Phylogenetic Mosaic of an Arms Race with Asymmetrical Sexual Conflict and Its Macroevolutionary Consequences in a Lineage of Small Water Striders

Reviewer #1 (Public review):

Summary:

This work explores the relationship between sexual conflict and species diversification in a clade of small water striders. They use multiple phylogenetic methods to establish a potential phylogenetic tree and explore incomplete lineage sorting and introgression. They then compare their calibrated species tree with phenotypic measures of many species to try to infer the relationship between species diversity and sexual conflict. They show evidence for both ILS and introgression within the broader clade. They also find evidence that male leg diversity is associated with speciation, potentially due to sexual conflict, and that male genital grasping structures as well as female anti-grasping traits have less evidence for an association with speciation. This paper seems like a generally rigorous and interesting addition to the literature on sexual conflict and speciation, and I believe the conclusions are well supported with only a few minor concerns with methodology.

Strengths:

This paper verifies results using multiple methodologies to ensure robustness to changes in software use, and presents convincing evidence for the hypotheses tested.

Weaknesses:

(1) Lines 299-304: For the categorization of sexual conflict traits, were categories chosen by a blinded participant or by a researcher who knew the species they were observing? If any of these traits are subtle, this could add bias to the categorisation of traits.

(2) Lines 325-327: It is unclear to me if you control for phylogenetic relationships in this model. Diversification rate could be lineage-specific regardless of sexual conflict, so it seems like potentially including phylogenetic relationships in a model would control for that. And similarly, lines 331-333, I may be mistaken, but it sounds like you are using lm() to model a binary outcome (presence/absence), but lm() doesn't do logistic regression as far as I know, so it may be better to model this as a logistic regression (controlled for phylogeny) using glm().

(3) There are a few areas where the reporting of inference or statistics could be improved, and throughout the manuscript there are often mentions of 'significant' without any measure of uncertainty such as confidence intervals (example on lines 449-451). In lines 385-390, because the intervals are so wide, I would suggest saying these clades diverged between X-mya and Y-mya, rather than giving an actual estimate. It seems to me like giving a specific date is a bit overconfident when the authors have such wide intervals. On line 436, I think the authors should report confidence intervals for their 5mya estimate. In lines 455-456, what is this correlation and what are the authors' uncertainties around the estimate?

(4) Lines 549-545: When the two possible explanations for this result are reported, it seems like the authors are saying that because they can't think of how to test this possibility, the other possibility is more likely. But I don't think that is an argument against the first possibility.

(5) Lines 591-602: This paragraph confuses me. I thought that much of the introduction and Figure 1 seemed to be putting forth that the terminal segment complexity was a conflict structure that we were interested in. However, here it is stated that it does not strongly influence mating success, so is it necessarily a conflict trait? Is it demonstrated that the leg structures influence mating success?

Reviewer #2 (Public review):

Summary:

This study uses comparative phylogenetic methods to examine the evolution of male and female antagonistic traits in a group of small water striders. Water striders have long been a model system for studies into the sexual conflict that arises through anisogamy, the differential investment in gametes by males and females. Here, the authors aimed to reveal the evolutionary rates and trajectories of male grasping and female anti-grasping traits across species of the minute water-strider subgenus Pseudovelia. This was done by combining multiple genomic techniques to generate phylogenies to test trait evolution, quantify rates of evolution, and identify instances of incomplete lineage sorting (a result of rapid diversification) and introgression (the result of interbreeding between genetically different populations/species).

Strengths:

The strengths of this study lie in its comparative macroevolutionary framework, in particular the generation of multiple phylogenetic hypotheses using different methods (mitochondrial genes, USCOs, and SNPs), and contrasting these to glean insights into evolutionary patterns across species.

Weaknesses:

The main weakness of the study is the lack of underlying experimental evidence to explicitly show the grasping and anti-grasping functions of the various male and female traits, relying instead on studies of similar structures in more distantly related taxa. Without explicitly showing the functional mechanisms and reproductive costs of these traits, the resulting interpretations are wholly speculative. However, I would argue that such macroevolutionary studies are still very useful, and provide the groundwork for future studies untangling the relative roles of sexual conflict, cryptic female choice, sperm competition and reproductive interference in trait evolution and ultimately in speciation.

Author response:

Response to Reviewer 1:

We thank the reviewer for their valuable and constructive suggestions.

(1) The categorization of morphological traits was performed by researchers who are familiar with the taxonomy and morphology of this taxa. We acknowledge that this may introduce some degree of subjectivity, and we will provide photographs of other morphological traits for each species in the Supplementary data.

(2) In our original analysis, we treated the PCAmix values derived from multiple discrete traits as continuous variables and used the lm () function to test the correlation between these values and net diversification fates (lines 352-357). We agree that a phylogenetic comparative approach would be more appropriate. We plan to re-analyze the data using either glm () or phylogenetic generalized least squares (PGLS) to properly account for phylogenetic relationships. In lines 331-333, we would clarify that this part refers to the HiSSE analysis based on discrete traits, which is independent of the linear regression analysis mentioned above. Nevertheless, we will ensure that both analyses are clearly distinguished and properly described in the revised Methods section. We will update the relevant sections accordingly.

(3) We will carefully re-examine the entire manuscript and add appropriate measures of uncertainty.

(4) We agree with the reviewer that two hypotheses are not mutually exclusive. In the revised manuscript, we will rephrase this paragraph to present both possibilities more neutrally.

(5) We have observed mating behaviors in this group and found that ASE function occurs after the male has successfully grasped the female using its legs. However, we acknowledge that our study did not include direct experiments to quantitatively test the effect of ASE complexity on mating success. Therefore, our discussion in this section is indeed somewhat speculative.

Response to Reviewer 2:

We thank the reviewer for their encouraging and constructive comments. We agree that our study lacks direct experimental evidence to explicitly demonstrate the grasping and anti-grasping functions of the various male and female traits in Pseudovelia, and that our interpretations currently rely on comparisons with functional studies in more distantly related taxa. In the revised manuscript, we will explicitly state this limitation and refer to these traits as “putative” grasping or anti-grasping traits throughout the text where appropriate.

We will also carefully address all minor editorial suggestions, including clarifying terminology, correcting typos, and improving figure legends.