Patterns and Processes of Genomic Evolution Inferred From the Ten Smallest Vertebrate Genomes

Pufferfish exhibit the smallest vertebrate genomes, making them ideal models for investigating evolutionary patterns and processes that affect genome size. While the Takifugu rubripes genome was fully sequenced two decades ago, key evolutionary drivers remain elusive. We sequenced 10 pufferfish genomes and generated 35 transcriptomes and 13 methylomes to understand genomic evolutionary mechanisms. Comparative genomics revealed that transposable element suppression-rather than lineage-specific conserved element loss-primarily underlies genome compaction. This is mediated by reductions in transposon-associated enzymes that limit transposable element propagation and modify DNA repair mechanisms that promote genomic streamlining. Based on resolved phylogeny among nine Takifugu, it is found that introgression drives speciation of T. niphobles and T. oblongus, while long-term linked selection dominates divergence in other species. Positive selection analyses highlighted mechanotransduction pathway genes (integrins, ion channel transport) that are functionally convergent with mammalian lung cell mechanisms, potentially supporting inflation-based anti-predatory strategies. Additionally, positive selection variants in genes that control lineage-specific skin patterning and coloration, which are either selected for or are rewired in regulatory processes, might suggest a role for pigmentation during the rapid speciation of this lineage. This findings shed light on mechanisms enabling extreme vertebrate genome compaction and provide insights for genome engineering applications.