Analysis of structural variation and sex differentiation associated phylogenetic signals in newly sequenced Rhodiola chloroplast genomes using a batch processing pipeline.

Rhodiola is one of the few genera in Crassulaceae that includes both dioecious and hermaphroditic species. However, previous studies have mainly relied on representative individuals or limited plastid fragments, which may not fully resolve evolutionary relationships within the genus, especially lineage divergence associated with sexual-system differentiation. In this study, we analyzed the complete chloroplast genomes of 89 newly sequenced samples representing 35 Rhodiola species. A batch-processing pipeline was used to integrate raw-read processing, automated chloroplast genome assembly and annotation, and downstream comparative genomic and phylogenetic analyses. The chloroplast genomes of Rhodiola were highly conserved in overall structure, gene content, and codon usage, while showing moderate sequence variation in several coding genes and intergenic regions. Highly variable regions included the coding genes ycf1, matK, rpoC2, and rpoB, as well as the intergenic regions trnR-UCU-atpA, trnH-GUG-psbA, and rpl14-rpl16. A rare 108-bp expansion in the ndhA-ndhH intergenic region was detected exclusively in R. discolor. Phylogenetic analysis supported two major clades broadly corresponding to hermaphroditic and dioecious lineages. However, the placements of R. sachalinensis, R. wallichiana, and R. wallichiana var. cholaensis indicate a more complex evolutionary history, potentially involving parallel evolution of sexual systems or transitional lineages. Lineage-specific polymorphisms in rps15, ndhI, rps3, and rpl20 were further identified as candidate chloroplast genome markers associated with maternal lineage divergence and sexual-system evolution. These findings demonstrate that complete chloroplast genome data provide higher resolution for understanding evolutionary relationships within Rhodiola. The identified highly variable regions and lineage-specific polymorphisms offer useful molecular markers for species identification and provide new insights into chloroplast genome evolution and reproductive-trait divergence in Rhodiola.