*4.6. Phylogenetic Analyses*

There were 26 plastid genomes used for phylogenetic analyses, including 2 outgroups (*Passiflora laurifolia* and *Passiflora ligularis*) and 24 Salicaceae *s.l.* species (Table 1). We extracted 63 protein-coding genes in all 26 species using a python script. All the genes aligned by MAFFT and used RAxML v8.1.24 [80] to conduct maximum likelihood (ML) analyses with the GTR + I + G model. The best scoring ML tree was obtained with 1000 bootstrap replicates. Then, we estimated divergence times of the plastome dataset, using an approximate likelihood method as implemented in MCMCtree (in PAML version 4) [81], with an independent relaxed clock and birth–death sampling [82]. The divergence between Salicaceae *s.l.* species and the outgroup, *Passiflora*, was assigned an age constraint of 87 to 97 Mya, as has been previously used [83–86].
