Comparative organelle genome analysis of three medicinal species of the genus Woodwardia (Blechnaceae): new insights into the phylogeny and molecular identification

Abstract

Plants of the genus Woodwardia have a long history of medicinal use in China. Among these plants, Woodwardia japonica, Woodwardia unigemmata , and Woodwardia prolifera are the primary source species of Guan Zhong, a traditional Chinese medicine. However, because of morphological similarity among their dried herbal materials and limited availability of organellar (mitochondrial and chloroplast) genomic data, research on species identification, molecular evolution, and phylogeny has been impeded. Organelle genomes of the three Woodwardia species were sequenced and assembled using the second- and third-generation sequencing technologies. In-depth analyses were conducted, including protein-coding genes (PCGs) presence/absence, non-synonymous/synonymous mutation ratios (Ka/Ks), codon usage bias, repeat sequence identification, RNA editing site prediction, synteny and mitochondrial–chloroplast homologous fragment (MTPT) identification, and molecular identification via mVISTA and nucleotide diversity. Finally, we employed both maximum likelihood (ML) and Bayesian (BI) methods, based on single-copy orthologous genes and secondary calibrations, to infer phylogenetic relationships and divergence times among Woodwardia and related ferns. The mitochondrial genomes of the three Woodwardia species exhibited notable differences in genome size and characteristics; while the chloroplast genomes were relatively conserved and highly similar. Phylogenetic and divergence time analyses indicated that W. unigemmata and W. prolifera are more closely related and diverged more recently. Integrated analyses of highly variable regions and phylogenetic revealed that the intergenic spacer rrn16–rps12_exon2 or rps12_exon2–rrn16 could effectively differentiate the three Guan Zhong-related Woodwardia species, suggesting their potential utility as reliable DNA barcodes for identifying medicinal materials. The results provide comprehensive insights and reliable, detailed organelle genomic data for the phylogeny and molecular identification of W. japonica, W. unigemmata , and W. prolifera .