Comparative analysis of mitochondrial genomes of Epimedium L. reveals heterogeneity in structure, synteny, intercellular gene transfer, and RNA editing

<i>Epimedium</i> L. is a taxonomically complex genus comprising 68 species worldwide, yet its mitochondrial genome (mitogenome) remains unexplored. The complete genomes of <i>Epimedium pubescens</i>, <i>Epimedium wushanense</i>, and <i>Epimedium sagittatum</i> were assembled using Illumina and Nanopore sequencing. The <i>Epimedium</i> mitogenomes displayed diverse structural variation, including a single circular molecule in <i>E. sagittatum</i> (324,345 bp), two circular molecules in <i>E. wushanense</i> (281,026 and 72,800 bp), and a multipartite structure with three circular chromosomes (171,784, 76,915, and 71,519 bp) and one linear chromosome (26,149 bp) in <i>E. pubescens</i>. Each species contained 58 unique genes, including 36 protein-coding genes (PCGs), 19 transfer RNAs (tRNAs), three ribosomal RNAs (rRNAs), and abundant repetitive elements [77-89 simple sequence repeats (SSRs) and 169-380 dispersed repeats]. A total of 642 cytidine (C)-to-uridine (U) RNA editing sites were predicted across 35-36 PCGs, with experimentally validated edits generating start and stop codons, revealing species-specific editing profiles. Nine mitochondrial plastid DNA (MTPT) fragments (4.4-7.5 kb) were identified per species, containing six to nine complete genes. Three <i>Epimedium</i> species and related taxa exhibited conserved mitogenome regions alongside extensive gene rearrangements and inversions. Phylogenetic analysis based on 29 conserved PCGs strongly supported a monophyletic <i>Epimedium</i> clade (100% bootstrap), with <i>E. sagittatum</i> and <i>E. pubescens</i> forming a sister group to <i>E. wushanense</i>. This study provides the first comprehensive view of <i>Epimedium</i> mitogenome architecture, RNA editing, and evolutionary relationships, enriching our understanding of its mitochondrial evolution and taxonomy.