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

<h4>Introduction</h4><i>Rhodiola</i> 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.<h4>Methods</h4>In this study, we analyzed the complete chloroplast genomes of 89 newly sequenced samples representing 35 <i>Rhodiola</i> 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.<h4>Results</h4>The chloroplast genomes of <i>Rhodiola</i> 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 <i>ycf1, matK, rpoC2</i>, and <i>rpoB</i>, as well as the intergenic regions <i>trnR</i>-UCU-<i>atpA</i>, <i>trnH</i>-GUG-<i>psbA</i>, and <i>rpl14-rpl16</i>. A rare 108-bp expansion in the <i>ndhA-ndhH</i> intergenic region was detected exclusively in <i>R. discolor</i>. Phylogenetic analysis supported two major clades broadly corresponding to hermaphroditic and dioecious lineages. However, the placements of <i>R. sachalinensis, R. wallichiana</i>, and <i>R. wallichiana</i> var. <i>cholaensis</i> indicate a more complex evolutionary history, potentially involving parallel evolution of sexual systems or transitional lineages. Lineage-specific polymorphisms in <i>rps15</i>, <i>ndhI</i>, <i>rps3</i>, and <i>rpl20</i> were further identified as candidate chloroplast genome markers associated with maternal lineage divergence and sexual-system evolution.<h4>Discussion</h4>These findings demonstrate that complete chloroplast genome data provide higher resolution for understanding evolutionary relationships within <i>Rhodiola</i>. 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 <i>Rhodiola</i>.