Comparatively analyzing of chloroplast genome and new insights into phylogenetic relationships regarding the genus Stephania.

The medicinal plant of the genus Stephania holds significant economic importance in the pharmaceutical industry. However, accurately classifying and subdividing this genus remains a challenge. Herein, the chloroplast (cp) genomes of Stephania and Cyclea were sequenced, and the primary characteristics, repeat sequences, inverted repeats regions, simple sequence repeats, and codon usage bias of 17 species were comparatively analyzed. Twelve markers were identified through genome alignment and sliding window analysis. Moreover, a molecular clock analysis revealed the divergence between subgenus (subg.) Botryodiscia and the combined Cyclea, subg. Stephania and Tuberiphania during the early Oligocene epoch. Notably, the raceme-type inflorescence represents the ancestral state of the Stephania and Cyclea. The genetic relationships inferred from the cp genome and protein-coding genes exhibited similar topologies. Additionally, the paraphyletic relationship between the genera Cyclea and Stephania was confirmed. Bayesian inference, maximum likelihood, and neighbor-joining trees consistently showed that section Tuberiphania and Transcostula were non-monophyletic. In conclusion, this research provides valuable insights for further investigations into species identification, evolution, and phylogenetics within the Stephania genus.