OrthoMCL (http://orthomcl.org/orthomcl/) was used to construct orthologous gene families between P. somniferum (CHM and HN1) and 12 other plant species. MUSCLE44 was utilized to construct multiple sequence alignments of 35 single-copy orthologs among 14 species. RAxML software45 (version 7.2.3) was carried out to construct the maximum likelihood tree with the PROTGAMMAAUTO model by using the sequence alignments with A. trichopoda as an outgroup (Supplementary Fig. S9). The MCMCTree program of PAML (http://abacus.gene.ucl.ac.uk/software/paml.html) was applied to estimate divergence time using CDS alignments transformed from protein alignments. Five calibration values were chosen from the Time Tree website (http://www.timetree.org). Expansions and contractions of orthologous gene families were determined using CAFÉ 2.2 (Computational Analysis of gene Family Evolution)46 (Supplementary Fig. S11). For whole-genome duplication (WGD) analysis, the syntenic region between and within P. somniferum (Pso), M. cordata (Mco), and A. coerulea (Aco) was determined by MCscanX47 based on the all-to-all BLASTP results. The protein sequences of homologous gene pairs in the syntenic region were extracted and aligned using the MUSCLE program44. Subsequently, the protein sequence alignments were converted into CDS files, and 4DTv values were calculated based on the CDS alignments, accompanying the correction of the HKY model.

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