Insulated Outlier Chromosomes Drive Metabolic and Evolutionary Innovation in Minimal Eukaryotic Algae
Marine picoeukaryotes of the order Mamiellales, including Ostreococcus tauri, the smallest known free-living eukaryote, possess compact genomes yet maintain enigmatic "outlier chromosomes" characterised by lower GC content and hypervariability. To determine the structural and functional nature of these regions, we applied chromosome conformation capture to O. tauri and conducted comparative multi-omics analyses across the Mamiellales order, presenting the first analysis of three-dimensional genome organisation in marine picoeukaryotes. We reveal that outlier regions form structures resembling topologically associating domains, with sharp boundaries that spatially insulate them from the standard chromosomes. These compartments are defined by a distinctive chromatin state characterised by hypomethylation and transcriptional hyperactivity, and are frequently, though not universally, enriched in transposable elements. Crucially, species that lack transposable element enrichment in their outlier chromosomes nonetheless retain the transcriptional hyperactivity and distinct nucleotide composition of these regions, indicating that the functional identity of these compartments persists independently of transposon accumulation. The dynamic nature of these insulated domains is highlighted by the presence of structurally diverse giant polyketide synthase loci. We identify convergent genomic organisation in other chlorophytes, as well as phylogenetically distant stramenopiles. Our results suggest that such compartmentalisation of rapidly evolving, dynamic genomic regions represents a fundamental architectural principle of minimal eukaryotic genomes.