Characterization of mitochondrial genomes from three medicinal species of rutaceae and comparative analysis within the family: insights into evolution

Rutaceae encompasses numerous medicinal plants with unclear genetic relationships and mitochondrial genome (mitogenome) characteristics, which hinders understanding of their evolutionary adaptation and medicinal trait development. Here, we aimed to investigate the conserved and divergent features of mitogenomes among closely related medicinal genera of Rutaceae, and to explore how mitogenomic variations correlate with their phylogenetic positions. Citrus aurantium, Toddalia asiatica, and Zanthoxylum nitidum (Roxb.) DC are medicinally valuable but understudied Rutaceae species belonging to different genera. We sequenced, assembled, and annotated their complete mitogenomes: C. aurantium (504,387 bp, 45.17% GC content) contained 62 genes (36 protein-coding genes (PCGs), 3 rRNAs, and 23 tRNAs), T. asiatica (566,784 bp, 45.37% GC content) harbored 58 genes (35 PCGs, 3 rRNAs, and 20 tRNAs), and Z. nitidum (539,013 bp, 45.48% GC content) possessed 61 genes (34 PCGs, 3 rRNAs, and 24 tRNAs). Comparative analyses with 12 published Rutaceae mitogenomes showed that the three species exhibited the highest number of RNA editing sites in PCGs among the examined species. Evaluation of selective pressure and gene clusters revealed fewer differences between C. aurantium and C. maxima cultivar Hirado Buntan than with the other two species, while C. sinensis displayed greater dissimilarities with other Citrus species (notably, C. maxima and C. maxima cultivar Hirado Buntan were conspecific and should have the least divergence). Phylogenetic trees constructed using 24 conserved mitochondrial PCGs clarified evolutionary relationships: C. maxima was closest to C. sinensis, followed by C. maxima cultivar Hirado Buntan and C. aurantium formed an unexpected separate clade, highlighting the need for more Rutaceae species' mitogenome in future phylogenetic analyses. This study expands the resource pool in Rutaceae mitogenomic resources, provides insights into features that may be associated with ecological adaptation, and offers valuable genomic data for future phylogenetic and comparative studies of Rutaceae medicinal plants.