Ning Sun ^1* Fuchuan Han ² Suyan Wang ¹ Wei Liu ³ Weishu Fan ^4,5* Changwei Bi ^1,6*

• ¹ College of Information Science and Technology & Artificial Intelligence, Nanjing Forestry University, Nanjing, China
• ² Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou, Zhejiang Province, China
• ³ College of Optical, Mechanical and Electrical Engineering, Zhejiang Agriculture and Forestry University, Hangzhou, Zhejiang Province, China
• ⁴ New Cornerstone Science Laboratory, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
• ⁵ Germplasm Bank of Wild Species in Southwest China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan Province, China
• ⁶ State Key Laboratory of Tree Genetics and Breeding, Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, China

Lycophytes and ferns represent one of the earliest-diverging lineages of vascular plants, with the Lycopodiaceae family constituting the basal clade among lycophytes. In this research, we successfully assembled and annotated the complete Lycopodium japonicum Thunb. (L. japonicum) mitochondrial genome (mitogenome) utilizing PacBio HiFi sequencing data, resulting in a single circular molecule with a size of 454,458 bp. 64 unique genes were annotated altogether, including 34 protein-coding genes, 27 tRNAs and 3 rRNAs. It also contains 32 group Ⅱ introns, all of which undergo cis-splicing. We identified 195 simple sequence repeats, 1,948 dispersed repeats, and 92 tandem repeats in the L. japonicum mitogenome. Collinear analysis indicated that the mitogenomes of Lycopodiaceae are remarkably conserved compared to those of other vascular plants. We totally identified 326 RNA editing sites in 31 unique protein-coding genes with 299 sites converting cytosine to uracil and 27 sites the reverse. Notably, the L. japonicum mitogenome has small amounts foreign DNA from plastid or nuclear origin, accounting for only 2.81% of the mitogenome. The maximum likelihood phylogenetic analysis based on 23 diverse land plant mitogenomes and plastid genomes supports the basal position of lycophytes within vascular plants and they form a sister clade to all other vascular lineages, which is consistent with the PPG I classification system. As the first reported mitogenome of Lycopodioideae subfamily, this study enriches our understanding of Lycopodium mitogenomes, and sets the stage for future research on mitochondrial diversity and evolution within the lycophytes and ferns.