The plastomes of Cypripedium (Orchidaceae: Cypripedioideae) exhibit atypical GC content and genome size based on different sequencing strategies.

Most of the sequenced plastomes of photosynthetic angiosperms exhibit conservation in size, gene content, gene order, and GC content. In contrast, the sequenced plastomes of Cypripedium are distinguished by genome size expansion, AT-biased base composition, structural variation, and a low substitution rate. Additionally, the impact of sequencing methods is seldom addressed in prior studies, and the species represented in these studies are underrepresented. These atypical plastome features render the genus an ideal candidate for investigating plastome evolution. Besides, the backbone relationships within the genus remain poorly resolved. In this study, we sequenced twelve Cypripedium plastomes using three distinct sequencing strategies and obtained an additional 27 sequences from GenBank for comparative analysis. We classified the plastomes of the genus into two types: one resembling those of most other angiosperms, and the other characterized by inverted repeat (IR) expansion and small single copy (SSC) contraction. The plastomes within this genus exhibit significant size variations (∼72 kb), variations in GC content, and structural differences at the genus level. Furthermore, our comparative analysis revealed that the choice of sequencing strategy significantly impacts the assembly results. The uncovered regions in samples sequenced with short-read technology are predominantly AT-rich, suggesting that short-read sequencing may lead to assembly errors in plastomes with AT-rich regions and long repeats. Additionally, we have reconstructed the phylogeny of the genus using plastome-level data. However, the phylogenetic relationships within the genus remain partially solved. This study provides new insights into the evolution of plastomes, particularly those with AT-rich base compositions and genomes containing long repeat regions.