Abstract
This study sequenced the complete chloroplast genomes of Stephania japonica var. timoriensis and Stephania japonica var. discolor using the Illumina NovaSeq and PacBio RSII platforms. Following sequencing, the genomes were assembled, annotated, comparatively analyzed, and used to construct a phylogenetic tree to explore their phylogenetic positions. Results indicated that the chloroplast genomes of S. japonica var. timoriensis and S. japonica var. discolor both displayed a typical double-stranded circular tetrameric structure, measuring 157,609 and 157,748 bp in length, respectively. Each genome contained 130 annotated genes, with similar total GC content and relative codon usage patterns, showing a distinct preference for A/U at the third codon position. Simple sequence repeat analysis identified 207 and 211 repeats in S. japonica var. timoriensis and S. japonica var. discolor, respectively, primarily the A/T type. Boundary condition analysis indicated no significant expansion or contraction in the inverted repeat regions with consistent gene types and locations across both varieties. Nucleotide polymorphism analysis highlighted greater variation in the intergenic regions than in the coding sequences of Stephania chloroplast genomes. Phylogenetic analyses demonstrated that the species Stephania clustered into a distinct, well-supported clade. Notably, Stephania japonica, along with S. japonica var. discolor and S. japonica var. timoriensis, established a monophyletic lineage. Within this lineage, S. japonica and S. japonica var. discolor were closely related, with S. japonica var. timoriensis serving as their sister taxon.