Direct and indirect effects of male genital elaboration in female seed beetles

Our understanding of coevolution between male genitalia and female traits remains incomplete. This is perhaps especially true for genital traits that cause internal injuries in females, such as the spiny genitalia of seed beetles where males with relatively long spines enjoy a high relative fertilization success. We report on a new set of experiments, based on extant selection lines, aimed at assessing the effects of long male spines on females in Callosobruchus maculatus. We first draw on an earlier study using microscale laser surgery, and demonstrate that genital spines have a direct negative (sexually antagonistic) effect on female fecundity. We then ask whether artificial selection for long versus short spines resulted in direct or indirect effects on female lifetime offspring production. Reference females mating with males from long-spine lines had higher offspring production, presumably due to an elevated allocation in males to those ejaculate components that are beneficial to females. Remarkably, selection for long male genital spines also resulted in an evolutionary increase in female offspring production as a correlated response. Our findings thus suggest that female traits that affect their response to male spines are both under direct selection to minimize harm but are also under indirect selection (a good genes effect), consistent with the evolution of mating and fertilization biases being affected by several simultaneous processes.

lacking. There is now a large literature focusing on sexual conflict in this species, and the results are complex. However, I feel some discussion of this complexity is useful for putting these results into context. For example, Cayetano & Bonduriansky (2015) explicitly test whether male genital spine length was condition dependent, but did not find an effect. This result is not mentioned when considering condition-dependence in the introduction (lines 88-89), or in the discussion (line 323). I would therefore like to see an expanded discussed of this and other relevant studies (incl. Hotzy et al., 2012) to better put these results into context. Including this context would not diminish the results of this study-indeed, the surprising result of this study (that both sexual conflict and good-genes effects may influence spine evolution) may partly explain why results of previous studies have been so mixed. 4.
I'm not entirely comfortable with the removal of the two outlier females in the phenotypic engineering experiment (lines 162-164), especially given that the result this pertains to is barely significant. Is R >2.5 a common metric? Regardless, I would prefer you to present the results without removing these females 5.
Several interesting results are presented in the supplementary material, but it is not obvious why. Does the manuscript as it stands overrun the Proc B space limits? If not, I think you should add these methods and results to the main text Other comments 1.
Lines 169-171: When were the experiments using the sexual-selection lines done? Around 2012? 4.
Line 213: please list the maximal value so readers do not have to access the reference 5.
Lines 238-239: why no figure for this significant effect? 6.
Lines 290-292: other authors have suggested that negative effects on female fitness may be hard to detect because they are very small when the species is at a coevolutionary equilibrium. Do these results counter that argument?
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 RSPB 2021-0578 Direct and indirect effects of male genital elaboration in seed beetles This paper examines the effect of male genital spine morphology on direct and indirect female fitness. The results are potentially very important because the effects of sexual conflict have been wrongly assumed to be only through direct fitness effects, while clearly there can be an impact on indirect fitness. The paper relies on spine ablation data from a previous published experiment adding body size as a novel aspect of the work, and from selection experiments for short and long spines. While the questions asked in this manuscript are indeed exciting, I had a hard time following the different experiments and their results. There is a lot of simplification here which seems necessary to make the paper more readable, but this comes at the expense of clarity of which crosses the authors are talking about in specific areas. I had to re-read the methods and result several times to try and figure out exactly what was done here. In addition, I have a hard time with the presentation of both direct costs and direct benefits, in that the direct benefits results come from experiments where females were kept with the same male in monogamy for life, a situation that is highly unlikely in nature, and may have resulted in males modifying other behaviors or seminal fluids, that would mitigate the damage that is normally inflicted by the spines in a single mating, and the authors present good evidence for this in the discussion. That means that the result of increased in offspring production could simply be the result of male modification of ejaculate fluid and have nothing to do with spine length. The section on indirect fitness is relies on female offspring production which is indistinguishable from fecundity as measured for direct benefits, with no justification of how these are interpreted as being different. While I fully understand the limitations of measuring the fitness of offspring produced by the experiments, that would have been the appropriate measure, and in that sense the results are oversold. The full results of all the statistical models should be included in supplementary materials.
Line 105-106: How does this experiment overcome these challenges though? You are still only manipulating males so the confounding effects of female resistance and other male behaviors remain.
Lines 127-128. If the experiments are distinct, how can the data be the same? This is very confusing! Line 139-140. This study should stand on its own. Please provide brief description of those procedures using the reference for further details, not instead of an explantion.
Line 163. Please conduct the analyses with and without your outliers. Outliers are part of the normal biological variation and in an experiment with relatively small sample sizes such as this one, they may change the outcome of the statistics altogether.
Lines 180-188. What is baseline female? A female from a non-selected line? And then what type of selected males? S, L both? There are 20 replicates per selection and then you selected one female to mate with males from each of these replicates? Why are there six lines? I thought there were 2 lines with 20 replicates each? Confusing! Please describe better.
Line 217. The questions that were meant to be addressed by this experiment are all important and relevant to the interpretation of the current results presented in this paper. This experiment should not be part of the supplementary materials, but should be included in full in the manuscript.
Line 233. Name the covariates here. I believe they are male body size, female body size and number of eggs laid between matings?
Line 248: Average lifetime fitness production is 17% higher in L mated females than S mated females? How does that happen when the females produce 15% fewer eggs when mated with L males?
Line 263: Females from L lines had a 6% higher production of offspring regardless of the phenotype of the male with whom they mated right?
Line 269-270. How would this genetic correlation come to be? It could be that in lines where males have longer spines, female defenses such as kicking become more important and larger females are better at kicking… The "direct benefits" need further justification. How likely would it be in nature that a female would become fully monogamous and end up mating with a single male per life over multiple reproductive bouts? This is one of the confounding factors that the authors mentioned at the beginning Line 286-287: What are those female traits? Important to include here.
Line 290: the phenotypic engineering did not elongate the spines! It shortened them!! Am I missing something here?
Line 302-327: This explanation confirms my suspicion that males are changing the composition of their ejaculate when they are in this artificially enforced monogamy for a lifetime situation, and therefore the effect of the spine length is completely unknown! The increase in offspring production by females mated to Long spined males for life could be mediated by changes in ejaculate fluid as agued here.
Line 334-336. Exactly the problem here. The indirect benefit has to be through the descendants, and that was not shown in this experiment. You measured the same thing as direct and indirect benefits.
Line 368-371: While I agree wholeheartedly with the idea, this is not shown in this study.

18-Apr-2021
Dear Professor Arnqvist: I am writing to inform you that your manuscript RSPB-2021-0578 entitled "Direct and indirect effects of male genital elaboration in female seed beetles" has, in its current form, been rejected for publication in Proceedings B.
This action has been taken on the advice of referees, who have recommended that substantial revisions are necessary. With this in mind we would be happy to consider a resubmission, provided the comments of the referees are fully addressed. However please note that this is not a provisional acceptance.
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Sincerely, Professor Gary Carvalho mailto: proceedingsb@royalsociety.org Associate Editor Board Member: 1 Comments to Author: The two reviewers were somewhat mixed in their enthusiasm, though both raise concerns over how much of the data and analyses presented here are novel, and how much has been previously published. I was also somewhat confused by this, and without a clearer understanding, I think it is quite difficult to determine the novelty presented in this manuscript by the authors. Both reviewers also ask for a more thorough context of previous empirical findings. Reviewer 1 also raises concerns about the removal of two outlier females from the analysis. Reviewer 2 is far more critical, and raises key points about the limits that the experimental design place on the interpretation. These are, in my opinion, valid, especially the lack of distinction between female fecundity and female offspring production.
Reviewer(s)' Comments to Author: Referee: 1 Comments to the Author(s) The manuscript reports a series of experiments examining the direct and indirect effects that male genital spines have on female fitness in the seed beetle Callosobruchus maculatus. The conflict between males and females that arises due to this harmful genital trait have been well-studied in this species, though results are mixed. By experimentally shortening male spines, the authors show for the first time that males with long spines significantly reduce female fecundity. However, artificial selection on male spine length finds the opposite result-reference females mating to males with long spines have higher fecundity, and selection for increased male spine length led to a correlated increase in female fecundity. These results suggest that females actually gain indirect benefits to mating with males with long spines, and that sexual coevolution in this species is driven by both sexual antagonism and good genes sexual selection.
Overall, this is a very well-presented study describing an elegant series of experiments testing these ideas. Further, the results significantly improve our understanding of sexual coevolution in this model species, and will be relevant to the study of sexual conflict and sexual selection more generally. However, I have some major concerns with how the study is presented, and how it links to previous studies, which I feel the authors need to address.
Main comments 1. This study builds heavily from a study from this group from 2012 (Hotzy et al., 2012). This means that several of the approaches used in this study are not novel (though some of the results are). Specifically, the previous study used the same phenotypic engineering of male spine length (but found no significant difference between manipulated and control males), and compared the same experimental evolution lines (though female fitness was not recorded in the earlier study). This in and of itself is not a problem. However, for me this relationship was not made very transparent in the present paper, at least in the introduction (e.g. lines 105-111). I would go so far as to say that, for a reader not familiar with this previous study, this link would very much not be apparent from a casual read of the paper. So, to give due credit to this earlier work, and to put this study in its proper context, I think the links between these two studies needs to be much clearer in the introduction, and throughout. 2. Related to above, at several points that manuscript mentions using partly the same data from Hotzy et al., 2012 (lines 128-130, 240-242). However, it is not clear to me exactly which data were re-used. This needs to be made clearer here and in the introduction, along with a justification for why this way done. 3. More generally, I found the discussion of previous empirical studies in this species to be lacking. There is now a large literature focusing on sexual conflict in this species, and the results are complex. However, I feel some discussion of this complexity is useful for putting these results into context. For example, Cayetano & Bonduriansky (2015) explicitly test whether male genital spine length was condition dependent, but did not find an effect. This result is not mentioned when considering condition-dependence in the introduction (lines 88-89), or in the discussion (line 323). I would therefore like to see an expanded discussed of this and other relevant studies (incl. Hotzy et al., 2012) to better put these results into context. Including this context would not diminish the results of this study-indeed, the surprising result of this study (that both sexual conflict and good-genes effects may influence spine evolution) may partly explain why results of previous studies have been so mixed. 4. I'm not entirely comfortable with the removal of the two outlier females in the phenotypic engineering experiment (lines 162-164), especially given that the result this pertains to is barely significant. Is R >2.5 a common metric? Regardless, I would prefer you to present the results without removing these females 5. Several interesting results are presented in the supplementary material, but it is not obvious why. Does the manuscript as it stands overrun the Proc B space limits? If not, I think you should add these methods and results to the main text Other comments 1. Line 104: remove extra 'of' 2. Lines 181-183: why were two males used here? 3. Lines 169-171: When were the experiments using the sexual-selection lines done? Around 2012? 4. Line 213: please list the maximal value so readers do not have to access the reference 5. Lines 238-239: why no figure for this significant effect? 6. Lines 290-292: other authors have suggested that negative effects on female fitness may be hard to detect because they are very small when the species is at a coevolutionary equilibrium. Do these results counter that argument?
Referee: 2 Comments to the Author(s) RSPB 2021-0578 Direct and indirect effects of male genital elaboration in seed beetles This paper examines the effect of male genital spine morphology on direct and indirect female fitness. The results are potentially very important because the effects of sexual conflict have been wrongly assumed to be only through direct fitness effects, while clearly there can be an impact on indirect fitness. The paper relies on spine ablation data from a previous published experiment adding body size as a novel aspect of the work, and from selection experiments for short and long spines. While the questions asked in this manuscript are indeed exciting, I had a hard time following the different experiments and their results. There is a lot of simplification here which seems necessary to make the paper more readable, but this comes at the expense of clarity of which crosses the authors are talking about in specific areas. I had to re-read the methods and result several times to try and figure out exactly what was done here. In addition, I have a hard time with the presentation of both direct costs and direct benefits, in that the direct benefits results come from experiments where females were kept with the same male in monogamy for life, a situation that is highly unlikely in nature, and may have resulted in males modifying other behaviors or seminal fluids, that would mitigate the damage that is normally inflicted by the spines in a single mating, and the authors present good evidence for this in the discussion. That means that the result of increased in offspring production could simply be the result of male modification of ejaculate fluid and have nothing to do with spine length. The section on indirect fitness is relies on female offspring production which is indistinguishable from fecundity as measured for direct benefits, with no justification of how these are interpreted as being different. While I fully understand the limitations of measuring the fitness of offspring produced by the experiments, that would have been the appropriate measure, and in that sense the results are oversold. The full results of all the statistical models should be included in supplementary materials.
Line 105-106: How does this experiment overcome these challenges though? You are still only manipulating males so the confounding effects of female resistance and other male behaviors remain.
Lines 127-128. If the experiments are distinct, how can the data be the same? This is very confusing! Line 139-140. This study should stand on its own. Please provide brief description of those procedures using the reference for further details, not instead of an explantion.
Line 163. Please conduct the analyses with and without your outliers. Outliers are part of the normal biological variation and in an experiment with relatively small sample sizes such as this one, they may change the outcome of the statistics altogether.
Lines 180-188. What is baseline female? A female from a non-selected line? And then what type of selected males? S, L both? There are 20 replicates per selection and then you selected one female to mate with males from each of these replicates? Why are there six lines? I thought there were 2 lines with 20 replicates each? Confusing! Please describe better.
Line 217. The questions that were meant to be addressed by this experiment are all important and relevant to the interpretation of the current results presented in this paper. This experiment should not be part of the supplementary materials, but should be included in full in the manuscript.
Line 233. Name the covariates here. I believe they are male body size, female body size and number of eggs laid between matings?
Line 248: Average lifetime fitness production is 17% higher in L mated females than S mated females? How does that happen when the females produce 15% fewer eggs when mated with L males?
Line 263: Females from L lines had a 6% higher production of offspring regardless of the phenotype of the male with whom they mated right?
Line 269-270. How would this genetic correlation come to be? It could be that in lines where males have longer spines, female defenses such as kicking become more important and larger females are better at kicking… The "direct benefits" need further justification. How likely would it be in nature that a female would become fully monogamous and end up mating with a single male per life over multiple reproductive bouts? This is one of the confounding factors that the authors mentioned at the beginning Line 286-287: What are those female traits? Important to include here.
Line 290: the phenotypic engineering did not elongate the spines! It shortened them!! Am I missing something here?
Line 302-327: This explanation confirms my suspicion that males are changing the composition of their ejaculate when they are in this artificially enforced monogamy for a lifetime situation, and therefore the effect of the spine length is completely unknown! The increase in offspring production by females mated to Long spined males for life could be mediated by changes in ejaculate fluid as agued here.
Line 334-336. Exactly the problem here. The indirect benefit has to be through the descendants, and that was not shown in this experiment. You measured the same thing as direct and indirect benefits.
Line 368-371: While I agree wholeheartedly with the idea, this is not shown in this study.

Quality of the paper: Is the overall quality of the paper suitable? Excellent
Is the length of the paper justified? Yes Should the paper be seen by a specialist statistical reviewer? No

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 I thank the authors for responding to my comments. Happily, I feel the authors have addressed all of my comments in a satisfactory way. On second reading, I think this really is a great paper, which has become much more apparent after the different sources of data have been clarified. I am also much happier with the exclusion of the one outlier female after her behaviour was described in more detail.
I have just a few small suggestions: 1.
You should add the species name somewhere in the abstract 2.
Lines 189-190: I think you should rephrase this to something like "both the original and additional experiments were run in 2012". As it is, the sentence is still a little ambiguous 3.
Lines 375-377. Would it be correct to spell out this result even more plainly as "females mated to long-spine males lines have daughters that are larger and have higher fecundity"?

Review form: Reviewer 3
Recommendation Accept as is Scientific importance: Is the manuscript an original and important contribution to its field? Excellent

General interest: Is the paper of sufficient general interest? Good
Quality of the paper: Is the overall quality of the paper suitable? Excellent 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 This paper examines the role of harmful male genital spines on female fitness in seed beetles, and presents significant results. I am happy the authors resurrected the data from this second set of experiments (following Hotzy et al 2012), as the results herein signigicantly improve our understanding the interplay of costs and benefits in sexual coevolution. It is exciting to see actual, measured indirect genetic benefits in females, especially when they are paired with direct costs. I believe this iteration of the manuscript has addressed the concerns raised in previous review. In particular I appreciate the efforts you have gone to in explaining the various experiments and how they differ from the Hotzy et al (2012) study. This has been a difficult paper to digest, but once digested highly rewarding. This is a significant contribution to our understanding of the interplay of competing selective forces in driving the evolution of mating systems. Thank you.

01-Jun-2021
Dear Professor Arnqvist I am pleased to inform you that your manuscript RSPB-2021-1068 entitled "Direct and indirect effects of male genital elaboration in female seed beetles" has been accepted for publication in Proceedings B.
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Sincerely, Professor Gary Carvalho mailto: proceedingsb@royalsociety.org Associate Editor Board Member Comments to Author: Many thanks to the authors for their careful revision and thoughtful response letter. The reviewers and I all agree that the revised manuscript is much clearer. Reviewer 1 has some very minor suggestions, but I am otherwise happy to recommend acceptance.
Reviewer(s)' Comments to Author: Referee: 1 Comments to the Author(s). I thank the authors for responding to my comments. Happily, I feel the authors have addressed all of my comments in a satisfactory way. On second reading, I think this really is a great paper, which has become much more apparent after the different sources of data have been clarified. I am also much happier with the exclusion of the one outlier female after her behaviour was described in more detail.
I have just a few small suggestions: 1. You should add the species name somewhere in the abstract 2. Lines 189-190: I think you should rephrase this to something like "both the original and additional experiments were run in 2012". As it is, the sentence is still a little ambiguous 3. Lines 375-377. Would it be correct to spell out this result even more plainly as "females mated to long-spine males lines have daughters that are larger and have higher fecundity"? Referee: 3 Comments to the Author(s). This paper examines the role of harmful male genital spines on female fitness in seed beetles, and presents significant results. I am happy the authors resurrected the data from this second set of experiments (following Hotzy et al 2012), as the results herein signigicantly improve our understanding the interplay of costs and benefits in sexual coevolution. It is exciting to see actual, measured indirect genetic benefits in females, especially when they are paired with direct costs. I believe this iteration of the manuscript has addressed the concerns raised in previous review. In particular I appreciate the efforts you have gone to in explaining the various experiments and how they differ from the Hotzy et al (2012) study. This has been a difficult paper to digest, but once digested highly rewarding. This is a significant contribution to our understanding of the interplay of competing selective forces in driving the evolution of mating systems. Thank you. You can expect to receive a proof of your article from our Production office in due course, please check your spam filter if you do not receive it. PLEASE NOTE: you will be given the exact page length of your paper which may be different from the estimation from Editorial and you may be asked to reduce your paper if it goes over the 10 page limit.
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Authors' detailed responses to comments given (MS RSPB-2021-0578)
We were very happy to see that all three readers of our manuscript found our contribution of great interest and potentially suitable for publication in Proc B.
We are also very grateful indeed for the thorough assessment of our work and for the effort spent by the reviewers on our behalf. We have now prepared a thoroughly revised version which, as you will see, is very closely aligned with the suggestions provided by the reviewers of the previous version. We feel that we have been able to accommodate all suggestions given and that our contribution has improved significantly as a result. We hope that you will agree. We stress here that if you in any case feel that our response is unclear or that additional revisions are needed to further improve our contribution, we would of course be more than happy to discuss such issues and to further revise our manuscript should you consider this desirable or necessary.
Please find our responses to all comments given detailed below. Here, the actual comments are in italicized and in blue font and our responses are given in normal black font, to increase clarity and to facilitate your editorial work. All changes and revisions are highlighted using track-changes in MS Word in a dedicated PDF copy of our manuscript, to make our revisions maximally transparent.

Associate Editor
The two reviewers were somewhat mixed in their enthusiasm, though both raise concerns over how much of the data and analyses presented here are novel, and how much has been previously published. I was also somewhat confused by this, and without a clearer understanding, I think it is quite difficult to determine the novelty presented in this manuscript by the authors. Both reviewers also ask for a more thorough context of previous empirical findings. Reviewer 1 also raises concerns about the removal of two outlier females from the analysis. Reviewer 2 is far more critical, and raises key points about the limits that the experimental design place on the interpretation. These are, in my opinion, valid, especially the lack of distinction between female fecundity and female offspring production.
Thank you for this summary. Indeed, although both readers saw much merit in our work, as the AE noted, both also requested a clearer delineation of exactly how our data relates to the previous study and data of Hotzy et al [ref 35]. We had actually strived to be clear over this in our original version, but the reviewers' reports shows that we had simply not succeeded in being sufficiently clear. We sincerely apologize for this. Please find the revised version substantially improved in clarity in this regard. Hopefully, it should now be clear to all readers exactly what our data and experiments are and how they relate to previous work in this model system. Please see our detailed responses to the reviewers below. As you will see, all data involving the selection lines are entirely novel and, of course, previously unpublished. We agree with the AE in that both reviewers gave series valid and constructive comments and we have revised our MS in close agreement with these comments. Again, please see our responses below.

Referee: 1
The manuscript reports a series of experiments examining the direct and indirect effects that male genital spines have on female fitness in the seed beetle Callosobruchus maculatus. The conflict between males and females that arises due to this harmful genital trait have been well-studied in this species, though results are mixed. By experimentally shortening male spines, the authors show for the first time that males with long spines significantly reduce female fecundity. However, artificial selection on male spine length finds the opposite resultreference females mating to males with long spines have higher fecundity, and selection for increased male spine length led to a correlated increase in female fecundity. These results suggest that females actually gain indirect benefits to mating with males with long spines, and that sexual coevolution in this species is driven by both sexual antagonism and good genes sexual selection.

Overall, this is a very well-presented study describing an elegant series of experiments testing these ideas. Further, the results significantly improve our understanding of sexual coevolution in this model species, and will be relevant to the study of sexual conflict and sexual selection more generally. However, I have some major concerns with how the study is presented, and how it links to previous studies, which I feel the authors need to address.
We were very happy to see that the reviewer also found our results interesting and important for our understanding of sexual coevolution in our model system as well as for sexual selection more generally. Needless to say, we share this view. We have addressed all of the reviewer's comments in our revision. (Hotzy et al., 2012). This means that several of the approaches used in this study are not novel (though some of the results are). Specifically, the previous study used the same phenotypic engineering of male spine length (but found no significant difference between manipulated and control males), and compared the same experimental evolution lines (though female fitness was not recorded in the earlier study). This in and of itself is not a problem. However, for me this relationship was not made very transparent in the present paper, at least in the introduction (e. g. lines 105-111). I would go so far as to say that, for a reader not familiar with this previous study, this link would very much not be apparent from a casual read of the paper. So, to give due credit to this earlier work, and to put this study in its proper context, I think the links between these two studies needs to be much clearer in the introduction, and throughout.

Main comments 1. This study builds heavily from a study from this group from 2012
We thank the reviewer for this general comment -as said above, we are most sympathetic to this request (see our comments to the AE above). We actually felt that this was clear in the original version, but on a second reading we know realize that these aspects were not at all sufficiently clear (in part, we note, reflecting an effort to restrict the length of the text). As a result of this comment, you will now find the text revised in many places in the MS (including but not restricted to former lines 105-111), with the explicit purpose of increasing clarity. Hopefully, you will now find the methods maximally transparent. Several sections have been entirely rewritten.
Briefly, as the reviewer correctly notes, the selection lines used are the same selection lines previously used in distinct assays focused on male competitive fertilization success. During the publication of that work (Hotzy et al., 2012), we conducted two additional, novel and independent assays of female fitness during 2012 (!) before the lines were discontinued. However, the results of the new experiments were, due to a series of unfortunate reasons, left in the freezer and the file drawer for several years. These have now been processed. It is the results of these additional experiments (none of which have previously been analysed or published, of course) that we now present here. We agree with the reviewer that the fact that some time has passed since the collection of data cannot, of course, in and of itself be a problem.
2. Related to above, at several points that manuscript mentions using partly the same data from Hotzy et al., 2012 (lines 128-130, 240-242). However, it is not clear to me exactly which data were re-used. This needs to be made clearer here and in the introduction, along with a justification for why this way done.
As said above, we agree entirely. Please find these issues and analyses greatly clarified and motivated in our revised version.
3. More generally, I found the discussion of previous empirical studies in this species to be lacking. There is now a large literature focusing on sexual conflict in this species, and the results are complex. However, I feel some discussion of this complexity is useful for putting these results into context. For example, Cayetano & Bonduriansky (2015) explicitly test whether male genital spine length was condition dependent, but did not find an effect. This result is not mentioned when considering condition-dependence in the introduction (lines 88-89), or in the discussion (line 323). I would therefore like to see an expanded discussed of this and other relevant studies (incl. Hotzy et al., 2012) to better put these results into context.

Including this context would not diminish the results of this study-indeed, the surprising result of this study (that both sexual conflict and good-genes effects may influence spine evolution) may partly explain why results of previous studies have been so mixed.
Point well taken. The original version reflected an effort to restrict the length of the text. We agree, however, and have thus somewhat expanded the discussion of these system-specific topics in our MS, for example in the positions highlighted by the reviewer. We note that we had already cited Cayetano & Bonduriansky (2015) in our original version, but we have now lifted this ref to the introduction as suggested. engineering experiment (lines 162-164), especially given that the result this pertains to is barely significant. Is R >2.5 a common metric? Regardless, I would prefer you to present the results without removing these females.

I'm not entirely comfortable with the removal of the two outlier females in the phenotypic
Assessing and dealing with outliers can, depending on the data, be a critical part of sound data analysis. If, for example, a sick or otherwise aberrant female is included in a study of the effects of treatment on a response, then the inclusion of this female can seriously bias the estimate of the effect of the treatment. In our original version, we used an absolute value of the standardized residual R > 2.5. This is a commonly used cutoff for medium sample sizes, and some 98.75% of all "sound" observations should fall within this delineation under the standard normal expectations. The probability of finding at least one "sound" female residing outside this delineation when sampling 48 females (as we did) is P = 0.45 [i.e., 1 -(0.9875^48)].
The reviewer is, however, "not entirely comfortable with this". To meet this concern, we have dramatically increased the delineation of outliers to, in effect, an absolute value of the standardized residual R > 5.0. Here, the probability of finding at least one "sound" female residing outside this new delineation when sampling 48 females is P = 0.00002. We can thus be absolutely certain that any such female is aberrant. This definition leaves a single female (instead of two as before) defined as an outlier (R = 5.6). This female layed only one single egg after her second mating, which was well outside the range of all other females (range = 13 -87; average = 42.2, SD = 15.6). We feel that including this clearly aberrant female would generate a biased analysis and we would not feel comfortable doing so. We hope that you can agree that this is reasonable.
Trimming data sets from outliers but failing to report this is a widely acknowledged problem in science. We feel that acknowledgement of the importance of dealing with outliers for sound data analysis and explicit definitions of outliers are both essential, as is maximal transparency and clarity in this regard. Please find this detailed in the revised results section. As you will see, defining only one instead of two females as outliers did in no case affect our ability to reject null hypotheses. In fact, the P value for the test of the focal hypotheses was reduced (from P = 0.046 to P = 0.029).

Several interesting results are presented in the supplementary material, but it is not obvious why. Does the manuscript as it stands overrun the Proc B space limits? If not, I think you should add these methods and results to the main text
We chose to place the details of these, admittedly interesting, experiments in the SM because (1) they are truly supplemental [i.e., they do not address how genital spines affect females] and (2) we wish not to exceed the Proc B page limit, as the reviewer suspected. We have nevertheless tried to accommodate this comment by including a brief account of the main results in the text of the revised version. We hope you feel that this is an acceptable compromise.

Lines 181-183: why were two males used here?
Primarily to elevate the net cost of mating.

Lines 169-171: When were the experiments using the sexual-selection lines done? Around 2012?
Yes, 2012. This has now been specified. Well, this would be a plot of two means (the numbers and SE's are given at the top of the paragraph), which we honestly feel would add little but length to our contribution.
6. Lines 290-292: other authors have suggested that negative effects on female fitness may be hard to detect because they are very small when the species is at a coevolutionary equilibrium. Do these results counter that argument?
Good point -we have included a brief discussion on this in this paragraph.

Referee: 2 Comments to the Author(s) RSPB 2021-0578 Direct and indirect effects of male genital elaboration in seed beetles
This paper examines the effect of male genital spine morphology on direct and indirect female fitness. The results are potentially very important because the effects of sexual conflict have been wrongly assumed to be only through direct fitness effects, while clearly there can be an impact on indirect fitness. The paper relies on spine ablation data from a previous published experiment adding body size as a novel aspect of the work, and from selection experiments for short and long spines.
We are very happy that the reviewer recognizes the importance of our results and the urgency to communicate these findings, as they modulate and supplement the interpretation of sexual antagonism in an important model system.
While the questions asked in this manuscript are indeed exciting, I had a hard time following the different experiments and their results. There is a lot of simplification here which seems necessary to make the paper more readable, but this comes at the expense of clarity of which crosses the authors are talking about in specific areas. I had to re-read the methods and result several times to try and figure out exactly what was done here.
We apologize for this confusion -we fully acknowledge that the original version was not written in a sufficiently clear manner (see comments above). Hopefully, you will find clarity significantly improved in the revised version.

In addition, I have a hard time with the presentation of both direct costs and direct benefits,
in that the direct benefits results come from experiments where females were kept with the same male in monogamy for life, a situation that is highly unlikely in nature, and may have resulted in males modifying other behaviors or seminal fluids, that would mitigate the damage that is normally inflicted by the spines in a single mating, and the authors present good evidence for this in the discussion. That means that the result of increased in offspring production could simply be the result of male modification of ejaculate fluid and have nothing to do with spine length.
Thank you for sharing these concerns. We make the following reflections. First, in the experiment on direct costs of spines, females were not "kept with the same male in monogamy for life": they were mated twice with two virgin males each and then kept in isolation for oviposition. This was necessary to provide sufficient control of potentially confounding variables in this experiment, and means that the results could not have been due to these sorts of complications. Second, in the experiments on the direct effects of the evolution of male spines, females were also not "kept with the same male in monogamy for life". In the experiment involving base line females, each female was kept with two males for life. In the experiment involving selection lines females, five males and five females were kept together for life. The two experiments showed congruent results (i.e., females kept with L males produced more offspring -see MS). Third, although the particular complications that the reviewer delineates can thus not be an issue, we agree that the effects of evolutionary elaboration of the spines are very likely mediated by seminal fluid, as we suggest and discuss at some length in the discussion.
The section on indirect fitness is relies on female offspring production which is indistinguishable from fecundity as measured for direct benefits, with no justification of how these are interpreted as being different. While I fully understand the limitations of measuring the fitness of offspring produced by the experiments, that would have been the appropriate measure, and in that sense the results are oversold. The full results of all the statistical models should be included in supplementary materials.
We are struggling to interpret this comment, and suspect that it might reflect a misunderstanding of what we did. The section on indirect effects in females is based on the correlated response in females to selection in males across multiple generations. It is an entirely genetic effect: when we select for males with long/short spines over successive generations, we see the evolution of males with long/short spines but also the simultaneous correlated evolution of females with high/low offspring production. This is definitely a highly appropriate (some would argue the ultimate) measure of indirect genetic effects in females. To our knowledge, ours is the first study to achieve this for any male trait.
Line 105-106: How does this experiment overcome these challenges though? You are still only manipulating males so the confounding effects of female resistance and other male behaviors remain.
Phenotypic manipulation/engineering of sexual traits is a common strategy to isolate the effects of the manipulated trait in one sex, while keeping "all else constant". To "only manipulate males", and to do so with only one trait, is in fact the very point of the experiment. The classic example is of course Malte Andersson's 1982 study of widowbirds (cited 1026 times), where he prolonged and shortened male tail length and showed that female prefer to mate with males with long tails (rather than some trait that correlated with tail length). See e.g. Travis & Reznick (1998) [Experimental approaches to the study of evolution. In: Experimental ecology: issues and perspectives, 437-459] for a discussion of the basic principles and strengths of phenotypic manipulation experiments.
Lines 127-128. If the experiments are distinct, how can the data be the same? This is very confusing!
We fully agree that this was confusing and we hope these points have now been greatly clarified in the revised version (see our responses above).
Line 139-140. This study should stand on its own. Please provide brief description of those procedures using the reference for further details, not instead of an explantion.
Thank you for this comment. This section has now been rewritten to improve clarity and transparency.
Line 163. Please conduct the analyses with and without your outliers. Outliers are part of the normal biological variation and in an experiment with relatively small sample sizes such as this one, they may change the outcome of the statistics altogether.
This has now been revised, and data has been re-analysed. Please see our detailed response to a similar comment made by reviewer 1 above. To reiterate our response to reviewer 1 above: "We chose to place the details of these, admittedly interesting, experiments in the SM because (1) they are truly supplemental [i.e., they do not address how genital spines affect females] and (2) we wish not to exceed the Proc B page limit, as the reviewer suspected. We have tried to accommodate this comment by including a summary of the main results in the text of the revised version. We hope you feel that this is an acceptable balance." Line 233. Name the covariates here. I believe they are male body size, female body size and number of eggs laid between matings?
Good point -done! Yes, that it correct.
Line 248: Average lifetime fitness production is 17% higher in L mated females than S mated females? How does that happen when the females produce 15% fewer eggs when mated with L males?
As we detail in the discussion, the former is a genetic response to the evolution of long spines in males, the latter a results of direct phenotypic manipulation of male spine length. Thus, the former must involve genetic effects which are correlated with male spine length.
Line 263: Females from L lines had a 6% higher production of offspring regardless of the phenotype of the male with whom they mated right?
Yes, this is averaged over all mates (i.e., are marginal means). This has now been clarified.

Line 269-270. How would this genetic correlation come to be? It could be that in lines where males have longer spines, female defenses such as kicking become more important and larger females are better at kicking…
Any of a number of scenarios could result in such a genetic covariance. The precise effect suggested here is unlikely, as the estimate of the genetic covariance is not based on the selection lines. This has now been clarified. But the general effect suggested (basically, a positive genetic covariance between male persistence and female resistance) is predicted from theory and could contribute to the sex-specific covariance seen. We would, however, prefer to not speculate on the many possible causes of this covariance in the interest of restricting the length of our contribution.
The "direct benefits" need further justification. How likely would it be in nature that a female would become fully monogamous and end up mating with a single male per life over multiple reproductive bouts? This is one of the confounding factors that the authors mentioned at the beginning As detailed above, our experiments were not based on assaying females that "mate with a single male per life". Please see our comments above.
Line 286-287: What are those female traits? Important to include here.
Unfortunately, we do not know what the cryptic female choice traits are. This is, as far as we know, not known in any system. This sentence is meant to be a general discussion and we have now edited the text to clarify this.
Line 290: the phenotypic engineering did not elongate the spines! It shortened them!! Am I missing something here?
Correct -thanks for pointing this out! This has now been rephrased.
Line 302-327: This explanation confirms my suspicion that males are changing the composition of their ejaculate when they are in this artificially enforced monogamy for a lifetime situation, and therefore the effect of the spine length is completely unknown! The increase in offspring production by females mated to Long spined males for life could be mediated by changes in ejaculate fluid as agued here.
Well, females were not kept under "enforced monogamy for a lifetime situation" (see our responses above) and the effects of spines per se is not "completely unknown" -we show here that the direct effect of spines in females is in fact negative. But, yes, the effects of evolution of long male spines in their mates (which is positive) very likely involves effects of male seminal fluid (as we suggest here).
Line 334-336. Exactly the problem here. The indirect benefit has to be through the descendants, and that was not shown in this experiment. You measured the same thing as direct and indirect benefits.
We are not sure what problem the reviewer is referring to here. The point and strength of our work, in this regard, is that we actually did measure indirect effects through the descendants. We suspect that the reviewer has somewhat misunderstood what we actually did, and have made efforts to clarify this in text of the revised version.
Line 368-371: While I agree wholeheartedly with the idea, this is not shown in this study.
Again, we suspect that this critical remark reflects at least a partial misunderstanding of what we achieved: we are measuring the indirect effect in females as a truly genetic effect (as a correlated response over many generations to sex-limited selection in males). incomplete. This is perhaps especially true for genital traits that cause internal injuries in 31 females, such as the spiny genitalia of seed beetles where males with relatively long spines 32 enjoy a high relative fertilization success. We report on a new set of experiments, based on 33 extant selection lines, aimed at assessing the effects of long male spines on females in 34 Callosobruchus maculatus. We first draw on an earlier study using microscale laser surgery, 35 and demonstrate that genital spines have a direct negative (sexually antagonistic) effect on 36 female fecundity. We then ask whether artificial selection for long vs short spines resulted in 37 direct or indirect effects on female lifetime offspring production. Reference females mating 38 with males from long-spine lines had higher offspring production, presumably due to an 39 elevated allocation in males to those ejaculate components that are beneficial to females. As predicted, we found that females mated with males with relatively long spines produced 255 on average some 17% fewer eggs after their second mating (LS mean = 38.3, SE = 2.51) than 256 did those mated to males with shorter spines (LS mean = 46.3, SE = 2.56). An analysis of 257 covariance showed a significant effect of genital spine treatment on female egg production 258 (F 1,42 = 5.09, P = 0.029). The homogeneity of slopes assumption of this model was verified as 259 the three interactions between the covariates (female body size, male body size and eggs 260 laid between matings) and the spine length treatment did not collectively improve model fit 261 to data (F 3,39 = 0.92, P = 0.44), and interactions were thus not included in the inferential 262 model. The three covariates collectively improved model fit (F 3,42 = 13.32, P < 0.0001). In 263 particular, the effect of female body size on female fecundity was sizeable (F 1,42 = 12.1, P = 264 0.001). The residuals of our inferential model were well behaved (Levene's test for 265 homogeneity of variance; P = 0.534. Kolmogorov-Smirnov test for normality; P = 0.326). This 266 result provides evidence for a direct cost to females of male genital spines per se. 267 268

Direct effects on female fitness of the evolution of male spines 269
We have previously shown that mating rate does not differ significantly between base line showed that average lifetime offspring production, primarily reflecting female fecundity 272 (see above), was some 17% higher in base line females mated to L males than in those 273 mated to S males ( Figure 1A) (paired t-test; t 2 = 7.72, P = 0.016) when kept for life with 274 males. A previous experiment performed in twice-only mated females, using males from the 275 same lines, found a non-significant effect in the same direction [35]. We note that male body 276 size was about 0.8% smaller in the L lines compared to the S lines (mean elytra length; S: In our second assay, we instead mated males and females from our selection lines in a fully 282 orthogonally crossed manner, and assayed female lifetime offspring production. Again, 283 females mated with L males enjoyed slightly higher lifetime offspring production ( Figure 1B) 284 (L: 107.1, SE = 1.53; S: 105.9, SE = 2.24), but not significantly so in this experiment ( Table 1; 285 effect of male selection line origin). 286 287

Indirect effects on female fitness of the evolution of male spines 288
The second assay showed that selection on male spine length significantly affected female 289 fitness as a correlated response: females derived from L lines showed a 6% elevation in 290 lifetime offspring production compared to females from S lines ( Figure 1B) (Table 1; effect of 291 female SLO), averaged over all mates, thus demonstrating a positive genetic covariance 292 between male genital morphology and female lifetime offspring production. Female body 293 size was positively related to lifetime offspring production (R 2 = 0.11, F 1,94 = 12.41, P < 0.001) 294 and females in L lines evolved to be 1.1% larger than females in S lines (mean elytra length; 295 S: 2.10, SE = 0.005 mm, L: 2.12, SE = 0.004 mm) [35], consistent with the positive intersexual 296 genetic correlation found between male spine length and female body size (see below). 297 Therefore, the indirect effect in females of the male spine selection treatment could 298 potentially be the result of an evolutionary increase in female body size. However, 299 statistically removing the effects of female body size on offspring production yielded very 300 similar results (male SLO: F 1,7 = 0.14, P = 0.717; female SLO: F 1,7 = 6.511, P = 0.038). This 301 assay thus demonstrated that there is a positive genetic association between male genital 302 spine length and female size-specific fitness. 303 304 Genetic architecture of male genital spines 305