Repeated loss of variation in insect ovary morphology highlights the role of development in life-history evolution

The number of offspring an organism can produce is a key component of its evolutionary fitness and life history. Here we perform a test of the hypothesized trade-off between the number and size of offspring using thousands of descriptions of the number of egg-producing compartments in the insect ovary (ovarioles), a common proxy for potential offspring number in insects. We find evidence of a negative relationship between egg size and ovariole number when accounting for adult body size. However, in contrast to prior claims, we note that this relationship is not generalizable across all insect clades, and we highlight several factors that may have contributed to this size-number trade-off being stated as a general rule in previous studies. We reconstruct the evolution of the arrangement of cells that contribute nutrients and patterning information during oogenesis (nurse cells), and show that the diversification of ovariole number and egg size have both been largely independent of their presence or position within the ovariole. Instead, we show that ovariole number evolution has been shaped by a series of transitions between variable and invariant states, with multiple independent lineages evolving to have almost no variation in ovariole number. We highlight the implications of these invariant lineages on our understanding of the specification of ovariole number during development, as well as the importance of considering developmental processes in theories of life-history evolution.

The methods to me appear robust, although I note that I'm not a methodological expert here; I'm a user, rather than a designer of such methods (and I have very limited experience with analyses of evolutionary rate). I would note that it is possible to account for variation within species in a phylogenetic regression within one analysis by using Bayesian mixed models in the MCMCglmm package, rather than using multiple GLS analyses and picking randomly among the data each time. Given that the authors have used Bayesian methods elsewhere in the MS, this might be a suggestion to consider. Nevertheless, I have no inherent problem with the data-shuffling method that the authors have used here. Finally, I also note that the data the authors use from their ref 10, a PhD thesis, are now published (Gilbert & Manica, 2010, American Naturalist; Gilbert 2011, Florida Entomologist), so these may be more appropriate citations here.
The finding that ovariole number and egg volume are not associated unless you account for body size is very interesting. Looking at figure 2a, though, I wonder whether these traits would be associated if you excluded the eusocial Hymenoptera and termites. I think you could make a good case for excluding eusocial queens given that reproductive trade-offs in these groups may be affected by the division of labour seen in eusocial colonies. More broadly on this point (tradeoffs against reproductive investment), I think this finding suggests further hypotheses to do with post-oviposition investment in offspring -whether the relationship between ovariole number and egg size might depend upon investment in each offspring by the parents -maybe a route for further research.
The finding about the evolution of variable versus invariant numbers of ovarioles is fantastically interesting. The authors suggest that "if a trade-off between egg size and fecundity exists, factors beyond variation in ovariole number must contribute to fecundity." I think a pertinent question here might possibly be whether ovariole number *mediates* a trade-off between egg size and lifetime fecundity, i.e. whether you'd see that tradeoff within lineages that carry invariant ovariole numbers (or whether variable numbers somewhat obscure the tradeoff). To this end, I think it might have been a good idea to include multiple regression analyses here with body size/lifetime fecundity included as well as just egg size and ovariole number, rather than a successive set of bivariate ones -I think a number of revealing interactions might have been detectable with the large sample sizes the authors have employed.

MINOR POINTS
Line 148: please define what you mean by maximum clade credibility tree Line 157: simulated Line 204-5 "the slope of the regression is lower" -please clarify whether you mean "lower in magnitude", or "more negative" here.
Line 224 There is no number for the figure or table specified.
LIne 310-4. Please indicate the position of the Lepidoptera within the Neuropteroidea on the figure.
Figure 5. I noticed there is one group within the Neuropteroidea which has apparently nearinvariant ovariole numbers (or at least few that vary) but which do not appear in black -is this worth mentioning or explaining?
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.

Do you have any ethical concerns with this paper? No
Comments to the Author The main aim of "Repeated loss of variation in insect ovary morphology highlights the role of developmental constraint in life-history evolution" by Church et al. is to test a well-known life history prediction, namely that there is a trade off between the number and size of offspring using developmental traits (ovariole number and egg size). The authors use an impressive comparative database that they have compiled from the literature and use sophisticated phylogenetic comparative methods to test this hypothesis. They conclude that the hypothesized relationship is not general across insects, but rather a characteristic of particular groups, like the Drosophilidae. They also explore the relationship between nurse cell arrangement and egg size, which was also insignificant, as well as the dynamics of ovariole number evolution across insects. The most interesting result is that ovariole number is significantly variable in some groups and invariant in others, and that the invariant groups evolved several times independently. This last result, in our view, will be of interest to the broad readership of PRS-B and we recommend its publication. However, we would like the Authors to address the following comments and critiques to help improve the clarity and quality of the manuscript: MAJOR COMMENTS: (1) In their analyses, Church et al. control for body size by calculating phylogenetically controlled residuals of egg size versus body size and residuals of ovariole number versus body size. They then use these residuals to assess the relationship between ovariole number versus body-size. The reason why body-size is frequently "controlled for" is to ensure that the correlation between two variables of interest (in this case ovariole number and egg size) is not due to a third variable (in this case body size). However, at the beginning of the manuscript, the reader is left to assume that there exists a significant relationship between body size and ovariole number and body size and egg size, which must be accounted for. But on lines 222 to 227 (and figure S5), the reader surprisingly learns that there is in fact no significant relationship between ovariole number and body size (with the exception of orthopterans). This leaves that reader wondering about whether there is in fact a significant relationship between egg size and body size, which is actually never shown or reported. Therefore, it becomes unclear whether it is actually necessary to control for body size and using residuals in the first place.
Furthermore, demonstrating whether there is (or is not) a relationship between egg size and body size is a key consideration because from our humble understanding of the trade-off between offspring number and offspring size, is that offspring size is assumed to be correlated to eventual adult size. So, the tradeoff as we understand it would be as follows: the more offspring, the less energy available to invest in the offspring and so the offspring develop into smaller adults (and vice versa). However, if there is no relationship between egg size and adult size, then egg size is not a good proxy of eventual adult size, and is therefore an indicator of another ecological trait that is independent of size in general. If there is a significant indicator of eventual adult size, then we find it confusing why we would 'control' for body size in this case when it is the very thing the tradeoff is trying to assess. Perhaps we have missed something in our logic, so it would be very helpful if this was made more clear in the manuscript and laid out more clearly both in the introduction and the methods.
(2) In some panels, the Authors represent species-level regressions, while in others they present genus-level regressions, and in the main text (lines 104 to 107) they refer to family-level regressions. There are two points for consideration: the first is that the figures should be better labeled as to not confuse readers at which level the regressions are being performed, and the second (more important) consideration is that in the phylogenetic comparative method literature, there is an important pattern they call the "taxon-level affect", which is discussed extensively in Harvey and Pagel (1991) and in the brain to body scaling literature. Therefore, the authors should be aware of the taxon-level effect, discuss it, and perhaps try to explain why at the genus level there appears to be a relationship but that the species-level there is not. This result was dismissed by the Authors as not significant, but it may be more telling than the Authors may realize.
(3) In the section on "Modeling ovariole number evolution" the Authors mainly evaluate a Brownian Motion model and conclude that "a multi-rate Brownian motion model far outperforms a single rate model in fitting the data..." While this is entirely valid, we feel that the analyses and results would be more compelling if the authors considered other models of evolutionary change to explain the existence of variable and invariant insect lineages.
(4) The title of the manuscript states that the Authors' study "highlights the role of developmental constraint in life-history evolution". However there is no mention of the term 'developmental constraint' within the manuscript. It would benefit the discussion to define the concept of 'developmental constraint' and contextualise its place in the literature on evolutionary theory. Particularly, the Authors' finding that low-variation lineages tend to have little variation in ovariole number not just between species, but also within species and between the two ovaries of an individual, is very interesting and warrants further discussion, perhaps in the context of an expanded discussion of developmental constraint. For instance, do the authors view a lack of variation in intraspecific and within-individual ovariole number as evidence of more robust/less plastic ovary development in these species (perhaps due to developmental mechanisms different from the Drosophila paradigm, as they discuss)? How do the authors propose that differences in ovary development might drive or bias evolution of ovariole number across species? MINOR COMMENTS: Introduction: (1) On lines 50 to 51, the Authors state that there is both intra-and interspecific variation of ovariole number across species. However, one of the main assumptions for phylogenetic comparative methods (which don't explicitly take into account intraspecific variation) is that interspecific variation is significantly greater than intraspecific variation. Therefore, the Authors should make this very clear.
(2) The Introduction is quite short. Perhaps Authors would like to mention / discuss whether there are other studies that have used developmental traits to assess the trade off between offspring number and size, and mention what the consensus is based on previous studies. Is this the first test? It would be useful for the reader to situate the results of this study in the context of previous work.
(2) In line 148, 'both' seems to be a typo.
(3) It is not clear how the Authors dealt with data on intra-specific ovariole number variation. In the methods there is no explicit mention of generating measures of intra-specific variation, nor is such data presented in any of the figures, but in lines 329-331 of the results the Authors state that "invariant lineages have near-zero variation when comparing between species, between individuals within a species, and between the left and right ovary within an individual." The Authors should point to the data that support their claim of lower within-species variation in these lineages.

Results:
(1) Lines 224 to 225 the figure and table number are missing.
(2) The Authors should ensure the link to their dataset is correct (doi:10.5061/dryad.59zw3r253).

Recommendation
Major revision is needed (please make suggestions in comments)

Scientific importance: Is the manuscript an original and important contribution to its field? Excellent
General interest: Is the paper of sufficient general interest? Excellent Quality of the paper: Is the overall quality of the paper suitable? Good Is the length of the paper justified? Yes Should the paper be seen by a specialist statistical reviewer? 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.

Do you have any ethical concerns with this paper? No
Comments to the Author The main aim of "Repeated loss of variation in insect ovary morphology highlights the role of developmental constraint in life-history evolution" by Church et al. is to test a well-known life history prediction, namely that there is a trade off between the number and size of offspring using developmental traits (ovariole number and egg size). The authors use an impressive comparative database that they have compiled from the literature and use sophisticated phylogenetic comparative methods to test this hypothesis. They conclude that the hypothesized relationship is not general across insects, but rather a characteristic of particular groups, like the Drosophilidae. They also explore the relationship between nurse cell arrangement and egg size, which was also insignificant, as well as the dynamics of ovariole number evolution across insects. The most interesting result is that ovariole number is significantly variable in some groups and invariant in others, and that the invariant groups evolved several times independently. This last result, in our view, will be of interest to the broad readership of PRS-B and we recommend its publication. However, we would like the Authors to address the following comments and critiques to help improve the clarity and quality of the manuscript: MAJOR COMMENTS: (1) In their analyses, Church et al. control for body size by calculating phylogenetically controlled residuals of egg size versus body size and residuals of ovariole number versus body size. They then use these residuals to assess the relationship between ovariole number versus body-size. The reason why body-size is frequently "controlled for" is to ensure that the correlation between two variables of interest (in this case ovariole number and egg size) is not due to a third variable (in this case body size). However, at the beginning of the manuscript, the reader is left to assume that there exists a significant relationship between body size and ovariole number and body size and egg size, which must be accounted for. But on lines 222 to 227 (and figure S5), the reader surprisingly learns that there is in fact no significant relationship between ovariole number and body size (with the exception of orthopterans). This leaves that reader wondering about whether there is in fact a significant relationship between egg size and body size, which is actually never shown or reported. Therefore, it becomes unclear whether it is actually necessary to control for body size and using residuals in the first place. Furthermore, demonstrating whether there is (or is not) a relationship between egg size and body size is a key consideration because from our humble understanding of the trade-off between offspring number and offspring size, is that offspring size is assumed to be correlated to eventual adult size. So, the tradeoff as we understand it would be as follows: the more offspring, the less energy available to invest in the offspring and so the offspring develop into smaller adults (and vice versa). However, if there is no relationship between egg size and adult size, then egg size is not a good proxy of eventual adult size, and is therefore an indicator of another ecological trait that is independent of size in general. If there is a significant indicator of eventual adult size, then we find it confusing why we would 'control' for body size in this case when it is the very thing the tradeoff is trying to assess. Perhaps we have missed something in our logic, so it would be very helpful if this was made more clear in the manuscript and laid out more clearly both in the introduction and the methods.
(2) In some panels, the Authors represent species-level regressions, while in others they present genus-level regressions, and in the main text (lines 104 to 107) they refer to family-level regressions. There are two points for consideration: the first is that the figures should be better labeled as to not confuse readers at which level the regressions are being performed, and the second (more important) consideration is that in the phylogenetic comparative method literature, there is an important pattern they call the "taxon-level affect", which is discussed extensively in Harvey and Pagel (1991) and in the brain to body scaling literature. Therefore, the authors should be aware of the taxon-level effect, discuss it, and perhaps try to explain why at the genus level there appears to be a relationship but that the species-level there is not. This result was dismissed by the Authors as not significant, but it may be more telling than the Authors may realize.
(3) In the section on "Modeling ovariole number evolution" the Authors mainly evaluate a Brownian Motion model and conclude that "a multi-rate Brownian motion model far outperforms a single rate model in fitting the data..." While this is entirely valid, we feel that the analyses and results would be more compelling if the authors considered other models of evolutionary change to explain the existence of variable and invariant insect lineages.
(4) The title of the manuscript states that the Authors' study "highlights the role of developmental constraint in life-history evolution". However there is no mention of the term 'developmental constraint' within the manuscript. It would benefit the discussion to define the concept of 'developmental constraint' and contextualise its place in the literature on evolutionary theory. Particularly, the Authors' finding that low-variation lineages tend to have little variation in ovariole number not just between species, but also within species and between the two ovaries of an individual, is very interesting and warrants further discussion, perhaps in the context of an expanded discussion of developmental constraint. For instance, do the authors view a lack of variation in intraspecific and within-individual ovariole number as evidence of more robust/less plastic ovary development in these species (perhaps due to developmental mechanisms different from the Drosophila paradigm, as they discuss)? How do the authors propose that differences in ovary development might drive or bias evolution of ovariole number across species?

MINOR COMMENTS:
Introduction: (1) On lines 50 to 51, the Authors state that there is both intra-and interspecific variation of ovariole number across species. However, one of the main assumptions for phylogenetic comparative methods (which don't explicitly take into account intraspecific variation) is that interspecific variation is significantly greater than intraspecific variation. Therefore, the Authors should make this very clear.
(2) The Introduction is quite short. Perhaps Authors would like to mention / discuss whether there are other studies that have used developmental traits to assess the trade off between offspring number and size, and mention what the consensus is based on previous studies. Is this the first test? It would be useful for the reader to situate the results of this study in the context of previous work.
(2) In line 148, 'both' seems to be a typo.
(3) It is not clear how the Authors dealt with data on intra-specific ovariole number variation. In the methods there is no explicit mention of generating measures of intra-specific variation, nor is such data presented in any of the figures, but in lines 329-331 of the results the Authors state that "invariant lineages have near-zero variation when comparing between species, between individuals within a species, and between the left and right ovary within an individual." The Authors should point to the data that support their claim of lower within-species variation in these lineages.

Results:
(1) Lines 224 to 225 the figure and table number are missing.
(2) The Authors should ensure the link to their dataset is correct (doi:10.5061/dryad.59zw3r253).

24-Feb-2021
Dear Dr Church: Your manuscript has now been peer reviewed and the reviews have been assessed by an Associate Editor. The reviewers' comments (not including confidential comments to the Editor) and the comments from the Associate Editor are included at the end of this email for your reference. As you will see, the reviewers and the Editors have raised some concerns with your manuscript and we would like to invite you to revise your manuscript to address them.
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Best wishes, Professor Gary Carvalho mailto: proceedingsb@royalsociety.org Associate Editor Board Member: 1 Comments to Author: Two independent sets of reviews have been provided (noting that one is jointly produced from the same lab), which are generally positive and agree that this presents an interesting and novel piece of research, of appeal to readers of Proceedings B. The finding of ovariole number being variable in some groups and invariant in others, with this invariance having evolved independently multiple times, was deemed to be of particular interest. I agree with this consensus, that this analysis presents a powerful test of a classic life history prediction, and generates a number of new theories and possibilities for future work. I also found the figures to be extremely clear and effective.
Both reviews provide a number of detailed and constructive suggestions to improve the manuscript, and I do not repeat these here. In addition to the reviewers' points, I make the following suggestions: -More clarity on rationale to study the evolution of nurse cells in the Introduction -this forms quite a significant part of the analysis, but only gets one sentence in the introduction (l.53-55). I also noticed that the term 'nurse cells' is not mentioned in the discussion. It likely relates to the point about precursor cells (l.363 onwards), but it would help for non-experts if there was more consistency in terminology. (Note that this point is related to Reviewer 2's general point 4 about more context for 'developmental constraints' mentioned in the title) -l.56-59, you could reiterate which 'hypotheses about reproductive evolution' are being tested here.
-I am not familiar with these phylogenetic regressions, but I was surprised that only p-values and n are reported, and no mention of effect size in the main results. I see that slope estimates are given in the supplementary tables, perhaps this is most efficient in terms of space but if there was some room then I would like to these ranges also provided in the main results.
Reviewer(s)' Comments to Author: Referee: 1 Comments to the Author(s) I think this is a wonderful paper that is unquestionably suitable for publication, subject to a couple of minor suggestions which I detail below. It is of broad appeal to the readers of Proceedings B as it deals with fundamental questions of reproductive allocation, evolution, tempo and mode, and more, for a large taxonomic group of wide appeal to researchers. The authors have compiled a large set of published data on insect ovariole numbers alongside various other published life history traits, and test hypotheses about the evolution of reproductive allocation and reproductive mode. Some of the results are (to me) astonishing, and are likely to stimulate a large number of new hypotheses and research. I would therefore recommend acceptance with minor revisions.
The methods to me appear robust, although I note that I'm not a methodological expert here; I'm a user, rather than a designer of such methods (and I have very limited experience with analyses of evolutionary rate). I would note that it is possible to account for variation within species in a phylogenetic regression within one analysis by using Bayesian mixed models in the MCMCglmm package, rather than using multiple GLS analyses and picking randomly among the data each time. Given that the authors have used Bayesian methods elsewhere in the MS, this might be a suggestion to consider. Nevertheless, I have no inherent problem with the data-shuffling method that the authors have used here. Finally, I also note that the data the authors use from their ref 10, a PhD thesis, are now published (Gilbert & Manica, 2010, American Naturalist; Gilbert 2011, Florida Entomologist), so these may be more appropriate citations here.
The finding that ovariole number and egg volume are not associated unless you account for body size is very interesting. Looking at figure 2a, though, I wonder whether these traits would be associated if you excluded the eusocial Hymenoptera and termites. I think you could make a good case for excluding eusocial queens given that reproductive trade-offs in these groups may be affected by the division of labour seen in eusocial colonies. More broadly on this point (tradeoffs against reproductive investment), I think this finding suggests further hypotheses to do with post-oviposition investment in offspring -whether the relationship between ovariole number and egg size might depend upon investment in each offspring by the parents -maybe a route for further research.
The finding about the evolution of variable versus invariant numbers of ovarioles is fantastically interesting. The authors suggest that "if a trade-off between egg size and fecundity exists, factors beyond variation in ovariole number must contribute to fecundity." I think a pertinent question here might possibly be whether ovariole number *mediates* a trade-off between egg size and lifetime fecundity, i.e. whether you'd see that tradeoff within lineages that carry invariant ovariole numbers (or whether variable numbers somewhat obscure the tradeoff). To this end, I think it might have been a good idea to include multiple regression analyses here with body size/lifetime fecundity included as well as just egg size and ovariole number, rather than a successive set of bivariate ones -I think a number of revealing interactions might have been detectable with the large sample sizes the authors have employed.

MINOR POINTS
Line 148: please define what you mean by maximum clade credibility tree Line 157: simulated Line 204-5 "the slope of the regression is lower" -please clarify whether you mean "lower in magnitude", or "more negative" here. Line 224 There is no number for the figure or table specified.  . I noticed there is one group within the Neuropteroidea which has apparently nearinvariant ovariole numbers (or at least few that vary) but which do not appear in black -is this worth mentioning or explaining?
Referee: 2 Comments to the Author(s) The main aim of "Repeated loss of variation in insect ovary morphology highlights the role of developmental constraint in life-history evolution" by Church et al. is to test a well-known life history prediction, namely that there is a trade off between the number and size of offspring using developmental traits (ovariole number and egg size). The authors use an impressive comparative database that they have compiled from the literature and use sophisticated phylogenetic comparative methods to test this hypothesis. They conclude that the hypothesized relationship is not general across insects, but rather a characteristic of particular groups, like the Drosophilidae. They also explore the relationship between nurse cell arrangement and egg size, which was also insignificant, as well as the dynamics of ovariole number evolution across insects. The most interesting result is that ovariole number is significantly variable in some groups and invariant in others, and that the invariant groups evolved several times independently. This last result, in our view, will be of interest to the broad readership of PRS-B and we recommend its publication. However, we would like the Authors to address the following comments and critiques to help improve the clarity and quality of the manuscript: MAJOR COMMENTS: (1) In their analyses, Church et al. control for body size by calculating phylogenetically controlled residuals of egg size versus body size and residuals of ovariole number versus body size. They then use these residuals to assess the relationship between ovariole number versus body-size. The reason why body-size is frequently "controlled for" is to ensure that the correlation between two variables of interest (in this case ovariole number and egg size) is not due to a third variable (in this case body size). However, at the beginning of the manuscript, the reader is left to assume that there exists a significant relationship between body size and ovariole number and body size and egg size, which must be accounted for. But on lines 222 to 227 (and figure S5), the reader surprisingly learns that there is in fact no significant relationship between ovariole number and body size (with the exception of orthopterans). This leaves that reader wondering about whether there is in fact a significant relationship between egg size and body size, which is actually never shown or reported. Therefore, it becomes unclear whether it is actually necessary to control for body size and using residuals in the first place.
Furthermore, demonstrating whether there is (or is not) a relationship between egg size and body size is a key consideration because from our humble understanding of the trade-off between offspring number and offspring size, is that offspring size is assumed to be correlated to eventual adult size. So, the tradeoff as we understand it would be as follows: the more offspring, the less energy available to invest in the offspring and so the offspring develop into smaller adults (and vice versa). However, if there is no relationship between egg size and adult size, then egg size is not a good proxy of eventual adult size, and is therefore an indicator of another ecological trait that is independent of size in general. If there is a significant indicator of eventual adult size, then we find it confusing why we would 'control' for body size in this case when it is the very thing the tradeoff is trying to assess. Perhaps we have missed something in our logic, so it would be very helpful if this was made more clear in the manuscript and laid out more clearly both in the introduction and the methods.
(2) In some panels, the Authors represent species-level regressions, while in others they present genus-level regressions, and in the main text (lines 104 to 107) they refer to family-level regressions. There are two points for consideration: the first is that the figures should be better labeled as to not confuse readers at which level the regressions are being performed, and the second (more important) consideration is that in the phylogenetic comparative method literature, there is an important pattern they call the "taxon-level affect", which is discussed extensively in Harvey and Pagel (1991) and in the brain to body scaling literature. Therefore, the authors should be aware of the taxon-level effect, discuss it, and perhaps try to explain why at the genus level there appears to be a relationship but that the species-level there is not. This result was dismissed by the Authors as not significant, but it may be more telling than the Authors may realize.
(3) In the section on "Modeling ovariole number evolution" the Authors mainly evaluate a Brownian Motion model and conclude that "a multi-rate Brownian motion model far outperforms a single rate model in fitting the data..." While this is entirely valid, we feel that the analyses and results would be more compelling if the authors considered other models of evolutionary change to explain the existence of variable and invariant insect lineages.
(4) The title of the manuscript states that the Authors' study "highlights the role of developmental constraint in life-history evolution". However there is no mention of the term 'developmental constraint' within the manuscript. It would benefit the discussion to define the concept of 'developmental constraint' and contextualise its place in the literature on evolutionary theory. Particularly, the Authors' finding that low-variation lineages tend to have little variation in ovariole number not just between species, but also within species and between the two ovaries of an individual, is very interesting and warrants further discussion, perhaps in the context of an expanded discussion of developmental constraint. For instance, do the authors view a lack of variation in intraspecific and within-individual ovariole number as evidence of more robust/less plastic ovary development in these species (perhaps due to developmental mechanisms different from the Drosophila paradigm, as they discuss)? How do the authors propose that differences in ovary development might drive or bias evolution of ovariole number across species?

MINOR COMMENTS:
Introduction: (1) On lines 50 to 51, the Authors state that there is both intra-and interspecific variation of ovariole number across species. However, one of the main assumptions for phylogenetic comparative methods (which don't explicitly take into account intraspecific variation) is that interspecific variation is significantly greater than intraspecific variation. Therefore, the Authors should make this very clear.
(2) The Introduction is quite short. Perhaps Authors would like to mention / discuss whether there are other studies that have used developmental traits to assess the trade off between offspring number and size, and mention what the consensus is based on previous studies. Is this the first test? It would be useful for the reader to situate the results of this study in the context of previous work.
(2) In line 148, 'both' seems to be a typo.
(3) It is not clear how the Authors dealt with data on intra-specific ovariole number variation. In the methods there is no explicit mention of generating measures of intra-specific variation, nor is such data presented in any of the figures, but in lines 329-331 of the results the Authors state that "invariant lineages have near-zero variation when comparing between species, between individuals within a species, and between the left and right ovary within an individual." The Authors should point to the data that support their claim of lower within-species variation in these lineages. Results: (1) Lines 224 to 225 the figure and table number are missing.
(2) The Authors should ensure the link to their dataset is correct (doi:10.5061/dryad.59zw3r253).

Referee: 3
Comments to the Author(s) The main aim of "Repeated loss of variation in insect ovary morphology highlights the role of developmental constraint in life-history evolution" by Church et al. is to test a well-known life history prediction, namely that there is a trade off between the number and size of offspring using developmental traits (ovariole number and egg size). The authors use an impressive comparative database that they have compiled from the literature and use sophisticated phylogenetic comparative methods to test this hypothesis. They conclude that the hypothesized relationship is not general across insects, but rather a characteristic of particular groups, like the Drosophilidae. They also explore the relationship between nurse cell arrangement and egg size, which was also insignificant, as well as the dynamics of ovariole number evolution across insects. The most interesting result is that ovariole number is significantly variable in some groups and invariant in others, and that the invariant groups evolved several times independently. This last result, in our view, will be of interest to the broad readership of PRS-B and we recommend its publication. However, we would like the Authors to address the following comments and critiques to help improve the clarity and quality of the manuscript: MAJOR COMMENTS: (1) In their analyses, Church et al. control for body size by calculating phylogenetically controlled residuals of egg size versus body size and residuals of ovariole number versus body size. They then use these residuals to assess the relationship between ovariole number versus body-size. The reason why body-size is frequently "controlled for" is to ensure that the correlation between two variables of interest (in this case ovariole number and egg size) is not due to a third variable (in this case body size). However, at the beginning of the manuscript, the reader is left to assume that there exists a significant relationship between body size and ovariole number and body size and egg size, which must be accounted for. But on lines 222 to 227 (and figure S5), the reader surprisingly learns that there is in fact no significant relationship between ovariole number and body size (with the exception of orthopterans). This leaves that reader wondering about whether there is in fact a significant relationship between egg size and body size, which is actually never shown or reported. Therefore, it becomes unclear whether it is actually necessary to control for body size and using residuals in the first place. Furthermore, demonstrating whether there is (or is not) a relationship between egg size and body size is a key consideration because from our humble understanding of the trade-off between offspring number and offspring size, is that offspring size is assumed to be correlated to eventual adult size. So, the tradeoff as we understand it would be as follows: the more offspring, the less energy available to invest in the offspring and so the offspring develop into smaller adults (and vice versa). However, if there is no relationship between egg size and adult size, then egg size is not a good proxy of eventual adult size, and is therefore an indicator of another ecological trait that is independent of size in general. If there is a significant indicator of eventual adult size, then we find it confusing why we would 'control' for body size in this case when it is the very thing the tradeoff is trying to assess. Perhaps we have missed something in our logic, so it would be very helpful if this was made more clear in the manuscript and laid out more clearly both in the introduction and the methods.
(2) In some panels, the Authors represent species-level regressions, while in others they present genus-level regressions, and in the main text (lines 104 to 107) they refer to family-level regressions. There are two points for consideration: the first is that the figures should be better labeled as to not confuse readers at which level the regressions are being performed, and the second (more important) consideration is that in the phylogenetic comparative method literature, there is an important pattern they call the "taxon-level affect", which is discussed extensively in Harvey and Pagel (1991) and in the brain to body scaling literature. Therefore, the authors should be aware of the taxon-level effect, discuss it, and perhaps try to explain why at the genus level there appears to be a relationship but that the species-level there is not. This result was dismissed by the Authors as not significant, but it may be more telling than the Authors may realize.
(3) In the section on "Modeling ovariole number evolution" the Authors mainly evaluate a Brownian Motion model and conclude that "a multi-rate Brownian motion model far outperforms a single rate model in fitting the data..." While this is entirely valid, we feel that the analyses and results would be more compelling if the authors considered other models of evolutionary change to explain the existence of variable and invariant insect lineages.
(4) The title of the manuscript states that the Authors' study "highlights the role of developmental constraint in life-history evolution". However there is no mention of the term 'developmental constraint' within the manuscript. It would benefit the discussion to define the concept of 'developmental constraint' and contextualise its place in the literature on evolutionary theory. Particularly, the Authors' finding that low-variation lineages tend to have little variation in ovariole number not just between species, but also within species and between the two ovaries of an individual, is very interesting and warrants further discussion, perhaps in the context of an expanded discussion of developmental constraint. For instance, do the authors view a lack of variation in intraspecific and within-individual ovariole number as evidence of more robust/less plastic ovary development in these species (perhaps due to developmental mechanisms different from the Drosophila paradigm, as they discuss)? How do the authors propose that differences in ovary development might drive or bias evolution of ovariole number across species?

MINOR COMMENTS:
Introduction: (1) On lines 50 to 51, the Authors state that there is both intra-and interspecific variation of ovariole number across species. However, one of the main assumptions for phylogenetic comparative methods (which don't explicitly take into account intraspecific variation) is that interspecific variation is significantly greater than intraspecific variation. Therefore, the Authors should make this very clear.
(2) The Introduction is quite short. Perhaps Authors would like to mention / discuss whether there are other studies that have used developmental traits to assess the trade off between offspring number and size, and mention what the consensus is based on previous studies. Is this the first test? It would be useful for the reader to situate the results of this study in the context of previous work.
(2) In line 148, 'both' seems to be a typo.
(3) It is not clear how the Authors dealt with data on intra-specific ovariole number variation. In the methods there is no explicit mention of generating measures of intra-specific variation, nor is such data presented in any of the figures, but in lines 329-331 of the results the Authors state that "invariant lineages have near-zero variation when comparing between species, between individuals within a species, and between the left and right ovary within an individual." The Authors should point to the data that support their claim of lower within-species variation in these lineages.

Results:
(1) Lines 224 to 225 the figure and table number are missing.
(2) The Authors should ensure the link to their dataset is correct (doi:10.5061/dryad.59zw3r253).

26-Mar-2021
Dear Dr Church I am pleased to inform you that your Review manuscript RSPB-2021-0150.R1 entitled "Repeated loss of variation in insect ovary morphology highlights the role of development in life-history evolution" has been accepted for publication in Proceedings B.
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Sincerely, Professor Gary Carvalho Editor, Proceedings B mailto:proceedingsb@royalsociety.org Associate Editor Board Member Comments to Author: I enjoyed reading this manuscript again and commend the authors on their efforts to respond to the reviewers' constructive suggestions and incorporate these, where feasible, into their report. Congratulations -I am confident that this will be of great interest to readers of Proceedings B.
I only have some very minor final comments on phrasing or to slightly improve clarity. I note also that Proceedings B require any data/code to be available during peer review (https://royalsociety.org/journals/authors/author-guidelines/#data), and Dryad allow for such sharing without public release of the dataset (https://datadryad.org/stash/faq#ppr). I encourage the authors to do this for future submissions.
Minor suggestions: -Add short phrase to explain 'nurse cells' in Abstract; for the broad readership of Proceedings B. -l.76 "The taxonomic groups used to search" -&gt; "The taxonomic groups used in the search process [or exercise]" -l.79 Should 'ten publications' be in parentheses? The sentence does not make sense otherwise. -It would be helpful in the methods or results to give summary of the dataset: ovariole number was established for how many unique species, how many genera across how many families? (this could help put the values of 3355 records across 448 publications into more relevant context, this could also be mentioned at l.376) -l.214 would add 'across genera' after 'negative relationship between egg size and ovariole number' (Note that Reviewer 2's major comment 2 had been more clarity on the level of analysis in the figure legends, this should also be reflected in the main text) -l.285 delete comma after 'nurse cells' Author's Response to Decision Letter for (RSPB-2021-0150.R1)

06-Apr-2021
Dear Dr Church I am pleased to inform you that your manuscript entitled "Repeated loss of variation in insect ovary morphology highlights the role of development in life-history evolution" has been accepted for publication in Proceedings B.
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Open Access You are invited to opt for Open Access, making your freely available to all as soon as it is ready for publication under a CCBY licence. Our article processing charge for Open Access is £1700. Corresponding authors from member institutions (http://royalsocietypublishing.org/site/librarians/allmembers.xhtml) receive a 25% discount to Introduction 32 Offspring number is a fundamental parameter in the study of life-history 1 . This number 33 differs widely between organisms 1 , and its variation is the foundation for several 34 hypotheses about life-history evolution, including the prediction that there is an 35 evolutionary trade-off between the number of offspring and their size (e.g. egg size) 1-3 . In 36 insects, the number of egg-producing compartments in the ovary, called ovarioles, has been 37 used as a proxy for potential offspring number in the study of life-history 4-6 . However, 38 without an understanding of the phylogenetic distribution of ovariole number, this 39 hypothesized relationship cannot be assessed across insects. Here we tested for the 40 presence of a general trade-off between ovariole number and egg size by collecting 41 thousands of records of ovariole number from the published literature, placing them in a 42 phylogenetic context, and comparing them to other datasets of insect reproductive 43 morphology. 44 The insect female reproductive system includes a pair of ovaries, each of which contains a 45 number of ovarioles 7 (Fig 1a). Each ovariole consists of an anterior germarium containing 46 the stem cell niche or resting oogonia, developing oocytes arranged in an ontogenic series 47 from anterior to posterior, and a posterior connection to a common oviduct. The number of 48 ovarioles varies across species 6 , and can vary across individuals in a population 4 , as well as 49 between the left and right ovary within a single individual 8 . Therefore total ovariole 50 number may be an even or odd integer for an individual female insect. In addition to 51 variation in the number of ovarioles, the tissue morphology within ovarioles varies across 52 insects, and has been classified into several modes of oogenesis based on the presence and 53 position of special nutritive cells called nurse cells 7 . 54 Here we compiled 3355 records of ovariole number from across 28 orders, 301 families, 55 and 2103 species of insects. We combined these data with published datasets of egg size 9 , 56 fecundity 10,11 , and body size 12 , to test hypotheses about the evolutionary trade-off between 57 offspring size and number. In these analyses we used an existing phylogeny of insects 13 to 58 analyze evolutionary patterns in ovariole number, and found that hypotheses about life-59 history evolution do not hold generally true across insects. We then combined these data 60 with published observations of the mode of oogenesis 7 , and reconstructed the evolutionary 61 history of the presence and position of nurse cells that contribute to the oocyte during 62 oogenesis. We tested whether patterns in the distribution of ovariole number, egg size, or 63 egg shape were driven by the evolution of nurse cells, and found no significant results. 64 Instead we observe that the phylogenetic distribution of ovariole number suggests a model 65 where the developmental mechanisms that govern ovariole number have shifted between 66 variable and invariant states several times over the course of insect evolution. Based on 67 this finding, we propose that the developmental mechanisms used to establish ovariole 68 number in well-studied insects such as Drosophila melanogaster are unlikely to regulate 69 ovariole number in all insects. 70

71
Gathering trait data 72 We searched the published literature for references to insect ovariole number using a 73 predetermined set of 131 search terms, entered into Google Scholar (scholar.google.com) 74 between June and October of 2019. Each search term was comprised of an insect taxonomic 75 group and the words "ovariole number". The taxonomic groups used to search included all 76 insect orders, many large insect families, and taxonomic groups that are well-represented 77 in the insect egg dataset 9 . For each Google Scholar search, we evaluated all publications in 78 the first page of results ten publications. For 61 search terms that had a large number of 79 informative hits, significant representation in the egg dataset, or that corresponded to very 80 speciose groups, we evaluated an additional 20 publications. tree (the tree with highest credibility score from the posterior distribution of the Bayesian 160 analysis). We considered a relationship significant when the p-value was below the 161 threshold 0.01. To assess the robustness of results to uncertainty in phylogenetic 162 relationships, we also repeated these analyses over the posterior distribution of 163 phylogenetic trees and report the number of regressions that gave a significant result (see 164  Table S1). 165 For two comparisons, we validated that our tests had sufficient statistical power using the 166 selected threshold by comparing the distribution of p-values from regressions of observed 167 data to regressions of data simulated under alternative hypotheses. We compared the 168 results of analyses of our observed to those based on simulated data to evaluate the 169 likelihood of false positives (comparing to data simulated under no correlation) and false 170 negatives (comparing to data simulated with strong correlation). 171 Model comparisons of trait evolution were also performed over a posterior distribution 172 and accounting for phenotypic uncertainty. For these analyses, we considered a model to 173 have significantly better fit the data than other models when the difference in the corrected 174 Akaike Information Criterion (AICc) was greater than two in every analysis iteration. 175

176
Ovariole number diversity 177 Ovariole number varies by at least four orders of magnitude across insect species (Fig. 1b). 178 We identify seven insect families with species that have been reported to have more than 179 1,000 total ovarioles, including several eusocial insects (e.g. queens of the termite species 180 Hypotermes obscuriceps, Blattodea: Termitidae 34 , and several ant species, Hymenoptera: 181 Formicidae) 35,36 and non-eusocial insects (e.g. the blister beetle Meloe proscarabaeus, 182 Coleoptera: Meloidae) 37 . We also find two independent lineages that have evolved to have 183 only one functional ovariole: dung beetles in the tribe Scarabaeinae (Coleoptera:  184 Scarabaeidae) 38 , and grass flies in the genus Pachylophus (Diptera: Chloropidae) 39,40 . In 185 these insects one of the two ovaries presumably established during embryogenesis is 186 reported to atrophy during development 40,41 , resulting in an asymmetric adult 187 reproductive system. We also evaluated intraspecific variation in ovariole number, and 188 found that, for species for which it has been reported, the average percent difference 189 number within species is between 10% and 100% of the median value (Fig. S1) comparing egg size and ovariole number across insect species (Fig. 2a, Table S1, p-value 203 0.195, n=306). We also compared egg size and ovariole number, combining data from 204 species within the same genus to increase sample size, and again did not observe a 205 significant relationship (Fig. S2, p-value 0.066, n=482). To verify this finding was not driven 206 by the high ovariole numbers seen in the queens of some eusocial insects, we repeated this 207 comparison excluding insects from families with eusocial representatives, with the same 208 result (Fig. S3, p-value 0.209, n=415). 209 Given that this predicted relationship is often conditioned on body size, which is predicted 210 to limit total potential reproductive investment 21,43 , we combined data on ovariole number 211 and egg size with data on insect adult body mass 10,11,17 and length 12 . When accounting for 212 adult body mass, we observed a significant negative relationship between egg size and 213 ovariole number (Fig. 2b, S4, p-value 0.003, slope -0.399, n=61). To evaluate the robustness 214 of this result, we repeated the analysis 1000 times, taking into account uncertainty in both 215 the phylogeny and trait measurements. Out of 1000 regressions, 995 indicated a significant 216 negative relationship (Table S1). We performed the same comparison accounting for adult 217 body length, and likewise observed a significant negative relationship (Fig. S5, p-value 218 <0.001, slope -0.52, n=126), supported by 966 of 1000 repeated analyses (Table S1). 219 We further explored these results using two methods: First, to evaluate our findings against 220 alternative evolutionary hypotheses, we compared these results to regressions based on 221 simulated data. Our results showed that when considering body size, the slope of the 222 regression of egg size and ovariole number is more negative than we would expect to 223 observe by chance, as assessed by comparing to data simulated with no evolutionary 224 correlation (Fig. S6). However, for both adult body length and dry mass, the slope of the 225 regressions on observed data are not within the range that would be expected under a 226 strong negative correlation (slope of -1 in log-log space, Fig. S6). This suggests the presence 227 of a weak evolutionary relationship between ovariole number and egg size, when 228 accounting for body size. 229 Second, we assessed the relationship between egg size and ovariole number, accounting for 230 body size, within four subclades of insects. We found that across Drosophilidae fly species, 231 egg size is indeed strongly negatively correlated with ovariole number when accounting for 232 body size (Fig. 2c, Table S2, p-value <0.001, slope -0.809, n=30). For grasshoppers and 233 crickets (Orthoptera), beetles (Coleoptera), and wasps (Hymenoptera), we observed no 234 significant relationship between ovariole number and egg size, even when accounting for 235 body size (Fig. 2d, S7, across insects, many of which lay eggs singly and continuously rather than in distinct 264 clutches. 265 Using a previously reported dataset of lifetime fecundity measurements across insects 10,11 , 266 we assessed the relationship between lifetime fecundity and ovariole number. We 267 observed a significant positive relationship (Fig. 3, p- Fig. 1b. 281

Evolution of nurse cells 282
In addition to the number of ovarioles, insect ovary morphology has been classified into 283 several modes of oogenesis based on the presence and position of nutritive cells called 284 nurse cells 7 , (Fig. 4a). Egg formation in the well-studied species D. melanogaster is an 285 example of a meroistic oogenesis mode, meaning that its ovarioles contain nurse cells of 286 germ line origin that are connected to developing oocytes via cytoplasmic bridges 48 . In 287 insects with a polytrophic arrangement, these nurse cells are clonally related and 288 immediately adjacent to each oocyte. An alternative arrangement is seen in telotrophic 289 meroistic ovaries, where oocytes in each ovariole are connected to a common pool of nurse 290 cells located in the germarium 7 . Meroistic ovaries are thought to have evolved from an 291 ancestral panoistic mode, meaning they lack nurse cells 7 . Using a previously published set 292 of descriptions of these oogenesis modes across insects 7 , we reconstructed the 293 evolutionary transitions between these states. Consistent with previous analyses 7 , we 294 found that the ancestral insect likely had panoistic ovaries (lacking nurse cells), with 295 several independent shifts to both telotrophic and polytrophic meroistic modes, and at 296 least two reversals from meroistic back to panoistic (Figs. 4b, S10). 297 Using this ancestral state reconstruction, we then compared models of trait evolution to 298 test whether evolutionary transitions in oogenesis mode helped explain the diversification 299 of ovariole number and egg morphology. We found that, for the traits studied here, models 300 that take into account evolutionary changes in mode of oogenesis do not consistently 301 demonstrate a significant improvement over models that do not take these changes into 302 account ( AIC < 2, Table S5). In other words, the evolution of nurse cells and their position 303 within the ovary do not explain the diversification of egg size, egg shape, or ovariole 304 number. 305 To analyze the robustness of these results to uncertainty in the tree topology and in the 306 inference of ancestral states, we repeated each analysis over a posterior distribution of 307 trees. For egg asymmetry and curvature, but not for volume or aspect ratio, we observed a 308 few iterations where a model that takes into account oogenesis mode evolution was 309 significantly favored over models that did not ( AIC > 2, Table S5). However, this result 310 was infrequent over 100 repetitions of the analysis. We therefore interpret these results as 311 suggestive of a possible relationship between mode of oogenesis and egg asymmetry and 312 curvature, but one which cannot be confirmed given the current data available. 313  Modeling ovariole number evolution 324 Using the dataset compiled here and a previously published phylogeny of insects (Fig.  325 5a) 13 , we modeled the rate of evolutionary change in ovariole number (Figs. S11, S12, S13, 326 S14). We observed substantial rate heterogeneity in the evolution of ovariole number (Fig.  327 S14), meaning that for some lineages ovariole number has evolved rapidly where in others, 328 ovariole number has evolved very slowly or not at all. The most striking example of this are 329 the multiple lineages which have independently evolved invariant or near-invariant 330 ovariole number across taxa (e.g., nearly all Lepidoptera have exactly eight ovarioles, Fig.  331 5b, Lepidoptera are part of Amphiesmenoptera, in cyan), from an ancestral variable state. 332 These invariant lineages were identified by finding regions of the phylogeny that 333 experience extremely low rates of ovariole number diversification (Figs. S14, S15). Using 334 this approach, we found that invariant ovariole numbers have evolved at least nine times 335 independently across insects, with several subsequent reversals from invariant to variable 336 states (Fig. 5a). 337 We find that the rate of evolutionary change in ovariole number is correlated with the 338 number of ovarioles: lineages with relatively low ovariole number also experience 339 relatively low degrees of ovariole number change (Fig. S11). This is evidenced by the fact 340 that, of the nine invariant lineages, none have greater than seven ovarioles per ovary (Fig.  341 5c). However we note that not all insects with low ovariole counts are in invariant lineages; 342 many insects with fewer than 14 total ovarioles are in lineages with relatively high rates of 343 intra-and interspecific ovariole number variation (Fig. 5) 344 The distribution of ovariole numbers across insects is enriched for even numbers of total 345 ovarioles (Fig. 5c). While many insects show asymmetries in the number of ovarioles 346 between the left and right ovaries, all of the invariant lineages are symmetric (at 4, 6, 8, 10, 347 12, and 14 total ovarioles). Additionally, for the insects identified as part of invariant 348 lineages, none have any reported intraspecific variation in ovariole number. Therefore, 349 invariant lineages have near-zero variation when comparing between species, between 350 individuals within a species, and between the left and right ovary within an individual. 351 Using these results, we propose a multi-rate model, where the rate of ovariole number 352 evolution differs based on the evolution of a discrete trait representing invariant or 353 variable status. We propose that the evolution of this discrete trait is governed by a model 354 where the likelihood of transitions from a variable to an invariant state is negatively 355 correlated with the current number of ovarioles. Here we demonstrate that a multi-rate 356 Brownian motion model far outperforms a single rate model in fitting the data ( AICc 357 1770.93). In addition, using a parametric bootstrap to evaluate model fit, we find evidence 358 that processes beyond Brownian Motion processes are likely at play (Fig. S11) 31 . We 359 suggest that as researchers continue to develop non-Gaussian models for continuous trait 360 evolution 49 , those models will be useful for describing the evolution of ovariole number. 361