The tree topology is derived from our presence/absence matrix (Supplementary Table S1; Supplementary Software) utilizing maximum parsimony and considering representatives of the key avian lineages1314. Black filled circles (branch A) are bird-specific retroposon insertions (exhibiting a 6-nt deletion that is found in some avian CR1s, but absent in all other avian and reptilian CR1s), and dark grey balls represent retroposon presence/absence markers that are congruent with one another. Light grey balls on grey gradient (label F) are retroposon markers that were probably inserted at the very beginning of the neoavian radiation and were subjected to incomplete lineage sorting of retroposon dimorphisms, as they exhibit presence/absence patterns that are incongruent with one another and with some of the retroposon markers on the dashed branches. Nodes without retroposon support are not collapsed (but highlighted by asterisks) if they received very strong support in nucleotide sequence analyses121314. Higher-ranking taxa are in red letters (English terms in orange letters and parentheses), including the new taxa Eufalconimorphae (falcons+parrots+passerines) and Psittacopasserae (parrots+passerines), and some recently introduced superordinal groupings145354. Bird names in bold letters belong to the nearest bird icon.

Potential target site duplications (direct repeats) are in black boxes, 5′ and 3′ ends of the retroposon insertions are shown in lower case letters in grey boxes.

Computational estimates of activity periods (normal distributions displayed as ovals29) of selected retroposon subtypes were calculated using the TinT model2529 and plotted on a simplified chronogram30 (black lines) using the experimentally verified retroposon insertion events (numbered blue or red balls, numbers indicate the respective retroposon subtype) of Figure 1 as temporal landmarks. Single capital letters correspond to the branch labels of Figure 1 (A, Aves; B, Neognathae; D, Neoaves; F, incongruent markers; G, 'landbirds'; H, 'landbirds' without mousebirds; I, Eufalconimorphae+seriemas; J, Eufalconimorphae; K, Psittacopasserae; L, Passeriformes). CR1 retroposons are highlighted in blue and LTR retroposons are shown in red. The dashed bracket consists of retroposon markers that were inserted during the neoavian radiation; the grey dashed vertical line indicates the estimated end of the Mesozoic Era at the Cretaceous/Tertiary boundary30. We note that the exceptionally long TinT activity range of the CR1-E_Pass element (no. 4) is most probably an overestimation because of CR1 subfamily misidentification, as only a few diagnostic nucleotides distinguish this retroposon from other CR1 subfamilies.

Schematic brain drawings (adapted from Jarvis et al.31) depict the hearing- and vocalizing-induced ZENK transcription factor expressions in hummingbirds, budgerigars (parrot representatives) and songbirds (passerine representatives). Our robust phylogenetic framework implies that some traits associated with vocal learning (for example, the anterior–medial vocal pathway (red)) are potentially homologous and thus evolved as an autapomorphic trait (black square) in the psittacopasseran ancestor, but also independently in the distantly related ancestor of hummingbirds (dashed lines indicate that several neoavian lineages are not shown). Neuroanatomical studies on early branching passerines (that is, Suboscines and Acanthisittidae) are necessary to infer that the posterior–lateral vocal pathway (green, located at different brain regions in parrots and oscine passerines) is either homologous or evolved independently in the two lineages. The caudal auditory pathway (blue) is a plesiomorphic trait (white square) and was probably inherited from a common avian ancestor32. The putative location of the auditory pathway in falcons and swifts is not shown, as ZENK expression patterns have, to our knowledge, not yet been investigated in these birds. Scale bar, 2 mm.