Divergence time estimations were computed on truncated datasets to allow completion of the analyses. AliStat v1.6 (63 ) was used to generate a subsampled dataset containing 195 Amphiesmenoptera species (all non-Amphiesmenoptera removed) and only including sites for which at least 80% of samples had unambiguous amino acids. MCMCTree and codeml (both part of the PAML software package, v4.9g) (64 (link)) were used to estimate divergence dates on this subsampled dataset. The best ML tree inferred from the concatenated amino acid dataset was used as the input tree. The input tree was first calibrated using age estimates of 16 carefully selected fossils, following the best-practice recommendations by Parham et al. (65 (link)). Although all 16 fossils have diagnostic morphological characteristics that enable reasonably confident placement on the tree, only 3 of these fossils have true synapomorphies. To strictly follow the guidelines of Parham et al. (65 (link)), additional analyses were performed using only these 3 fossils. We applied a conservative age constraint on the root of the input tree, with a minimum age of 201 Ma, based on the stem Glossata scale fossils discovered by van Eldijk et al. (66 (link)) and a maximum age of 314.4 Ma, based on the absence of Amphiesmenoptera fossils in the Late Carboniferous. We used 2 well-established approaches to convert fossil ages into calibrations on tree nodes. For the conservative strategy, fossil calibrations were treated with uniform distributions constrained between the corresponding fossil age (the minimum bound) and a hard maximum equal to the maximum root age. For the second strategy, the truncated-Cauchy distribution (67 (link)) was used to set calibrations for internal nodes younger than 80 Ma. (Additional information on this approach is provided in SI Appendix, section 10.) We applied both uncorrelated rates and autocorrelated rates to estimate divergence, for a total of 8 analyses: 2 sets of fossil calibrations (16 fossils and 3 fossils) × 2 fossil calibration strategies (uniform and Cauchy priors for nodes <80 Ma) × 2 rate types (independent and autocorrelated). To compare the radiation of Lepidoptera and flowering plants, estimates of the mean age of the ancestral angiosperm were compiled from the literature; these angiosperm ages are presented in Dataset S12 and shown in Fig. 2. Since the estimated timespan between divergence of angiosperms and gymnosperms is large, and it is possible that flowering plants existed long before the crown of angiosperms, we present the interval between the mean age of the crown (node Angiosperm) and the mean age of the stem (node Angiosperm + Gymnosperm) from the abovementioned studies (Dataset S12). Additional information and justification for our approaches to divergence time estimation is available in SI Appendix, section 10.