ABSTRACT: We report the complete chloroplast genomes of four Viola species (V. mirabilis, V. phalacrocarpa, V. raddeana, and V. websteri) and the results of a comparative analysis between these species and the published plastid genome of the congeneric species V. seoulensis. The total genome length of the five Viola species, including the four species analyzed in this study and the species analyzed in the previous study, ranged from 156,507 (V. seoulensis) to 158,162 bp (V. mirabilis). The overall GC contents of the genomes were almost identical (36.2-36.3%). The five Viola plastomes each contained 111 unique genes comprising 77 protein-coding genes, 30 transfer RNA (tRNA) genes, and 4 ribosomal RNA (rRNA) genes. Among the annotated genes, 16 contained one or two introns. Based on the results of a chloroplast genome structure comparison using MAUVE, all five Viola plastomes were almost identical. Additionally, the large single copy (LSC), inverted repeat (IR), and small single copy (SSC) junction regions were conserved among the Viola species. A total of 259 exon, intron, and intergenic spacer (IGS) fragments were compared to verify the divergence hotspot regions. The nucleotide diversity (Pi) values ranged from 0 to 0.7544. The IR region was relatively more conserved than the LSC and SSC regions. The Pi values in ten noncoding regions were relatively high (>0.03). Among these regions, all but rps19-trnH, petG-trnW, rpl16-rps3, and rpl2-rpl23 represent useful molecular markers for phylogenetic studies and will be helpful to resolve the phylogenetic relationships of Viola. The phylogenetic tree, which used 76 protein-coding genes from 21 Malpighiales species and one outgroup species (Averrhoa carambola), revealed that Malpighiales is divided into five clades at the family level: Erythroxylaceae, Chrysobalanaceae, Euphorbiaceae, Salicaceae, and Violaceae. Additionally, Violaceae was monophyletic, with a bootstrap value of 100% and was divided into two subclades.
