Comparative chloroplast genomes and phylogenetic analysis of the genus Stephania (Ranunculales: Menispermaceae): implications for molecular marker development.
TL;DR
The complete chloroplast genomes of Stephania exhibited a high degree of conservation, with no significant large-scale gene rearrangements or inversions observed, and the boundaries are relatively stable.
Abstract
The genus Stephania is a member of the family Menispermaceae within the angiosperms, possesses remarkable species diversity across south of China. To elucidate phylogenetic relationships within the genus Stephania at the chloroplast genome level, we sequenced the complete chloroplast genomes of four Stephania species, namely Stephania longipes, Stephania lincangensis, Stephania longa, and Stephania herbacea, using a combination of second- and third-generation sequencing technologies. The complete chloroplast genomes of four Stephania species sequenced in this study exhibited a conserved quadripartite circular structure, with total lengths ranging from 157,727 bp to 158,138 bp. These genomes comprised 130 functional genes, with a Guanine-cytosine (GC) content ranging from 38.24% to 38.28%. Among these, protein-coding genes displayed a significant A/U preference at the third codon position. Repetitive sequence analysis detected a total of 31 to 43 long repeat sequences and 74 to 96 simple sequence repeats (SSRs), with A/T type mononucleotide repeats constituting the largest proportion. The complete chloroplast genomes of Stephania exhibited a high degree of conservation, with no significant large-scale gene rearrangements or inversions observed, and the boundaries are relatively stable. Nucleotide diversity (Pi) analysis showed that the shared intergenic regions generally exhibited higher nucleotide polymorphism than the shared coding regions, and nine highly variable regions could serve as candidate barcode regions for species identification in Stephania. DNA barcoding analysis showed that the trnK-UUU-matK region could be successfully amplified in all tested samples, and the phylogenetic tree based on this region was highly consistent with that inferred from complete chloroplast genomes, indicating its potential as a candidate DNA barcode for Stephania. Phylogenetic analysis of chloroplast genomes revealed that the genus Stephania formed a distinct monophyletic clade, with S. longipes and S. lincangensis closely related, while S. longa and S. herbacea clustered together in another lineage. The phylogenetic relationships in the genus Stephania reflect the division of subgenera, but do not reveal section division within the genus. It is indicated that inflorescence and fruit morphological characteristics in Stephania are useful for taxonomic identification but may provide limited phylogenetic signal. Additionally, Stephania japonica var. timoriensis and Stephania japonica var. discolor showed a certain degree of differentiation from typical Stephania japonica, suggesting that their taxonomic status warrants further evaluation.