Nucleotide based validation of Ocimum species by evaluating three candidate barcodes of the chloroplast region

The genus Ocimum comprises of several medicinally important species which frequently fall prey to adulteration due to misidentification. A proficient method is hence required to solve the problems that exist in differentiating its various morphotypes. In plants, candidate DNA barcodes of the chloroplast and nuclear regions have proved to be a great success in the validation of several plant families. Hence, this study involves the use of the molecular‐based DNA barcoding method to identify some of the most common and useful species of the genus Ocimum (Tulsi). Here, DNA amplification of three candidate barcodes of the chloroplast genome viz. matK, rbcL and psbA‐trnH was performed, to access their ability to produce high sequence variability. The discrimination among species was performed using the Kimura 2‐parameter and maximum composite likelihood methods. On analysing the sequence data, the psbA‐trnH region proved to be the most suitable candidate barcode and gave an overall variation of 7.3% at the interspecies level. A clear differentiation was found at the species level, showing a maximum distance of 0.264 between dissimilar species. Also, phylogenetic analysis led to the successful identification of hybrids, while it failed to do so at the variety level. Hence, it can be inferred that DNA barcoding is ideal for species‐level identification of the genus Ocimum.     

DNA barcoding of economically important freshwater fish species from north‐central Nigeria uncovers cryptic diversity

This study examines the utility of morphology and DNA barcoding in species identification of freshwater fishes from north‐central Nigeria. We compared molecular data (mitochondrial cytochrome c oxidase subunit I (COI) sequences) of 136 de novo samples from 53 morphologically identified species alongside others in GenBank and BOLD databases. Using DNA sequence similarity‐based (≥97% cutoff) identification technique, 50 (94.30%) and 24 (45.30%) species were identified to species level using GenBank and BOLD databases, respectively. Furthermore, we identified cases of taxonomic problems in 26 (49.00%) morphologically identified species. There were also four (7.10%) cases of mismatch in DNA barcoding in which our query sequence in GenBank and BOLD showed a sequence match with different species names. Using DNA barcode reference data, we also identified four unknown fish samples collected from fishermen to species level. Our Neighbor‐joining (NJ) tree analysis recovers several intraspecific species clusters with strong bootstrap support (≥95%). Analysis uncovers two well‐supported lineages within Schilbe intermedius. The Bayesian phylogenetic analyses of Nigerian S. intermedius with others from GenBank recover four lineages. Evidence of genetic structuring is consistent with geographic regions of sub‐Saharan Africa. Thus, cryptic lineage diversity may illustrate species’ adaptive responses to local environmental conditions. Finally, our study underscores the importance of incorporating morphology and DNA barcoding in species identification. Although developing a complete DNA barcode reference library for Nigerian ichthyofauna will facilitate species identification and diversity studies, taxonomic revisions of DNA sequences submitted in databases alongside voucher specimens are necessary for a reliable taxonomic and diversity inventory.     

Identification of species in the angiosperm family Apiaceae using DNA barcodes

Apiaceae (Umbelliferae) is a large angiosperm family that includes many medicinally important species. The ability to identify these species and their adulterants is important, yet difficult to do so because of their subtle fruit morphological differences and often lack of diagnostic features in preserved specimens. Moreover, dried roots are often the official medical organs, making visual identification to species almost impossible. DNA barcoding has been proposed as a powerful taxonomic tool for species identification. The Consortium for the Barcode of Life (CBOL) Plant Working Group has recommended the combination of rbcL+matK as the core plant barcode. Recently, the China Plant BOL Group proposed that the nuclear ribosomal DNA internal transcribed spacer (ITS), as well as a subset of this marker (ITS2), be incorporated alongside rbcL+matK into the core barcode for seed plants, particularly angiosperms. In this study, we assess the effectiveness of these four markers plus psbA‐trnH as Apiaceae barcodes. A total of 6032 sequences representing 1957 species in 385 diverse genera were sampled, of which 211 sequences from 50 individuals (representing seven species) were newly obtained. Of these five markers, ITS and ITS2 showed superior results in intra‐ and interspecific divergence and DNA barcoding gap assessments. For the matched data set (173 samples representing 45 species in five genera), the ITS locus had the highest identification efficiency (73.3%), yet ITS2 also performed relatively well with 66.7% identification efficiency. The identification efficiency increased to 82.2% when using an ITS+psbA‐trnH marker combination (ITS2+psbA‐trnH was 80%), which was significantly higher than that of rbcL+matK (40%). For the full sample data set (3052 ITS sequences, 3732 ITS2 sequences, 1011 psbA‐trnH sequences, 567 matK sequences and 566 rbcL sequences), ITS, ITS2, psbA‐trnH, matK and rbcL had 70.0%, 64.3%, 49.5%, 38.6% and 32.1% discrimination abilities, respectively. These results confirm that ITS or its subset ITS2 be incorporated into the core barcode for Apiaceae and that the combination of ITS/ITS2+psbA‐trnH has much potential value as a powerful, standard DNA barcode for Apiaceae identification.     

Assessing the potential of candidate DNA barcodes for identifying non‐flowering seed plants

In plants, matK and rbcL have been selected as core barcodes by the Consortium for the Barcode of Life (CBOL) Plant Working Group (PWG), and ITS/ITS2 and psbA‐trnH were suggested as supplementary loci. Yet, research on DNA barcoding of non‐flowering seed plants has been less extensive, and the evaluation of DNA barcodes in this division has been limited thus far. Here, we evaluated seven markers (psbA‐trnH, matK, rbcL, rpoB, rpoC1, ITS and ITS2) from non‐flowering seed plants. The usefulness of each region was assessed using four criteria: the success rate of PCR amplification, the differential intra‐ and inter‐specific divergences, the DNA barcoding gap and the ability to discriminate species. Among the seven loci tested, ITS2 produced the best results in the barcoding of non‐flowering seed plants. In addition, we compared the abilities of the five most‐recommended markers (psbA‐trnH, matK, rbcL, ITS and ITS2) to identify additional species using a large database of gymnosperms from GenBank. ITS2 remained effective for species identification in a wide range of non‐flowering seed plants: for the 1531 samples from 608 species of 80 diverse genera, ITS2 correctly authenticated 66% of them at the species level. In conclusion, the ITS2 region can serve as a useful barcode to discriminate non‐flowering seed plants, and this study will contribute valuable information for the barcoding of plant species.     

Higher efficiency of ISSR markers over plastid psbA‐trnH region in resolving taxonomical status of genus Ocimum L.

High level of morphological as well as chemical variability exists within the genus Ocimum, and its taxonomy and phylogenetic relationships are still doubtful. For evaluating interspecific genetic relationships among the Ocimum species, genotyping with intersimple sequence repeat (ISSR) markers and sequence analyses of noncoding psbA‐trnH intergenic region belonging to chloroplast DNA were carried out. Although ISSR markers are highly efficient and reproducible, they have not been used extensively in phylogenetic studies. The use of the plastidial barcode candidate was expected to provide more variable and informative insight into evolutionary rates, and was thus employed as a phylogenetic marker to assess interspecific relationships. This study revealed that the ISSR markers were more efficient than psbA‐trnH sequences in resolving the current status of Ocimum L. genus. Distance‐ and character‐based methodological approaches applied on the molecular data with biparental and maternal inheritance were used for deducing the phylogenetic relationships among Ocimum species. Average polymorphic information content (0.344) and resolving power (6.285) depicted through ISSR markers proved to be efficient in discriminating the studied species of Ocimum. The primers used in this study revealed 99.585% polymorphism across the species demonstrating the polymorphic nature of ISSR markers.     

DNA barcoding of Rhododendron (Ericaceae), the largest Chinese plant genus in biodiversity hotspots of the Himalaya–Hengduan Mountains

The Himalaya–Hengduan Mountains encompass two global biodiversity hotspots with high levels of biodiversity and endemism. This area is one of the diversification centres of the genus Rhododendron, which is recognized as one of the most taxonomically challenging plant taxa due to recent adaptive radiations and rampant hybridization. In this study, four DNA barcodes were evaluated on 531 samples representing 173 species of seven sections of four subgenera in Rhododendron, with a high sampling density from the Himalaya–Hengduan Mountains employing three analytical methods. The varied approaches (nj , pwg and blast ) had different species identification powers with blast performing best. With the pwg analysis, the discrimination rates for single barcodes varied from 12.21% to 25.19% with ITS < rbcL < matK < psbA‐trnH. Combinations of ITS + psbA‐trnH + matK and the four barcodes showed the highest discrimination ability (both 41.98%) among all possible combinations. As a single barcode, psbA‐trnH performed best with a relatively high performance (25.19%). Overall, the three‐marker combination of ITS + psbA‐trnH + matK was found to be the best DNA barcode for identifying Rhododendron species. The relatively low discriminative efficiency of DNA barcoding in this genus (~42%) may possibly be attributable to too low sequence divergences as a result of a long generation time of Rhododendron and complex speciation patterns involving recent radiations and hybridizations. Taking the morphology, distribution range and habitat of the species into account, DNA barcoding provided additional information for species identification and delivered a preliminary assessment of biodiversity for the large genus Rhododendron in the biodiversity hotspots of the Himalaya–Hengduan Mountains.     

New information for Ilex phylogenetics based on the plastid psbA‐trnH intergenic spacer (Aquifoliaceae)

The aim of the present work was to clarify the origin and phylogenetic position of the species belonging to the genus Ilex (Aquifoliaceae), especially the South American species. Phylogenetic relationships of the genus Ilex were investigated using the plastid psbA‐trnH intergenic spacer and parsimony and Bayesian analyses. The psbA‐trnH intergenic spacer was shown to evolve slowly within Ilex, but a major gap present in this region was useful in the phylogenetic study of the genus. To obtain more potentially parsimonious characters, atpB‐rbcL intergenic spacer data were combined with those for psbA‐trnH. Many gaps present in the psbA‐trnH region were useful in the phylogenetic study of the genus Ilex. The topology of the trees showed that, in general, the clades are strongly related to geographical areas, a fact especially evident in certain different Asian lineages. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 182–193.     

Assessment of a 16S rRNA amplicon Illumina sequencing procedure for studying the microbiome of a symbiont‐rich aphid genus

The bacterial communities inhabiting arthropods are generally dominated by a few endosymbionts that play an important role in the ecology of their hosts. Rather than comparing bacterial species richness across samples, ecological studies on arthropod endosymbionts often seek to identify the main bacterial strains associated with each specimen studied. The filtering out of contaminants from the results and the accurate taxonomic assignment of sequences are therefore crucial in arthropod microbiome studies. We aimed here to validate an Illumina 16S rRNA gene sequencing protocol and analytical pipeline for investigating endosymbiotic bacteria associated with aphids. Using replicate DNA samples from 12 species (Aphididae: Lachninae, Cinara) and several controls, we removed individual sequences not meeting a minimum threshold number of reads in each sample and carried out taxonomic assignment for the remaining sequences. With this approach, we show that (i) contaminants accounted for a negligible proportion of the bacteria identified in our samples; (ii) the taxonomic composition of our samples and the relative abundance of reads assigned to a taxon were very similar across PCR and DNA replicates for each aphid sample; in particular, bacterial DNA concentration had no impact on the results. Furthermore, by analysing the distribution of unique sequences across samples rather than aggregating them into operational taxonomic units (OTUs), we gained insight into the specificity of endosymbionts for their hosts. Our results confirm that Serratia symbiotica is often present in Cinara species, in addition to the primary symbiont, Buchnera aphidicola. Furthermore, our findings reveal new symbiotic associations with Erwinia‐ and Sodalis‐related bacteria. We conclude with suggestions for generating and analysing 16S rRNA gene sequences for arthropod‐endosymbiont studies.     

Quantitative multi‐locus metabarcoding and waggle dance interpretation reveal honey bee spring foraging patterns in Midwest agroecosystems

We explored the pollen foraging behaviour of honey bee colonies situated in the corn and soybean dominated agroecosystems of central Ohio over a month‐long period using both pollen metabarcoding and waggle dance inference of spatial foraging patterns. For molecular pollen analysis, we developed simple and cost‐effective laboratory and bioinformatics methods. Targeting four plant barcode loci (ITS2, rbcL, trnL and trnH), we implemented metabarcoding library preparation and dual‐indexing protocols designed to minimize amplification biases and index mistagging events. We constructed comprehensive, curated reference databases for hierarchical taxonomic classification of metabarcoding data and used these databases to train the metaxa 2 DNA sequence classifier. Comparisons between morphological and molecular palynology provide strong support for the quantitative potential of multi‐locus metabarcoding. Results revealed consistent foraging habits between locations and show clear trends in the phenological progression of honey bee spring foraging in these agricultural areas. Our data suggest that three key taxa, woody Rosaceae such as pome fruits and hawthorns, Salix, and Trifolium provided the majority of pollen nutrition during the study. Spatially, these foraging patterns were associated with a significant preference for forests and tree lines relative to herbaceous land cover and nonflowering crop fields.     

DNA barcoding of Corydalis,the most taxonomically complicated genus of Papaveraceae

The genus Corydalis is recognized as one of the most taxonomically challenging plant taxa. It is mainly distributed in the Himalaya–Hengduan Mountains, a global biodiversity hotspot. To date, no effective solution for species discrimination and taxonomic assignment in Corydalis has been developed. In this study, five nuclear and chloroplast DNA regions, ITS, ITS2, matK, rbcL, and psbA‐trnH, were preliminarily assessed based on their ability to discriminate Corydalis to eliminate inefficient regions, and the three regions showing good performance (ITS, ITS2 and matK) were then evaluated in 131 samples representing 28 species of 11 sections of four subgenera in Corydalis using three analytical methods (NJ, ML, MP tree; K2P‐distance and BLAST). The results showed that the various approaches exhibit different species identification power and that BLAST shows the best performance among the tested approaches. A comparison of different barcodes indicated that among the single barcodes, ITS (65.2%) exhibited the highest identification success rate and that the combination of ITS + matK (69.6%) provided the highest species resolution among all single barcodes and their combinations. Three Pharmacopoeia‐recorded medicinal plants and their materia medica were identified successfully based on the ITS and ITS2 regions. In the phylogenetic analysis, the sections Thalictrifoliae, Sophorocapnos, Racemosae, Aulacostigma, and Corydalis formed well‐supported separate lineages. We thus hypothesize that the five sections should be classified as an independent subgenus and that the genus should be divided into three subgenera. In this study, DNA barcoding provided relatively high species discrimination power, indicating that it can be used for species discrimination in this taxonomically complicated genus and as a potential tool for the authentication of materia medica belonging to Corydalis.     

GB‐to‐TNT: facilitating creation of matrices from GenBank and diagnosis of results in TNT

This paper presents a pipeline, implemented in an open‐source program called GB→TNT (GenBank‐to‐TNT), for creating large molecular matrices, starting from GenBank files and finishing with TNT matrices which incorporate taxonomic information in the terminal names. GB→TNT is designed to retrieve a defined genomic region from a bulk of sequences included in a GenBank file. The user defines the genomic region to be retrieved and several filters (genome, length of the sequence, taxonomic group, etc.); each genomic region represents a different data block in the final TNT matrix. GB→TNT first generates Fasta files from the input GenBank files, then creates an alignment for each of those (by calling an alignment program), and finally merges all the aligned files into a single TNT matrix. The new version of TNT can make use of the taxonomic information contained in the terminal names, allowing easy diagnosis of results, evaluation of fit between the trees and the taxonomy, and automatic labelling or colouring of tree branches with the taxonomic groups they represent. © The Willi Hennig Society 2012.     

DNA barcoding of Orchidaceae in Korea

Species of Orchidaceae are under severe threat of extinction mainly due to overcollection and habitat destruction; accurate identification of orchid species is critical in conservation biology and sustainable utilization of orchids as plant resources. We examined 647 sequences of the cpDNA regions rbcL, matK, atpF‐atpH IGS, psbK‐psbI IGS and trnH‐psbA IGS from 89 orchid species (95 taxa) and four outgroup taxa to develop an efficient DNA barcode for Orchidaceae in Korea. The five cpDNA barcode regions were successfully amplified and sequenced for all chlorophyllous taxa, but the amplification and sequencing of the same regions in achlorophyllous taxa produced variable results. psbK‐psbI IGS showed the highest mean interspecific K2P distance (0.1192), followed by matK (0.0803), atpF‐atpH IGS (0.0648), trnH‐psbA IGS (0.0460) and rbcL (0.0248). The degree of species resolution for individual barcode regions ranged from 60.5% (rbcL) to 83.5% (trnH‐psbA IGS). The degree of species resolution was significantly enhanced in multiregion combinations of the five barcode regions. Of the 26 possible combinations of the five regions, six provided the highest degree of species resolution (98.8%). Among these, a combination of atpF‐atpH IGS, psbK‐psbI IGS and trnH‐psbA IGS, which comprises the least number of DNA regions, is the best option for barcoding of the Korean orchid species.     

DNA barcoding in a biodiversity hot spot: potential value for the identification of Malagasy Euphorbia L. listed in CITES Appendices I and II

The island of Madagascar is a key hot spot for the genus Euphorbia, with at least 170 native species, almost all endemic. Threatened by habitat loss and illegal collection of wild plants, nearly all Malagasy Euphorbia are listed in CITES Appendices I and II. The absence of a reliable taxonomic revision makes it particularly difficult to identify these plants, even when fertile, and thereby compromises the application of CITES regulations. DNA barcoding, which can facilitate species‐level identification irrespective of developmental stage and the presence of flowers or fruits, may be a promising tool for monitoring and controlling trade involving threatened species. In this study, we test the potential value of barcoding on 41 Euphorbia species representative of the genus in Madagascar, using the two widely adopted core barcode markers (matK and rbcL), along with two additional DNA regions, nuclear internal transcribed spacer (ITS) and the chloroplastic intergenic spacer psbA‐trnH. For each marker and for selected marker combinations, inter‐ and intraspecific distance estimates and species discrimination rates are calculated. Results using just the ‘official’ barcoding markers yield overlapping inter‐ and intraspecific ranges and species discrimination rates below 60%. When ITS is used, whether alone or in combination with the core markers, species discrimination increases to nearly 100%, whereas the addition of psbA‐trnH produces less satisfactory results. This study, the first ever to test barcoding on the large, commercially important genus Euphorbia shows that this method could be developed into a powerful identification tool and thereby contribute to more effective application of CITES regulations.     

Phylogenetic relationships within Adiaphanida (phylum Platyhelminthes) and the status of the crustacean‐parasitic genus Genostoma

The existence of the platyhelminth clade Adiaphanida—an assemblage comprising the well‐studied order Tricladida as well as two lesser known taxa, Prolecithophora and the obligate parasitic Fecampiida—is among the more surprising results of flatworm molecular systematics. Each of these three clades is itself largely well‐defined from a morphological point of view, although Adiaphanida at large, despite its strong support in molecular phylogenetic analyses, lacks known morphological synapomorphies. However, one taxon, the genus Genostoma, a parasite of the leptostracan crustacean Nebalia, rests uneasily within its current classification within the fecampiid family Genostomatidae; ultrastructural investigations on this taxon have uncovered a spermatogenesis reminiscent of Kalyptorhynchia, and a dorsal syncytium resembling the neodermatan tegument. Here, we provide molecular sequence data (nearly complete 18S and 28S rRNA) from a representative of Genostoma, with which we test hypotheses on the phylogenetic position of this taxon within Platyhelminthes, expanding upon a recently published phylum‐wide analysis, and applying novel alignment algorithms and substitution models. These analyses unequivocally position Genostoma as the sister group of Prolecithophora. However, even in taxon‐rich analyses, support for the position of the root of Adiaphanida is lacking, highlighting the need for new data types to study the phylogeny of this clade. Interestingly, our analyses also do not recover the monophyly of several taxa previously proposed, notably Continenticola within Tricladida and Protomonotresidae within Prolecithophora. In light of this phylogeny and the distinctive morphology (especially, spermatogenesis) of Genostoma, we advocate for a redefinition of the family Genostomatidae, outside of both Fecampiida and Prolecithophora, to encompass the members of this unique genus of parasites. Within Fecampiida, the family Piscinquilinidae fam. nov. is erected to accommodate the vertebrate‐parasitic Piscinquilinus, formerly Genostomatidae.     

Classification and differentiation of the genus Peganum indigenous to China based on chloroplast trnL-F and psbA-trnH sequences and seed coat morphology

The genus Peganum (Zygophyllaceae) consists of six species and one subspecies; three of which are distributed in China, P. harmala Linn, P. nigellastrum Bunge and P. multisectum (Maxim.) Bobr. A probable new or intermediate species, Peganum sp., has been suggested in the wild in northwest China. Traditional classification in genus Peganum has focused on hairs on the plant surface. In this study, seed coat characteristics of Peganum species were investigated using light and scanning electron microscopy, demonstrating clear differences in morphology between species. In addition, DNA sequence data from two sequences (chloroplast: trnL‐F, psbA‐trnH) were used to differentiate Peganum sp. and study polygenetic relationships. Diversity in DNA sequences among Peganum species was found, with inter‐specific sequence divergence ranging from 0.6% to 5.6% in psbA‐trnH, and 0.0% to 1.8% in trnL‐F. The variations within species were low: from 0.0% to 0.4% in psbA‐trnH and 0.0% to 0.4% in trnL‐F. Therefore, Peganum species can now be easily identified as separate entities based on variations in DNA sequences. Phylogenetic trees were constructed from the combined data set for the two gene fragments, and the results indicate that Peganum sp. is monophyletic and sister to P. harmala and P. nigellastrum, while P. multisectum is also monophyletic. DNA data further confirmed that P. multisectum is an independent species and that a new species, Peganum sp., exists within the genus Peganum growing wild in China.     

Phylogeny and species delineation in the marine diatom Pseudo‐nitzschia (Bacillariophyta) using cox1, LSU,and ITS2 rRNA genes: A perspective in character evolution

Analyses of the mitochondrial cox1, the nuclear‐encoded large subunit (LSU), and the internal transcribed spacer 2 (ITS2) RNA coding region of Pseudo‐nitzschia revealed that the P. pseudodelicatissima complex can be phylogenetically grouped into three distinct clades (Groups I–III), while the P. delicatissima complex forms another distinct clade (Group IV) in both the LSU and ITS2 phylogenetic trees. It was elucidated that comprehensive taxon sampling (sampling of sequences), selection of appropriate target genes and outgroup, and alignment strategies influenced the phylogenetic accuracy. Based on the genetic divergence, ITS2 resulted in the most resolved trees, followed by cox1 and LSU. The morphological characters available for Pseudo‐nitzschia, although limited in number, were overall in agreement with the phylogenies when mapped onto the ITS2 tree. Information on the presence/absence of a central nodule, number of rows of poroids in each stria, and of sectors dividing the poroids mapped onto the ITS2 tree revealed the evolution of the recently diverged species. The morphologically based species complexes showed evolutionary relevance in agreement with molecular phylogeny inferred from ITS2 sequence–structure data. The data set of the hypervariable region of ITS2 improved the phylogenetic inference compared to the cox1 and LSU data sets. The taxonomic status of P. cuspidata and P. pseudodelicatissima requires further elucidation.     

Evaluation of bias on the assessment of diet breadth of herbivorous insects using molecular methods

The interactions between herbivores and their host plants play a key role in ecological processes. Understanding the width and nature of these interactions is fundamental to ecology and conservation. Recent research on DNA‐based inference of trophic associations suggests that the host range of phytophagous insects in the tropics may be wider than previously thought based on traditional observation. However, the reliability of molecular inference of ecological associations, still strongly dependent on PCR and thus exposed to the risk of contamination with environmental DNA, is under debate. Here, we explored alternative procedures to reduce the chance of amplification of external, nondiet DNA, including surface decontamination and analysis of mid/hind guts, comparing the results with those obtained using the standard protocol. We studied 261 specimens in eight species of Neotropical Chrysomelidae that yielded 316 psbA‐trnH intergenic spacer sequences (cpDNA marker of putative diets) from unique and multiple‐band PCR results. The taxonomic identity of these sequences was inferred using the automated pipeline BAGpipe, yielding results consistent with 31 plant families. Regardless of the protocol used, a wide taxonomic spectrum of food was inferred for all chrysomelid species. Canonical Correspondence Analysis using these data revealed significant differences attributed mainly to species (expectedly, since they represent different ecologies), but also to treatment (untreated vs. cleaned/gut samples) and PCR results (single vs. multiple bands). Molecular identification of diets is not straightforward and, regardless of the species’ niche breadth, combining approaches that reduce external contamination and studying multiple individuals per species may help increasing confidence in results.     

Two New Computational Methods for Universal DNA Barcoding: A Benchmark Using Barcode Sequences of Bacteria,Archaea, Animals,Fungi, and Land Plants

Taxonomic identification of biological specimens based on DNA sequence information (a.k.a. DNA barcoding) is becoming increasingly common in biodiversity science. Although several methods have been proposed, many of them are not universally applicable due to the need for prerequisite phylogenetic/machine-learning analyses, the need for huge computational resources, or the lack of a firm theoretical background. Here, we propose two new computational methods of DNA barcoding and show a benchmark for bacterial/archeal 16S, animal COX1, fungal internal transcribed spacer, and three plant chloroplast (rbcL, matK, and trnH-psbA) barcode loci that can be used to compare the performance of existing and new methods. The benchmark was performed under two alternative situations: query sequences were available in the corresponding reference sequence databases in one, but were not available in the other. In the former situation, the commonly used “1-nearest-neighbor” (1-NN) method, which assigns the taxonomic information of the most similar sequences in a reference database (i.e., BLAST-top-hit reference sequence) to a query, displays the highest rate and highest precision of successful taxonomic identification. However, in the latter situation, the 1-NN method produced extremely high rates of misidentification for all the barcode loci examined. In contrast, one of our new methods, the query-centric auto-k-nearest-neighbor (QCauto) method, consistently produced low rates of misidentification for all the loci examined in both situations. These results indicate that the 1-NN method is most suitable if the reference sequences of all potentially observable species are available in databases; otherwise, the QCauto method returns the most reliable identification results. The benchmark results also indicated that the taxon coverage of reference sequences is far from complete for genus or species level identification in all the barcode loci examined. Therefore, we need to accelerate the registration of reference barcode sequences to apply high-throughput DNA barcoding to genus or species level identification in biodiversity research.