Project Summary The eukaryotic innovation of genetic diversification through sex and recombination provides significant adaptive advantages. Still, the recognition that some animals are capable of asexual reproduction dates back to at least 1740 CE, and asexuality has arisen independently multiple times across the animal kingdom. This transition is often accompanied by modified meiosis and genome organization. Since meiotic defects underlie age-related decline in human fertility, and genomic instability is a hallmark of aging cells, studying how successful asexual lineages can thrive in light of major modifications to these core cellular programs can provide new molecular insights into the mechanisms of aging and infertility. To characterize genomic signatures of asexual reproduction, we previously sequenced the genome and transcriptome of Diploscapter pachys, a nematode from an unusually persistent asexual lineage estimated to have originated 18 million years ago. This work showed that D. pachys lacks key meiotic genes and the first (reductional) meiotic division, enabling reconstitution of a diploid genome in the oocyte without fertilization. Strikingly, its nuclear genome is packaged into exactly one pair of chromosomes, which we showed derives from the full fusion of all ancestral chromosomes. However, the genome assembly still contains many gaps that limit our ability to answer key questions about D. pachys evolution: how and when genome fusions and abridged meiosis arose, how a high level of sequence diversity is maintained without genetic recombination, and which molecular changes drove the transition to asexual reproduction remain a mystery. We propose to use the power of long-read sequencing and comparative genomics to address these questions. In Aim 1, we will generate a highly contiguous, chromosome-level genome assembly for D. pachys. This will reveal the pattern of ancestral chromosome fusions, whether major genome rearrangements likely preclude meiotic crossovers, and the nature of chromosome fusion sites and telomeres. In Aim 2, we will produce chromosome-level genome assemblies of four additional parthenogens and their closest known sexual relative in the same phylogenetic clade. This will allow a comparative analyses of genome architecture and enable evolutionary reconstruction of molecular genetic changes linked to asexual reproduction. In Aim 3, we will analyze chromatin accessibility and changes in regulatory sequences and coding regions in all five species to uncover whether genetic and/or epigenetic mechanisms underlie differences in expression levels between alleles, as seen in D. pachys, which may enable these animals to overcome potentially high loads of deleterious alleles. In summary, this study presents a unique opportunity to explore the evolution of asexual reproduction in animals, a centuries-old mystery in biology, whose molecular underpinning may provide new insights into molecular processes underlying aging in hu...