Abstract
Cypripedium macranthos Swartz, C. × ventricosum Swartz, and C. shanxiense S. C. Chen were highly promising ornamental plants. In this study, the latest complete chloroplast genome sequence of C. macranthos, C. × ventricosum, and C. shanxiense were reported using correct sample material from their native range, and their phylogenetic relationships with other related species were investigated preliminarily. The whole chloroplast genome lengths of C. macranthos, C. × ventricosum, and C. shanxiense were 181,030 bp, 175,385 bp, and 177,627 bp, respectively, with total GC contents of 34.56%, 34.48%, and 34.42%, respectively. Based on the maximum likelihood phylogenetic tree from the chloroplast genome sequences and internal transcribed spacer (ITS) sequences, it was confirmed that C. × ventricosum is most closely related to C. calceolus, and the idea that C. × ventricosum is an interspecific hybrid between C. calceolus and C. macranthos is supported. By integrating the results of phylogenetic analysis, genomic structural comparison, and considerations of sampling locations, it is evident that the former chloroplast genome of C. macranthos is inaccurate. This study provides crucial information for research on the phylogeny, genetics, and conservation of C. macranthos, C. × ventricosum, and C. shanxiense.