Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it is difficult to amplify in fungi, often includes large introns, and can be insufficiently variable. Three subunits from the nuclear ribosomal RNA cistron were compared together with regions of three representative protein-coding genes (largest subunit of RNA polymerase II, second largest subunit of RNA polymerase II, and minichromosome maintenance protein). Although the protein-coding gene regions often had a higher percent of correct identification compared with ribosomal markers, low PCR amplification and sequencing success eliminated them as candidates for a universal fungal barcode. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter- and intraspecific variation. The nuclear ribosomal large subunit, a popular phylogenetic marker in certain groups, had superior species resolution in some taxonomic groups, such as the early diverging lineages and the ascomycete yeasts, but was otherwise slightly inferior to the ITS. The nuclear ribosomal small subunit has poor species-level resolution in fungi. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups.

/pdf/nuclear-ribosomal-internal-transcribed-spacer-its-region-as-9rhgb6gvih.pdf

Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi

Within uncharacterized groups, DNA barcodes, short DNA sequences that are present in a wide range of species, can be used to assign organisms into species. We propose an automatic procedure that sorts the sequences into hypothetical species based on the barcode gap, which can be observed whenever the divergence among organisms belonging to the same species is smaller than divergence among organisms from different species. We use a range of prior intraspecific divergence to infer from the data a model-based one-sided confidence limit for intraspecific divergence. The method, called Automatic Barcode Gap Discovery (ABGD), then detects the barcode gap as the first significant gap beyond this limit and uses it to partition the data. Inference of the limit and gap detection are then recursively applied to previously obtained groups to get finer partitions until there is no further partitioning. Using six published data sets of metazoans, we show that ABGD is computationally efficient and performs well for standard prior maximum intraspecific divergences (a few per cent of divergence for the five data sets), except for one data set where less than three sequences per species were sampled. We further explore the theoretical limitations of ABGD through simulation of explicit speciation and population genetics scenarios. Our results emphasize in particular the sensitivity of the method to the presence of recent speciation events, via (unrealistically) high rates of speciation or large numbers of species. In conclusion, ABGD is fast, simple method to split a sequence alignment data set into candidate species that should be complemented with other evidence in an integrative taxonomic approach.

ABGD, Automatic Barcode Gap Discovery for primary species delimitation

Environmental DNA - An emerging tool in conservation for monitoring past and present biodiversity

Background
The plant working group of the Consortium for the Barcode of Life recommended the two-locus combination of rbcL + matK as the plant barcode, yet the combination was shown to successfully discriminate among 907 samples from 550 species at the species level with a probability of 72%. The group admits that the two-locus barcode is far from perfect due to the low identification rate, and the search is not over.

/pdf/validation-of-the-its2-region-as-a-novel-dna-barcode-for-4wtt0ffynn.pdf

Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species.

Virtually all empirical ecological studies require species identification during data collection. DNA metabarcoding refers to the automated identification of multiple species from a single bulk sample containing entire organisms or from a single environmental sample containing degraded DNA (soil, water, faeces, etc.). It can be implemented for both modern and ancient environmental samples. The availability of next-generation sequencing platforms and the ecologists' need for high-throughput taxon identification have facilitated the emergence of DNA metabarcoding. The potential power of DNA metabarcoding as it is implemented today is limited mainly by its dependency on PCR and by the considerable investment needed to build comprehensive taxonomic reference libraries. Further developments associated with the impressive progress in DNA sequencing will eliminate the currently required DNA amplification step, and comprehensive taxonomic reference libraries composed of whole organellar genomes and repetitive ribosomal nuclear DNA can be built based on the well-curated DNA extract collections maintained by standardized barcoding initiatives. The near-term future of DNA metabarcoding has an enormous potential to boost data acquisition in biodiversity research.

Towards next‐generation biodiversity assessment using DNA metabarcoding

DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. However, there has been no agreement on which region(s) should be used for barcoding land plants. To provide a community recommendation on a standard plant barcode, we have compared the performance of 7 leading candidate plastid DNA regions (atpF–atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK–psbI spacer, and trnH–psbA spacer). Based on assessments of recoverability, sequence quality, and levels of species discrimination, we recommend the 2-locus combination of rbcL+matK as the plant barcode. This core 2-locus barcode will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of land plants.

/pdf/a-dna-barcode-for-land-plants-3fs5bo85qv.pdf

A DNA barcode for land plants.

Many species in the genus Guettarda are known to exert anti-inflammatory effects and are used as traditional medicinal plants to treat various inflammatory symptoms. However, no studies on the inflammatory activities of Guettarda crispiflora Vahl have been reported. The aim of the study was to investigate in vitro and in vivo the anti-inflammatory effects of a methanol extract of Guettarda crispiflora Vahl (Gc-ME). To determine the anti-inflammatory activity of Gc-ME, lipopolysaccharide (LPS)-, poly(I:C)-, or Pam3CSK4-treated RAW264.7 cells, HCl/EtOH- and LPS-treated mice were employed for in vitro and in vivo tests. LPS-induced nitric oxide production in RAW264.7 cells was determined by Griess assays and cytokine gene expression in LPS-activated RAW264.7 cells, confirmed by RT- and real-time PCR. Transcriptional activation was evaluated by luciferase reporter gene assay. Target protein validation was assessed by Western blot analysis and cellular thermal shift assays (CETSA) with LPS-treated RAW264.7 and gene-transfected HEK293 cells. Using both a HCl/EtOH-induced gastritis model and an LPS-induced lung injury model, inflammatory states were checked by scoring or evaluating gastric lesions, lung edema, and lung histology. Phytochemical fingerprinting of Gc-ME was observed by using liquid chromatography–mass spectrometry. Nitric oxide production induced by LPS and Pam3CSK4 in RAW264.7 cells was revealed to be reduced by Gc-ME. The LPS-induced upregulation of iNOS, COX-2, IL-6, and IL-1β was also suppressed by Gc-ME treatment. Gc-ME downregulated the promotor activities of AP-1 and NF-κB triggered by MyD88- and TRIF induction. Upstream signaling proteins for NF-κB activation, namely, p-p50, p-p65, p-IκBα, and p-Src were all downregulated by Ch-EE. Moreover, Src was revealed to be directly targeted by Gc-ME. This extract, orally treated strongly, attenuated the inflammatory symptoms in HCl/EtOH-treated stomachs and LPS-treated lungs. Therefore, these results strongly imply that Guettarda crispiflora can be developed as a promising anti-inflammatory remedy with Src-suppressive properties.

/pdf/guettarda-crispiflora-vahl-methanol-extract-ameliorates-3njxt188.pdf

Guettarda crispiflora Vahl Methanol Extract Ameliorates Acute Lung Injury and Gastritis by Suppressing Src Phosphorylation

Abstract Caesalpinia eriostachys Benth. (CE) is native to the Mexico and multiple effects have been observed from several plants belonging to the same family. CE was subjected to extraction with 95% ethanol, and the components were isolated through column chromatography. The structure of the compound was elucidated based on nuclear magnetic resonance (NMR) spectral data, electron ionization–mass (EI‐MS) spectroscopy, and liquid chromatography–mass (LC‐MS) spectroscopy. In vivo antinociceptive studies were conducted using writhing, 5% formalin, tail‐flick, hot‐plate, and von Frey filament tests. The ethanolic extract showed a significant effect in the acetic acid‐induced pain model and nociceptive behavior in the formalin model (second phase). In hot‐plate test and tail‐flick test, the results showed no difference compared to the control group. The results suggest that the ethanolic extract may act peripherally to reduce pain. In the streptozotocin (STZ)‐induced pain model, the ethanolic extract showed significant effect in the von Frey test model. The n‐Hex (Hexane) and MC (Methylene chloride) fractions and isolated compounds, ellagic acid and agathisflavone, showed increased effect. Based on these results, we confirmed that the CE ethanolic extract and their compounds, ellagic acid and agathisflavone, have antinociceptive effect on diabetes mellitus‐induced pain. Furthermore, the results of this study might be valuable for identifying compounds with antinociceptive activity from natural products.

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/fsn3.2846

Dong-Keun Yi

Papers

A DNA barcode for land plants.

Guettarda crispiflora Vahl Methanol Extract Ameliorates Acute Lung Injury and Gastritis by Suppressing Src Phosphorylation

Antinociceptive activity of the Caesalpinia eriostachys Benth. ethanolic extract, fractions, and isolated compounds in mice