A deep-time perspective on Polycladida (Platyhelminthes) through integrated phylogenetic and molecular clock analyses

This study presents a comprehensive phylogenetic and molecular clock analysis of Polycladida based on the mitochondrial marker cytochrome c oxidase subunit I (cox1). By including a broad representation of taxa across both suborders (Acotylea and Cotylea) and integrating evolutionary rate estimates, this work offers new insights into the evolutionary history of the group. The phylogenetic reconstruction supports previous studies while refining the relationships among key lineages. Our analyses strongly support Boniniidae as the earliest-diverging lineage of Polycladida. Cestoplanidae and Pericelidae are consistently recovered together as the sister group to Acotylea, highlighting their key position in the early diversification of the order. Within Cotylea, Anonymidae forms a well-supported monophyletic clade, though its placement varies between analyses. Within Cotylea, the monophyly of major families is challenged, particularly in Euryleptidae and Pseudocerotidae, indicating cryptic lineages and convergent traits. Acotylea displays greater phylogenetic instability, with Stylochoidea and Discoceloidea showing limited support, in contrast to the more cohesive Leptoplanoidea. For the first time, a molecular clock analysis was conducted in Polycladida, revealing that Boniniidae diverged approximately 550 million years ago, acting as a stem-group lineage that retains ancestral traits and bridges the evolutionary transition between Cotylea and Acotylea. Major diversification events within Polycladida occurred between 360 and 350 Mya, aligning with late Devonian–early Carboniferous marine expansions. Cotylea likely originated around 250 Mya, coinciding with reef diversification in the Mesozoic, while the divergence of Cotylea and Acotylea species occurred during the early Mesozoic (100–50 Mya), following the Cretaceous–Paleogene mass extinction. These findings underscore the importance of integrating molecular, morphological, and temporal data, with the molecular clock analyses providing a coherent framework that resolves long-standing uncertainties in the early diversification of Polycladida.