Molecular dating and diversification analysis

The transcriptome sequencing reads from 339 cycad species were generated in the current study. Clean reads were assembled with TRINITY⁸⁹, and the longest transcripts were selected and translated with TRANSDECODER (https://github.com/TransDecoder). OrthoFinder⁸² was then used to construct orthogroups for all the cycad species using Ginkgo as the outgroup. The software KinFin⁸³ was used to select the mostly single-copy genes for phylogenetic reconstruction with default settings. TranslatorX⁸⁴, IQ-TREE 2 (ref. ⁸⁵) and ASTRAL⁸⁶ were used to align the sequences and to infer the species tree for cycads as aforementioned. The software SORTADATE⁹⁰ was used to select genes with mostly concordant evolutionary histories for dating analyses using MCMCTREE within the software PAML 4 (ref. ⁹¹). Rate priors and time priors were set following the method of Morris et al.⁹². A total of 27 fossils were used to calibrate the chronogram of seed plants, and six fossils for the chronogram of cycads. The diversification pattern for cycads were analysed with Bayesian analysis of macroevolutionary mixture (www.bamm-project.org) following Condamine et al.⁹³

See Supplementary Note 5 for details on organellar phylogenetic reconstruction, evaluation of the impact of RNA editing and investigation of cyto-nuclear incongruences.