these two

populations. Instead, the authors should clearly state the implications of your study to climate change.

The title is also misleading as it suggests that these species have already shown epigenetic responses to

climate change.

>Thank you for pointing out this out and we agree. We have now altered the title from including

“climate change” to simply “climate”. Moreover we now place our study more in the context of

environmental variability and emphasize the implications under climate change scenarios.

7. The study supports epigenetic adaptation across relatively short time scales. The authors may want to

consider the implications of their study and the fact that chondrichthyes, in general, having persisted

relatively evolutionary unchanged through many eras and through vast changes in climate (e.g., ice

ages).

> This is a very nice observation and we have now added a section before the conclusion detailing this

suggestion.

8. It would be useful to include a table to explain the linkages of the specific epigenetic responses

(up/down regulation of a certain process) mean to the adaptation to environmental changes. It is not

apparent in the manuscript. As the manuscript aims to build such linkages, it would be helpful for

readers if the author could include such a table e.g., the first column is the process, second column as

up/down regulation or no change, and then final column as inferred links to adaptation to climate.

>Thank you. We initially excluded this table and relied on the main text to describe these links. We agree

that a table would be useful and so have added it as suggested to Table S3-5

The following minor changes to be addressed:

9. Page 5, line 121. Consider changing “K-strategy” to “K-strategist”.

>changed as suggested

10. Page 6, line 173. Grammar: “manufactures” to “manufacturer’s”.


11. Page 9, line 262. Grammar: “an 95%” to “a 95%”.




12. Page 11, line 311. Spelling: “exposer” to “exposure”.


13. Page 21, figure 1. Identify study sites on map. This is an integral component to your study, showing

the geographical and environmental differences between the populations.

> Thank you. This was an initial oversight that we have now corrected.

14. Page 22-23, figure 2. This figure is not properly labelled and referenced in text.

>Amended

15. Page 24-26, figure 3-5. These figures were not labelled properly and did not correspond to labels in

text (e.g., ‘a’, ‘b’).

>Amended

Reviewer: 2


The manuscript reports results from a study comparing transcriptomes of two populations of the winter

skates. The topic is timely and the protocols appear to be executed appropriately. The scope of the

study is somewhat limited to two populations, and I do not quite understand why the study was not

extended to investigate also other, more southern, populations.

> The study was funded by the Department of Fisheries and Oceans Canada to determine the ecological

status of skates at risk in Canadian waters. The central question was “how is the southern Gulf

population of skate different from the Scotian shelf population”. Given our findings we agree that it

would be very interesting to look at range wide variation in gene expression of skates that inhabit

different environments, which was unfortunately outside of the scope of this study. Hopefully we

provide impetus for other researchers to take this approach and have suggested this may be valuable in

order to understand skate evolution and conservation.

Most importantly, the authors need to clarify the collection methods,

>We have now clarified that live skate were sampled from trawling at particular depths



and they need to include a broader reference to the literature on the effect of temperature on skates in

light of climate change beyond the fisheries studies.

>The direct effect of climate change on skates, beyond fisheries studies (i.e. range size shifts etc.), is

extremely poorly understood. We have included a few additional citations that relate to the effect of

temperature on reproductive traits, and growth. Beyond these, skates are much less well studied than

other fishes. If there are specific studies that the referee is referring to then we would happily include

them.

I recommend that these issues are addressed in a revision.

Minor comments and suggestions:

L.52: “factor” instead of condition


L.57: some species shrunk their range, i.e. didn’t shift towards higher latitudes, but they rather lost the

southern population

> Thank you for pointing out this potential source of confusion. We have now clarified by describing a

northward shift in mean latitude which accounts for both northward expansions, and southern

extinctions.

“Indeed, rising water temperatures can affect fish by limiting their ability to efficiently deliver oxygen to

tissues (7, 8), and as ocean temperatures have risen, the mean latitude of some species ranges have

started to shift towards the poles in response to changing conditions in their native habitat (9–11).”

L.60-61: I’m confused. If you’re talking about plastic physiological responses it is not “adaptation” but

“acclimation”, please clarify.

> “Acclimation” is suggestive of physical mechanisms to cope with different environmental conditions

when an individual is placed in a different environment, e.g. shivering in the cold, or sweating in the

heat. Furthermore, “acclimation” does not suggest genetic or epigenetic mechanisms, and hence, we

think that “physiological adaption” is more appropriate.



L.63-65: rephrase this whole sentence, it’s awkward.

> Changed from “Although contemporary climate change has been accelerated through increased

carbon emissions, still, natural historical episodes of temperature change have occurred within a blink of

the eye across geological time scales (13)”

to

“Even though contemporary climate change has been accelerated through increased carbon emissions,

natural episodes of temperature change have still occurred relatively quickly across geological time

scales (13)”

Line 74 and on.. : if you are talking about phenotypic plasticity, then I think you need to report also

physiological studies (see general comments).

>Although we are talking about changing phenotypes, this is in an adaptive epigenetic sense instead of a

physiological or metabolic regulatory response to changes in conditions i.e. acclimation. We have now

changed the wording throughout to try to reflect ‘adaptation’ rather than ‘acclimation’.

L.121, 280,291: “elasmobranchs” misspelled

>Thanks. Corrected

L.123 and on: perhaps because many species of skates don’t seem to travel very far?

> An Interesting point. However, individual skates can actually move quite far (hundreds of kilometers).

We are not aware of any evidence that suggest a sedentary lifestyle has promoted biodiversity in skates,

or other fishes. The evidence in the cited studies suggestS that skate ranges and patterns of biodiversity

are significantly affected by ecosystem composition.

L.150: how did you collect? Could the collection methods have affected gene expression? How many

skates? And at which temperature?

>We now clarify that skate were collected live from trawl fishing surveys and sampled immediately after

landing. Unfortunately, the fishing gear did not have temperature sensors attached, however we believe

the map provided in figure 1 provides adequate information on depth temperature differences between

the two habitats during the time of year when the samples were collected.

L.287-289: rephrase, some of the wording -I think- is not so appropriate for a paper.

> Agreed. Changed from “These observations suggest that resilient species with a modest evolutionary

potential such as skate must carry an evolutionary ‘trump card’ that enables them to persist over macro-

evolutionary time”



To “These observations suggest that resilient species with a modest evolutionary potential such as skate

may possess an eco-evolutionary strategy that enables them to persist over macro-evolutionary time”

L.302: need to explain these effects – expand this paragraph

>We explain these effects below this paragraph and now in Table S3-S5. We now direct the reader to

these tables and the section below.

L.375 “affect”

> We believe “effects” is correctly used here. However we have changed the wording from “epigenetic

effects of adaptation” to “epigenetic effects on adaptation”

Fig.1 It would be useful to see the 2 sampling spots on the map

>We added the sampling locations to the map

Reviewer: 3


Dear authors,

Although the manuscript could have potential, in its current form it is incomprehensible to the wider

audience. Although it is possible that it can be understood by scientists who already have a fairly

extensive knowledge on (epi)genetics, this would not be the case for a majority of readers. The

Introduction and Discussion sections can be fairly well understood, but there is no good connect with

the (highly technical) Methods and Results sections.

>Thank you for pointing this out. We have attempted to add a little more explanation to the methods

and results. We purposefully kept the results section short so that they can be explained in context

within the discussion. However, the methods are in fact quite technical and to ensure repeatability of

the protocol for other researchers we aimed to include all the necessary detail. We have again tried to

include wording which explains to the non-expert reader the reasons for, and the outcomes of,

particular approaches where possible.

The key findings cannot be grasped. The key findings seem to be mentioned in Discussion, lines 289-293

("Our study suggests that a phenotypically plastic response through epigenetic regulation ... take place

over time." However, it remains unclear how the actual results section really supports this. The Methods

and Results sections require significant clarification.

>We believe the methods and results are now clarified and direct the reader to the discussion for

interpretation of the results.



The figures are also of insufficient quality (e.g., the map in Fig. 1 should have the names of places

referred to in text included - the reader cannot be expected to look up in an atlas to check where

Northumberland Strait is).

>We have improved the resolution of the figures and included place names on the map. This was an

initial oversight on our part.

It is, moreover, important to realise that, although during summer the shallow southern Gulf of St

Lawrence is warmer than the open Bay of Fundy, the reverse is true during winter. At least this point -

which might interfere with the hypothesis to be tested - needs to be acknowledged.

>We did not include individuals from the Bay of Fundy. Our comparisons were made between those the

Northumberland Strait and the Scotian Shelf. We have clarified the fact that skate in the

Northumberland Strait over-winter in the much deeper Laurentian Channel to avoid the much colder

temperatures of the Northumberland Strait in the winter:

Line 139 “Alternatively, the epigenetic induced phenotypic adaptation may be acquired at the start of

each generation that is born in the warmer sGSL, after adults have over-wintered in the much deeper

Laurentian channel which has similar winter bottom temperatures to the Scotian Shelf.”

Although the hypothesis on epigenetics is interesting and potentially widely relevant, the paper, as is,

does not allow the typical reader of Royal Society Open Science to understand whether the results

presented actually do, or do not, support it. Significantly improved presentation is needed, where the

authors should focus to make this article more reader-friendly.

>We believe that the discussion section puts the differences in gene expression (epigenetics) in to

context with the differences seen between populations in eco-physiology. We now recognize that

further work with more biological replicates and focus on the system is required to fully understand the

epigenetics of skate adaptation.

Specific points:

Simplify wherever possible throughout document - e.g. 7000 years ago (instead 7ka);


avoid introducing difficult terms if used once or twice only (e.g. 'epigenetic augmentation').

>We have clarified the use of this terminology with the addition of a simpler descriptor

“We argue that the epigenetic augmentation of species evolutionary potential (its regulation though gene expression) can enable K-strategists to survive and adapt in times of rapid environmental change.”

Line 54 simplify; warmer temperatures mean higher oxygen demand due to higher metabolism, and

lower oxygen levels in water. Note that oxygen levels also tend to be lower deeper down in the ocean.

>Thank you for pointing this out. We have now simplified in reference to metabolic rates and dissolved

oxygen at different temperatures.



Line 95. Reference (17) appears to be about mammals, not about marine fish that can very quickly

switch genes on or off (?)

>Corrected

Line 117. These references (17, 18) appear incorrect.

>Corrected

Line 151. Correct explanation DFO.

>Corrected

Line 156. Probably some more information on Eurofins MWG needed (e.g. location, what sort of

company it is).

> Changed to “Eurofins Genomics” to better reflect their services. They are among the largest

international companies providing DNA molecular analysis and reside in 39 different countries. We

believe the improved company name is now sufficient.

Lines 177-236. I could not comment on these sections owing to the technical nature, but would suggest

to go through this and make this more easily accessible to wider readership. The broad readership

should be able to get a feel, although not necessarily understand the detail.

> Thank you for this suggestion. We believe to have now achieved this.

Line 258 the reference to table S1 seems incorrect (I could not find this table in suppl. mat.). Likewise,

the figures 68.6% and 70.5% could not be found anywhere in suppl. mat.

>Apologies for this oversight, which has now been corrected with the proper inclusion of these tables.

The two quoted percentages are not in the supplementary materials, which details assembly statistics,

as they describe re-mapping. We believe this is sufficiently presented.

Line 252, this did not seem supported by figure S2a-b. There was no caption for figure S2a-b (or c or d).

> The figure does have a caption but maybe the transfer to PDF caused an error? We have now included

reference to Table S1 (alongside the figures) to fully support all of the observations stated in this

sentence.

Lines 268-277: I could not understand this (and would worry that most potential readers would neither).

Does this support adaptive phenotypic plasticity?

>We found it better to describe the results alongside their interpretation within the discussion. This

small section merely provides a high level summary of the results. We then direct the reader to the

discussion for details. We found this the best way to present the results in light of their relevance with

the eco-evolutionary system.

Lines 272. I could not see how this statement was supported by figures 3-5 (which were lacking a

caption).

>We have now included reference to Table 1, which describes the differences, and referred the reader

to the Discussion for details



Lines 280-288. This is introductory text, and does not seem to fit at a start of discussion.

> In this text we do not repeat any introductory information but instead build the picture for the reader

to follow on and understand the relevance of the rest of the discussion. We believe that this small

amount of background would be valuable to the general reader in reminding them of the context within

which the story we tell is held.

Lines 289-293. It is not clear how this message is derived from the results. The results are stated too

technically and this conclusion is not evident.

>The results are quite technical (i.e. many differential expressions of transcripts) and need to be put into

context with the ecological and physiological differences between the population. Because of this we

ask the reader to compare Table 1 and Fig 3-5 and we give full details in the discussion. We refer the

reader to this now at the end of this section i.e. an explanation follows.

Lines 297-300. please clarify.

>We do in the next section. However, we agree that the subheading to the next section disrupts

clarification of these two statements, and is superfluous. We removed the subheading “Epigenetic

adaptation to new environment”.

P.S. recommend to do a better spelling check; for example, this single sentence (caption of table S2)

contains 4 spelling mistakes: "Statistics of de novo transcriptome assembly for the southern Gulf of St.

Lawrence (sGSL) and Scotian Shelf Winter Skate (Leucoraja ocellata) compared to statistics from the

published transcriptome of the closely related Little Skate (Leucoraja erinacea)"

> Apologies. Now remedied.



SECOND REVIEW OF MS “Adaptive phenotypic response to climate change enabled by epigenetics in a

K-strategy species, the fish Leucoraja ocellata (Rajidae).” – Lighten et al., 2016

Reviewed by Dr Georg H. Engelhard

Reviewer: 3


Dear authors,

Although the manuscript could have potential, in its current form it is incomprehensible to the wider

audience. Although it is possible that it can be understood by scientists who already have a fairly

extensive knowledge on (epi)genetics, this would not be the case for a majority of readers. The

Introduction and Discussion sections can be fairly well understood, but there is no good connect with

the (highly technical) Methods and Results sections.

>Thank you for pointing this out. We have attempted to add a little more explanation to the methods

and results. We purposefully kept the results section short so that they can be explained in context

within the discussion. However, the methods are in fact quite technical and to ensure repeatability of

the protocol for other researchers we aimed to include all the necessary detail. We have again tried to

include wording which explains to the non-expert reader the reasons for, and the outcomes of,

particular approaches where possible.

It was great to see the additions provided to Methods in lines 178-185, 207-213, 228-231 etc. These

additions do really help to understand the Methods. I was positively surprised to see that Reviewers 1

and 2 had much less difficulty to follow the paper than I had, and combined with the revisions done by

the authors this gives greater comfort that the paper is understandable to a majority of readers. Still,

some concerns remain (see below), and would require to be addressed – as in my earlier review, below

suggestions really aim at making the paper better understandable to the wider audience – I think it is

already reasonably accessible to the more specialist readership. This is, of course, relevant to the wide

readership of a journal like Royal Society Open Science.

I think that some wording is still neede to make Results better link with Discussion.

The key findings cannot be grasped. The key findings seem to be mentioned in Discussion, lines 289-293

("Our study suggests that a phenotypically plastic response through epigenetic regulation ... take place

over time." However, it remains unclear how the actual results section really supports this. The Methods

and Results sections require significant clarification.

>We believe the methods and results are now clarified and direct the reader to the discussion for

interpretation of the results.

As stated above I disagree – this remains unclear (to the reader more naive on the subject) although the

manuscript has been improved here. Perhaps, after line 344, add something like: “We base this on the

very different gene expression observed between two populations which evolutionarily speaking,

diverged only recently” or something simple along these lines.

Appendix B on June 29, 2018http://rsos.royalsocietypublishing.org/Downloaded from


The figures are also of insufficient quality (e.g., the map in Fig. 1 should have the names of places

referred to in text included - the reader cannot be expected to look up in an atlas to check where

Northumberland Strait is).

>We have improved the resolution of the figures and included place names on the map. This was an

initial oversight on our part.

It is, moreover, important to realise that, although during summer the shallow southern Gulf of St

Lawrence is warmer than the open Bay of Fundy, the reverse is true during winter. At least this point -

which might interfere with the hypothesis to be tested - needs to be acknowledged.

>We did not include individuals from the Bay of Fundy. Our comparisons were made between those the

Northumberland Strait and the Scotian Shelf. We have clarified the fact that skate in the

Northumberland Strait over-winter in the much deeper Laurentian Channel to avoid the much colder

temperatures of the Northumberland Strait in the winter:

The figure caption 1 remains somewhat unclear. Perhaps you should write that the “sGSL population of

winter skate is concentrated in the relatively warm Northumberland Strait (NSL) during summer, when

samples were collected here, and in the winter when this shallow area is ice-scoured, moves to the

deeper Laurentian Channel where temperatures are milder then. The Scotian Shelf winter skate

population lives in a more oceanic area where temperatures are colder in summer, and comparable to

the sGSL population during winter.”

Line 139 “Alternatively, the epigenetic induced phenotypic adaptation may be acquired at the start of

each generation that is born in the warmer sGSL, after adults have over-wintered in the much deeper

Laurentian channel which has similar winter bottom temperatures to the Scotian Shelf.”

Although the hypothesis on epigenetics is interesting and potentially widely relevant, the paper, as is,

does not allow the typical reader of Royal Society Open Science to understand whether the results

presented actually do, or do not, support it. Significantly improved presentation is needed, where the

authors should focus to make this article more reader-friendly.

>We believe that the discussion section puts the differences in gene expression (epigenetics) in to

context with the differences seen between populations in eco-physiology. We now recognize that

further work with more biological replicates and focus on the system is required to fully understand the

epigenetics of skate adaptation.

Most of the points below were addressed well.

Specific points:

Simplify wherever possible throughout document - e.g. 7000 years ago (instead 7ka);


avoid introducing difficult terms if used once or twice only (e.g. 'epigenetic augmentation').

>We have clarified the use of this terminology with the addition of a simpler descriptor



“We argue that the epigenetic augmentation of species evolutionary potential (its regulation though

gene expression) can enable K-strategists to survive and adapt in times of rapid environmental change.”

Line 54 simplify; warmer temperatures mean higher oxygen demand due to higher metabolism, and

lower oxygen levels in water. Note that oxygen levels also tend to be lower deeper down in the ocean.

>Thank you for pointing this out. We have now simplified in reference to metabolic rates and dissolved

oxygen at different temperatures.

Line 95. Reference (17) appears to be about mammals, not about marine fish that can very quickly

switch genes on or off (?)

>Corrected

Line 117. These references (17, 18) appear incorrect.

>Corrected

Line 151. Correct explanation DFO.

>Corrected

Line 156. Probably some more information on Eurofins MWG needed (e.g. location, what sort of

company it is).

> Changed to “Eurofins Genomics” to better reflect their services. They are among the largest

international companies providing DNA molecular analysis and reside in 39 different countries. We

believe the improved company name is now sufficient.

Lines 177-236. I could not comment on these sections owing to the technical nature, but would suggest

to go through this and make this more easily accessible to wider readership. The broad readership

should be able to get a feel, although not necessarily understand the detail.

> Thank you for this suggestion. We believe to have now achieved this.

Line 258 the reference to table S1 seems incorrect (I could not find this table in suppl. mat.). Likewise,

the figures 68.6% and 70.5% could not be found anywhere in suppl. mat.

>Apologies for this oversight, which has now been corrected with the proper inclusion of these tables.

The two quoted percentages are not in the supplementary materials, which details assembly statistics,

as they describe re-mapping. We believe this is sufficiently presented.

Line 252, this did not seem supported by figure S2a-b. There was no caption for figure S2a-b (or c or d).

> The figure does have a caption but maybe the transfer to PDF caused an error? We have now included

reference to Table S1 (alongside the figures) to fully support all of the observations stated in this

sentence.

Lines 268-277: I could not understand this (and would worry that most potential readers would neither).

Does this support adaptive phenotypic plasticity?



>We found it better to describe the results alongside their interpretation within the discussion. This

small section merely provides a high level summary of the results. We then direct the reader to the

discussion for details. We found this the best way to present the results in light of their relevance with

the eco-evolutionary system.

Lines 272. I could not see how this statement was supported by figures 3-5 (which were lacking a

caption).

>We have now included reference to Table 1, which describes the differences, and referred the reader

to the Discussion for details

Lines 280-288. This is introductory text, and does not seem to fit at a start of discussion.

> In this text we do not repeat any introductory information but instead build the picture for the reader

to follow on and understand the relevance of the rest of the discussion. We believe that this small

amount of background would be valuable to the general reader in reminding them of the context within

which the story we tell is held.

Lines 289-293. It is not clear how this message is derived from the results. The results are stated too

technically and this conclusion is not evident.

>The results are quite technical (i.e. many differential expressions of transcripts) and need to be put into

context with the ecological and physiological differences between the population. Because of this we

ask the reader to compare Table 1 and Fig 3-5 and we give full details in the discussion. We refer the

reader to this now at the end of this section i.e. an explanation follows.

See above for suggested clarification.

Lines 297-300. please clarify.

>We do in the next section. However, we agree that the subheading to the next section disrupts

clarification of these two statements, and is superfluous. We removed the subheading “Epigenetic

adaptation to new environment”.

P.S. recommend to do a better spelling check; for example, this single sentence (caption of table S2)

contains 4 spelling mistakes: "Statistics of de novo transcriptome assembly for the southern Gulf of St.

Lawrence (sGSL) and Scotian Shelf Winter Skate (Leucoraja ocellata) compared to statistics from the

published transcriptome of the closely related Little Skate (Leucoraja erinacea)"

> Apologies. Now remedied.

Do check the spelling of captions in Fig. S1.



K-strategy species, the fish Leucoraja ocellata (Rajidae).” – Lighten et al., 2016 Reviewed by Dr Georg H. Engelhard

Reviewer: 3


Dear authors, Although the manuscript could have potential, in its current form it is incomprehensible to the wider audience. Although it is possible that it can be understood by scientists who already have a fairly extensive knowledge on (epi)genetics, this would not be the case for a majority of readers. The Introduction and Discussion sections can be fairly well understood, but there is no good connect with the (highly technical) Methods and Results sections.

>Thank you for pointing this out. We have attempted to add a little more explanation to the methods and results. We purposefully kept the results section short so that they can be explained in context within the discussion. However, the methods are in fact quite technical and to ensure repeatability of the protocol for other researchers we aimed to include all the necessary detail. We have again tried to include wording which explains to the non-expert reader the reasons for, and the outcomes of, particular approaches where possible.

It was great to see the additions provided to Methods in lines 178-185, 207-213, 228-231 etc. These additions do really help to understand the Methods. I was positively surprised to see that Reviewers 1 and 2 had much less difficulty to follow the paper than I had, and combined with the revisions done by the authors this gives greater comfort that the paper is understandable to a majority of readers. Still, some concerns remain (see below), and would require to be addressed – as in my earlier review, below suggestions really aim at making the paper better understandable to the wider audience – I think it is already reasonably accessible to the more specialist readership. This is, of course, relevant to the wide readership of a journal like Royal Society Open Science. I think that some wording is still need to make Results better link with Discussion.

The key findings cannot be grasped. The key findings seem to be mentioned in Discussion, lines 289-293 ("Our study suggests that a phenotypically plastic response through epigenetic regulation ... take place over time." However, it remains unclear how the actual results section really supports this. The Methods and Results sections require significant clarification.

>We believe the methods and results are now clarified and direct the reader to the discussion for interpretation of the results.

As stated above I disagree – this remains unclear (to the reader more naive on the subject) although the manuscript has been improved here. Perhaps, after line 344, add something like: “We base this on the very different gene expression observed between two populations which evolutionarily speaking, diverged only recently” or something simple along these lines.

We have adding in the suggested clarification, however before line 344 and at the end of the opening Discussion paragraph.

Appendix C on June 29, 2018http://rsos.royalsocietypublishing.org/Downloaded from


“We base this on the very different gene expression profiles observed between the two populations, which evolutionary speaking, remain very similar at the DNA nucleotide level due to only recent separation within approximately the last 7 thousand years.”

The figures are also of insufficient quality (e.g., the map in Fig. 1 should have the names of places referred to in text included - the reader cannot be expected to look up in an atlas to check where Northumberland Strait is). >We have improved the resolution of the figures and included place names on the map. This was an initial oversight on our part. It is, moreover, important to realise that, although during summer the shallow southern Gulf of St Lawrence is warmer than the open Bay of Fundy, the reverse is true during winter. At least this point - which might interfere with the hypothesis to be tested - needs to be acknowledged. >We did not include individuals from the Bay of Fundy. Our comparisons were made between those the Northumberland Strait and the Scotian Shelf. We have clarified the fact that skate in the Northumberland Strait over-winter in the much deeper Laurentian Channel to avoid the much colder temperatures of the Northumberland Strait in the winter: The figure caption 1 remains somewhat unclear. Perhaps you should write that the “sGSL population of winter skate is concentrated in the relatively warm Northumberland Strait (NSL) during summer, when samples were collected here, and in the winter when this shallow area is ice-scoured, moves to the deeper Laurentian Channel where temperatures are milder then. The Scotian Shelf winter skate population lives in a more oceanic area where temperatures are colder in summer, and comparable to the sGSL population during winter.”

Thank you again for highlighting improvements in clarity. If fact if we were to be more accurate, many or even most of the sGSL winter skate occupy colder (near freezing -1.7 C) waters in winter than those on the Scotian shelf. So they actually occupy a greater range of extreme temperatures. We have changed the figure legend to reflect this: “Figure 1. Map showing variation in mean summer bottom temperatures of oceanic waters in

Atlantic Canada (Nova Scotia – NS; Québec – QC; Newfoundland – NL). The southern Gulf of St.

Lawrence (sGSL) population of Winter Skate (Leucoraja ocellata) is concentrated in the

Northumberland Strait (NLS) during summer, when samples were collected here, and when

this shallow area is ice-scoured, moves to deeper water towards the Laurentian Channel, which

still experiences near freezing temperatures (-1.7°) (26). The Scotian Shelf population lives in a

more oceanic area where winter temperatures are milder and summer temperatures cooler.

The summer bottom temperatures vary between these regions by circa 10°C. Although some

warmer shallow waters are also found in the Bay of Fundy (NS), skates tend to move in and out

of shallow regions into cooler deeper regions during the summer, compared to the NLS, which

is surrounded by much deeper and colder water.”



Line 139 “Alternatively, the epigenetic induced phenotypic adaptation may be acquired at the start of each generation that is born in the warmer sGSL, after adults have over-wintered in the much deeper Laurentian channel which has similar winter bottom temperatures to the Scotian Shelf.” Although the hypothesis on epigenetics is interesting and potentially widely relevant, the paper, as is, does not allow the typical reader of Royal Society Open Science to understand whether the results presented actually do, or do not, support it. Significantly improved presentation is needed, where the authors should focus to make this article more reader-friendly. >We believe that the discussion section puts the differences in gene expression (epigenetics) in to context with the differences seen between populations in eco-physiology. We now recognize that further work with more biological replicates and focus on the system is required to fully understand the epigenetics of skate adaptation. Most of the points below were addressed well. Thank you Specific points: Simplify wherever possible throughout document - e.g. 7000 years ago (instead 7ka); >changed as suggested avoid introducing difficult terms if used once or twice only (e.g. 'epigenetic augmentation'). >We have clarified the use of this terminology with the addition of a simpler descriptor “We argue that the epigenetic augmentation of species evolutionary potential (its regulation though gene expression) can enable K-strategists to survive and adapt in times of rapid environmental change.” Line 54 simplify; warmer temperatures mean higher oxygen demand due to higher metabolism, and lower oxygen levels in water. Note that oxygen levels also tend to be lower deeper down in the ocean. >Thank you for pointing this out. We have now simplified in reference to metabolic rates and dissolved oxygen at different temperatures. Line 95. Reference (17) appears to be about mammals, not about marine fish that can very quickly switch genes on or off (?) >Corrected Line 117. These references (17, 18) appear incorrect. >Corrected Line 151. Correct explanation DFO.



>Corrected Line 156. Probably some more information on Eurofins MWG needed (e.g. location, what sort of company it is). > Changed to “Eurofins Genomics” to better reflect their services. They are among the largest international companies providing DNA molecular analysis and reside in 39 different countries. We believe the improved company name is now sufficient. Lines 177-236. I could not comment on these sections owing to the technical nature, but would suggest to go through this and make this more easily accessible to wider readership. The broad readership should be able to get a feel, although not necessarily understand the detail. > Thank you for this suggestion. We believe to have now achieved this. Line 258 the reference to table S1 seems incorrect (I could not find this table in suppl. mat.). Likewise, the figures 68.6% and 70.5% could not be found anywhere in suppl. mat. >Apologies for this oversight, which has now been corrected with the proper inclusion of these tables. The two quoted percentages are not in the supplementary materials, which details assembly statistics, as they describe re-mapping. We believe this is sufficiently presented. Line 252, this did not seem supported by figure S2a-b. There was no caption for figure S2a-b (or c or d). > The figure does have a caption but maybe the transfer to PDF caused an error? We have now included reference to Table S1 (alongside the figures) to fully support all of the observations stated in this sentence. Lines 268-277: I could not understand this (and would worry that most potential readers would neither). Does this support adaptive phenotypic plasticity? >We found it better to describe the results alongside their interpretation within the discussion. This small section merely provides a high level summary of the results. We then direct the reader to the discussion for details. We found this the best way to present the results in light of their relevance with the eco-evolutionary system. Lines 272. I could not see how this statement was supported by figures 3-5 (which were lacking a caption). >We have now included reference to Table 1, which describes the differences, and referred the reader to the Discussion for details Lines 280-288. This is introductory text, and does not seem to fit at a start of discussion. > In this text we do not repeat any introductory information but instead build the picture for the reader to follow on and understand the relevance of the rest of the discussion. We believe that this small amount of background would be valuable to the general reader in reminding them of the context within which the story we tell is held.



Lines 289-293. It is not clear how this message is derived from the results. The results are stated too technically and this conclusion is not evident. >The results are quite technical (i.e. many differential expressions of transcripts) and need to be put into context with the ecological and physiological differences between the population. Because of this we ask the reader to compare Table 1 and Fig 3-5 and we give full details in the discussion. We refer the reader to this now at the end of this section i.e. an explanation follows. See above for suggested clarification. Given the addition of the suggested clarifying sentence at the end of the first Discussion paragraph, we assume this has now been addressed. Lines 297-300. please clarify. >We do in the next section. However, we agree that the subheading to the next section disrupts clarification of these two statements, and is superfluous. We removed the subheading “Epigenetic

adaptation to new environment”. P.S. recommend to do a better spelling check; for example, this single sentence (caption of table S2) contains 4 spelling mistakes: "Statistics of de novo transcriptome assembly for the southern Gulf of St. Lawrence (sGSL) and Scotian Shelf Winter Skate (Leucoraja ocellata) compared to statistics from the published transcriptome of the closely related Little Skate (Leucoraja erinacea)" > Apologies. Now remedied. Do check the spelling of captions in Fig. S1.

We cannot find any spelling mistakes but do not describe the abbreviations in the legend



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Adaptive phenotypic response to climate enabled by ...rsos.royalsocietypublishing.org/content/royopensci/3/10/160299...epigenetics in a K-strategy species, the fish Leucoraja ocellata