Charophyta, initially classified as ferns and later within green algae, were recognized as an independent division in the twentieth century. The taxonomic status of Charophyta has evolved considerably over time and phylogenomic studies elucidating the evolutionary relationships within Charophyta remain limited. This study sequenced and analyzed the mitochondrial genomes of eight charophyte plants, including genome size, gene content, and codon usage bias, and compared these features with those of other algal lineages and higher plants. Total DNA was extracted from the plant samples and subjected to single-molecule sequencing and genomic sequencing, respectively. The successfully sequenced rbcL and genomic sequences were used to reconstruct the phylogenetic tree. The single-gene phylogeny indicated that the sample sxuxm2407072 (xm2) is a close relative of Chara canescens Loiseleur, while the remaining samples are affiliated with Chara vulgaris Linn. Additionally, xm2 exhibited distinct spine cell morphology compared to other samples. Eight mitochondrial genomes were assembled in this study, ranging from 66,695 to 70,447 bp in size and exhibiting slightly lower GC content than AT content, yet a significantly greater GC skew compared to the AT skew. RSCU analysis revealed consistent codon usage patterns across samples, with AGA representing the most frequently used codon. Analysis of codon usage bias in the eight mitochondrial genomes yielded values for CAI, CBI, GC12, GC3s, and ENC. The results showed extremely low CAI, consistently negative CBI, a lack of correlation between GC12 and GC3s, and a significant positive correlation between ENC and GC3s. Phylogenetic reconstruction based on mitochondrial genomic data consistently revealed eight plant samples as a sister group to the C. vulgaris clade and highly congruent topological arrangements. The newly assembled eight Chara mitochondrial genomes in our study revealed complex structures and relatively conserved sequences, exhibiting limited divergence across the entire genome while displaying elevated variability in specific gene regions. The mitochondrial genomes underwent nearly neutral evolution in their codon usage patterns. Based on mitochondrial genome phylogenetic analysis, the mitochondrial genomes of Chara plants in this study showed consistent phylogenetic signals, indicating a similar evolutionary pathway. The mitochondrial data generated in this study provide new insights into the evolutionary history of charophytes, enrich the genomic resources for this group, and establish a solid foundation for future research on this important lineage.
Xie et al. (2026) studied this question.