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Concept: DNA barcoding

513

Herbal products available to consumers in the marketplace may be contaminated or substituted with alternative plant species and fillers that are not listed on the labels. According to the World Health Organization, the adulteration of herbal products is a threat to consumer safety. Our research aimed to investigate herbal product integrity and authenticity with the goal of protecting consumers from health risks associated with product substitution and contamination.

Concepts: Organism, Species, Herbalism, DNA barcoding, World Health Organization, Herbal, Consumer protection, Consumer

170

DNA barcoding is a molecular tool that exploits a unique DNA sequence of a standardized gene or non-coding region for the species identification of unknown individuals. The investigation into a suitable barcode for diatoms is ongoing and there are several promising candidates including mitochondrial, plastidial and nuclear markers. We analyzed 272 sequences from 76 diatoms species in the orders Thalassiosirales, Lithodesmiales and Cymatosirales, using distance and character based approaches, to assess the applicability of a DNA barcode based on the hypervariable V4 region of the nuclear 18S rRNA gene. We show that the proposed V4 barcode separated ca. 97% of all centric diatom taxa tested using a threshold p-distance of 0.02 and that many problem pairs were further separated using a character based approach. The reliability of amplification, extensive reference library and variability seen in the V4 region make it the most promising candidate to date for a barcode marker for diatoms particularly when combined with DNA character analysis.

Concepts: DNA, Gene, Molecular biology, Species, Ribosomal RNA, DNA barcoding, Identification, Diatom

166

DNA barcoding as a tool for species identification has been successful in animals and other organisms, including certain groups of plants. The exploration of this new tool for species identification, particularly in tree species, is very scanty from biodiversity-rich countries like India. and are standard barcode loci while ITS, and are considered as supplementary loci for plants.

Concepts: DNA, Gene, Biology, Organism, Eukaryote, Species, Plant, DNA barcoding

47

During acoustic communication, an audible message is transmitted from a sender to a receiver, often producing changes in behavior. In a system where evolutionary changes of the sender do not result in a concomitant adjustment in the receiver, communication and species recognition could fail. However, the possibility of an evolutionary decoupling between sender and receiver has rarely been studied. Frog populations in the Allobates femoralis cryptic species complex are known for their extensive morphological, genetic and acoustic variation. We hypothesized that geographic variation in acoustic signals of A. femoralis was correlated with geographic changes in communication through changes in male-male recognition. To test this hypothesis, we quantified male call recognition using phonotactic responses to playback experiments of advertisement calls with two, three and four notes in eight localities of the Amazonian basin. Then, we reconstructed the ancestral states of call note number in a phylogenetic framework and evaluated whether the character state of the most recent common ancestor predicted current relative responses to two, three and four notes. The probability of a phonotactic response to advertisement calls of A. femoralis males was strongly influenced by the call mid-frequency and the number of notes in most populations. Positive phonotaxis was complete for calls from each individual’s population, and in some populations, it was also partial for allotopic calls; however, in two populations, individuals equally recognized calls with two, three or four notes. This evidence, in conjunction with our results from phylogenetic comparative methods, supports the hypothesis of decoupled evolution between sender and receiver in the male-male communication system of the A. femoralis complex. Thus, signal recognition appears to evolve more slowly than the calls.

Concepts: Evolution, Biology, Species, Phylogenetics, Speciation, DNA barcoding, Cryptic species complex, Phylogenetic comparative methods

30

We present the first national DNA barcode resource that covers the native flowering plants and conifers for the nation of Wales (1143 species). Using the plant DNA barcode markers rbcL and matK, we have assembled 97.7% coverage for rbcL, 90.2% for matK, and a dual-locus barcode for 89.7% of the native Welsh flora. We have sampled multiple individuals for each species, resulting in 3304 rbcL and 2419 matK sequences. The majority of our samples (85%) are from DNA extracted from herbarium specimens. Recoverability of DNA barcodes is lower using herbarium specimens, compared to freshly collected material, mostly due to lower amplification success, but this is balanced by the increased efficiency of sampling species that have already been collected, identified, and verified by taxonomic experts. The effectiveness of the DNA barcodes for identification (level of discrimination) is assessed using four approaches: the presence of a barcode gap (using pairwise and multiple alignments), formation of monophyletic groups using Neighbour-Joining trees, and sequence similarity in BLASTn searches. These approaches yield similar results, providing relative discrimination levels of 69.4 to 74.9% of all species and 98.6 to 99.8% of genera using both markers. Species discrimination can be further improved using spatially explicit sampling. Mean species discrimination using barcode gap analysis (with a multiple alignment) is 81.6% within 10×10 km squares and 93.3% for 2×2 km squares. Our database of DNA barcodes for Welsh native flowering plants and conifers represents the most complete coverage of any national flora, and offers a valuable platform for a wide range of applications that require accurate species identification.

Concepts: Bioinformatics, Biology, Species, Plant, Taxonomy, DNA barcoding, Barcode, Identification

28

A potential DNA barcode, ITS2, was studied to discriminate herbal materials to confirm their identities and ensure their safe application in pharmaceuticals. Here, a total of 4385 samples of 2431 species were collected, and these samples are from 61 commonly used herbs and their closely related species or adulterants. Based on assessments of the extent of genetic divergence, the DNA barcoding gap and the ability for species discrimination, our results suggest that ITS2 is a powerful tool for distinguishing herbs. For the first dataset including 61 herbs, ITS2 correctly identified 100 % of them. For the second dataset containing 51 herbs and their 2382 closely related species, ITS2 could discriminate correctly 48 herbs from their closely related species. For the third dataset comprising 34 herbs and their 111 adulterants, ITS2 could distinguish successfully all the herbs from their adulterants. In conclusion, the ITS2 region is an efficient marker for the authentication of herbal materials, and our study will accelerate the process of the application of the DNA barcoding technique in differentiating herbs.

Concepts: DNA, Genetics, Bioinformatics, Species, Botany, DNA barcoding, Cryptic species complex, Identification

28

Due to the difficulty in morphological identification the development of reliable molecular tools for species distinction is a priority for piroplasma. Previous studies based on 18S rRNA and other gene sequences provided a backbone for the phylogeny of piroplasma. However, it is difficult to discriminate species in a comprehensive sample. Here, the abilities of eight DNA regions including 18S rRNA, 28S rRNA, internal transcribed spacer (ITS) regions and COI genes, have been compared as candidates of DNA barcodes for piroplasma. In total, 484 sequences of piroplasma were collected from this study and GenBank. The eight proposed DNA regions were evaluated according to the criterion of Consortium for the Barcode of Life (CBOL). From this evaluation, ITS2 had 100% PCR amplification efficiency, an ideal sequence length, the largest gap between the intra- and inter-specific divergence, 98% identification efficiency at the genus level, and 92% at the species level. Thus, we propose that ITS2 is the most ideal DNA barcode based on the current database for piroplasma.

Concepts: DNA, Gene, Molecular biology, Biology, Organism, Species, Ribosomal RNA, DNA barcoding

27

Indian snakeroot (Rauvolfia serpentina) is a valuable forest product, root extracts of which are used as an antihypertensive drug. Increasing demand led to overharvesting in the wild. Control of international trade is hampered by the inability to identify root samples to the species level. We therefore evaluated the potential of molecular identification by searching for species-specific DNA polymorphisms. We found two species-specific indels in the rps16 intron region for R. serpentina. Our DNA barcoding method was tested for its specificity, reproducibility, sensitivity and stability. We included samples of various tissues and ages, which had been treated differently for preservation. DNA extractions were tested in a range of amplification settings and dilutions. Species-specific rps16 intron sequences were obtained from 79 herbarium accessions and one confiscated root, encompassing 39 different species. Our results demonstrate that molecular analysis provides new perspectives for forensic identification of Indian snakeroot.

Concepts: DNA, Species, Sensitivity and specificity, DNA barcoding, Identification, Rauvolfia, Rauwolfia serpentina

27

As for many other regions, environmental and biodiversity monitoring of the brackish Baltic Sea suffers from low species resolution for several taxa. One such case is the benthic larvae of midges Chironomidae (Diptera), which are estimated to constitute about 30% of the macrozoobenthos species of the Baltic Sea and are important indicators of environmental quality. We assessed the usefulness of COI (cytochrome oxidase I) gene barcoding to improve species resolution and its potential for implementation in monitoring programmes. Neighbour-Joining, Maximum parsimony and Bayesian-inference analyses all provided high congruency with morphological analyses of adult males for almost all 42 species studied. Barcoding was helpful to elucidate some cases of taxonomical difficulties, such as synonyms. In contrast to the high identification accuracy when using our local database, there were a number of cases where matching with GenBank and BOLD provided puzzling results. For reliable species identification at least 15-30 specimens from 5-10 well-distributed sites within the geographical range of the species might be needed in a database to adequately cover the intraspecific variability of chironomids. Implementation of DNA barcoding, as applied here, in monitoring would result in an increase from at present less than 10% to more than 90% successful chironomid species identification of Baltic Sea benthic samples, as it also would for many nearby lakes. Routine monitoring of benthic environmental samples based on Next-Generation sequencing techniques would provide a cost effective way to obtain a taxonomically much more complete assessment of environmental quality and biodiversity, as required by EU directives and national legislation.

Concepts: Species, Taxonomy, Baltic Sea, Estonia, DNA barcoding, Identification, Poland, Latvia

26

We sampled 14,603 geometrid moths along a forested elevational gradient from 1020-3021 m in the southern Ecuadorian Andes, and then employed DNA barcoding to refine decisions on species boundaries initially made by morphology. We compared the results with those from an earlier study on the same but slightly shorter gradient that relied solely on morphological criteria to discriminate species. The present analysis revealed 1857 putative species, an 80% increase in species richness from the earlier study that detected only 1010 species. Measures of species richness and diversity that are less dependent on sample size were more than twice as high as in the earlier study, even when analysis was restricted to an identical elevational range. The estimated total number of geometrid species (new dataset) in the sampled area is 2350. Species richness at single sites was 32-43% higher, and the beta diversity component rose by 43-51%. These impacts of DNA barcoding on measures of richness reflect its capacity to reveal cryptic species that were overlooked in the first study. The overall results confirmed unique diversity patterns reported in the first investigation. Species diversity was uniformly high along the gradient, declining only slightly above 2800 m. Species turnover also showed little variation along the gradient, reinforcing the lack of evidence for discrete faunal zones. By confirming these major biodiversity patterns, the present study establishes that incomplete species delineation does not necessarily conceal trends of biodiversity along ecological gradients, but it impedes determination of the true magnitude of diversity and species turnover.

Concepts: Biodiversity, Conservation biology, Evolution, Species, Ecology, DNA barcoding, Cryptic species complex, Species diversity