I appreciate the authors spent the time to collect sexual dimorphism data and performed the correlation analyses with and without considering the phylogeny. I think their answers addressed my previous questions and the ms should be accepted for publication.

This manuscript examines sex chromosome evolution in strepsirrhine primates, specifically looking for evidence of changes in the extent of the pseudoautosomal region. They find evidence that this boundary has been very stable over time, suggesting little decrease in the extent of recombination between the X and Y over time, especially compared to other primates. They interpret this finding as being consistent with low levels of sexual antagonism within strepsirrhine primates, and support this interpretation with an analysis showing that the degree of sexual dimorphism is lower in strepsirrhines than in other primates.

I was not one of the original reviewers of this manuscript, but after reading their comments and the authors' response to them, it seems like the revised version has successfully addressed the criticisms that were raised in the first round of review. I did feel that the last sentence of the abstract is a bit strongly worded ("Our work supports the view that sexually antagonistic mutations have influenced the evolution of sex chromosomes in primates"), but the main text provides a more nuanced view and acknowledges that other explanations are possible.

Unfortunately I was not able to find any information about the analysis of sexual dimorphism and testes size anywhere other than the response to reviewers. Table S2 with the results of the analysis was missing from the supplementary information files, and there was no detailed description of how the analysis was carried out. This is presumably an oversight on the part of the authors, but it's a shame since this seems like it was a nice addition to the manuscript, and I currently can't evaluate the robustness of these results.

This is an interesting analysis of the evolution of the pseudo-autosomal region in primates, which demonstrates that the suppression of recombination between the X and the Y chromosomes has proceeded along different trajectories in the two main lineages of primates. In haplorrhines, there has been further suppression of recombination leading to the formation of two additional strata, while in strepsirrhines, there has been no further suppression of recombination since these lineages diverged. The patterns of recombination suppression are consistent with the presence of high levels of sexual dimorphism in haplorrhines and low levels of sexual dimorphism in strepsirrhines. Using levels of sexual dimorphism as a proxy for the intensity of sexually antagonistic selection, the authors conclude that selection for linkage between the sex-determination locus and alleles with sexually antagonistic effects might have driven suppression of recombination and the formation of the two additional strata in haplorrhines. I find this an intriguing hypothesis that lays the groundwork for additional studies. Although these conclusions remain speculative, the authors are careful in their conclusions and clear about the limitations. They have responded well to the previous reviews.

Nonetheless, the manuscript could be more clearly written in places, and so I provide some suggestions here for revisions to improve the clarity of the presentation.

1. P4, L25 - P5, L1: change “a ratio of 0.5 the X-specific region” to “a ratio of 0.5 should indicate the X-specific region”
2. P5, L15 and P6, L2: perhaps change “human strata 4, 5 and PAR1” to “PAR1 and human strata 4 and 5”
3. P5, L17: change “and agreeing with” to “in agreement with”
4. P5, L23 - P6, L1: the sentences here seem out of order. Perhaps start by saying why the fragmented assembly cannot identify the size of the PAR or autosomal to PAR translocations, and then state that an improved, de novo assembly based on long-read sequencing would allow you to confirm one of these alternatives.
5. P6, L3: “the location of the PAB is conserved” – conserved to what? The PAB is different in grey mouse lemur and humans. Either delete this phrase or be more specific.
6. P6, L5: change “only available one” to “only one available”
7. P6, L12: change “PAR1 and strata 4 and 5 in humans”
8. P6, L13: Interestingly, in Figure S2, the location of the PAB seems to differ between lorises and lemurs, but I assume this is simply due to differences in the X assembly?
9. P6, L14: change “no recombination suppressing event” to “no suppression of recombination between the X and the Y”
10. P6, L16 - P7, L15: here, I suggest changing the order of presentation; currently it is very confused. First, I would present the density of SNPs to show that you did not miss any strata. Simply move this paragraph up. Then, I would have a separate paragraph describing the rates of strata formation, with the conclusion that the rate is higher in haplorrhines. Finally, you can end with a paragraph describing the analyses of sexual dimorphism and say that the higher levels of sexual dimorphism in haplorrhines is consistent with sexual antagonism driving higher rates of strata formation in this group. For me, this is a nice logical presentation that builds the results to a clear statement of your major conclusions at the end.
11. Discussion, P7, L21: delete “and this is validated by a statistical test”. Also, in this paragraph, the biggest caveat of all these analyses is that it is a comparative study, and correlation does not equal causation! Even if you had a large phylogenetic study, this remains a caveat.
12. P8, L6-9: the evidence for expansion of loss of recombination on the guppy Y chromosome in high predation populations is not supported by the work of the Charlesworth group, and I think this needs to be acknowledged here (e.g. Bergero et al. 2019 PNAS).
13. P8, L13: please define “a process of attrition” and change “DNA repeats accumulation” to “accumulation of DNA repeats”.
14. P8, L14-15: this is a challenging sentence to read. Perhaps change to “In Microbotryum violaceum, strata are found on the mating type chromosomes despite the fact that this species only has mating types and not sexes, such that sexual antagonism is absent”.
15. P8, L22-23: What is the evidence that there is more suppression of recombination in red-bellied lemur than in the other species? I do not see this in Figure 1 or the supplementary figures?
16. P9, L24-25: Perhaps cite a nice recent paper in Science that identifies the genetic basis of sexual dichromatism in canaries (Gazda et al Science 12 June 2020)
17. In Figure 1 (as well as supplementary figures), can you add the common names of the species (used in the manuscript) to the scientific names (used in the figures) so that the reader can easily find the appropriate panel without needing to look at Table S1.
18. P12, L5-6: How can a major allele frequency be 0.3x the site coverage? For example, if the site coverage is 10, then the major allele frequency would be 3. This does not make sense. Also, these analyses do not seem very robust, as the older strata are also not identified these analyses. Most of the SNP densities across the X chromosome seem to fall within the autosomal range?

Shearn et al conduct a comparative analysis of sex chromosome evolution across two primate groups to test the relationship between sexual dimorphism, a proxy for sexual conflict, and recombination suppression. This is a hot topic at the moment. They find that there is a higher rate of strata formation in haplorrhine primates than strepsirrhines, a direction consistent with the role of sexual conflict in halting recombination. I enjoyed reading the manuscript and think it makes a valuable contribution to sex chromosome research. My comments are below:

Can the authors comment about when sexual dimorphism evolved in haplorrhines? This essential to interpret the results as the argument that sexual conflict promoted the evolution of S4 and S5 only holds if the common ancestor of haplorrhines was sexually dimorphic.

The discussion of alternative processes promoting recombination suppression is a bit light. Eg. Úbeda F, Patten MM, Wild G (2015) On the origin of sex chromosomes from meiotic drive. Proc Biol Sci 282:20141932 Matsumoto T, Yoshida K, Kitano J (2017) Contribution of gene flow to the evolution of recombination suppression in sex chromosomes. J Theor Biol 431:25–31

I don’t quite follow the use of SNP density to identify recent strata. Why is the sum of SNPs divided by four? Do you really expect 0.5 for the old strata? I am bit perplexed by the results in the SI. I agree that in regions where the Y has largely degenerated, we might expect SNP density to be lower in males as the X is effectively hemizygous in males. But this will depend on the level of standing genetic variation in the population, and whether females are homozygous for the X. I don’t understand how all of the X has a SNP density ratio of 0.5? I would expect the ratio to be much closer to 1.

For the methods further detail is necessary on: a. Whether any trimming steps on the reads was taken b. What thresholds were imposed on SNP calling ie minor allele frequency

The colour scheme of Fig 1 need to be improved for clarity. a. Firstly, mPAR needs to be defined in the figure legend, I assume mPAR refers to the ancestral PAR (what does the m mean)? At the moment it is red, which is the same colour as s5. Therefore, it looks like s5 is at the beginning of the X for the 7 species studied when I believe the authors are trying to show this is ancestral PAR. b. Similarly, misA needs to be defined, I assume it means misassembled autosomal scaffolds? But currently it is grey which makes these regions look like PAR.

Abstract L13 ‘Moved’ seems a bit unspecific in this sentence, I would replace with extended PAR. P3 L12 I’m not aware of any direct evidence that recombination between the primate X and Y stopped instantaneously as ‘at once’ suggests. I would remove this. P3 L14 I am not sure what process is referred to in this sentence. P7 L12 I believe Wright et al showed extension of recombination suppression not additional strata P7 L17 What does ‘process of erosion’ refer to? P8 L24 Typo – should be in not on P9 L6 Typo – should be cell P9 L10 Typo – should be samples P9 L14 Typo – should be on not onto P10 L4 Should be gap after bracket P10 L6 Remove ; P10 L15 Remove ) Fig 1 Legend Missing of after Identification Fig 1 Legend What does the sentence starting ‘Strata in humans using definition of strata shown …’ mean? Why does coverage ratio approach 1.5 in some species?

Sex chromosomes show a dynamic composition even between related species regarding their regions with or without recombination. The sex-linked regions with recombination is called pseudoautosomal regions (PAR), and this work by Rylan et al. found that strepsirrhines (lemurs and lorises) share a longer PAR than other primates (apes and monkeys), and they explained this pattern by suggesting a lower level of sexual antagonistic selection in the former than the latter. It is a great effort to characterize the PAR of all seven species, and the topic should be of broad interest to evolutionary biologists. However, I hope the authors provide more quantitative measurements of the level of sexual antagonistic (SA) selection ongoing in the strepsirrhines. Because after all, the experimental evidence for SA selection causing the recombination loss is quite rare. Guppy is an excellent example because the previous comparison by Alison et al was performed between two closely related populations with a very clear indication of sexual selection (the color), thus the association between the expanded recombination suppression region vs. different color (different degree of SA selection) can be interpreted as the suggestive evidence. The author talked briefly in the introduction about sexual size dimorphism, and how exactly was the degree of strepsirrhines compared to that of haplorrhines? How about the mating-system in these two groups of haplorrhines? I only know the mating system is associated with the relative testis size of primates, which is indicative of the strength of sexual selection. Second, I am not sure if the author has identified a scaffold that shows a switch between the M:F ratio of 1 vs. 0.5. The authors mentioned scaffolds that show a M:F ratio of 1 correspond to strata 4 & 5 in human, this only indicate there were no recombination loss after strata 3 formed in strepsirrhines. A process called attrition, which as mentioned by the authors is the gradual erosion of PAR by repetitive elements has been reported in cow, and in fact, many mammalian PABs are characterized by insertion of TEs (reviewed by Terje et al. 2015). Has the author found a similar pattern? Or are the PABs of the seven species actually aligned to each other? Finally, just curious, has the author managed to assemble any Y-linked genes, and are they any different from those that have been assembled for other mammals?

Some other minor comments:
introduction: line 1, 'strongly' maybe changed to 'very';
line 9, the master male-determining gene in Eutherian mammals, as platypus does not share the same sex chromosome and master sex determining gene;
line 15, I suggest to emphasize here that all eutherian mammals share strata 1-3;
page 4, line 21, the same copy number of autosomes;
page 5, line 16, are there any read pairs that span the X-specific region and the autosome like region? If so, there should help to clarify if there are potential recent fusion or just assembly errors;
page 6, I am not sure about the N50 of gray mouse lemur, but there is also a blue-eyed black lemur genome published with about 400kb N50 size, would it be better to use that as reference?