The first complete multichromosomal mitogenome of Cunninghamia lanceolata (Chinese fir): assembly, characterization...

Cunninghamia lanceolata is a key native timber species in southern China, yet the absence of mitochondrial genomic data has limited molecular breeding and phylogenetic studies in this species. Here, the complete mitochondrial genome of C. lanceolata was assembled via a hybrid Illumina/Nanopore approach. The genome adopts a complex architecture comprising one linear contig and a multi-branched structure formed by six additional linear contigs, as verified by targeted PCR across junctions and Sanger sequencing of the products. Genome annotation identified 31 protein-coding genes (PCGs), 3 rRNA genes, and 11 tRNA genes. We further detected 613 predicted C‑to‑U RNA editing sites across all PCGs. To validate our findings, we performed PCR amplification, Sanger sequencing, and alignment of PCR products with genomic DNA sequences, which confirmed RNA editing at atp6 (ACG → AUG/Thr → Met), ccmFN (CGU → UGU/Arg → Cys), nad6 (CGA → UGA/Arg → End), and another atp6 site (CAA → UAA/Gln → End). Comparative analysis with the chloroplast genome revealed six mitochondrial plastid DNA sequences, including fragments of two rRNA genes and one tRNA gene. Phylogenetic analysis using maximum likelihood and Bayesian methods based on PCGs strongly supported (bootstrap = 100, posterior probability = 1.0) a monophyletic clade comprising two traditional Taxodiaceae species (C. lanceolata and Metasequoia glyptostroboides) and four Cupressaceae s.s. species (Cupressus sempervirens, Hesperocyparis glabra, Platycladus orientalis, and Thuja sutchuenensis). Cupressaceae s.l. was resolved as sister to Taxaceae, while Podocarpaceae and Araucariaceae together formed a distinct sister group. Selection pressure analysis (Ka/Ks) indicated that nad3 and rps4 exhibited Ka/Ks ratios > 1, suggesting positive selection. Notably, pairwise comparisons between Th. sutchuenensis and other Cupressaceae species consistently yielded Ka/Ks > 1, indicating accelerated adaptive evolution in this lineage. This study provides the first mitochondrial genome resource for C. lanceolata, offering a foundation for molecular breeding and investigations into conifer mitochondrial evolution.