Phylogenomics confirms Palaeoptera monophyly and illuminates the origin of winged insects.

The origin and early radiation of winged insects (Pterygota) represent one of the most transformative events in terrestrial evolution, yet phylogenetic relationships among their basalmost lineages (Ephemeroptera, Odonata and Neoptera) continue to be a central debate within insect systematics. Here, we present the most taxonomically comprehensive phylogenomic analysis to date, leveraging genomic and transcriptomic data from 101 species, including 16 sequenced ephemeropteran genomes. Employing 1,367 universal single-copy orthologs across two supermatrices, we used concatenation- and coalescent-based frameworks under homogeneous and heterogeneous, and multispecies coalescent models. Our analyses support Palaeoptera monophyly. Although coalescent-based methods initially suggested a Chiastomyaria (Ephemeroptera + Neoptera) topology, subsequent signal dissection and topology tests confirm that the preponderance of genomic signal favors a monophyletic Palaeoptera. Furthermore, we identify incomplete lineage sorting and gene tree estimation error as the primary drivers of previous topological conflicts. Our molecular dating results point to an Early Devonian origin of crown-Pterygota, whereas crown-Palaeoptera first emerged in the Late Devonian. By reconciling molecular evidence with morphological synapomorphies, such as wing-base sclerite homologies, this study provides a coherent framework for the early evolution of winged insects, supports a single origin of wings, and identifies the neopteran wing-folding mechanism as a singular evolutionary innovation.