Chun-Xia Zeng

Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants

A two-marker combination of plastid rbcL and matK has previously been recommended as the core plant barcode, to be supplemented with additional markers such as plastid trnH–psbA and nuclear ribosomal internal transcribed spacer (ITS). To assess the effectiveness and universality of these barcode markers in seed plants, we sampled 6,286 individuals representing 1,757 species in 141 genera of 75 families (42 orders) by using four different methods of data analysis. These analyses indicate that (i) the three plastid markers showed high levels of universality (87.1–92.7%), whereas ITS performed relatively well (79%) in angiosperms but not so well in gymnosperms; (ii) in taxonomic groups for which direct sequencing of the marker is possible, ITS showed the highest discriminatory power of the four markers, and a combination of ITS and any plastid DNA marker was able to discriminate 69.9–79.1% of species, compared with only 49.7% with rbcL + matK; and (iii) where multiple individuals of a single species were tested, ascriptions based on ITS and plastid DNA barcodes were incongruent in some samples for 45.2% of the sampled genera (for genera with more than one species sampled). This finding highlights the importance of both sampling multiple individuals and using markers with different modes of inheritance. In cases where it is difficult to amplify and directly sequence ITS in its entirety, just using ITS2 is a useful backup because it is easier to amplify and sequence this subset of the marker. We therefore propose that ITS/ITS2 should be incorporated into the core barcode for seed plants.

Chloroplast phylogenomic analyses resolve deep-level relationships of an intractable bamboo tribe Arundinarieae (poaceae).

The temperate woody bamboos constitute a distinct tribe Arundinarieae (Poaceae: Bambusoideae) with high species diversity. Estimating phylogenetic relationships among the 11 major lineages of Arundinarieae has been particularly difficult, owing to a possible rapid radiation and the extremely low rate of sequence divergence. Here, we explore the use of chloroplast genome sequencing for phylogenetic inference. We sampled 25 species (22 temperate bamboos and 3 outgroups) for the complete genome representing eight major lineages of Arundinarieae in an attempt to resolve backbone relationships. Phylogenetic analyses of coding versus noncoding sequences, and of different regions of the genome (large single copy and small single copy, and inverted repeat regions) yielded no well-supported contradicting topologies but potential incongruence was found between the coding and noncoding sequences. The use of various data partitioning schemes in analysis of the complete sequences resulted in nearly identical topologies and node support values, although the partitioning schemes were decisively different from each other as to the fit to the data. Our full genomic data set substantially increased resolution along the backbone and provided strong support for most relationships despite the very short internodes and long branches in the tree. The inferred relationships were also robust to potential confounding factors (e.g., long-branch attraction) and received support from independent indels in the genome. We then added taxa from the three Arundinarieae lineages that were not included in the full-genome data set; each of these were sampled for more than 50% genome sequences. The resulting trees not only corroborated the reconstructed deep-level relationships but also largely resolved the phylogenetic placements of these three additional lineages. Furthermore, adding 129 additional taxa sampled for only eight chloroplast loci to the combined data set yielded almost identical relationships, albeit with low support values. We believe that the inferred phylogeny is robust to taxon sampling. Having resolved the deep-level relationships of Arundinarieae, we illuminate how chloroplast phylogenomics can be used for elucidating difficult phylogeny at low taxonomic levels in intractable plant groups.

Complex evolution in Arundinarieae (Poaceae: Bambusoideae): Incongruence between plastid and nuclear GBSSI gene phylogenies

The monophyly of tribe Arundinarieae (the temperate woody bamboos) has been unequivocally recovered in previous molecular phylogenetic studies. In a recent phylogenetic study, 10 major lineages in Arundinarieae were resolved based on eight non-coding plastid regions, which conflicted significantly with morphological classifications both at the subtribal and generic levels. Nevertheless, relationships among and within the 10 lineages remain unclear. In order to further unravel the evolutionary history of Arundinarieae, we used the nuclear GBSSI gene sequences along with those of eight plastid regions for phylogenetic reconstruction, with an emphasis on Chinese species. The results of the plastid analyses agreed with previous studies, whereas 13 primary clades revealed in the GBSSI phylogeny were better resolved at the generic level than the plastid phylogeny. Our analyses also revealed many inconsistencies between the plastid DNA and the nuclear GBSSI trees. These results implied that the nuclear genome and the plastid genome had different evolutionary trajectories. The patterns of incongruence suggested that lack of informative characters, incomplete lineage sorting, and/or hybridization (introgression) could be the causes. Seven putative hybrid species were hypothesized, four of which are discussed in detail on the basis of topological incongruence, chromosome numbers, morphology, and distribution patterns, and those taxa probably resulted from homoploid hybrid speciation. Overall, our study indicates that the tribe Arundinarieae has undergone a complex evolution.

A New Type of Monoterpenoid Indole Alkaloid Precursor from Alstonia rostrata

Currently, all monoterpenoid indole alkaloids (MIAs) have been derived from strictosidine, which originates from the condensation of tryptophan with secologanin in a 1:1 ratio. However, our phytochemical research on Alstonia rostrata revealed a potential new precursor for these compounds. We isolated the alstrostines A and B, and it was determined that they were derived from tryptophan and secologanin in a 1:2 ratio, which supported the presence of a new type of MIA precursor.

Fifteen novel universal primer pairs for sequencing whole chloroplast genomes and a primer pair for nuclear ribosomal DNAs

Chloroplast genome information helps improve the phylogenetic resolution and can act as organelle‐scale barcodes in recently radiated plant groups. Previously we reported that nine universal primer pairs could amplify angiosperm whole chloroplast genomes by long‐range polymerase chain reaction and using next‐generation sequencing. Although these primers show high universality and efficiency for sequencing whole chloroplast genomes in angiosperms, they did not fully resolve the following two issues surrounding sequencing angiosperm chloroplast genomes: (i) approximately 30% of angiosperms cannot be amplified successfully; and (ii) only fresh leaves can be applied. In this study, we designed another set of 15 universal primer pairs for amplifying angiosperm whole chloroplast genomes to complement the original nine primer pairs. Furthermore, we designed a primer pair for nuclear ribosomal DNAs (nrDNAs). To validate the functionality of the primers, we tested 44 species with silica gel‐dried leaves and 15 species with fresh leaves that have been shown to not be amplified with the original nine primer pairs. The result showed that, in 65.9% and 88.6% of the 44 species with silica gel‐dried leaves, the whole chloroplast genome and nrDNAs could be amplified, respectively. In addition, all 15 fresh leaf samples could have the whole chloroplast genome successfully amplified. The nrDNAs comprise partial sequences of 18S and 26S, along with the complete sequence of 5.8S and the internal transcribed spacers ITS1 and ITS2. The mean size of nrDNA was 5800 bp. This study shows that the 15 universal primer set is an indispensable tool for amplifying whole chloroplast genomes in angiosperms, and these are an important supplement to the nine reported primer pairs.

Dimeric Erythrina Alkaloids from the Flower of Erythrina variegata

Unprecedented dimeric Erythrina alkaloids, spirocyclic (6/5/6/6) erythrivarine A (1) and spiro-fused (6/5/7/6) rings erythrivarine B (2), were isolated from the cultivated plant, E. variegata. The structures were determined on the basis of 1D and 2D NMR, FTIR, UV, and mass spectroscopic data and X-ray crystal diffraction. The biogenetic relationship of 1 and 2 is proposed.

Isolation and characterization of 15 microsatellite markers from the spring orchid (Cymbidium goeringii) (Orchidaceae).

PREMISE OF THE STUDY Microsatellite primers were developed for the important and popular ornamental spring orchid, Cymbidium goeringii, to investigate its genetic diversity and population genetic structure. METHODS AND RESULTS Using the Fast Isolation by AFLP of Sequences Containing (FIASCO) repeats protocol, 15 primer sets were identified in two wild populations. The number of alleles per locus ranged from 2 to 5, with a mean of 3.3. The observed and expected heterozygosities varied from 0.167 to 0.917 and from 0.159 to 0.822, respectively. All these primers successfully amplified in the congener C. tortisepalum, and 12 primers were found useful in C. faberi and C. sinense. CONCLUSIONS These markers will facilitate further studies on the population genetics of Cymbidium goeringii and other congeneric species.

Identification of putative orthologous genes for the phylogenetic reconstruction of temperate woody bamboos (Poaceae: Bambusoideae)

The temperate woody bamboos (Arundinarieae) are highly diverse in morphology but lack a substantial amount of genetic variation. The taxonomy of this lineage is intractable, and the relationships within the tribe have not been well resolved. Recent studies indicated that this tribe could have a complex evolutionary history. Although phylogenetic studies of the tribe have been carried out, most of these phylogenetic reconstructions were based on plastid data, which provide lower phylogenetic resolution compared with nuclear data. In this study, we intended to identify a set of desirable nuclear genes for resolving the phylogeny of the temperate woody bamboos. Using two different methodologies, we identified 209 and 916 genes, respectively, as putative single copy orthologous genes. A total of 112 genes was successfully amplified and sequenced by next‐generation sequencing technologies in five species sampled from the tribe. As most of the genes exhibited intra‐individual allele heterozygotes, we investigated phylogenetic utility by reconstructing the phylogeny based on individual genes. Discordance among gene trees was observed and, to resolve the conflict, we performed a range of analyses using BUCKy and HybTree. While caution should be taken when inferring a phylogeny from multiple conflicting genes, our analysis indicated that 74 of the 112 investigated genes are potential markers for resolving the phylogeny of the temperate woody bamboos.

Monoterpenoid indole alkaloids from Alstonia rostrata

Four new monoterpenoid indole alkaloids, alstrostines C-F together with thirteen known alkaloids were isolated from the leaves and twigs of Alstonia rostrata. All structures of new compounds were elucidated based on NMR, FTIR, UV, and MS spectroscopic data. Alstrostines C-E might originate from keto-enol tautomerism of preakummicine during biogenetic pathway of akummicine.

Two new lignans from twigs of Aglaia odorata.

HPLC-guided separation of twigs of Aglaia odorata led to the isolation of eight lignans, including two new ones, 3′-methoxy-N-demethylrocaglamide (1) and 4′-O-demethyl-deacetylaglaxiflorin A (2). Compound 1 showed excellent cytotoxicity against three human cancer cell lines, HeLa, SGC-7901 gastric cancer, and A-549 lung cancer with the values of 0.32, 0.12, and 0.25 μM, respectively.