Many species of Corydalis (Papaveraceae) have been used as medicinal plants in East Asia, and the most well-known species are Corydalis yanhusuo and C. decumbens in the Pharmacopoeia of China. However, authentication of these species remains problematic because of their high morphological variation. Here, we selected 14 closely related species and five genomic regions (chloroplast: matK, trnG, rbcL, psbA-trnH; nuclear: ITS) to explore the utility of DNA barcoding for authenticating these herbs. In addition, the Poisson tree process (PTP) and automatic barcode gap discovery (ABGD) were also used and compared with DNA barcoding. Our results showed that the ITS region was not suitable for molecular analysis because of its heterogeneous nature in Corydalis. In contrast, matK was an ideal region for species identification because all species could be resolved when matK was used along with the other three chloroplast regions. We found that at least five traditional identified species were lumped into one genetic species by ABGD and PTP methods; thus, highlighting the overestimation of species diversity using the morphological approach. In conclusion, our first attempt of molecular analysis of Corydalis herbs presented here successfully resolved the identification of medicinal species and encouraged their taxonomic re-assessment.

The crucial function of the internal transcribed spacer 2 (ITS2) region in ribosome biogenesis depends on its secondary and tertiary structures. Despite rapidly evolving, ITS2 is under evolutionary constraints to maintain the specific secondary structures that provide functionality. A link between function, structure and evolution could contribute an understanding to each other and recently has created a growing point of sequence-structure phylogeny of ITS2. Here we briefly review the current knowledge of ITS2 processing in ribosome biogenesis, focusing on the conservative characteristics of ITS2 secondary structure, including structure form, structural motifs, cleavage sites, and base-pair interactions. We then review the phylogenetic implications and applications of this structure information, including structure-guiding sequence alignment, base-pair mutation model, and species distinguishing. We give the rationale for why incorporating structure information into tree construction could improve reliability and accuracy, and some perspectives of bioinformatics coding that allow for a meaningful evolutionary character to be extracted. In sum, this review of the integration of function, structure and evolution of ITS2 will expand the traditional sequence-based ITS2 phylogeny and thus contributes to the tree of life. The generality of ITS2 characteristics may also inspire phylogenetic use of other similar structural regions.

A compensatory base change (CBC) that coevolves in the secondary structure of ribosomal internal transcribed spacer 2 (ITS2) influences the estimation of genetic distance and thus challenges the phylogenetic use of this most popular genetic marker. To date, however, the CBC effect on ITS2 genetic distance is still unclear. Here, ITS2 sequences of 46 more recent angiosperm lineages were screened from 5677 genera and phylogenetically analyzed in sequence-structure format, including secondary structure prediction, structure-based alignment and sequence partition of paired and unpaired regions. ITS2 genetic distances were estimated comparatively by using both conventional DNA substitution models and RNA-specific models, which were performed in the PHASE package. Our results showed that the existence of the CBC substitution inflated the ITS2 genetic distances to different extents, and the deviation could be 180% higher if the relative ratio of substitution rate in ITS2 secondary structure stems was threefold higher than that in the loops. However, the CBC effect was minor if that ratio was below two, indicating that the DNA model is still applicable in recent lineages in which few CBCs occur. We thus provide a general empirical threshold to take account of CBC before ITS2 phylogenetic analyses.

The great threat of microbes carried by ballast water calls for figuring out the species composition of the ballast-tank microbial community, where the dark, cold, and anoxic tank environment might select special taxa. In this study, we reconstructed 103 metagenome-assembled genomes (MAGs), including 102 bacteria and one archaea, from four vessels on international voyages. Of these MAGs, 60 were ‘near complete’ (completeness >90%), 34 were >80% complete, and nine were >75% complete. Phylogenomic analysis revealed that over 70% (n = 74) of these MAGs represented new taxa at different taxonomical levels, including one order, three families, 12 genera, and 58 species. The species composition of these MAGs was most consistent with the previous reports, with the most abundant phyla being Proteobacteria (n = 69), Bacteroidota (n = 17), and Actinobacteriota (n = 7). These draft genomes provided novel data on species diversity and function in the ballast-tank microbial community, which will facilitate ballast water and sediments management.

Guanine and cytosine (GC) content is a fundamental component of genetic diversity and essential for phylogenetic analyses. However, the GC content of the ribosomal internal transcribed spacer 2 (ITS2) remains unknown, despite the fact that ITS2 is a widely used phylogenetic marker. Here, the ITS2 was high-throughput sequenced from 29 Corydalis species, and their GC contents were comparatively investigated in the context of ITS2’s characteristic secondary structure and concerted evolution. Our results showed that the GC contents of ITS2 were 131% higher than those of their adjacent 5.8S regions, suggesting that ITS2 underwent GC-biased evolution. These GCs were distributed in a heterogeneous manner in the ITS2 secondary structure, with the paired regions being 130% larger than the unpaired regions, indicating that GC is chosen for thermodynamic stability. In addition, species with homogeneous ITS2 sequences were always GC-rich, supporting GC-biased gene conversion (gBGC), which occurred with ITS2’s concerted evolution. The RNA substitution model inferred also showed a GC preference among base pair transformations, which again supports gBGC. Overall, structurally based GC investigation reveals that ITS2 evolves under structural stability and gBGC selection, significantly increasing its GC content.