Comparative chloroplast genomes and phylogenetic analysisy of the Phlegmariurus (Lycopodiaceae) from China and neighboring regions

Ruichen Xiang^1,2,3Jiayu Hu^1,4Javzandolgor Chuluunbat^1,2,3,5Fei Wu^2,6Bo Qin⁷Xianchun Zhang^1,2Rihong Jiang^7*

• ¹State Key Laboratory of Plant Diversity and Specialty Crops, Institute of Botany, Chinese Academy of Sciences, Beijing, China
• ²China National Botanical Garden, Beijing, China
• ³College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China
• ⁴Key Laboratory of South Subtropical Plant Diversity, Fairy Lake Botanical Garden (CAS), Shenzhen, China
• ⁵Laboratory of Plant Systematics and Phylogenetic, Botanic Garden and Research Institute, Mongolian Academy of Sciences, Ulaanbaatar, Mongolia
• ⁶Beijing Floriculture Engineering Technology Research Centre, Beijing, China
• ⁷Guangxi Key Laboratory of Special Non-wood Forest Cultivation and Utilization, Guangxi Engineering and Technology Research Center for Woody Spices, Guangxi Forestry Research Institute, Nanning, Guangxi Zhuang Region, China

The lycophyte genus Phlegmariurus (Herter) Holub (Huperzioideae, Lycopodiaceae) is ecologically and pharmaceutically significant, notably as a natural source of Huperzine A—a promising therapeutic candidate for Alzheimer’s disease. Despite its medicinal potential, taxonomic ambiguities on species delimitation and infrageneric classification have impeded conservation and sustainable utilization efforts. Here, we assembled 40 Phlegmariurus complete chloroplast genomes, including all taxa from China, most of which were reported for the first time. Our results revealed the conserved quadripartite architectures and little variation in genome size and GC content in the genus. Comparative analyses on genome sequences identified seven hypervariable loci as prospective DNA barcodes for species discrimination. The phylogenetic toopologies reconstructed from nuclear ribosomal DNA and chloroplast genome data consistently resolved four monophyletic clades, further validated by SNP-based discriminant analysis of principal components. They are well corresponding to the four sections’ classification on Chinese taxa (sect. Squarrosurus, sect. Phlegmariurus, sect. Fargesiani, sect. Hamiltoniani). Notably, nuclear and chloroplast data congruently yielded a sister relationship between sect. Squarrosurus and sect. Phlegmariurus. However, the phylogenetic positions of sect. Fargesiani and sect. Hamiltoniani conflicted across different datasets. The diversification of the Chinese Phlegmariurus was traced back to the Oligocene (ca. 26.04 Ma). The comprehensive genetic resources generated herein provide a foundation for future research on species identification, population genomics and genetic diversity preservation in this medicinally significant vital genus.