High rates of evolution preceded shifts to sex-biased gene expression in Leucadendron, the most sexually dimorphic angiosperms
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Evaluation Summary:
This is one of the first studies to investigate sex-biased gene expresion in a broad phylogenetic context, and the first in a plant genus. The findings go against the classical view that sex-biased gene expression is driven by sex-specific selection for sexual dimorphism, and instead suggests that sex-bias preverentially evolved in genes that already had the highest expression variance to begin with. It will broadly appeal to researchers interested in the evolution of sexual dimorphism.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Differences between males and females are usually more subtle in dioecious plants than animals, but strong sexual dimorphism has evolved convergently in the South African Cape plant genus Leucadendron . Such sexual dimorphism in leaf size is expected largely to be due to differential gene expression between the sexes. We compared patterns of gene expression in leaves among 10 Leucadendron species across the genus. Surprisingly, we found no positive association between sexual dimorphism in morphology and the number or the percentage of sex-biased genes (SBGs). Sex bias in most SBGs evolved recently and was species specific. We compared rates of evolutionary change in expression for genes that were sex biased in one species but unbiased in others and found that SBGs evolved faster in expression than unbiased genes. This greater rate of expression evolution of SBGs, also documented in animals, might suggest the possible role of sexual selection in the evolution of gene expression. However, our comparative analysis clearly indicates that the more rapid rate of expression evolution of SBGs predated the origin of bias, and shifts towards bias were depleted in signatures of adaptation. Our results are thus more consistent with the view that sex bias is simply freer to evolve in genes less subject to constraints in expression level.
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Author Response:
Reviewer #1:
*A summary of what the authors were trying to achieve.
The study takes advantage of the interesting plant genus Leucadendron to compare gene expression between male vs. female in species with more or less sexual dimorphism. This question was addressed in a somewhat comparable manner in only one previous paper by Harrison et al. 2015 across six bird species. The overarching question is the role of natural selection in sexual dimorphism.
*An account of the major strengths and weaknesses of the methods and results.
-Beside the genus-wide comparison of whole transcriptomes across related species, which makes in itself a strong dataset, the major strength of the analysis is the phylogenetic framework that allows the authors to track the evolution of sex bias through several tens of million years of …
Author Response:
Reviewer #1:
*A summary of what the authors were trying to achieve.
The study takes advantage of the interesting plant genus Leucadendron to compare gene expression between male vs. female in species with more or less sexual dimorphism. This question was addressed in a somewhat comparable manner in only one previous paper by Harrison et al. 2015 across six bird species. The overarching question is the role of natural selection in sexual dimorphism.
*An account of the major strengths and weaknesses of the methods and results.
-Beside the genus-wide comparison of whole transcriptomes across related species, which makes in itself a strong dataset, the major strength of the analysis is the phylogenetic framework that allows the authors to track the evolution of sex bias through several tens of million years of evolutionary history. Despite ancestral dioecy in the genus, very few genes show consistent sex bias across several species, with sex-bias being mostly species-specific. Two striking negative results will be of special interest to the community : 1) species with more pronounced sexual dimorphism at the morphological level do not tend to exhibit more pronounced sex-biased gene expression 2) the few genes that do show sex-biased expression were apparently recruited among those with the highest expression variance to begin with, strongly suggesting that sexual selection has not been the main force driving their expression divergence.
-In my view, the main limitation of the work is the use of leaf rather than reproductive tissues, making the comparison to other studies less straightforward to interpret. It is especially important that the expectations for somatic vs gonadic tissues be made a lot clearer in the text.
We have added a full paragraph to the Introduction that lays out the expected differences between reproductive and non-reproductive tissues (traits) in the intensity of sex-specific (or sexual) selection, sexual dimorphism and sex-biased gene expression. We have also taken care to state the reproductive or non-reproductive tissues in cited references.
Also, the fact that a single leaf phenotype is measured (specific leaf area) seems arbitrary : one could imagine sexual dimorphism on many other characteristics, yet they are not considered here. The text on p.324 mentions "striking convergence in aspects of morphological dimorphism across the genus", but there is no way for the reader to appreciate the extent of this convergence. Finally, it would be useful to at least make some mention of the sex-determination system in these species, since the expectations would differ if some of the sex-biased genes were linked to sex chromosomes.
Indeed, Leucadendron can be sexually dimorphic for many phenotypes ranging from plant architecture to phenology. We cite more specialised studies on this topic throughout the manuscript, including works on physiology, ecology, and trait evolution (convergence). The focus on only two traits (specific leaf area and leaf area) is justified by two arguments: 1) we precluded any ambiguity in correspondence by measuring both morphology and gene expression in the same organ / material, and 2) the focus of this study was on the question whether sex-biased gene expression evolved adaptively, rather than discovering new macroscopic sexually dimorphic traits.
*An appraisal of whether the authors achieved their aims, and whether the results support their conclusions.
The analysis is mostly sound, but I am a bit concerned by the arbitrary threshold used to define SBGE. The text on p.305 says that "This result is extremely robust to the choice of threshold", but 1) the results are not reported so it is impossible for the readers to evaluate the basis of this assertion and 2) it is not clear whether robustness of the other results has been evaluated at all. This aspect clearly deserves more attention.
We added two new Appendices, and a directory with data uploaded to the Dryad repository to support these assertions. Appendix 1 presents key results under very permissive (no minimum fold-change, uncorrected p-value <= 0.05) and very stringent thresholds (minimum 3-fold change, FDR <= 0.001) for the definition of sex-bias. Appendix 2 shows how the choice of thresholds for the delta-x analysis (the assertion in original submission line 305) affects the conclusion that expression shifts to sex-bias are depleted in signatures of adaptation of expression levels. The patterns and conclusions of our study are generally robust to the choice of threshold to define sex-biased expression.
*A discussion of the likely impact of the work on the field, and the utility of the methods and data to the community.
This work will be of interest to the community, as rapid rates of expression evolution would generally be interpreted as the consequence of sex bias, whereas the phylogenetic analysis presented here instead supports the idea that the expression of genes that end up being sex biased were instead intrinsically less constrained to begin with.
Reviewer #2:
Scharmann et al. present a study of sex-biased gene expression as a function of sexual dimorphism in leaf tissue in the genus Leucadendron. Comparative studies of sex-biased expression across clades are still relatively rare, and this analysis tests some core findings of a recent paper (Harrison et al. 2015). Overall, I like the analysis and think it could be a valuable addition to the literature on sex-biased genes. This is particularly true given the difficulty of cross-species expression comparisons and the paucity of them in plants.
However, there are some critical differences between the Harrison paper and the one here, and I think it would be helpful if the authors present them early in the text. Specifically, Harrison et al. (2015) was primarily focused on gonad tissue, which in animals is the site of the vast majority of sex-biased genes. In contrast, the authors here focus on vegetative (leaf) tissue, which is analogous to animal somatic tissue. None of the patterns that Harrison et al. (2015) observed and report from the gonad were evidence in the somatic tissue they assessed. Also, by looking at gonadal tissue, Harrison et al. (2015) focused on the tissue that produces gametes, which are thought to be subject to some of the strongest sexual selection pressures. The fairest comparison would be flower tissue in plants, so I am unsure how much of the Harrison results would be expected to hold up in leaf samples. This doesn't mean the authors should do the analyses they present, just that they should be a little more upfront about what they might reasonably expect to find.
We have added a full paragraph to the Introduction that lays out the expected differences between reproductive and non-reproductive tissues (traits) in the intensity of sex-specific (or sexual) selection, sexual dimorphism and sex-biased gene expression. We have also taken care to state the reproductive or non-reproductive tissues in cited references.
There is also a conflation at times in the paper between sexual dimorphism, which the authors can quantify in their leaf samples, and sexual selection. I explain this in more detail below, but to summarize here, I think the expectations for the relationship between sex-biased gene expression and sexual selection versus sexual dimorphism are somewhat distinct.
We added a new paragraph in the Introduction to clarify the key differences between Harrison et al.s' (2015) reasoning and ours, and the expectations how leaf sexual dimorphism could be related to sexual selection and sex-biased gene expression. We argue that sexual selection is but one of several components of sex-specific selection promoting sexual dimorphism in vegetative organs of plants. Please see also our reply to the more detailed comment below.
Finally, I am a little concerned that the low numbers of sex-biased genes, expected from leaf tissue, offer limited power for some of the tests the authors want to do. Harrison et al. (2015) had hundreds of sex-biased genes from the gonad, and this power made it possible to detect subtle patterns. The authors have a few dozen sex-biased genes, and this makes it difficult to know whether their negative results are the result of low statistical power. That they find clear associations between pre-sex-biased genes and rates of evolution is quite impressive given this low power.
Indeed, we found fewer sex-biased gene per species than Harrison et al. (2015), but over all species together we discovered 650 sex-biased genes. For comparisons of the properties and evolution of sex-biased (or pre-sex-biased) versus unbiased genes, this sample size of about 4% of the total 16,194 genes is acceptable. The test for a correlation of sex-biased expression and morphological dimorphism should not be affected by low numbers (or proportions) of sex-biased genes; rather, these numbers or proportions themselves constitute the test. Our RNA-seq and differential expression testing (6 males versus 6 (or 5) females) was certainly powerful enough to discover thousands of sex-biased genes in each species, but these were not found. Furthermore, we have added a new Appendix 1, in which we explore results for a three times larger sample of SBGs (1,973). Although the larger sample of SBGs is obtained by unconventionally lax thresholds to define SBGs, the patterns and conclusions drawn are fully consistent with those from the smaller set of 650 SBGs. No changes made to the main text.
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Reviewer #3 (Public Review):
The authors study the leaf transcriptomes of males and females in 10 species of Leucadendron and infer genes expressed significantly differently between males and females (sex-biased genes, hereafter SBGs). Most SBGs in Leucadendron leaves evolved recently, suggesting that SBGs turnover (evolution and reversion) is very high because the genus is ancestrally dioecious since >10My. Using species in which the genes orthologous to SBGs are not sex-biased, the authors show that SBGs have high rates of expression evolution already before becoming SBGs. This suggests that most SBGs evolved under drift and the majority of SBGE (sex-biased gene expression) is evolving neutrally. This is confirmed by the estimated small proportion of SBGs evolving under adaptation (about 20% of SBGs have 5 fold higher expression …
Reviewer #3 (Public Review):
The authors study the leaf transcriptomes of males and females in 10 species of Leucadendron and infer genes expressed significantly differently between males and females (sex-biased genes, hereafter SBGs). Most SBGs in Leucadendron leaves evolved recently, suggesting that SBGs turnover (evolution and reversion) is very high because the genus is ancestrally dioecious since >10My. Using species in which the genes orthologous to SBGs are not sex-biased, the authors show that SBGs have high rates of expression evolution already before becoming SBGs. This suggests that most SBGs evolved under drift and the majority of SBGE (sex-biased gene expression) is evolving neutrally. This is confirmed by the estimated small proportion of SBGs evolving under adaptation (about 20% of SBGs have 5 fold higher expression divergence compared to polymorphism divergence, a mark of relatively recent adaptation). Also, SBGs are more tissue specific (less pleiotropic). Finally, the percentage of SBG is not correlated to the intensity of morphological dimorphism. All these findings go against the classical view that SBGE is driven by sex-specific selection for sexual dimorphism.
The analyses are very cautious with well designed controls and randomizations.
The results support well the conclusions.
This study puts forward the role of drift in sex-biased gene expression, offering a new interpretation of this common evolutionary phenomenon.
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Reviewer #2 (Public Review):
Scharmann et al. present a study of sex-biased gene expression as a function of sexual dimorphism in leaf tissue in the genus Leucadendron. Comparative studies of sex-biased expression across clades are still relatively rare, and this analysis tests some core findings of a recent paper (Harrison et al. 2015). Overall, I like the analysis and think it could be a valuable addition to the literature on sex-biased genes. This is particularly true given the difficulty of cross-species expression comparisons and the paucity of them in plants.
However, there are some critical differences between the Harrison paper and the one here, and I think it would be helpful if the authors present them early in the text. Specifically, Harrison et al. (2015) was primarily focused on gonad tissue, which in animals is the site of …
Reviewer #2 (Public Review):
Scharmann et al. present a study of sex-biased gene expression as a function of sexual dimorphism in leaf tissue in the genus Leucadendron. Comparative studies of sex-biased expression across clades are still relatively rare, and this analysis tests some core findings of a recent paper (Harrison et al. 2015). Overall, I like the analysis and think it could be a valuable addition to the literature on sex-biased genes. This is particularly true given the difficulty of cross-species expression comparisons and the paucity of them in plants.
However, there are some critical differences between the Harrison paper and the one here, and I think it would be helpful if the authors present them early in the text. Specifically, Harrison et al. (2015) was primarily focused on gonad tissue, which in animals is the site of the vast majority of sex-biased genes. In contrast, the authors here focus on vegetative (leaf) tissue, which is analogous to animal somatic tissue. None of the patterns that Harrison et al. (2015) observed and report from the gonad were evidence in the somatic tissue they assessed. Also, by looking at gonadal tissue, Harrison et al. (2015) focused on the tissue that produces gametes, which are thought to be subject to some of the strongest sexual selection pressures. The fairest comparison would be flower tissue in plants, so I am unsure how much of the Harrison results would be expected to hold up in leaf samples. This doesn't mean the authors should do the analyses they present, just that they should be a little more upfront about what they might reasonably expect to find.
There is also a conflation at times in the paper between sexual dimorphism, which the authors can quantify in their leaf samples, and sexual selection. I explain this in more detail below, but to summarize here, I think the expectations for the relationship between sex-biased gene expression and sexual selection versus sexual dimorphism are somewhat distinct.
Finally, I am a little concerned that the low numbers of sex-biased genes, expected from leaf tissue, offer limited power for some of the tests the authors want to do. Harrison et al. (2015) had hundreds of sex-biased genes from the gonad, and this power made it possible to detect subtle patterns. The authors have a few dozen sex-biased genes, and this makes it difficult to know whether their negative results are the result of low statistical power. That they find clear associations between pre-sex-biased genes and rates of evolution is quite impressive given this low power.
-
Reviewer #1 (Public Review):
*A summary of what the authors were trying to achieve.
The study takes advantage of the interesting plant genus Leucadendron to compare gene expression between male vs. female in species with more or less sexual dimorphism. This question was addressed in a somewhat comparable manner in only one previous paper by Harrison et al. 2015 across six bird species. The overarching question is the role of natural selection in sexual dimorphism.
*An account of the major strengths and weaknesses of the methods and results.
-Beside the genus-wide comparison of whole transcriptomes across related species, which makes in itself a strong dataset, the major strength of the analysis is the phylogenetic framework that allows the authors to track the evolution of sex bias through several tens of million years of evolutionary …
Reviewer #1 (Public Review):
*A summary of what the authors were trying to achieve.
The study takes advantage of the interesting plant genus Leucadendron to compare gene expression between male vs. female in species with more or less sexual dimorphism. This question was addressed in a somewhat comparable manner in only one previous paper by Harrison et al. 2015 across six bird species. The overarching question is the role of natural selection in sexual dimorphism.
*An account of the major strengths and weaknesses of the methods and results.
-Beside the genus-wide comparison of whole transcriptomes across related species, which makes in itself a strong dataset, the major strength of the analysis is the phylogenetic framework that allows the authors to track the evolution of sex bias through several tens of million years of evolutionary history. Despite ancestral dioecy in the genus, very few genes show consistent sex bias across several species, with sex-bias being mostly species-specific. Two striking negative results will be of special interest to the community : 1) species with more pronounced sexual dimorphism at the morphological level do not tend to exhibit more pronounced sex-biased gene expression 2) the few genes that do show sex-biased expression were apparently recruited among those with the highest expression variance to begin with, strongly suggesting that sexual selection has not been the main force driving their expression divergence.
-In my view, the main limitation of the work is the use of leaf rather than reproductive tissues, making the comparison to other studies less straightforward to interpret. It is especially important that the expectations for somatic vs gonadic tissues be made a lot clearer in the text. Also, the fact that a single leaf phenotype is measured (specific leaf area) seems arbitrary : one could imagine sexual dimorphism on many other characteristics, yet they are not considered here. The text on p.324 mentions "striking convergence in aspects of morphological dimorphism across the genus", but there is no way for the reader to appreciate the extent of this convergence. Finally, it would be useful to at least make some mention of the sex-determination system in these species, since the expectations would differ if some of the sex-biased genes were linked to sex chromosomes.
*An appraisal of whether the authors achieved their aims, and whether the results support their conclusions.
The analysis is mostly sound, but I am a bit concerned by the arbitrary threshold used to define SBGE. The text on p.305 says that "This result is extremely robust to the choice of threshold", but 1) the results are not reported so it is impossible for the readers to evaluate the basis of this assertion and 2) it is not clear whether robustness of the other results has been evaluated at all. This aspect clearly deserves more attention.
*A discussion of the likely impact of the work on the field, and the utility of the methods and data to the community.
This work will be of interest to the community, as rapid rates of expression evolution would generally be interpreted as the consequence of sex bias, whereas the phylogenetic analysis presented here instead supports the idea that the expression of genes that end up being sex biased were instead intrinsically less constrained to begin with.
-
Evaluation Summary:
This is one of the first studies to investigate sex-biased gene expresion in a broad phylogenetic context, and the first in a plant genus. The findings go against the classical view that sex-biased gene expression is driven by sex-specific selection for sexual dimorphism, and instead suggests that sex-bias preverentially evolved in genes that already had the highest expression variance to begin with. It will broadly appeal to researchers interested in the evolution of sexual dimorphism.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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