Assembly and comparative analysis of the complete multichromosomal mitochondrial genome of an endangered orchid species, Calanthe sieboldii

Introduction Plant mitochondrial genomes (mitogenomes) are known for their structural complexity, particularly within the Orchidaceae. To understand the evolutionary dynamics in the endangered genus Calanthe , this study provides the first complete mitogenome assembly for the endangered species Calanthe sieboldii , a species of horticultural and conservation importance. Methods A hybrid sequencing approach combining Nanopore long reads and BGI short reads was used for denovo assembly. The genome was annotated, and we performed comparative analyses of repetitive sequences, interorganellar DNA transfer, codon usage, RNA editing, synteny, and phylogeny. Results The 644,236 bp mitogenome exhibits a highly fragmented architecture, comprising 21 independent circular chromosomes ranging from 19.9 to 48.7 kb. We annotated 39 unique protein-coding genes, 23 tRNA genes, and 3 rRNA genes. The genome is characterized by a high density of repetitive sequences and a massive influx of chloroplast DNA, with mitochondrial–plastid sequences accounting for 12.72% of the total length. Comparative synteny analysis with other orchid species revealed an almost complete loss of gene order, highlighting extreme structural rearrangement. Despite this plasticity, core molecular features, such as codon usage and predicted RNA editing patterns, remain conserved. Phylogenetic analysis robustly placed C. sieboldii within the Orchidaceae. Discussion This study decodes a complex multichromosomal mitogenome, reinforcing the paradigm of dynamic structural evolution in orchids and providing a vital genomic resource to support conservation efforts and evolutionary research on the Calanthe genus.