Genetic and karyotype divergence between parents affect clonality and sterility in hybrids

Reviewer #1 (Public Review):

This paper provides new evidence on the relationship between genetic/chromosome divergence and capacity for asexual reproduction (via unreduced, clonal gametes) in hybrid males or females. Whereas previous studies have focussed just on the hybrid combinations that have yielded asexual lineages in nature, the authors take an experimental approach, analysing meiotic processes in F1 hybrids for combinations of species spanning different levels of divergence, whether or not they form asexual lineages in nature. As such, the findings here are a substantial advance towards understanding how new asexual lineages form.

The quality of the work is high, the analyses are sound, and the authors sensibly link their observations to the speciation continuum. I should also add that the cytogenetic work here is just beautiful!

A key finding is that the precondition for asexual reproduction - the formation of unreduced gametes - is not unusual among hybrid females, so that we have to consider other factors to explain the rarity of asexual species - a major unresolved issue in evolutionary biology. This work also highlights a previously overlooked effect of chromosome organisation on speciation.

Reviewer #2 (Public Review):

The authors investigate the origin of asexual reproduction through hybridization between species. In loaches, diploid, polyploid, and asexual forms have been described in natural populations. The authors experimentally cross multiple species of loaches and conduct an impressively detailed characterization of gametogenesis using molecular cytogenetics to show that although meiosis arrests early in male hybrids, a subset of cells in females undergo endoreplication before meiosis, producing diploid eggs. This only occurred in hybrids of parental species that were of intermediate divergence. This work supports an expanding view of speciation where asexuality could emerge during a narrow evolutionary window where genomic divergence between species is not too high to cause hybrid inviability, but high enough to disrupt normal meiotic processes.

I enjoyed reading this study and I appreciate the amount of work it takes to conduct these types of cytogenetic experiments. But, my main concern with this study is I was left wondering if the sample sizes are large enough to get a sense how variable endoreplication is in these loach species. Most of the hybrids between species are the result of crosses between 1-2 families. Within males and females, meiocyte observations are limited to a handful of pachytene and diplotene stages. I think it would be helpful to be more transparent about the sample sizes in the main text.

Along these lines, the authors argue against the possibility that endoreplication may be predisposed to occur at a higher rate in some species (line 291). Instead, they suggest that endoreplication is a result of perturbing the cell cycle by combining the genomes of two different species. Their main argument is based on gonocyte counts from parental females in a previous reference. It is essential to include counts from the parents used in this study to make a clear comparison with the F1s.

In the discussion (lines 320-333), the authors postulate the sex-specific clonality they observe could be a result of Haldane's rule. Given these fish do not have known sex chromosomes, I do not find this argument strong. Haldane's rule refers to the exposure of recessive incompatibilities with the sex chromosomes in the hybrid heterogametic sex. This effect would therefore be limited to degenerated sex chromosomes where much of the sequence content on the Y or W has been lost. These species may have homomorphic sex chromosomes, but if this is the case, they likely are not very degenerated. Instead, it seems more plausible that the sex-specific effect the authors observe is due to intrinsic differences of spermatogenesis and oogenesis. Is there any information about sex-specific differences in the fidelity of gametogenesis from other species that would support a higher likelihood of endoreplication?

The final thing I was left wondering about was this missing link between endoreplication and activating embryonic development of the diploid egg. In these loach species, a sperm is required to activate egg development, but the sperm genome is discarded (line 100). What is the mechanism of this and how does it evolve concurrently during hybridization?