Chinese herbal tea, also known as Liang Cha or cooling beverage, is popular in South China. It is regarded as a quick-fix remedy to relieve minor health problems. Hedyotis diffusa Willd. (colloquially Baihuasheshecao) is a common ingredient of cooling beverages. H. diffusa is also used to treat cancer and bacterial infections. Owing to the high demand for H. diffusa, two common adulterants, Hedyotis brachypoda (DC.) Sivar and Biju (colloquially Nidingjingcao) and Hedyotis corymbosa (L.) Lam. (colloquially Shuixiancao), are commonly encountered in the market. Owing to the close similarity of their morphological characteristics, it is difficult to differentiate them. Here, we sequenced the complete chloroplast genomes of the three species of Hedyotis using next-generation sequencing (NGS). By comparing the complete chloroplast genomes, we found that they are closely related in the subfamily Rubioideae. We also discovered that there are significant differences in the number and repeating motifs of microsatellites and complex repeats and revealed three divergent hotspots, rps16-trnQ intergenic spacer, ndhD and ycf1. By using these species-specific sequences, we propose new DNA barcoding markers for the authentication of H. diffusa and its two common adulterants.

Ilex is a monogeneric plant group (containing approximately 600 species) in the Aquifoliaceae family and one of the most commonly used medicinal herbs. However, its taxonomy and phylogenetic relationships at the species level are debatable. Herein, we obtained the complete chloroplast genomes of all 19 Ilex types that are native to Hong Kong. The genomes are conserved in structure, gene content and arrangement. The chloroplast genomes range in size from 157,119 bp in Ilex graciliflora to 158,020 bp in Ilex kwangtungensis. All these genomes contain 125 genes, of which 88 are protein-coding and 37 are tRNA genes. Four highly varied sequences (rps16-trnQ, rpl32-trnL, ndhD-psaC and ycf1) were found. The number of repeats in the Ilex genomes is mostly conserved, but the number of repeating motifs varies. The phylogenetic relationship among the 19 Ilex genomes, together with eight other available genomes in other studies, was investigated. Most of the species could be correctly assigned to the section or even series level, consistent with previous taxonomy, except Ilex rotunda var. microcarpa, Ilex asprella var. tapuensis and Ilex chapaensis. These species were reclassified; I. rotunda was placed in the section Micrococca, while the other two were grouped with the section Pseudoaquifolium. These studies provide a better understanding of Ilex phylogeny and refine its classification.

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Dalbergia L.f. is a pantropical genus consisting of 269 species of trees, shrubs, and woody lianas. This genus is listed in CITES Appendices because of illegal logging and trafficking driven by the high economic value of its heartwood. Some species are also used medicinally. Species identification of Dalbergia timber and herbs is challenging but essential for CITES implementation. Molecular methods had been developed for some timber species, mostly from Madagascar and Southeast Asia, but medicinal species in south China were usually not included in those studies. Here, we sequenced and assembled the chloroplast genomes of five Dalbergia species native to Hong Kong, four of which are medicinal plants. Our aim is to find potential genetic markers for the identification of medicinal Dalbergia species based on divergence hotspots detected in chloroplast genomes after comparative and phylogenetic analysis. Dalbergia chloroplast genomes displayed the typical quadripartite structure, with the 50 kb inversion found in most Papilionoideae lineages. Their sizes and gene content are well conserved. Phylogenetic tree of Dalbergia chloroplast genomes showed an overall topology similar to that of ITS sequences. Four divergence hotspots (trnL(UAA)-trnT(UGU), ndhG-ndhI, ycf1a and ycf1b) were identified and candidate markers for identification of several Dalbergia species were suggested.

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