Over the lifetime of the project, all major objectives described in the Description of the Action were achieved. The work combined genome sequencing, transcriptomics, cytology, comparative genomics, and theory-driven evolutionary analyses, resulting in a uniquely comprehensive resource for studying genomic conflict in animals.
A major initial effort focused on building the genomic and experimental foundation required for all subsequent analyses. Chromosome-level genome assemblies were generated for the large majority of focal species, complemented by whole-genome alignments across all ten Timema species and the identification of approximately 12,000 one-to-one orthologous genes. In parallel, an extensive RNA-seq dataset was assembled, covering multiple tissues, developmental stages, sexes, and reproductive modes. These resources enabled direct and robust comparisons between sexual and asexual lineages at unprecedented resolution. In addition, new molecular tools were developed, including custom antibodies against key centromere and kinetochore proteins, allowing detailed cytological and chromatin-based analyses of chromosome behavior.
Using these resources, the project first addressed how sexual antagonism is expressed and resolved across development. By tracking sex-biased gene expression from early juvenile stages to adulthood, it revealed that sexual dimorphism in gene expression emerges much earlier than previously appreciated, particularly in developing gonads. Comparisons with asexual sister species showed that most sexual antagonism over gene expression is efficiently resolved in sexual species, with only a restricted set of genes retaining signatures of unresolved conflict.
The project then examined the fate of sex chromosome regulation under relaxed selection. By combining developmental transcriptomics with cytological analyses, it produced the first complete picture of dosage compensation and meiotic sex chromosome inactivation in a hemimetabolous insect. Contrary to long-standing theoretical expectations, both mechanisms were found to be remarkably stable in asexual lineages that no longer experience selection on males.
One of the most striking outcomes of the project emerged from work on centromere evolution. While developing tools to study centromere drive, the project uncovered a previously unknown form of chromosome organization and meiosis in Timema. These insects possess chromosomes that are functionally monocentric yet display hallmarks of holocentricity. This discovery revealed an unanticipated degree of flexibility in centromere organization and established Timema as a new model for studying chromosome evolution. Ongoing comparative analyses of centromere sequences in sexual and asexual species are now providing the first empirical tests of centromere-drive theory across repeated, independent transitions in reproductive mode.
Finally, the project investigated how transposable elements and other repetitive sequences evolve when genomic conflict is reduced. Long-read genome assemblies and improved annotation pipelines allowed reconstruction of transposable element turnover across the Timema phylogeny.
Overall, the work performed not only fulfilled but substantially exceeded the original aims of the project. The remaining effort at the end of the action concerns the synthesis and writing of two very large comparative genomics papers that integrate these results across work packages and are expected to have particularly broad impact.
