A proteo-transcriptomic investigation of toxin evolution in planarians and their role in flatworm terrestrialization

The transition from aquatic to terrestrial environments represents a major evolutionary transition in animals, requiring significant adaptations in physiology and defense mechanisms to the challenges presented by the harsh terrestrial environment. Platyhelminthes, which include both aquatic and terrestrial species with a single terrestrialization event in the family Geoplaniidae, serve as excellent model organisms for studying the evolutionary adaptations required for terrestrialization. This study investigates the evolutionary dynamics of toxin orthologous groups (as a proxy to gene families) in aquatic and terrestrial flatworms, together with mucus composition, focusing on their role in terrestrialization from a molecular ecology perspective. Using a proteo-transcriptomic approach, we predicted and identified a broader toxin gene repertoire in terrestrial flatworms compared to freshwater ones. Although most toxins in flatworms arose before terrestrial planarian diversification—gaining a novel evolutionary origin at the Tricladida and Continenticola nodes—the mucus protein repertoire appears to have a far older evolutionary origin in both species. Moreover, distinct orthologous groups underpin the toxin gene repertoire and mucus composition in each lineage, highlighting the contrasting evolutionary trajectories of these two functional components. While toxin families in both aquatic and terrestrial flatworms revealed overall common functions, including cytokine modulation and ion channel regulation, terrestrial flatworms exhibited specific expansions of lectin-like proteins and pro-inflammatory responses, highlighting their potential key role to respond to land-based threats. This study provides new insights into the differential evolutionary trajectories of toxin and mucus proteins in planarians, offering a deeper understanding of the genetic innovations that facilitated flatworm terrestrialization.