Comparative Analysis of the Chloroplast Genomes of Cypripedium: Assessing the Roles of SSRs and TRs in the Non-Coding Regions of LSC in Shaping Chloroplast Genome Size

<i>Cypripedium</i> is renowned for its high morphological diversity and complex genetic and evolutionary characteristics. The chloroplast genome serves as a valuable tool for investigating phylogenetic relationships and evolutionary processes in plants. Currently, research on the evolution of the chloroplast genome within the <i>Cypripedium</i> genus is limited due to insufficient large-scale sampling and a lack of comprehensive understanding. Consequently, the mechanisms underlying the significant differences in chloroplast genome size among <i>Cypripedium</i> species remain poorly understood. In this study, we conducted a comprehensive comparative analysis of the chloroplast genomes of 29 <i>Cypripedium</i> species. The lengths of these genomes ranged from 162,092 to 246,177 base pairs (bp) and contained between 127 and 134 genes. Our results indicate that, while the overall structure of the chloroplast genomes in <i>Cypripedium</i> species is relatively conserved, significant differences were observed among the large single-copy (LSC), small single-copy (SSC), and inverted repeat (IR) regions. Several genes, including <i>psaC</i>, <i>rpl32</i>, <i>ycf1</i>, and <i>psbK</i>, exhibited higher levels of variability and may serve as molecular markers in taxonomic studies. The results of our correlation analysis suggest that the expansion of the LSC region, the increase in simple sequence repeats (SSRs), and tandem repeats (TRs) have significantly enlarged the size of the chloroplast genome in <i>Cypripedium</i> species. Phylogenetic signal testing supports the notion that genetic variation has driven species divergence within the genus. Overall, our findings provide insights into the substantial differences in chloroplast genome length observed among <i>Cypripedium</i> species. However, the relationship between diversification and the evolutionary mechanisms affecting <i>Cypripedium</i>, including ecological adaptive evolution, incomplete lineage sorting (ILS), hybridization, and reticulate events, requires further investigation.