Splitting the sexes : The birth and senescence of sex chromosomes

Abstract: The evolution of gonochorism from hermaphroditism can be gradual by increasing investment in one sex role while decreasing in the other, or rapid through the fixation of sex-role sterility mutations, eventually leading to the evolution of sex chromosomes. It is expected that the transition will involve a temporary state of gynodioecy or androdioecy as the mutations are not expected to take place at the same time. If the first mutation is a dominant female-sterility mutation, later accompanied by a recessive male sterility mutation, then an XY sex chromosome system evolves, while the opposite combination of mutations will result in a ZW system. Later on sexually antagonistic (SA) genes can be linked to the newly established sex-determining regions on the sex chromosomes. This is followed by recombination arrest in the region, so that the inheritance pattern is sex-limited for all these sex-specific genes. However, the lack of recombination leads to degeneration of the genetic content on the sexlimited chromosomes, since recombination is important for repairing mutations. Nevertheless, recombination arrest does not necessarily mean a dead-end for the sex-limited chromosomes. As our understanding of the very early stages of sex chromosome evolution is mainly based on theory and comparative evidence, we developed a system which we hoped would make it possible to observe in real time what happens after the acquisition of a new sex-determining gene. We used a previously established green fluorescent protein (GFP) line of the simultaneous hermaphrodite Macrostomum lignano. We used the GFP locus as a dominant sterility mutation, which is inherited in a Mendelian fashion. By allowing the GFP allele to be inherited only through sperm, we created male-limited selection lines (resembling the early stages in XY chromosome evolution), and by allowing the GFP allele to be inherited only through egg cells, we created female-limited selection lines (resembling the early stages in ZW chromosome evolution). We also created control lines, where the inheritance pattern was equally mixed. After tens of generations, we investigated how these lines have responded on the level of the genome, the transcriptome, and the phenotype. We sequenced genomes and analysed changes in SNP frequency and structural variant (SV) distribution in pairwise comparisons to see changes across the genome, but particularly on the scaffold where the GFP is located. We also sequenced transcriptomes and performed pairwise comparisons to detect differentially expressed genes, and analysed significant GO terms and KEGG pathways to see how the gene regulation has changed. Besides genomic analyses, we also looked at how mating behaviour (copulation frequency and duration, as well as probability of post-copulatory sucking behaviour) and sexual anatomy (gonad size and morphology of the male copulatory organ called stylet) has changed.We observed that the female-selected lines seemed to have responded the most at the genomic level. For example, the number of significantly differentially expressed transcripts was largest between the female-selected lines and the control lines. These changes seemed to involve downregulation of testes-biased genes. In addition, we observed the highest number of SVs in the female-selected lines, which could be related to changes in recombination rate. In contrast, the male-selected lines seemed to have responded the most at the phenotypic level, since we observed a decrease in the ovary size and body size in the male-selected lines, as well as behavioural changes that may be related to changes in the ejaculate. Both sex-specific selection regimes showed evidence of alterations in the shape of the stylet. Based on these results, we can conclude that our worms have indeed responded to the sex-limited selection in a way that is generally consistent with our expectations from other young sex chromosome systems. The evidence of a decrease in the testes function in the female-selected lines resembles adaptation towards gynodioecy, and the evidence of a decrease in the ovary size in the male-selected lines resembles adaptation towards androdioecy.