Genomic Exaptation and Regulatory Landscape Shifts as Key Mechanisms Enabling Flatworm Terrestrialization.

Understanding the genomic toolkit that enabled animal terrestrialization, the shift from aquatic to terrestrial habitats, is key to uncovering the evolutionary origins of land biodiversity. Yet, the genomic basis of the physiological and metabolic adaptations required for life on land remains poorly understood across most terrestrial animal phyla. Planarians (Platyhelminthes) offer a powerful model, as only one terrestrial lineage, the Geoplanidae (order Tricladida), is known. Here, we integrated genomics, transcriptomics, and proteomics to explore the genetic changes potentially supporting terrestrial adaptation. We identified a major burst of gene gain in the lineage leading to Tricladida, preceding the radiation of terrestrial planarians. Upon abiotic stress exposure, terrestrial and freshwater species exhibited distinct responses: most differentially expressed genes belonged to orthogroups gained in Tricladida, with over half under strong directional selection in terrestrial flatworms, suggesting their adaptive relevance. Transcriptomic profiles revealed divergent strategies: terrestrial species upregulated ancient genes, while freshwater species downregulated a separate set of ancestral genes. Across all datasets, the abiotic stress-response toolkit in terrestrial planarians was markedly different from freshwater relatives, with significant regulatory divergence. Our results highlight gene gain and co-option, rather than lineage-specific innovations, as key drivers of terrestrial flatworm evolution, emphasizing genomic exaptation and regulatory shifts as central to terrestrialization in Platyhelminthes. This study provides the first genome-wide view of the genetic basis of flatworm terrestrialization and sheds light on broader patterns of animal terrestrial adaptation.