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Recent advances in the field of environmental DNA (eDNA) metabarcoding have produced a promising platform for early detection and broad‐spectrum monitoring of plant pests and pathogens. To date, the majority of fungal metabarcoding assays have relied upon universal primers amplifying segments of the ribosomal DNA, to ensure broad taxonomic coverage while studying changes in community composition, or more recently, to screen for presence/absence of target species. In a diagnostics framework however, single, universal molecular barcodes do not discriminate accurately enough for many groups of important fungal phytopathogens at the required species level. Here, a modular, multi‐barcode amplicon sequencing pipeline was established to provide rapid and reliable diagnostics targeting the Ophiostomatales, an order containing economically important phytopathogens vectored by bark and ambrosia beetles (Curculionidae). Using compositionally varied mock communities, we evaluated five barcoding loci: ITS1, ITS2, the large ribosomal sub‐unit (LSU), translation elongation factor 1‐alpha (TEF1α) and calmodulin (CAL), for their ability to provide species‐level resolution. The sensitivity and detection limit of the assay was established by spiking genomic DNA of the exotic Dutch elm disease pathogen, Ophiostoma novo‐ulmi Brasier, into both the controlled mock communities and eDNA sampled from Ips grandicollis (Eichhoff) beetles collected in New South Wales, Australia. The TEF1α barcode successfully detected the Dutch elm pathogen DNA with 100% accuracy down to approx. 5 pg/μl, while the ITS1 barcode had a 100% accuracy down to approx. 0.4 pg/μl within a given sample. Our results demonstrate that a dual‐barcode approach targeting the ITS1 and TEF1α regions simultaneously is sufficient for accurate species level detection and characterisation of the Ophiostomatales and offers confident detection of low‐abundance taxa (relative read abundances of 0.1%–0.01%). Multi‐barcode metabarcoding provides a framework which can quickly and efficiently be adapted to new targets when establishing high throughput diagnostics for post‐border biosecurity surveillance of fungal phytopathogens. In a diagnostics framework, metabarcoding of single, universal molecular barcodes does not discriminate accurately enough for many groups of important fungal phytopathogens at the required species level. In this study, a modular, multi‐barcode amplicon sequencing pipeline was established to provide rapid and reliable diagnostics targeting the Ophiostomatales. Our results demonstrate that a dual‐barcode approach targeting the ITS1 and TEF1a regions simultaneously is sufficient for accurate species‐level detection and characterization of the Ophiostomatales and offers confident detection of low‐abundance taxa (relative read abundances of 0.1%–0.01%). Multi‐barcode metabarcoding provides a framework, which can quickly and efficiently be adapted to new targets when establishing high‐throughput diagnostics for post‐border biosecurity surveillance of fungal phytopathogens.

A broadband, high efficiency polarized beam splitter (PBS) metagrating based on integrated resonant units (IRUs) to enable simultaneous polarization analysis, spectral dispersion, and spatial imaging in the near infrared (NIR) is developed. A PBS metagrating with a diameter of 60 mm is the key technology component of the high‐resolution multiple‐species atmospheric profiler in the NIR (HiMAP‐NIR), which is a spaceborne instrument concept crafted to be a core payload of NASA's new generation Earth System Observatory. HiMAP‐NIR will enable the aerosol profiling in Earth's planetary boundary layer (from surface to2 km altitude) by simultaneously measuring four spatial‐spectral‐polarimetric images from 680 to 780 nm. Through detailed optimization of hybridized resonant modes in IRUs, the PBS metagrating shows a diffraction efficiency of 70% (or better) for all four linear‐polarized incident light, and polarization contrasts between orthogonal states are 0.996 (or better) from 680 to 780 nm. It meets the stringent performance required by the HiMAP‐NIR exploiting a new paradigm for the broad applications of metasurfaces. An polarized beam splitter, enabling to simultaneously measure four spatial‐spectral‐polarimetric images with an efficiency ≈70% in the entire near‐infrared oxygen bands and a polarization contrast greater than 0.996, are realized by hybridized gap‐plasmonic metasurfaces. This meets the standard of NASA's high‐resolution multiple‐species atmospheric profiler instrument for aerosol profiling in the planetary boundary layer.

Background Calligonum (Polygonaceae) is distributed from southern Europe through northern Africa to central Asia, and is typically found in arid, desert regions. Previous studies have revealed that standard DNA barcodes fail to discriminate Calligonum species. In this study, the complete plastid genomes (plastome) for 32 accessions of 21 Calligonum species is sequenced to not only generate the first complete plastome sequence for the genus Calligonum but to also 1) Assess the ability of the complete plastome sequence to discern species within the group, and 2) screen the plastome sequence for a cost-effective DNA barcode that can be used in future studies to resolve taxonomic relationships within the group. Results The whole plastomes of Calligonum species possess a typical quadripartite structure. The size of the Calligonum plastome is approximately 161 kilobase pairs (kbp), and encodes 113 genes, including 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. Based on ML phylogenetic tree analyses, the complete plastome has higher species identification (78%) than combinations of standard DNA barcodes ( rbcL  +  matK  + nrITS, 56%). Five newly screened gene regions ( ndhF , trnS-G , trnC-petN , ndhF-rpl32 , rpl32-trnL ) had high species resolution, where ndhF and trnS - G were able to distinguish the highest proportion of Calligonum species (56%). Conclusions The entire plastid genome was the most effective barcode for the genus Calligonum , although other gene regions showed great potential as taxon-specific barcodes for species identification in Calligonum .

The Eurasian otter Lutra lutra is a generalist carnivore that is widely distributed in many aquatic ecosystems. Based on its inherent attributes of opportunistic foraging behaviour and broad dietary range, it is naturally considered a potential sampler of the diversity of aquatic vertebrates. To test the ability and efficiency of otters as a diversity sampler, we used DNA metabarcoding to investigate the composition in vertebrates of the diet of otters that inhabit a forest stream area in northeast China. Twenty vertebrate prey taxa were detected in 98 otter spraints. Otter diet mainly comprised aquatic fishes (59.4%) and amphibians (39.0%). We also used traditional approaches to investigate fish communities at 60 sampling sites in the same area to determine the relationship between fish population composition in the environment and otter diet. The comparison revealed that 28 species of fish were distributed in this area, of which five are simultaneously detected in otter spraints. This indicates that molecular analysis of the diet of otters is not an ideal approach for investigating fish diversity, at least when using the 12SV5 primer pair. Based on a review of the available molecular research on otter diet, we conclude that the low species resolution may be due to the presence of many closely-related prey species in native habitats and lack of suitable barcodes. Considering the remarkable power of diet metabarcoding analysis in capturing elusive and rare species, it represents an approach that can compensate for the defects associated with fishing methods and we suggest that it can be used as an auxiliary means of measuring traditional fish diversity.

Aim Rapid and accurate species identification is the foundation for biodiversity assessment. DNA barcoding has been shown to be an effective tool to overcome the taxonomic impediment to facilitate biodiversity conservation in temperate forests. However, this tool has rarely been considered for use in tropical forests. This study aims to investigate the utility and species resolution of DNA barcoding in a subtropical region. Location The Dinghushan National Nature Reserve (DNNR) in China. Methods A DNA barcoding database was constructed for 531 trees present in the DNNR. We used a phylogenetic method (neighbour-joining trees) and sequence similarity (all-to-all BLASTn searches) to evaluate the utility and species resolution of five DNA barcode regions (rbcL, matK, ITS, ITS2 and trnH-psbA), both singly and in combinations of two or three region. Results The combination of rbcL + matK + ITS had the highest species resolution (94.19%). However, when considering the difficulty of sequence recoverability, rbcL + ITS2 performed best (64.64%). Species resolution for large genera containing more than two species was substantially lower than that for small genera with one and two species per genus. Local small spatial scales (1-ha quadrats) resulted in moderately improved species resolution (70.82% for rbcL + ITS2) compared to larger spatial scales (20 and 1133 ha). We document incongruent signals between nuclear and cpDNA regions and the challenges associated with barcoding large genera inherent to subtropical floras. Main conclusions This study considerably expands the global DNA barcode database for subtropical trees. Based on cost-effectiveness and the trade-off between sequence recovery and species resolution, we suggest that the rbcL + ITS2 barcode combination is an effective tool for documenting plant diversity in the DNNR. This study also sheds some light on the limitations and challenges for the application of barcoding to conservation biogeography in subtropical forests.

Paphiopedilum is among the most popular ornamental orchid genera due to its unique slipper flowers and attractive leaf coloration. Most of the Paphiopedilum species are in critical danger due to over-exploitation. They were listed in Appendix I of the Convention on International Trade in Endangered Species of Wild Fauna and Flora, which prevents their being traded across borders. While most Paphiopedilum species are distinctive, owing to their respective flowers, their vegetative features are more similar and undistinguished. Hence, the conservation of these species is challenging, as most traded specimins are immature and non-flowered. An urgent need exists for effective identification methods to prevent further illegal trading of Paphiopedilum species. DNA barcoding is a rapid and sensitive method for species identification, at any developmental stage, using short DNA sequences. In this study, eight loci, i.e., ITS, LEAFY, ACO, matK, trnL, rpoB, rpoC1, and trnH-psbA, were screened for potential barcode sequences on the Vietnamese Paphiopedilum species. In total, 17 out of 22 Paphiopedilum species were well identified. The studied DNA sequences were deposited to GenBank, in which Paphiopedilum dalatense accessions were introduced for the first time. ACO, LEAFY, and trnH-psbA were limited in amplification rate for Paphiopedilum. ITS was the best single barcode. Single ITS could be used along with nucleotide polymorphism characteristics for species discrimination. The combination of ITS + matK was the most efficient identification barcode for Vietnamese Paphiopedilum species. This barcode also succeeded in recognizing misidentified or wrongly-named traded samples. Different bioinformatics programs and algorithms for establishing phylogenetic trees were also compared in the study to propose quick, simple, and effective tools for practical use. It was proved that both the Bayesian Inference method in the MRBAYES program and the neighbor-joining method in the MEGA software met the criteria. Our study provides a barcoding database of Vietnamese Paphiopedilum which may significantly contribute to the control and conservation of these valuable species.

Background Sequencing of the 16S rRNA gene has been the standard for studying the composition of microbial communities. While it allows identification of bacteria at the level of species, this method does not usually provide sufficient information to resolve communities at the sub-species level. Species-level resolution is not adequate for studies of transmission or stability or for exploring subspecies variation in disease association. Strain level analysis using whole metagenome shotgun sequencing has significant limitations that can make it unsuitable for large-scale studies. Achieving sufficient depth of sequencing can be cost-prohibitive, and even with adequate coverage, deconvoluting complex communities such as the oral microbiota is computationally very challenging. Thus, there is a need for high-resolution, yet cost-effective, high-throughput methods for characterizing microbial communities. Results Significant improvement in resolution for amplicon-based bacterial community analysis was achieved by combining amplicon sequencing of a high-diversity marker gene, the ribosomal 16-23S intergenic spacer region (ISR), with a probabilistic error modeling based denoising algorithm, DADA2. The resolving power of this new approach was compared to that of both standard and high-resolution 16S-based approaches using a set of longitudinal subgingival plaque samples. The ISR strategy resulted in a 5.2-fold increase in community resolution compared to reference-based 16S rRNA gene analysis and showed 100% accuracy in predicting the correct source of a clinical sample. Individuals’ microbial communities were highly personalized, and although they exhibited some drift in membership and levels over time, that difference was always smaller than the differences between any two subjects, even after 1 year. The construction of an ISR database from publicly available genomic sequences allowed us to explore genomic variation within species, resulting in the identification of multiple variants of the ISR for most species. Conclusions The ISR approach resulted in significantly improved resolution of communities and revealed a highly personalized human oral microbiota that was stable over 1 year. Multiple ISR types were observed for all species examined, demonstrating a high level of subspecies variation in the oral microbiota. The approach is high-throughput, high-resolution yet cost-effective, allowing subspecies-level community fingerprinting at a cost comparable to that of 16S rRNA gene amplicon sequencing. It will be useful for a range of applications that require high-resolution identification of organisms, including microbial tracking, community fingerprinting, and potentially for identification of virulence-associated strains.

At present, molecular ecological studies of arbuscular mycorrhizal fungi (AMF) are only possible above species level when targeting entire communities. To improve molecular species characterization and to allow species level community analyses in the field, a set of newly designed AMF specific PCR primers was successfully tested. Nuclear rDNA fragments from diverse phylogenetic AMF lineages were sequenced and analysed to design four primer mixtures, each targeting one binding site in the small subunit (SSU) or large subunit (LSU) rDNA. To allow species resolution, they span a fragment covering the partial SSU, whole internal transcribed spacer (ITS) rDNA region and partial LSU. The new primers are suitable for specifically amplifying AMF rDNA from material that may be contaminated by other organisms (e.g., samples from pot cultures or the field), characterizing the diversity of AMF species from field samples, and amplifying a SSU-ITS-LSU fragment that allows phylogenetic analyses with species level resolution. The PCR primers can be used to monitor entire AMF field communities, based on a single rDNA marker region. Their application will improve the base for deep sequencing approaches; moreover, they can be efficiently used as DNA barcoding primers.

BACKGROUND: Because arctic plant communities are highly vulnerable to climate change, shifts in their composition require rapid, accurate identifications, often for specimens that lack diagnostic floral characters. The present study examines the role that DNA barcoding can play in aiding floristic evaluations in the arctic by testing the effectiveness of the core plant barcode regions (rbcL, matK) and a supplemental ribosomal DNA (ITS2) marker for a well-studied flora near Churchill, Manitoba. RESULTS: This investigation examined 900 specimens representing 312 of the 354 species of vascular plants known from Churchill. Sequencing success was high for rbcL: 95% for fresh specimens and 85% for herbarium samples (mean age 20 years). ITS2 worked equally well for the fresh and herbarium material (89% and 88%). However, sequencing success was lower for matK, despite two rounds of PCR amplification, which reflected less effective primer binding and sensitivity to the DNA degradation (76% of fresh, 45% of herbaria samples). A species was considered as taxonomically resolved if its members showed at least one diagnostic difference from any other taxon in the study and formed a monophyletic clade. The highest species resolution (69%) was obtained by combining information from all three genes. The joint sequence information for rbcL and matK distinguished 54% of 286 species, while rbcL and ITS2 distinguished 63% of 285 species. Discrimination of species within Salix, which constituted 8% of the flora, was particularly problematic. Despite incomplete resolution, the barcode results revealed 22 misidentified herbarium specimens, and enabled the identification of field specimens which were otherwise too immature to identify. Although seven cases of ITS2 paralogy were noted in the families Cyperaceae, Juncaceae and Juncaginaceae, this intergenic spacer played an important role in resolving congeneric plant species at Churchill. CONCLUSIONS: Our results provided fast and cost-effective solution to create a comprehensive, effective DNA barcode reference library for a local flora.

For vaginal microbiota studies, diverse 16S rRNA gene regions were applied for amplification and sequencing, which affect the comparability between different studies as well as the species-level resolution of taxonomic classification. We conducted comprehensive evaluation on the methods which influence the accuracy for the taxonomic classification and established an optimal pipeline to achieve high species-level resolution for vaginal microbiota with short amplicons, which will facilitate future studies.