2.3. Sequence analysis and genome comparison

The GC content was conducted by using the cusp program provided by EMBOSS version 6.3.1 (Rice et al. 2000). IRscope (https://irscope.shinyapps.io/irapp/) was used for visualizing the IR boundaries in these cp genomes (Amiryousefi et al. 2018). A total of 78 orthologous genes and 89 intergenic spacer regions (IGSs) among 14 Scutellaria species were identified and extracted by using Phylosuite version 1.2.1 (Zhang et al. 2020). The corresponding nucleotide sequences were aligned by using MAFFT version 7.450 (https://mafft.cbrc.jp/alignment/server/) (Rozewicki et al. 2019) implemented in Phylosuite. We used MEGA version 6.0 (Tamura et al. 2013) to calculate the percentage of variable sites (PV) in protein-coding genes and the pairwise K2-P distance in IGSs. Then, we used DnaSP version 6.0 (Rozas et al. 2017) to calculate the nucleotide diversity (Pi) among the protein-coding sequence.