Alex V. Borisenko

The role of DNA barcodes in understanding and conservation of mammal diversity in southeast Asia.

Background Southeast Asia is recognized as a region of very high biodiversity, much of which is currently at risk due to habitat loss and other threats. However, many aspects of this diversity, even for relatively well-known groups such as mammals, are poorly known, limiting ability to develop conservation plans. This study examines the value of DNA barcodes, sequences of the mitochondrial COI gene, to enhance understanding of mammalian diversity in the region and hence to aid conservation planning. Methodology and Principal Findings DNA barcodes were obtained from nearly 1900 specimens representing 165 recognized species of bats. All morphologically or acoustically distinct species, based on classical taxonomy, could be discriminated with DNA barcodes except four closely allied species pairs. Many currently recognized species contained multiple barcode lineages, often with deep divergence suggesting unrecognized species. In addition, most widespread species showed substantial genetic differentiation across their distributions. Our results suggest that mammal species richness within the region may be underestimated by at least 50%, and there are higher levels of endemism and greater intra-specific population structure than previously recognized. Conclusions DNA barcodes can aid conservation and research by assisting field workers in identifying species, by helping taxonomists determine species groups needing more detailed analysis, and by facilitating the recognition of the appropriate units and scales for conservation planning.

DNA barcoding in surveys of small mammal communities: a field study in Suriname.

The performance of DNA barcoding as a tool for fast taxonomic verification in ecological assessment projects of small mammals was evaluated during a collecting trip to a lowland tropical rainforest site in Suriname. We also compared the performance of tissue sampling onto FTA CloneSaver cards vs. liquid nitrogen preservation. DNA barcodes from CloneSaver cards were recovered from 85% of specimens, but DNA degradation was apparent, because only 36% of sequence reads were long (over 600 bp). In contrast, cryopreserved tissue delivered 99% barcode recovery (97% > 600 bp). High humidity, oversampling or tissue type may explain the poor performance of CloneSaver cards. Comparison of taxonomic assignments made in the field and from barcode results revealed inconsistencies in just 3.4% of cases and most of the discrepancies were due to field misidentifications (3%) rather than sampling/analytical error (0.5%). This result reinforces the utility of DNA barcoding as a tool for verification of taxonomic identifications in ecological surveys, which is especially important when the collection of voucher specimens is not possible.

The front-end logistics of DNA barcoding: challenges and prospects

Building a global library of DNA barcodes will require efficient logistics of pre‐laboratory specimen processing and seamless interfacing with molecular protocols. If not addressed properly, the task of aggregating specimens may become the biggest bottleneck in the analytical chain. Three years of experience in developing a collection management system to facilitate high‐throughput DNA barcoding have allowed the Canadian Centre for DNA Barcoding to recognize and resolve the most common logistical obstacles. Dealing with these challenges on a larger scale will be an important step towards building a solid collection‐based foundation for the international DNA barcoding effort.

Express barcodes: racing from specimen to identification.

Although devices combining microfluidic and advanced sequencing technologies promise a future where one can generate a DNA barcode in minutes, current analytical regimes typically involve workflows that extend over 2 days. Here we describe simple protocols enabling the advance from a specimen to barcode‐based identification in less than 2 h. The protocols use frozen or lyophilized reagents that can be prepackaged into ‘kits’ and support barcode analysis across the animal kingdom. The analytical procedure allows 5 min for DNA extraction, 25 min for polymerase chain reaction amplification of the barcode region, 25 min for cycle‐sequencing, 10 min for cleanup, 45 min for capillary sequencing and 5 min for trace file analysis to complete DNA‐based identification. This study involved the comparison of varied DNA preservation and extraction methods, and evaluated Taq polymerases with high processivity and resistance to inhibitors.

Authentication of Herbal Supplements Using Next-Generation Sequencing

Background DNA-based testing has been gaining acceptance as a tool for authentication of a wide range of food products; however, its applicability for testing of herbal supplements remains contentious. Methods We utilized Sanger and Next-Generation Sequencing (NGS) for taxonomic authentication of fifteen herbal supplements representing three different producers from five medicinal plants: Echinacea purpurea, Valeriana officinalis, Ginkgo biloba, Hypericum perforatum and Trigonella foenum-graecum. Experimental design included three modifications of DNA extraction, two lysate dilutions, Internal Amplification Control, and multiple negative controls to exclude background contamination. Ginkgo supplements were also analyzed using HPLC-MS for the presence of active medicinal components. Results All supplements yielded DNA from multiple species, rendering Sanger sequencing results for rbcL and ITS2 regions either uninterpretable or non-reproducible between the experimental replicates. Overall, DNA from the manufacturer-listed medicinal plants was successfully detected in seven out of eight dry herb form supplements; however, low or poor DNA recovery due to degradation was observed in most plant extracts (none detected by Sanger; three out of seven–by NGS). NGS also revealed a diverse community of fungi, known to be associated with live plant material and/or the fermentation process used in the production of plant extracts. HPLC-MS testing demonstrated that Ginkgo supplements with degraded DNA contained ten key medicinal components. Conclusion Quality control of herbal supplements should utilize a synergetic approach targeting both DNA and bioactive components, especially for standardized extracts with degraded DNA. The NGS workflow developed in this study enables reliable detection of plant and fungal DNA and can be utilized by manufacturers for quality assurance of raw plant materials, contamination control during the production process, and the final product. Interpretation of results should involve an interdisciplinary approach taking into account the processes involved in production of herbal supplements, as well as biocomplexity of plant-plant and plant-fungal biological interactions.

Genetic Diversity of Northeastern Palaearctic Bats as Revealed by DNA Barcodes

Sequences of the DNA barcode region of the cytochrome oxidase subunit I gene were obtained from 3 8 species of northeastern Palaearctic bats to assess patterns of genetic diversity. These results confirmed earlier findings of deep phylogeographic splits in four pairs of vicariant species (Myotis daubentoniilpetax, M. nattererilbombinus, Plecotus aurituslognevi and Miniopterus schreibersiil fuliginosus) and suggested previously unreported splits within Eptesicus nilssoni and Myotis aurascens. DNA barcodes support all taxa raised to species rank in the past 25 years and suggest that an additional species — Myotis sibiricus — should be separated from Myotis brandtii. Major phylogeographic splits occur between European and Asian populations of Myotis aurascens, Rhinolophus ferrumequinum and Myotis frater; smaller scale splits are observed between insular and mainland populations in the Far East (M. frater, Myotis ikonnikovi and Murina ussuriensis) and also between southeastern Europe and Ciscaucasia (Myotis daubentonii, Plecotus auritus, and Pipistrellus pipistrellus). One confirmed case of sequence sharing was observed in our dataset — Eptesicus nilssoni/serotinus. This study corroborates the utility of DNA barcodes as a taxonomic assessment tool for bats.

Molecular Phylogenetics and Taxonomy of the =Subgenus pika (Ochotona, Lagomorpha)

Abstract A phylogenetic analysis based on partial sequences of 2 mitochondrial genes (cytochrome b and cytochrome c oxidase subunit I) confirmed that Ochotona alpina (Pallas, 1773) and O. turuchanensis Naumov, 1934, are sister taxa to all other Palearctic species of the subgenus Pika. O. hyperborea (Pallas, 1811) contains sufficient genetic heterogeneity to define 2 or 3 races within this species. Examination of genetic data supports the recognition of O. scorodumovi Skalon, 1935, as a distinct species. The proper name of the taxon (either O. scorodumovi Skalon, 1935, or O. mantchurica Thomas, 1909) remains to be established. O. hoffmanni Formozov et al., 1996, is probably the closest relative of O. scorodumovi. These 2 taxa constitute a sister group to O. hyperborea. All the above-mentioned Palearctic taxa constitute the monophyletic alpina–hyperborea group. The pallasi group contains at least 3 taxa: O. (p.) pallasi (Gray, 1867), O. (p.) pricei Thomas, 1911, and O. (p.) argentata Howell, 1928. The taxonomic rank of these 3 taxa requires additional careful investigation. Nearctic pikas O. princeps (Richardson, 1828) and O. collaris (Nelson, 1893) constitute a monophyletic group separate from Palearctic taxa.

DNA Barcoding in Mammals

DNA barcoding provides an operational framework for mammalian taxonomic identification and cryptic species discovery. Focused effort to build a reference library of genetic data has resulted in the assembly of over 35 K mammalian cytochrome c oxidase subunit I sequences and outlined the scope of mammal-related barcoding projects. Based on the above experience, this chapter recounts three typical methodological pathways involved in mammalian barcoding: routine methods aimed at assembling the reference sequence library from high quality samples, express approaches used to attain cheap and fast taxonomic identifications for applied purposes, and forensic techniques employed when dealing with degraded material. Most of the methods described are applicable to a range of vertebrate taxa outside Mammalia.

Status and prospects of DNA barcoding in medically important parasites and vectors

For over 10 years, DNA barcoding has been used to identify specimens and discern species. Its potential benefits in parasitology were recognized early, but its utility and uptake remain unclear. Here we review studies using DNA barcoding in parasites and vectors affecting humans and find that the technique is accurate (accords with author identifications based on morphology or other markers) in 94-95% of cases, although aspects of DNA barcoding (vouchering, marker implicated) have often been misunderstood. In a newly compiled checklist of parasites, vectors, and hazards, barcodes are available for 43% of all 1403 species and for more than half of 429 species of greater medical importance. This is encouraging coverage that would improve with an active campaign targeting parasites and vectors.

Implications of hybridization, NUMTs, and overlooked diversity for DNA Barcoding of Eurasian ground squirrels.

The utility of DNA Barcoding for species identification and discovery has catalyzed a concerted effort to build the global reference library; however, many animal groups of economical or conservational importance remain poorly represented. This study aims to contribute DNA barcode records for all ground squirrel species (Xerinae, Sciuridae, Rodentia) inhabiting Eurasia and to test efficiency of this approach for species discrimination. Cytochrome c oxidase subunit 1 (COI) gene sequences were obtained for 97 individuals representing 16 ground squirrel species of which 12 were correctly identified. Taxonomic allocation of some specimens within four species was complicated by geographically restricted mtDNA introgression. Exclusion of individuals with introgressed mtDNA allowed reaching a 91.6% identification success rate. Significant COI divergence (3.5–4.4%) was observed within the most widespread ground squirrel species (Spermophilus erythrogenys, S. pygmaeus, S. suslicus, Urocitellus undulatus), suggesting the presence of cryptic species. A single putative NUMT (nuclear mitochondrial pseudogene) sequence was recovered during molecular analysis; mitochondrial COI from this sample was amplified following re-extraction of DNA. Our data show high discrimination ability of 100 bp COI fragments for Eurasian ground squirrels (84.3%) with no incorrect assessments, underscoring the potential utility of the existing reference librariy for the development of diagnostic ‘mini-barcodes’.