Abstract
The transition from marine to freshwater environments represents a remarkable evolutionary shift in cetaceans, yet the genomic underpinnings of this adaptation remain poorly understood. This study investigates the genomic signatures of freshwater adaptation in riverine cetaceans through comparative evolutionary analyses of six species: Inia geoffrensis , Lipotes vexillifer, Platanista gangetica , Platanista minor , Neophocaena asiaeorientalis asiaeorientalis , and Sotalia fluviatilis . By integrating positive selection analysis, gene family dynamics, and evolutionary rate convergence, we identified key molecular adaptations associated with freshwater colonization. The analysis revealed that five of the six species exhibited positive selection in the NSMAF and CTRL genes, suggesting widespread selective pressures related to inflammatory responses and digestive adaptations, respectively. Functional enrichment analyses revealed adaptive signatures in hematopoiesis, osmoregulation, skeletal development, and immune responses, reflecting the physiological challenges of freshwater environments. Gene family evolution analyses using CAFE identified dynamic patterns of expansions and contractions in immune-related genes, transcriptional regulation, and cell adhesion pathways across riverine lineages. Relative evolutionary rate (RER) analysis using RERconverge identified 95 genes showing convergent rate shifts associated with freshwater adaptation, including genes involved in cellular nitrogen compound responses and transcriptional regulation. Despite positively selected genes overlap was limited and did not follow simple clade-wide patterns, our results demonstrate that freshwater adaptation in cetaceans involves putative convergent evolution of fundamental biological systems, including immune responses, metabolic regulation, and morphological development. These findings provide new insights into the molecular mechanisms underlying speciation in aquatic mammals and highlight critical biological pathways that have enabled the successful colonization of freshwater ecosystems by multiple independent cetacean lineages.