Genetic structure in the endosymbiont Breviolum ‘muscatinei’ is correlated with geographical location, environment and host species

Corals and cnidarians form symbioses with dinoflagellates across a wide range of habitats from the tropics to temperate zones. Notably, these partnerships create the foundation of coral reef ecosystems and are at risk of breaking down due to climate change. This symbiosis couples the fitness of the partners, where adaptations in one species can benefit the holobiont. However, the scales over which each partner can match their current—and future—environment are largely unknown. We investigated population genetic patterns of temperate anemones (Anthopleura spp.) and their endosymbiont Breviolum ‘muscatinei’, across an extensive geographical range to identify the spatial scales over which local adaptation is possible. Similar to previously published results, two solitary host species exhibited isolation by distance across hundreds of kilometres. However, symbionts exhibited genetic structure across multiple spatial scales, from geographical location to depth in the intertidal zone, and host species, suggesting that symbiont populations are more likely than their hosts to adaptively mitigate the impact of increasing temperatures.


Do you have any concerns about statistical analyses in this paper? If so, please specify them explicitly in your report. No
It is a condition of publication that authors make their supporting data, code and materials available -either as supplementary material or hosted in an external repository. Please rate, if applicable, the supporting data on the following criteria.

Comments to the Author Summary
Cornwell & Hernandez analyzed the population genetics of congeneric sea anemones and their shared endosymbiont along the west coast of North America using a RADseq approach. They find little population structure in the hosts, instead identifying an isolation-by-distance pattern. But the symbionts exhibit more structure, with at least seven populations across the biogeographic range. Genetic structure in the symbiont is influence by macroscale (geography), mesoscale (intertidal zonation), and microscale (host specificity) processes. Given the symbiont's shorter generation time and greater structure (due to low gene flow and/or strong selection), the authors conclude that the symbionts are better positioned to adapt to climate change, which may influence the adaptive capacity of the holobiont. This is an excellent manuscript: thoughtful, easy to read, and interesting. The methods are sound and the conclusions follow from the data. I wouldn't say the results are particularly surprising (many of the patterns have been identified previously, albeit among symbiont species, not within species), but there are few studies in this intra-species space and even fewer that take advantage of high-throughput approaches to look at population structure. This focus on within-species variation does provide novel insight into the relative importance of coevolution among partners. I was particularly impressed with the analyses of correlations between genetic and environmental variation in the symbiont, as well as covariance between host and symbiont loci. I have not seen such efforts in the Symbiodiniaceae literature before; I hope this manuscript will popularize them. The Anthopleura-Breviolum association is a fantastic system; it's fairly unique in that it allows for consideration of macro/meso/micro-scale processes simultaneously. I really don't have much to critique here-just a few suggestions for minor improvements. Great job.

Minor Comments
You should probably include the Symbiodiniaceae revision in the introduction, as it provides context for the within/between genus discussion. LaJeunesse et al. 2018 Current Biology ("Systematic revision of Symbiodiniaceae highlights the antiquity and diversity of coral endosymbionts"). I found two instances the taxonomic rank should be adjusted. At L301: change "within-clade" to "within-genus." At L447: change "within symbiont genera" to "within symbiont species" (because you are talking about variation within B. muscatinei; because the statement holds for intra-specific variation; and because ITS2 is a species-level marker, though it also gives you the genus at the same time).
I found the Supplemental Methods to be quite helpful for understanding the analyses and they're only two pages long-if space is not an issue, I would recommend adding them back in. I realize Proceedings has a pretty stringent page limit. The discussion is a bit long, so it might be worth trying to cut it down somewhat to make more space for the methods. You might also condense figures to free up space. Figs 1 and 2 (and possibly 3) could be combined, as could Figs 4, 5, and 6.
When discussing the BBA A. elegantissima symbiont population whose reads did not align with the B. minutum genome, it is worth mentioning that this likely represents a unique species, rather than just a distinct population of B. muscatinei.
For the paragraph on specific environments favoring particular symbiont strains (L413-423), consider incorporating Grupstra et al. 2017 Coral Reefs ("Evidence for coral range expansion accompanied by reduced diversity of Symbiodinium genotypes"), as it's relevant.

Review form: Reviewer 2
Recommendation Accept with minor revision (please list in comments)

Scientific importance: Is the manuscript an original and important contribution to its field? Good
General interest: Is the paper of sufficient general interest? Good Quality of the paper: Is the overall quality of the paper suitable? Good Is the length of the paper justified? Yes

Do you have any concerns about statistical analyses in this paper? If so, please specify them explicitly in your report. No
It is a condition of publication that authors make their supporting data, code and materials available -either as supplementary material or hosted in an external repository. Please rate, if applicable, the supporting data on the following criteria.

Comments to the Author
This manuscript is an original study tracking genetic changes in symbiotic dinoflagellates over a latitudinal gradient. It findings the and the Authors' conclusions are consistent with their data and related studies as well as biological theory. Moreover this work sets the bar higher for future approaches in how investigators aught to study symbiotic animals and their mutualistic microbes. Their most significant finding, that the symbiont is more evolutionary responsive to selection from external environmental pressures, is substantiated by the phylogenetic differences where most symbiont lineages have evolved recently vs. the older longevity apparent in many host lineages. This is a solid contribution and with some revision should make for a nice publication.
The Sanders and Palumbi 2011 paper provided a nice study showing many of the genetic differences in the Breviolum symbiont found in Anthopleura along the coastline from Washington to California. I would like to see more effort to reconcile the present work with this paper. From the preliminary genetic work from this earlier paper, it appears that the northern population in Washington and Oregon may be a different species than that found southern California (and possibly a third). I think that the present work would have benefited from providing at least one phylogentic marker (LSU?) to help provide a phylogenetic framework for better perspective of their findings. This would also reveal the identity of the symbiont found in the most southern anemones. Clearly that symbiont is of another genus.
There are a few papers that also show how the genetics of the host does not match with that of the genetic structure of the symbiont, please read and include this in your citations: Baums, I.B., Devlin-Durante, M.K., and LaJeunesse, T.C. (2014). New insights into the dynamics between reef corals and their associated dinoflagellate endosymbionts from population genetic studies. Mol. Ecol. 23, 4203-4215.
I think that the discussion could be shortened as it is redundant in a few places and too speculative in other sections. When possible, please limit speculation to subjects where there is sufficient supporting data to warrant consideration.
Breviolum muscatinei is presently not a valid species as there is no formal charactirization. So it should be written in this paper as: Breviolum 'muscatinei' A thorough systematic treatment is needed for this dinoflagellate to determine if it indeed constitutes one species over the entire geographic range of the U.S. coastline or comprises two or more.
Please delete the Stat et al. 2008 paper in PNAS, cited at the end of the discussion. This is a misleading work and erroneous at all levels from the experimental design, data collection, to interpretation of results. It is a poster child of how to do bad science and get it published in PNAS if you know a member who is willing to see it though without much review (that practice has since been discontinued for reasons such as this paper). Sorry to be so blunt.

19-Jan-2021
Dear Dr Cornwell I am pleased to inform you that your Review manuscript RSPB-2020-2896 entitled "Genetic structure in the endosymbiont Breviolum muscatinei is correlated with geographic location, environment, and host species" has been accepted for publication in Proceedings B.
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Sincerely, Dr Daniel Costa mailto:proceedingsb@royalsociety.org Associate Editor Board Member: 1 Comments to Author: Dear Brendan and Luis Your manuscript has been reviewed by two experts in the field. They both like your manuscript but highlight a number of points that should be addressed before the manuscript is ultimately published. I hope you will find their reviews useful in the final edit of this ms.

Warm Regards
Line Reviewer(s)' Comments to Author: Referee: 1

Comments to the Author(s) Summary
Cornwell &amp; Hernandez analyzed the population genetics of congeneric sea anemones and their shared endosymbiont along the west coast of North America using a RADseq approach. They find little population structure in the hosts, instead identifying an isolation-by-distance pattern. But the symbionts exhibit more structure, with at least seven populations across the biogeographic range. Genetic structure in the symbiont is influence by macroscale (geography), mesoscale (intertidal zonation), and microscale (host specificity) processes. Given the symbiont's shorter generation time and greater structure (due to low gene flow and/or strong selection), the authors conclude that the symbionts are better positioned to adapt to climate change, which may influence the adaptive capacity of the holobiont. This is an excellent manuscript: thoughtful, easy to read, and interesting. The methods are sound and the conclusions follow from the data. I wouldn't say the results are particularly surprising (many of the patterns have been identified previously, albeit among symbiont species, not within species), but there are few studies in this intra-species space and even fewer that take advantage of high-throughput approaches to look at population structure. This focus on within-species variation does provide novel insight into the relative importance of coevolution among partners. I was particularly impressed with the analyses of correlations between genetic and environmental variation in the symbiont, as well as covariance between host and symbiont loci. I have not seen such efforts in the Symbiodiniaceae literature before; I hope this manuscript will popularize them. The Anthopleura-Breviolum association is a fantastic system; it's fairly unique in that it allows for consideration of macro/meso/micro-scale processes simultaneously. I really don't have much to critique here-just a few suggestions for minor improvements. Great job.

Minor Comments
You should probably include the Symbiodiniaceae revision in the introduction, as it provides context for the within/between genus discussion. LaJeunesse et al. 2018 Current Biology ("Systematic revision of Symbiodiniaceae highlights the antiquity and diversity of coral endosymbionts"). I found two instances the taxonomic rank should be adjusted. At L301: change "within-clade" to "within-genus." At L447: change "within symbiont genera" to "within symbiont species" (because you are talking about variation within B. muscatinei; because the statement holds for intra-specific variation; and because ITS2 is a species-level marker, though it also gives you the genus at the same time). I found the Supplemental Methods to be quite helpful for understanding the analyses and they're only two pages long-if space is not an issue, I would recommend adding them back in. I realize Proceedings has a pretty stringent page limit. The discussion is a bit long, so it might be worth trying to cut it down somewhat to make more space for the methods. You might also condense figures to free up space. Figs 1 and 2 (and possibly 3) could be combined, as could Figs 4, 5, and 6.
When discussing the BBA A. elegantissima symbiont population whose reads did not align with the B. minutum genome, it is worth mentioning that this likely represents a unique species, rather than just a distinct population of B. muscatinei. For the paragraph on specific environments favoring particular symbiont strains (L413-423), consider incorporating Grupstra et al. 2017 Coral Reefs ("Evidence for coral range expansion accompanied by reduced diversity of Symbiodinium genotypes"), as it's relevant.

Referee: 2
Comments to the Author(s) This manuscript is an original study tracking genetic changes in symbiotic dinoflagellates over a latitudinal gradient. It findings the and the Authors' conclusions are consistent with their data and related studies as well as biological theory. Moreover this work sets the bar higher for future approaches in how investigators aught to study symbiotic animals and their mutualistic microbes. Their most significant finding, that the symbiont is more evolutionary responsive to selection from external environmental pressures, is substantiated by the phylogenetic differences where most symbiont lineages have evolved recently vs. the older longevity apparent in many host lineages. This is a solid contribution and with some revision should make for a nice publication.
The Sanders and Palumbi 2011 paper provided a nice study showing many of the genetic differences in the Breviolum symbiont found in Anthopleura along the coastline from Washington to California. I would like to see more effort to reconcile the present work with this paper. From the preliminary genetic work from this earlier paper, it appears that the northern population in Washington and Oregon may be a different species than that found southern California (and possibly a third). I think that the present work would have benefited from providing at least one phylogentic marker (LSU?) to help provide a phylogenetic framework for better perspective of their findings. This would also reveal the identity of the symbiont found in the most southern anemones. Clearly that symbiont is of another genus.
There are a few papers that also show how the genetics of the host does not match with that of the genetic structure of the symbiont, please read and include this in your citations: Baums, I.B., Devlin-Durante, M.K., and LaJeunesse, T.C. (2014). New insights into the dynamics between reef corals and their associated dinoflagellate endosymbionts from population genetic studies. Mol. Ecol. 23, 4203-4215. Pettay, D.T., and LaJeunesse, T.C. (2013). Long-range dispersal and high-latitude environments influence the population structure of a "stress-tolerant" dinoflagellate endosymbiont. PLoS One 8, e79208.
I think that the discussion could be shortened as it is redundant in a few places and too speculative in other sections. When possible, please limit speculation to subjects where there is sufficient supporting data to warrant consideration.
Breviolum muscatinei is presently not a valid species as there is no formal charactirization. So it should be written in this paper as: Breviolum 'muscatinei' A thorough systematic treatment is needed for this dinoflagellate to determine if it indeed constitutes one species over the entire geographic range of the U.S. coastline or comprises two or more.
Please delete the Stat et al. 2008 paper in PNAS, cited at the end of the discussion. This is a misleading work and erroneous at all levels from the experimental design, data collection, to interpretation of results. It is a poster child of how to do bad science and get it published in PNAS if you know a member who is willing to see it though without much review (that practice has since been discontinued for reasons such as this paper). Sorry to be so blunt.

10-Feb-2021
Dear Dr Cornwell I am pleased to inform you that your manuscript entitled "Genetic structure in the endosymbiont Breviolum 'muscatinei' is correlated with geographic location, environment, and host species" has been accepted for publication in Proceedings B.
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Sincerely, Editor, Proceedings B mailto: proceedingsb@royalsociety.org Associate Editor Board Member: 1 Comments to Author: Dear Brendan and Luis Your manuscript has been reviewed by two experts in the field. They both like your manuscript but highlight a number of points that should be addressed before the manuscript is ultimately published. I hope you will find their reviews useful in the final edit of this ms.

Warm Regards
Line Reviewer(s)' Comments to Author: Referee: 1

Comments to the Author(s) Summary
Cornwell & Hernandez analyzed the population genetics of congeneric sea anemones and their shared endosymbiont along the west coast of North America using a RADseq approach. They find little population structure in the hosts, instead identifying an isolation-by-distance pattern. But the symbionts exhibit more structure, with at least seven populations across the biogeographic range. Genetic structure in the symbiont is influence by macroscale (geography), mesoscale (intertidal zonation), and microscale (host specificity) processes. Given the symbiont's shorter generation time and greater structure (due to low gene flow and/or strong selection), the authors conclude that the symbionts are better positioned to adapt to climate change, which may influence the adaptive capacity of the holobiont. This is an excellent manuscript: thoughtful, easy to read, and interesting. The methods are sound and the conclusions follow from the data. I wouldn't say the results are particularly surprising (many of the patterns have been identified previously, albeit among symbiont species, not within species), but there are few studies in this intra-species space and even fewer that take advantage of highthroughput approaches to look at population structure. This focus on within-species variation does provide novel insight into the relative importance of coevolution among partners. I was particularly impressed with the analyses of correlations between genetic and environmental variation in the symbiont, as well as covariance between host and symbiont loci. I have not seen such efforts in the Symbiodiniaceae literature before; I hope this manuscript will popularize them. The Anthopleura-Breviolum association is a fantastic system; it's fairly unique in that it allows for consideration of macro/meso/micro-scale processes simultaneously. I really don't have much to critique here-just a few suggestions for minor improvements. Great