The Populations of Two Differently Medicine-Used Plants of Hedyotis diffusa and Hedyotis corymbosa Shoot-Assembling Rich Bacterial and Fungal Communities with Varied Compositions but Conserved Structures.

The plant-colonized microbial communities have closely micro-ecological effects on host plant growth and health. There are many medicinal plants in the genus Hedyotis, but it is yet unclear about the shoot-assembled bacterial and fungal communities (SBFC) of Hedyotis plants. Hence, eight plant populations of Hedyotis diffusa (HD) and H. corymbosa (HC) were evaluated with 16S rRNA gene and ITS sequences, for comparing the types, abundance, or/and potential functions of SBFC at plant species- and population levels. In tested HD- and HC-SBFC, 682 fungal operational taxonomic units and 1,329 bacterial zero-radius operational taxonomic units were identified, with rich species compositions and varied alpha diversities. Notably, the SBFC compositions of HD and HC plant populations were exhibited with partly different types and abundances at phylum and genus levels but without significantly different beta diversities at plant species and population levels. Typically, the SBFC of HD and HC plant populations were presented with abundance-different biomarkers, such as Frankiaceae and Bryobacteraceae, and with similar micro-ecological functions of microbial metabolisms of lipids, terpenoids,and xenobiotics. Taken together, HD- and HC-SBFC possessed with varied rich compositions, conservative taxonomic structures, and similar metabolic functions, but with small-scale type and abundance differences at plant species- and population- levels.

Application of novel loop-mediated isothermal amplification (LAMP) for rapid authentication of the herbal tea ingredient Hedyotis diffusa Willd.

Hedyotis diffusa Willd. (Baihuasheshecao) is an ingredient of herbal teas commonly consumed in the Orient and tropical Asia for cancer treatment and health maintenance. In the market, this ingredient is frequently adulterated by the related species Hedyotis corymbosa (L.) Lam. The objective of this study is to develop a novel loop-mediated isothermal amplification (LAMP) technique to differentiate H. diffusa from its adulterant H. corymbosa. A set of four internal control primers (F3, FIP, BIP and B3) were designed based on six loci in the internal transcribed spacer (ITS) for LAMP of both H. diffusa and H. corymbosa. Two specific primers (S_F3 and S_FIP) were designed for specific LAMP detection of H. diffusa only. Our data showed that LAMP was successful for both H. diffusa and H. corymbosa in internal control. In contrast, only H. diffusa was detected in specific LAMP using the specific primers S_F3 and S_FIP. This study showed that LAMP was useful to differentiate H. diffusa from its adulterant H. corymbosa. This study is significant for the verification of the authenticity for better quality control of this common herbal tea ingredient. The strategy of including an internal control assures the quality of the concerned DNA region for LAMP.

Phylogeny of the Asian Hedyotis-Oldenlandia complex (Spermacoceae, Rubiaceae): evidence for high levels of polyphyly and the parallel evolution of diplophragmous capsules.

Generic delimitation in the Hedyotis-Oldenlandia complex has a long taxonomically confused history because of the controversy of lumping or splitting these two taxa. Previous morphological and phylogenetic studies with a paucity of Asian taxa suggested that Hedyotis should include only Asian species characterized by diplophragmous capsules. In order to test the reliability of this conclusion, assess the phylogenetic value of capsular characters, and evaluate generic circumscriptions in this complex, a phylogenetic study based on expanded inclusion of 63 Asian species was performed using two nuclear regions and eight plastid regions with parsimony and likelihood analyses. The results show that the Hedyotis-Oldenlandia complex is a highly polyphyletic group. Hedyotis should only include most Asian species with erect, robust herbs or shrubs and diplophragmous capsules. Oldenlandia s. str. consists primarily of African species, including the type O. corymbosa, that are characterized by small herbs, paniculate or corymbose inflorescences, inserted styles and stamens, and loculicidally dehiscent capsules. Dimetia, Scleromitrion and Thecagonum are proposed to be resurrected to accommodate three newly resolved clades. Morphological character optimizations indicate that the diplophragmous capsule evolved independently twice within this complex. Plant habit, stipule shape, and capsular dehiscent pattern are of great value in generic circumscriptions.

Genetic effects of recent habitat fragmentation in the Thousand-Island Lake region of southeast China on the distylous herb Hedyotis chrysotricha (Rubiaceae).

PREMISE OF THE STUDY: Known-age artificial-lake islands provide ideal model systems to elucidate the genetic and evolutionary consequences of anthropogenic habitat fragmentation on very recent time scales. Here, we studied a distylous herb, Hedyotis chrysotricha (Rubiaceae), in the artificially created Thousand-Island Lake (TIL) region of southeast China to explore the genetic consequences of islanding for this species. • METHODS: Seven microsatellite loci were used to genotype 384 individuals of H. chrysotricha from 18 populations to estimate genetic diversity, population structure, and demographic parameters. • KEY RESULTS: Island populations had significantly lower mean genetic diversity than those from the western/eastern mainland (e.g., H(E) = 0.381 vs. 0.461) and also displayed higher mean subdivision (F(ST) = 0.12 vs. 0.042/0.051). BayesAss analyses indicated moderate levels of migration rates among most populations, whereas Bottleneck did not provide strong evidence for such effects. In consequence, 2MOD strongly favored a gene flow-drift model over a pure drift model in the study area, but concomitantly revealed a relatively greater influence of drift in the island populations as evidenced by their significantly higher probabilities of allelic coancestry (F = 0.184 vs. 0.085). • CONCLUSIONS: The observed genetic patterns in H. chrysotricha indicate that recent anthropogenic habitat fragmentation in the TIL region can lead to significant loss of genetic diversity in isolated fragments (islands) due to ongoing drift. By contrast, patterns of random mating, gene flow, and population connectivity have not greatly been modified yet, possibly owing to the species' fruit (seed) dispersal capabilities providing resilience in the face of habitat fragmentation.

Development of microsatellite markers in heterostylous Hedyotis chrysotricha (Rubiaceae).

PREMISE OF THE STUDY: Microsatellite primers were developed for a heterostylous herb, Hedyotis chrysotricha to investigate the effect of habitat fragmentation on its genetic diversity and population structure. METHODS AND RESULTS: Twelve primer sets were developed and their polymorphisms were tested on 47 individuals from two island populations of H. chrysotricha in Thousand Island Lake of China. The number of alleles per locus ranged from five to 10, with an average of seven alleles. Expected heterozygosity per locus ranged from 0.284 to 0.821 and observed heterozygosity ranged from 0.191 to 0.851. CONCLUSIONS: We showed that all of the 12 microsatellite markers developed for H. chrysotricha are polymorphic within populations, which should provide a powerful tool for assessing population structure and genetic diversity across fragmented and continuous populations, and for studying the genetic effects of habitat fragmentation on this species.

Discovery of a linear cyclotide from the bracelet subfamily and its disulfide mapping by top-down mass spectrometry.

Cyclotides are heat-stable macrocyclic peptides from plants that display a wide range of biological activities. They can be divided into two subfamilies: Möbius or bracelet, based on the presence or absence of a cis-proline residue in loop 5, respectively. Currently, over 150 cyclotides have been discovered, but only four linear variants of the Möbius subfamily have been hitherto isolated. In this study, we report the discovery of two novel cyclotides, hedyotide B1 and hedyotide B2, from the aerial parts of Hedyotis biflora. Hedyotide B1 has a cyclic cystine knot structure typical of cyclotides. Interestingly, hedyotide B2 possesses a linear backbone and is the first linear representative of the bracelet subfamily. Disulfide mapping of hedyotide B2 by a top-down MS/MS approach showed that it shares the same knotted disulfide arrangement as conventional cyclotides. Its unfolding pathway also showed that the penetrating disulfide bond Cys III-VI is the most stable disulfide linkage. Cloning of the gene encoding hedyotide B2 revealed a nonsense mutation that introduces a premature stop codon at the conserved Asn residue position, which is essential for an end-to-end backbone ligation. Biophysical characterization showed that hedyotide B2 was more susceptible to exopeptidase degradation as compared with hedyotide B1. Hedyotide B2 was also inactive against all four tested bacterial strains, whereas hedyotide B1 was bactericidal to Escherichia coli and Streptococcus salivarius at low micromolar concentration. Our results provide a deeper understanding of the structures, functions, and biosynthetic processing of cyclotides and uncyclotides in plants.

[Application of allele-specific primer in the identificatin of Hedyotis diffusa].

OBJECTIVE: To develop a convenient and effective method for the identification of Hedyotis diffusa. METHODS: DNA templates were extracted from H. diffusa and its adulterants samples on commercial markets. And DNA fragments of rDNA ITS-2 regions were amplified and sequenced subsequently. ITS-2 sequences of all samples were aligned. The allele-specific primer was designed for distinguishing H. diffusa. RESULTS: The nucleotide difference between H. diffusa and 8 other species is obvious in the ITS-2 region. In addition, the allele-specific primers were employed to amplify the DNA from H. diffusa and 8 other species. The result indicated that a 392 bp DNA fragment was amplified from H. diffusa, whereas no any fragment was amplified from 8 other species under the same reaction condition. CONCLUSION: The primers designed in the present study were highly specific for H. diffusa. They could be used as key components in the H. diffusa identification kit.