Many species of Tetrastigma (Miq.) Planch. (Vitaceae) have long been used as medicinal plants in China, and some are endangered due to overexploitation. Although adulterants are often added to traditional Chinese medicines, there is no reliable or practical method for identifying them. In this study, we used four markers (rbcL, matK, trnH-psbA, and internal transcribed spacer [ITS]) as DNA barcodes to test their ability to distinguish species of Tetrastigma. The results indicated that the best barcode was ITS, which showed significant inter-specific genetic variability, and thus its potential as a DNA barcode for identifying Tetrastigma. Multiple loci provided a greater ability to distinguish species than single loci. We recommend using the combined rbcL+matK+ITS barcode for the genus. Phylogenetic trees from each barcode were compared. Analyses using the unweighted pair group method with arithmetic mean discriminated an equal or greater percentage of resolvable species than did neighbor joining, maximum likelihood, or maximum parsimony analyses. Additionally, five medicinal species of Tetrastigma, especially T. hemsleyanum, could be identified precisely using DNA barcoding.

BackgroundTetrastigma hemsleyanum is of great medicinal importance and used as a model system to address the evolutionary history of warm-temperate evergreen (WTE) forest biomes in East Asia over Neogene time scales. However, further studies on the neutral and adaptive divergence processes of T. hemsleyanum are currently impeded by a lack of genomic resources. In this study, we de novo assembled and annotated a reference transcriptome for two cpDNA lineages (Central-South-East vs. Southwest) of T. hemsleyanum. We further used comparative genomic and multilocus coalescent approaches to investigate the tempo and mode of lineage diversification in T. hemsleyanum.ResultsA total of 52,838 and 65,197 unigenes with an N50 of 1,667 and 1,841 bp for Central-South-East (CSE) and Southwest (SW) lineages, respectively, were recovered, and 6,692 putative orthologs were identified between the two lineages. Estimation of Ka/Ks ratios for these orthologs revealed that ten genes had Ka/Ks values significantly greater than 0.5 (P < 0.05), whereas 2,099 (Ka/Ks < 0.5, P < 0.05) were inferred to be under purifying selection. Based on three bioinformatic strategies, we identified a total of 1,018 single-copy nuclear genes (SCNGs) from the orthologs. We successfully designed eight nuclear gene primer pairs with high intraspecific variation (e.g. hT = 0.923, πT = 1.68×10-3), when surveyed across a subset of T. hemsleyanum individuals. Concordant with the previous cpDNA data, the haplotype networks constructed for most nuclear gene loci clearly identified the two lineages. A multilocus coalescence analysis suggested that the separation between the two lineages appears to have occurred during the mid-Pliocene. Despite their ancient divergence, both lineages experienced expansion at rather localized scales and have continued to exchange genes at a low rate.ConclusionsThis study demonstrated the utility of transcriptome sequencing as a basis for SCNG development in non-model species and the advantages of integrating multiple nuclear loci for phylogeographic and phylogenetic studies.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1429-8) contains supplementary material, which is available to authorized users.

Dysosma versipellis (Hance) M. Cheng is an endangered plant due to overharvesting for the extraction of podophyllotoxin. Thus, the in vitro technique is valuable for the propagation of this species. When the explants of rhizome buds were cultured on Murashige and Skoog's (MS) medium with 6-benzyladenine (BA) (1.0 mg l -1 ), gibberellic acid (GA 3 ) (0.5 mg l -1 ) and zeatin (Zea) (0.5 mg l -1 ), multiple buds were regenerated directly on the explants without callusing within 6 weeks. Callus was induced from the leaf segment cultures on MS basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.5 mg l -1 ) and BA (0.2 mg l -1 ) within 4 weeks. The adventitious buds were differentiated when the calli were subcultured on MS medium supplemented with BA (1.0 mg l -1 ) and thidiazuron (TDZ) (0.2 mg l -1 ) within 6 weeks. The adventitious buds obtained from callus and the rhizome-buds rooted with a frequency of 100% on half strength MS medium fortified with indole-3-butyric acid (IBA) 0.5 mg l -1 and activated charcoal (AC) 0.5 g l -1 for 4 weeks. The rooted shoots were successfully transplanted from a mixture of vermiculite:soil (1:1 v/v) to the field with a survival rate of 85%. Podophyllotoxin production in calli, cultured rhizomes, rhizomes of transplanting plants from the garden and rhizomes in the wild field was confirmed by high-performance liquid chromatography (HPLC) analysis. Our results suggest that calli, cultured rhizomes and rhizomes of transplanting plants would be the potential sources of podophyllotoxin.

Oresitrophe and Mukdenia (Saxifragaceae) are epilithic sister genera used in traditional Chinese medicine. The taxonomy of Mukdenia, especially of M. acanthifolia, has been controversial. To address this, we produced plastid and mitochondrial data using genome skimming for Mukdenia acanthifolia and Mukdenia rossii, including three individuals of each species. We assembled complete plastomes, mitochondrial CDS and nuclear ribosomal ETS/ITS sequences using these data. Comparative analysis shows that the plastomes of Mukdenia and Oresitrophe are relatively conservative in terms of genome size, structure, gene content, RNA editing sites and codon usage. Five plastid regions that represent hotspots of change (trnH‐psbA, psbC‐trnS, trnM‐atpE, petA‐psbJ and ccsA‐ndhD) are identified within Mukdenia, and six regions (trnH‐psbA, petN‐psbM, trnM‐atpE, rps16‐trnQ, ycf1 and ndhF) contain a higher number of species‐specific parsimony‐informative sites that may serve as potential DNA barcodes for species identification. To infer phylogenetic relationships between Mukdenia and Oresitrophe, we combined our data with published data based on three different datasets. The monophyly of each species (Oresitrophe rupifraga, M. acanthifolia and M. rossii) and the inferred topology ((M. rossii, M. acanthifolia), O. rupifraga) are well supported in trees reconstructed using the complete plastome sequences, but M. acanthifolia and M. rossii did not form a separate clade in the trees based on ETS + ITS data, while the mitochondrial CDS trees are not well‐resolved. We found low recovery of genes in the Angiosperms353 target enrichment panel from our unenriched genome skimming data. Hybridization or incomplete lineage sorting may be the cause of discordance between trees reconstructed from organellar and nuclear data. Considering its morphological distinctiveness and our molecular phylogenetic results, we strongly recommend that M. acanthifolia be treated as a distinct species.

Dysosma versipellis (Hance) M. Cheng ex Ying (Berberidaceae) is a rare and vulnerable, perennial herb endemic to China with pharmaceutical significance. Increasing habitat loss and over-exploitation of the plant has severely affected the plant's in situ conservation, necessitating ex situ conservation and commercial cultivation. The light regime is a critical environmental factor contributing to successful ex situ conservation via efficient production of biomass and secondary metabolites. We investigated the responses of 2-year-old D. versipellis plants to a light gradient in terms of leaf morphology, growth, biomass production, photosynthesis and podophyllotoxin (PTOX) accumulation. D. versipellis responded to the light gradient in terms of plant height, leaf size, leaf photosynthesis and PTOX accumulation. Plants demonstrated optimal vegetative growth, photosynthetic capability, and PTOX production, under a light intensity equal to 30% full sunlight. Half full sunlight led to the lowest production of PTOX despite having no obviously different effects on growth. Full sunlight was able to cause leaf death in mid-summer (July), although these plantlets did not significantly differ in growth or biomass production before July and new leaves emerged in August. D. versipellis is therefore proposed to be a shade-tolerant plant, well adapted to variations in irradiance. Irradiance of 10%-50%, particularly ca. 30% full sunlight is strongly recommended for ex situ conservation and commercial cultivation.