Application of chloroplast genome in the identification of Traditional Chinese Medicine Viola philippica.

BACKGROUND: Viola philippica Cav. is the only source plant of "Zi Hua Di Ding", which is a Traditional Chinese Medicine (TCM) that is utilized as an antifebrile and detoxicant agent for the treatment of acute pyogenic infections. Historically, many Viola species with violet flowers have been misused in "Zi Hua Di Ding". Viola have been recognized as a taxonomically difficult genera due to their highly similar morphological characteristics. Here, all common V. philippica adulterants were sampled. A total of 24 complete chloroplast (cp) genomes were analyzed, among these 5 cp genome sequences were downloaded from GenBank and 19 cp genomes, including 2 "Zi Hua Di Ding" purchased from a local TCM pharmacy, were newly sequenced. RESULTS: The Viola cp genomes ranged from 156,483 bp to 158,940 bp in length. A total of 110 unique genes were annotated, including 76 protein-coding genes, 30 tRNAs, and four rRNAs. Sequence divergence analysis screening identified 16 highly diverged sequences; these could be used as markers for the identification of Viola species. The morphological, maximum likelihood and Bayesian inference trees of whole cp genome sequences and highly diverged sequences were divided into five monophyletic clades. The species in each of the five clades were identical in their positions within the morphological and cp genome tree. The shared morphological characters belonging to each clade was summarized. Interestingly, unique variable sites were found in ndhF, rpl22, and ycf1 of V. philippica, and these sites can be selected to distinguish V. philippica from samples all other Viola species, including its most closely related species. In addition, important morphological characteristics were proposed to assist the identification of V. philippica. We applied these methods to examine 2 "Zi Hua Di Ding" randomly purchased from the local TCM pharmacy, and this analysis revealed that the morphological and molecular characteristics were valid for the identification of V. philippica. CONCLUSIONS: This study provides invaluable data for the improvement of species identification and germplasm of V. philippica that may facilitate the application of a super-barcode in TCM identification and enable future studies on phylogenetic evolution and safe medical applications.

Plastid and mitochondrial phylogenomics reveal correlated substitution rate variation in Koenigia (Polygonoideae, Polygonaceae) and a reduced plastome for Koenigia delicatula including loss of all ndh genes.

Koenigia, a genus proposed by Linnaeus, has a contentious taxonomic history. In particular, relationships among species and the circumscription of the genus relative to Aconogonon remain uncertain. To explore phylogenetic relationships of Koenigia with other members of tribe Persicarieae and to establish the timing of major evolutionary diversification events, genome skimming of organellar sequences was used to assemble plastomes and mitochondrial genes from 15 individuals representing 13 species. Most Persicarieae plastomes exhibit a conserved structure and content relative to other flowering plants. However, Koenigia delicatula has lost functional copies of all ndh genes and the intron from atpF. In addition, the rpl32 gene was relocated in the K. delicatula plastome, which likely occurred via overlapping inversions or differential expansion and contraction of the inverted repeat. The highly supported but conflicting relationships between plastome and mitochondrial trees and among gene trees complicates the circumscription of Koenigia, which could be caused by rapid diversification within a short period. Moreover, the plastome and mitochondrial trees revealed correlated variation in substitution rates among Persicarieae species, suggesting a shared underlying mechanism promoting evolutionary rate variation in both organellar genomes. The divergence of dwarf K. delicatula from other Koenigia species may be associated with the well-known Eocene Thermal Maximum 2 or Early Eocene Climatic Optimum event, while diversification of the core-Koenigia clade associates with the Mid-Miocene Climatic Optimum and the uplift of Qinghai-Tibetan Plateau and adjacent areas.

Plastid phylogenomics sheds light on divergence time and ecological adaptations of the tribe Persicarieae (Polygonaceae).

Southwestern China, adjacent to the Qinghai-Tibetan Plateau (QTP), is known as a hotspot for plant diversity and endemism, and it is the origin and diversification center of Persicarieae. As one of the major lineages in Polygonaceae, Persicarieae represents a diverse adaptation to various habitats. As a result of morphological plasticity and poorly resolving molecular markers, phylogenetic relationships and infrageneric classification within Persicarieae have long been controversial. In addition, neither plastome phylogenomic studies nor divergence time estimates on a larger sample of Persicarieae species have been made thus far. We sequenced and assembled 74 complete plastomes, including all of the recognized genera within Persicarieae and their relatives. We conducted a comprehensive phylogenetic study of the major clades within Persicarieae and, based on the thus obtained robust phylogeny, also estimated divergence time and the evolution of diagnostic morphological traits. Major relationships found in previous phylogenetic studies were confirmed, including those of the backbone of the tree, which had been a major problem in previous phylogenies of the tribe. Phylogenetic analysis revealed strong support for Koenigia as sister to Bistorta, and together they were sister to the robustly supported Persicaria. Based on the phylogenetic and morphological evidence, we recognize five sections in Persicaria: Persicaria, Amphibia, Tovara, Echinocaulon, and Cephalophilon. It is estimated that the divergence of the Persicarieae began around the late Paleocene, with diversification concentrated in the Eocene and Miocene. In addition, it is suggested that the increasing westerly and monsoon winds in conjunction with the uplift of the QTP may be the driving force for origin and diversification of Persicarieae species. These results provide a valuable evolutionary framework for the study of adaptation in Polygonaceae and insights into plant diversification on the QTP and adjacent areas.