Publications by authors named "Evgeny V Zakharov"

24 Publications

  • Page 1 of 1

A DNA barcode library for the butterflies of North America.

PeerJ 2021 19;9:e11157. Epub 2021 Apr 19.

Department of Integrative Biology, University of Guelph, Guelph, Ontario, Canada.

Although the butterflies of North America have received considerable taxonomic attention, overlooked species and instances of hybridization continue to be revealed. The present study assembles a DNA barcode reference library for this fauna to identify groups whose patterns of sequence variation suggest the need for further taxonomic study. Based on 14,626 records from 814 species, DNA barcodes were obtained for 96% of the fauna. The maximum intraspecific distance averaged 1/4 the minimum distance to the nearest neighbor, producing a barcode gap in 76% of the species. Most species (80%) were monophyletic, the others were para- or polyphyletic. Although 15% of currently recognized species shared barcodes, the incidence of such taxa was far higher in regions exposed to Pleistocene glaciations than in those that were ice-free. Nearly 10% of species displayed high intraspecific variation (>2.5%), suggesting the need for further investigation to assess potential cryptic diversity. Aside from aiding the identification of all life stages of North American butterflies, the reference library has provided new perspectives on the incidence of both cryptic and potentially over-split species, setting the stage for future studies that can further explore the evolutionary dynamics of this group.
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http://dx.doi.org/10.7717/peerj.11157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8061581PMC
April 2021

Torix Rickettsia are widespread in arthropods and reflect a neglected symbiosis.

Gigascience 2021 Mar;10(3)

Institute of Infection, Veterinary and Ecological Sciences, Faculty of Health and Life Sciences, University of Liverpool, Leahurst Campus, Chester High Road, Neston, Wirral CH64 7TE, UK.

Background: Rickettsia are intracellular bacteria best known as the causative agents of human and animal diseases. Although these medically important Rickettsia are often transmitted via haematophagous arthropods, other Rickettsia, such as those in the Torix group, appear to reside exclusively in invertebrates and protists with no secondary vertebrate host. Importantly, little is known about the diversity or host range of Torix group Rickettsia.

Results: This study describes the serendipitous discovery of Rickettsia amplicons in the Barcode of Life Data System (BOLD), a sequence database specifically designed for the curation of mitochondrial DNA barcodes. Of 184,585 barcode sequences analysed, Rickettsia is observed in ∼0.41% of barcode submissions and is more likely to be found than Wolbachia (0.17%). The Torix group of Rickettsia are shown to account for 95% of all unintended amplifications from the genus. A further targeted PCR screen of 1,612 individuals from 169 terrestrial and aquatic invertebrate species identified mostly Torix strains and supports the "aquatic hot spot" hypothesis for Torix infection. Furthermore, the analysis of 1,341 SRA deposits indicates that Torix infections represent a significant proportion of all Rickettsia symbioses found in arthropod genome projects.

Conclusions: This study supports a previous hypothesis that suggests that Torix Rickettsia are overrepresented in aquatic insects. In addition, multiple methods reveal further putative hot spots of Torix Rickettsia infection, including in phloem-feeding bugs, parasitoid wasps, spiders, and vectors of disease. The unknown host effects and transmission strategies of these endosymbionts make these newly discovered associations important to inform future directions of investigation involving the understudied Torix Rickettsia.
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http://dx.doi.org/10.1093/gigascience/giab021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7992394PMC
March 2021

Temporal Detection Limits of Remnant Larval Bloodmeals in Nymphal Ixodes scapularis (Say, Ixodida: Ixodidae) Using Two Next-Generation Sequencing DNA Barcoding Assays.

J Med Entomol 2021 03;58(2):821-829

Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada.

Using next-generation sequencing DNA barcoding, we aimed to determine: 1) if the larval bloodmeal can be detected in Ixodes scapularis nymphs and 2) the post-moult temporal window for detection of the larval bloodmeal. Subsets of 30 nymphs fed on a domestic rabbit (Oryctolagus cuniculus Linnaeus, Lagomorphia: Leporidae) as larvae were reared and frozen at 11 time points post-moult, up to 150 d. Vertebrate DNA was amplified using novel universal (UP) and species-specific primers (SSP) and sequenced for comparison against cytochrome c oxidase subunit I barcodes to infer host identification. Detectable bloodmeals decreased as time since moult increased for both assays. For the SSP assay, detection of bloodmeals decreased from 96.7% (n = 29/30) in day 0 nymphs to 3.3% (n = 1/30) and 6.7% (n = 2/30) at 4- and 5-mo post-moult, respectively. A shorter temporal detection period was achieved with the UP assay, declining from 16.7% (n = 5/30) in day 0 nymphs to 0/30 in 3-d-old nymphs. Bloodmeal detection was nonexistent for the remaining cohorts, with the exception of 1/30 nymphs at 2-mo post-moult. Host detection was significantly more likely using the SSP assay compared to the UP assay in the first three time cohorts (day 0: χ 2 = 39.1, P < 0.005; day 2: χ 2 = 19.2, P < 0.005; day 3: χ 2 = 23.3, P < 0.005). Regardless of the primer set used, the next-generation sequencing DNA barcoding assay was able to detect host DNA from a larval bloodmeal in the nymphal life stage; however, a short window with a high proportion of detection post-moult was achieved.
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http://dx.doi.org/10.1093/jme/tjaa192DOI Listing
March 2021

A reference library for Canadian invertebrates with 1.5 million barcodes, voucher specimens, and DNA samples.

Sci Data 2019 12 6;6(1):308. Epub 2019 Dec 6.

Centre for Biodiversity Genomics, University of Guelph, Guelph, Ontario, Canada.

The reliable taxonomic identification of organisms through DNA sequence data requires a well parameterized library of curated reference sequences. However, it is estimated that just 15% of described animal species are represented in public sequence repositories. To begin to address this deficiency, we provide DNA barcodes for 1,500,003 animal specimens collected from 23 terrestrial and aquatic ecozones at sites across Canada, a nation that comprises 7% of the planet's land surface. In total, 14 phyla, 43 classes, 163 orders, 1123 families, 6186 genera, and 64,264 Barcode Index Numbers (BINs; a proxy for species) are represented. Species-level taxonomy was available for 38% of the specimens, but higher proportions were assigned to a genus (69.5%) and a family (99.9%). Voucher specimens and DNA extracts are archived at the Centre for Biodiversity Genomics where they are available for further research. The corresponding sequence and taxonomic data can be accessed through the Barcode of Life Data System, GenBank, the Global Biodiversity Information Facility, and the Global Genome Biodiversity Network Data Portal.
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http://dx.doi.org/10.1038/s41597-019-0320-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6897906PMC
December 2019

Validation of COI metabarcoding primers for terrestrial arthropods.

PeerJ 2019 7;7:e7745. Epub 2019 Oct 7.

Centre for Biodiversity Genomics, University of Guelph, Guelph, ON, Canada.

Metabarcoding can rapidly determine the species composition of bulk samples and thus aids biodiversity and ecosystem assessment. However, it is essential to use primer sets that minimize amplification bias among taxa to maximize species recovery. Despite this fact, the performance of primer sets employed for metabarcoding terrestrial arthropods has not been sufficiently evaluated. This study tests the performance of 36 primer sets on a mock community containing 374 insect species. Amplification success was assessed with gradient PCRs and the 21 most promising primer sets selected for metabarcoding. These 21 primer sets were also tested by metabarcoding a Malaise trap sample. We identified eight primer sets, mainly those including inosine and/or high degeneracy, that recovered more than 95% of the species in the mock community. Results from the Malaise trap sample were congruent with the mock community, but primer sets generating short amplicons produced potential false positives. Taxon recovery from both mock community and Malaise trap sample metabarcoding were used to select four primer sets for additional evaluation at different annealing temperatures (40-60 °C) using the mock community. The effect of temperature varied by primer pair but overall it only had a minor effect on taxon recovery. This study reveals the weak performance of some primer sets employed in past studies. It also demonstrates that certain primer sets can recover most taxa in a diverse species assemblage. Thus, based our experimental set up, there is no need to employ several primer sets targeting the same gene region. We identify several suitable primer sets for arthropod metabarcoding, and specifically recommend BF3 + BR2, as it is not affected by primer slippage and provides maximal taxonomic resolution. The fwhF2 + fwhR2n primer set amplifies a shorter fragment and is therefore ideal when targeting degraded DNA (e.g., from gut contents).
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http://dx.doi.org/10.7717/peerj.7745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6786254PMC
October 2019

Metabarcoding a diverse arthropod mock community.

Mol Ecol Resour 2019 May;19(3):711-727

Centre for Biodiversity Genomics, University of Guelph, Guelph, Ontario, Canada.

Although DNA metabarcoding is an attractive approach for monitoring biodiversity, it is often difficult to detect all the species present in a bulk sample. In particular, sequence recovery for a given species depends on its biomass and mitome copy number as well as the primer set employed for PCR. To examine these variables, we constructed a mock community of terrestrial arthropods comprised of 374 species. We used this community to examine how species recovery was impacted when amplicon pools were constructed in four ways. The first two protocols involved the construction of bulk DNA extracts from different body segments (Bulk Abdomen, Bulk Leg). The other protocols involved the production of DNA extracts from single legs which were then merged prior to PCR (Composite Leg) or PCR-amplified separately (Single Leg) and then pooled. The amplicons generated by these four treatments were then sequenced on three platforms (Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5). The choice of sequencing platform did not substantially influence species recovery, although the Miseq delivered the highest sequence quality. As expected, species recovery was most efficient from the Single Leg treatment because amplicon abundance varied little among taxa. Among the three treatments where PCR occurred after pooling, the Bulk Abdomen treatment produced a more uniform read abundance than the Bulk Leg or Composite Leg treatment. Primer choice also influenced species recovery and evenness. Our results reveal how variation in protocols can have substantial impacts on perceived diversity unless sequencing coverage is sufficient to reach an asymptote.
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http://dx.doi.org/10.1111/1755-0998.13008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6850013PMC
May 2019

Characterization and comparison of poorly known moth communities through DNA barcoding in two Afrotropical environments in Gabon .

Genome 2019 Mar 2;62(3):96-107. Epub 2018 Oct 2.

o Centre d'Ecologie Fonctionnelle et Evolutive (CEFE UMR 5175, CNRS-Université de Montpellier-Université Paul-Valéry Montpellier-EPHE), 1919 Route de Mende, F-34293 Montpellier, France.

Biodiversity research in tropical ecosystems-popularized as the most biodiverse habitats on Earth-often neglects invertebrates, yet invertebrates represent the bulk of local species richness. Insect communities in particular remain strongly impeded by both Linnaean and Wallacean shortfalls, and identifying species often remains a formidable challenge inhibiting the use of these organisms as indicators for ecological and conservation studies. Here we use DNA barcoding as an alternative to the traditional taxonomic approach for characterizing and comparing the diversity of moth communities in two different ecosystems in Gabon. Though sampling remains very incomplete, as evidenced by the high proportion (59%) of species represented by singletons, our results reveal an outstanding diversity. With about 3500 specimens sequenced and representing 1385 BINs (Barcode Index Numbers, used as a proxy to species) in 23 families, the diversity of moths in the two sites sampled is higher than the current number of species listed for the entire country, highlighting the huge gap in biodiversity knowledge for this country. Both seasonal and spatial turnovers are strikingly high (18.3% of BINs shared between seasons, and 13.3% between sites) and draw attention to the need to account for these when running regional surveys. Our results also highlight the richness and singularity of savannah environments and emphasize the status of Central African ecosystems as hotspots of biodiversity.
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http://dx.doi.org/10.1139/gen-2018-0063DOI Listing
March 2019

Expedited assessment of terrestrial arthropod diversity by coupling Malaise traps with DNA barcoding .

Genome 2019 Mar 26;62(3):85-95. Epub 2018 Sep 26.

a Centre for Biodiversity Genomics, Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada.

Monitoring changes in terrestrial arthropod communities over space and time requires a dramatic increase in the speed and accuracy of processing samples that cannot be achieved with morphological approaches. The combination of DNA barcoding and Malaise traps allows expedited, comprehensive inventories of species abundance whose cost will rapidly decline as high-throughput sequencing technologies advance. Aside from detailing protocols from specimen sorting to data release, this paper describes their use in a survey of arthropod diversity in a national park that examined 21 194 specimens representing 2255 species. These protocols can support arthropod monitoring programs at regional, national, and continental scales.
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http://dx.doi.org/10.1139/gen-2018-0093DOI Listing
March 2019

Finding the pond through the weeds: eDNA reveals underestimated diversity of pondweeds.

Appl Plant Sci 2018 May 5;6(5):e01155. Epub 2018 Jun 5.

Centre for Biodiversity Genomics University of Guelph 50 Stone Road East Guelph Ontario N1G2W1 Canada.

Premise Of The Study: The detection of environmental DNA (eDNA) using high-throughput sequencing has rapidly emerged as a method to detect organisms from environmental samples. However, eDNA studies of aquatic biomes have focused on surveillance of animal species with less emphasis on plants. Pondweeds are important bioindicators of freshwater ecosystems, although their diversity is underestimated due to difficulties in morphological identification and monitoring.

Methods: A protocol was developed to detect pondweeds in water samples using - and ITS2 markers. The water samples were collected from the Grand River within the rare Charitable Research Reserve, Ontario (RARE). Short fragments were amplified using primers targeting pondweeds, sequenced on an Ion Torrent Personal Genome Machine, and assigned to the taxonomy using a local DNA reference library and GenBank.

Results: We detected two species earlier documented at the experimental site during ecological surveys ( and ) and three species new to the RARE checklist (, , and ).

Discussion: Our targeted approach to track the species composition of pondweeds in freshwater ecosystems revealed underestimation of their diversity. This result suggests that eDNA is an effective tool for monitoring plant diversity in aquatic habitats.
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http://dx.doi.org/10.1002/aps3.1155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5991581PMC
May 2018

A Sequel to Sanger: amplicon sequencing that scales.

BMC Genomics 2018 Mar 27;19(1):219. Epub 2018 Mar 27.

Centre for Biodiversity Genomics, University of Guelph, Guelph, ON, N1G 2W1, Canada.

Background: Although high-throughput sequencers (HTS) have largely displaced their Sanger counterparts, the short read lengths and high error rates of most platforms constrain their utility for amplicon sequencing. The present study tests the capacity of single molecule, real-time (SMRT) sequencing implemented on the SEQUEL platform to overcome these limitations, employing 658 bp amplicons of the mitochondrial cytochrome c oxidase I gene as a model system.

Results: By examining templates from more than 5000 species and 20,000 specimens, the performance of SMRT sequencing was tested with amplicons showing wide variation in GC composition and varied sequence attributes. SMRT and Sanger sequences were very similar, but SMRT sequencing provided more complete coverage, especially for amplicons with homopolymer tracts. Because it can characterize amplicon pools from 10,000 DNA extracts in a single run, the SEQUEL can reduce greatly reduce sequencing costs in comparison to first (Sanger) and second generation platforms (Illumina, Ion).

Conclusions: SMRT analysis generates high-fidelity sequences from amplicons with varying GC content and is resilient to homopolymer tracts. Analytical costs are low, substantially less than those for first or second generation sequencers. When implemented on the SEQUEL platform, SMRT analysis enables massive amplicon characterization because each instrument can recover sequences from more than 5 million DNA extracts a year.
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http://dx.doi.org/10.1186/s12864-018-4611-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5870082PMC
March 2018

Using herbarium-derived DNAs to assemble a large-scale DNA barcode library for the vascular plants of Canada.

Appl Plant Sci 2017 Dec 22;5(12). Epub 2017 Dec 22.

Centre for Biodiversity Genomics, University of Guelph, 50 Stone Road East, Guelph, Ontario N1G 2W1, Canada.

Premise Of The Study: Constructing complete, accurate plant DNA barcode reference libraries can be logistically challenging for large-scale floras. Here we demonstrate the promise and challenges of using herbarium collections for building a DNA barcode reference library for the vascular plant flora of Canada.

Methods: Our study examined 20,816 specimens representing 5076 of 5190 vascular plant species in Canada (98%). For 98% of the specimens, at least one of the DNA barcode regions was recovered from the plastid loci and and from the nuclear ITS2 region. We used beta regression to quantify the effects of age, type of preservation, and taxonomic affiliation (family) on DNA sequence recovery.

Results: Specimen age and method of preservation had significant effects on sequence recovery for all markers, but influenced some families more (e.g., Boraginaceae) than others (e.g., Asteraceae).

Discussion: Our DNA barcode library represents an unparalleled resource for metagenomic and ecological genetic research working on temperate and arctic biomes. An observed decline in sequence recovery with specimen age may be associated with poor primer matches, intragenomic variation (for ITS2), or inhibitory secondary compounds in some taxa.
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http://dx.doi.org/10.3732/apps.1700079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5749818PMC
December 2017

Probing planetary biodiversity with DNA barcodes: The Noctuoidea of North America.

PLoS One 2017 1;12(6):e0178548. Epub 2017 Jun 1.

Centre for Biodiversity Genomics, Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada.

This study reports the assembly of a DNA barcode reference library for species in the lepidopteran superfamily Noctuoidea from Canada and the USA. Based on the analysis of 69,378 specimens, the library provides coverage for 97.3% of the noctuoid fauna (3565 of 3664 species). In addition to verifying the strong performance of DNA barcodes in the discrimination of these species, the results indicate close congruence between the number of species analyzed (3565) and the number of sequence clusters (3816) recognized by the Barcode Index Number (BIN) system. Distributional patterns across 12 North American ecoregions are examined for the 3251 species that have GPS data while BIN analysis is used to quantify overlap between the noctuoid faunas of North America and other zoogeographic regions. This analysis reveals that 90% of North American noctuoids are endemic and that just 7.5% and 1.8% of BINs are shared with the Neotropics and with the Palearctic, respectively. One third (29) of the latter species are recent introductions and, as expected, they possess low intraspecific divergences.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0178548PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5453547PMC
September 2017

Testing the Efficacy of DNA Barcodes for Identifying the Vascular Plants of Canada.

PLoS One 2017 10;12(1):e0169515. Epub 2017 Jan 10.

Centre for Biodiversity Genomics, Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada.

Their relatively slow rates of molecular evolution, as well as frequent exposure to hybridization and introgression, often make it difficult to discriminate species of vascular plants with the standard barcode markers (rbcL, matK, ITS2). Previous studies have examined these constraints in narrow geographic or taxonomic contexts, but the present investigation expands analysis to consider the performance of these gene regions in discriminating the species in local floras at sites across Canada. To test identification success, we employed a DNA barcode reference library with sequence records for 96% of the 5108 vascular plant species known from Canada, but coverage varied from 94% for rbcL to 60% for ITS2 and 39% for matK. Using plant lists from 27 national parks and one scientific reserve, we tested the efficacy of DNA barcodes in identifying the plants in simulated species assemblages from six biogeographic regions of Canada using BLAST and mothur. Mean pairwise distance (MPD) and mean nearest taxon distance (MNTD) were strong predictors of barcode performance for different plant families and genera, and both metrics supported ITS2 as possessing the highest genetic diversity. All three genes performed strongly in assigning the taxa present in local floras to the correct genus with values ranging from 91% for rbcL to 97% for ITS2 and 98% for matK. However, matK delivered the highest species discrimination (~81%) followed by ITS2 (~72%) and rbcL (~44%). Despite the low number of plant taxa in the Canadian Arctic, DNA barcodes had the least success in discriminating species from this biogeographic region with resolution ranging from 36% with rbcL to 69% with matK. Species resolution was higher in the other settings, peaking in the Woodland region at 52% for rbcL and 87% for matK. Our results indicate that DNA barcoding is very effective in identifying Canadian plants to a genus, and that it performs well in discriminating species in regions where floristic diversity is highest.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0169515PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5224991PMC
August 2017

Counting animal species with DNA barcodes: Canadian insects.

Philos Trans R Soc Lond B Biol Sci 2016 09;371(1702)

Centre for Biodiversity Genomics, Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada N1G 2W1.

Recent estimates suggest that the global insect fauna includes fewer than six million species, but this projection is very uncertain because taxonomic work has been limited on some highly diverse groups. Validation of current estimates minimally requires the investigation of all lineages that are diverse enough to have a substantial impact on the final species count. This study represents a first step in this direction; it employs DNA barcoding to evaluate patterns of species richness in 27 orders of Canadian insects. The analysis of over one million specimens revealed species counts congruent with earlier results for most orders. However, Diptera and Hymenoptera were unexpectedly diverse, representing two-thirds of the 46 937 barcode index numbers (=species) detected. Correspondence checks between known species and barcoded taxa showed that sampling was incomplete, a result confirmed by extrapolations from the barcode results which suggest the occurrence of at least 94 000 species of insects in Canada, a near doubling from the prior estimate of 54 000 species. One dipteran family, the Cecidomyiidae, was extraordinarily diverse with an estimated 16 000 species, a 10-fold increase from its predicted diversity. If Canada possesses about 1% of the global fauna, as it does for known taxa, the results of this study suggest the presence of 10 million insect species with about 1.8 million of these taxa in the Cecidomyiidae. If so, the global species count for this fly family may exceed the combined total for all 142 beetle families. If extended to more geographical regions and to all hyperdiverse groups, DNA barcoding can rapidly resolve the current uncertainty surrounding a species count for the animal kingdom. A newly detailed understanding of species diversity may illuminate processes important in speciation, as suggested by the discovery that the most diverse insect lineages in Canada employ an unusual mode of reproduction, haplodiploidy.This article is part of the themed issue 'From DNA barcodes to biomes'.
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http://dx.doi.org/10.1098/rstb.2015.0333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4971185PMC
September 2016

Authentication of Herbal Supplements Using Next-Generation Sequencing.

PLoS One 2016 26;11(5):e0156426. Epub 2016 May 26.

Centre for Biodiversity Genomics, Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0156426PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4882080PMC
July 2017

DNA barcoding reveals twelve lineages with properties of phylogenetic and biological species within Melitaea didyma sensu lato (Lepidoptera, Nymphalidae).

Zookeys 2015 19(538):35-46. Epub 2015 Nov 19.

Department of Karyosystematics, Zoological Institute of the Russian Academy of Sciences, Universitetskaya emb. 1, 199034 St. Petersburg, Russia; Department of Entomology, Faculty of Biology, St. Petersburg State University, Universitetskaya emb. 7/9, 199034 St. Petersburg, Russia; McGuire Center for Lepidoptera and Biodiversity, Florida Museum of Natural History, McGuire Hall, 3215 Hull Road, PO Box 112710, University of Florida, Gainesville FL 32611-2710, USA.

The complex of butterfly taxa close to Melitaea didyma includes the traditionally recognized species Melitaea didyma, Melitaea didymoides and Melitaea sutschana, the taxa that were recognized as species only relatively recently (Melitaea latonigena, Melitaea interrupta, Melitaea chitralensis and Melitaea mixta) as well as numerous described subspecies and forms with unclear taxonomic status. Here analysis of mitochondrial DNA barcodes is used to demonstrate that this complex is monophyletic group consisting of at least 12 major haplogroups strongly differentiated with respect to the gene COI. Six of these haplogroups are shown to correspond to six of the above-mentioned species (Melitaea didymoides, Melitaea sutschana, Melitaea latonigena, Melitaea interrupta, Melitaea chitralensis and Melitaea mixta). It is hypothesized that each of the remaining six haplogroups also represents a distinct species (Melitaea mauretanica, Melitaea occidentalis, Melitaea didyma, Melitaea neera, Melitaea liliputana and Melitaea turkestanica), since merging these haplogroups would result in a polyphyletic assemblage and the genetic distances between them are comparable with those found between the other six previously recognized species.
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http://dx.doi.org/10.3897/zookeys.538.6605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4722858PMC
January 2016

Biodiversity inventories in high gear: DNA barcoding facilitates a rapid biotic survey of a temperate nature reserve.

Biodivers Data J 2015 30(3):e6313. Epub 2015 Aug 30.

Biodiversity Institute of Ontario, Guelph, Canada.

Background: Comprehensive biotic surveys, or 'all taxon biodiversity inventories' (ATBI), have traditionally been limited in scale or scope due to the complications surrounding specimen sorting and species identification. To circumvent these issues, several ATBI projects have successfully integrated DNA barcoding into their identification procedures and witnessed acceleration in their surveys and subsequent increase in project scope and scale. The Biodiversity Institute of Ontario partnered with the rare Charitable Research Reserve and delegates of the 6th International Barcode of Life Conference to complete its own rapid, barcode-assisted ATBI of an established land trust in Cambridge, Ontario, Canada.

New Information: The existing species inventory for the rare Charitable Research Reserve was rapidly expanded by integrating a DNA barcoding workflow with two surveying strategies - a comprehensive sampling scheme over four months, followed by a one-day bioblitz involving international taxonomic experts. The two surveys resulted in 25,287 and 3,502 specimens barcoded, respectively, as well as 127 human observations. This barcoded material, all vouchered at the Biodiversity Institute of Ontario collection, covers 14 phyla, 29 classes, 117 orders, and 531 families of animals, plants, fungi, and lichens. Overall, the ATBI documented 1,102 new species records for the nature reserve, expanding the existing long-term inventory by 49%. In addition, 2,793 distinct Barcode Index Numbers (BINs) were assigned to genus or higher level taxonomy, and represent additional species that will be added once their taxonomy is resolved. For the 3,502 specimens, the collection, sequence analysis, taxonomic assignment, data release and manuscript submission by 100+ co-authors all occurred in less than one week. This demonstrates the speed at which barcode-assisted inventories can be completed and the utility that barcoding provides in minimizing and guiding valuable taxonomic specialist time. The final product is more than a comprehensive biotic inventory - it is also a rich dataset of fine-scale occurrence and sequence data, all archived and cross-linked in the major biodiversity data repositories. This model of rapid generation and dissemination of essential biodiversity data could be followed to conduct regional assessments of biodiversity status and change, and potentially be employed for evaluating progress towards the Aichi Targets of the Strategic Plan for Biodiversity 2011-2020.
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http://dx.doi.org/10.3897/BDJ.3.e6313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4568406PMC
September 2015

Is DNA barcoding actually cheaper and faster than traditional morphological methods: results from a survey of freshwater bioassessment efforts in the United States?

PLoS One 2014 22;9(4):e95525. Epub 2014 Apr 22.

Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada.

Taxonomic identification accounts for a substantial portion of cost associated with bioassessment programs across the United States. New analytical approaches, such as DNA barcoding have been promoted as a way to reduce monitoring costs and improve efficiency, yet this assumption has not been thoroughly evaluated. We address this question by comparing costs for traditional morphology-based bioassessment, the standard Sanger sequencing-based DNA barcoding approach, and emerging next-generation (NGS) molecular methods. Market demand for molecular approaches is also assessed through a survey of the level of freshwater bioassessment effort in the United States across multiple habitat types (lakes, streams, wetlands) and indicators (benthic invertebrates, fish, algae). All state and regional level programs administered by public agencies and reported via agency web sites were included in the survey. Costs were based on surveys of labs and programs willing to provide such information. More than 19,500 sites are sampled annually across the United States, with the majority of effort occurring in streams. Benthic invertebrates are the most commonly used indicator, but algae and fish comprise between 35% and 21% of total sampling effort, respectively. We estimate that between $104 and $193 million is spent annually on routine freshwater bioassessment in the United States. Approximately 30% of the bioassessment costs are comprised of the cost to conduct traditional morphology-based taxonomy. Current barcoding costs using Sanger sequencing are between 1.7 and 3.4 times as expensive as traditional taxonomic approaches, excluding the cost of field sampling (which is common to both approaches). However, the cost of NGS methods are comparable (or slightly less expensive) than traditional methods depending on the indicator. The promise of barcoding as a cheaper alternative to current practices is not yet realized, although molecular methods may provide other benefits, such as a faster sample processing and increased taxonomic resolution.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0095525PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3995707PMC
January 2015

A transcontinental challenge--a test of DNA barcode performance for 1,541 species of Canadian Noctuoidea (Lepidoptera).

PLoS One 2014 25;9(3):e92797. Epub 2014 Mar 25.

Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada.

This study provides a first, comprehensive, diagnostic use of DNA barcodes for the Canadian fauna of noctuoids or "owlet" moths (Lepidoptera: Noctuoidea) based on vouchered records for 1,541 species (99.1% species coverage), and more than 30,000 sequences. When viewed from a Canada-wide perspective, DNA barcodes unambiguously discriminate 90% of the noctuoid species recognized through prior taxonomic study, and resolution reaches 95.6% when considered at a provincial scale. Barcode sharing is concentrated in certain lineages with 54% of the cases involving 1.8% of the genera. Deep intraspecific divergence exists in 7.7% of the species, but further studies are required to clarify whether these cases reflect an overlooked species complex or phylogeographic variation in a single species. Non-native species possess higher Nearest-Neighbour (NN) distances than native taxa, whereas generalist feeders have lower NN distances than those with more specialized feeding habits. We found high concordance between taxonomic names and sequence clusters delineated by the Barcode Index Number (BIN) system with 1,082 species (70%) assigned to a unique BIN. The cases of discordance involve both BIN mergers and BIN splits with 38 species falling into both categories, most likely reflecting bidirectional introgression. One fifth of the species are involved in a BIN merger reflecting the presence of 158 species sharing their barcode sequence with at least one other taxon, and 189 species with low, but diagnostic COI divergence. A very few cases (13) involved species whose members fell into both categories. Most of the remaining 140 species show a split into two or three BINs per species, while Virbia ferruginosa was divided into 16. The overall results confirm that DNA barcodes are effective for the identification of Canadian noctuoids. This study also affirms that BINs are a strong proxy for species, providing a pathway for a rapid, accurate estimation of animal diversity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0092797PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3965468PMC
December 2014

A DNA 'barcode blitz': rapid digitization and sequencing of a natural history collection.

PLoS One 2013 10;8(7):e68535. Epub 2013 Jul 10.

Biodiversity Institute of Ontario, University of Guelph, Guelph, ON, Canada.

DNA barcoding protocols require the linkage of each sequence record to a voucher specimen that has, whenever possible, been authoritatively identified. Natural history collections would seem an ideal resource for barcode library construction, but they have never seen large-scale analysis because of concerns linked to DNA degradation. The present study examines the strength of this barrier, carrying out a comprehensive analysis of moth and butterfly (Lepidoptera) species in the Australian National Insect Collection. Protocols were developed that enabled tissue samples, specimen data, and images to be assembled rapidly. Using these methods, a five-person team processed 41,650 specimens representing 12,699 species in 14 weeks. Subsequent molecular analysis took about six months, reflecting the need for multiple rounds of PCR as sequence recovery was impacted by age, body size, and collection protocols. Despite these variables and the fact that specimens averaged 30.4 years old, barcode records were obtained from 86% of the species. In fact, one or more barcode compliant sequences (>487 bp) were recovered from virtually all species represented by five or more individuals, even when the youngest was 50 years old. By assembling specimen images, distributional data, and DNA barcode sequences on a web-accessible informatics platform, this study has greatly advanced accessibility to information on thousands of species. Moreover, much of the specimen data became publically accessible within days of its acquisition, while most sequence results saw release within three months. As such, this study reveals the speed with which DNA barcode workflows can mobilize biodiversity data, often providing the first web-accessible information for a species. These results further suggest that existing collections can enable the rapid development of a comprehensive DNA barcode library for the most diverse compartment of terrestrial biodiversity - insects.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0068535PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3707885PMC
March 2014

"Darwin's butterflies"? DNA barcoding and the radiation of the endemic Caribbean butterfly genus Calisto (Lepidoptera, Nymphalidae, Satyrinae).

Comp Cytogenet 2011 24;5(3):191-210. Epub 2011 Aug 24.

McGuire Center for Lepidoptera and Biodiversity, Florida Museum of Natural History, University of Florida, Gainesville, FL 32611, USA.

The genus Calisto Hübner, 1823 is the only member of the diverse, global subfamily Satyrinae found in the West Indies, and by far the richest endemic Caribbean butterfly radiation. Calisto species occupy an extremely diverse array of habitats, suggestive of adaptive radiation on the scale of other classic examples such as the Galápagos or Darwin's finches. However, a reliable species classification is a key requisite before further evolutionary or ecological research. An analysis of 111 DNA 'barcodes' (655 bp of the mitochondrial gene COI) from 29 putative Calisto species represented by 31 putative taxa was therefore conducted to elucidate taxonomic relationships among these often highly cryptic and confusing taxa. The sympatric, morphologically and ecologically similar taxa Calisto confusa Lathy, 1899 and Calisto confusa debarriera Clench, 1943 proved to be extremely divergent, and we therefore recognize Calisto debarriera stat. n. as a distinct species, with Calisto neiba Schwartz & Gali, 1984 as a junior synonym syn. n. Species status of certain allopatric, morphologically similar sister species has been confirmed: Calisto hysius (Godart, 1824) (including its subspecies Calisto hysius aleucosticha Correa et Schwartz, 1986, stat. n.), and its former subspecies Calisto batesi Michener, 1943 showed a high degree of divergence (above 6%) and should be considered separate species. Calisto lyceius Bates, 1935/Calisto crypta Gali, 1985/Calisto franciscoi Gali, 1985 complex, also showed a high degree of divergence (above 6%), confirming the species status of these taxa. In contrast, our data suggest that the Calisto grannus Bates, 1939 species complex (including Calisto grannus dilemma González, 1987, Calisto grannus amazona González, 1987, stat. n., Calisto grannus micrommata Schwartz & Gali, 1984, stat. n., Calisto grannus dystacta González, 1987, stat. n., Calisto grannus phoinix González, 1987, stat. n., Calisto grannus sommeri Schwartz & Gali, 1984, stat. n., and Calisto grannus micheneri Clench, 1944, stat. n.) should be treated as a single polytypic species, as genetic divergence among sampled populations representing these taxa is low (and stable morphological apomorphies are absent). A widely-distributed pest of sugar cane, Calisto pulchella Lathy, 1899 showed higher diversification among isolated populations (3.5%) than expected, hence supporting former separation of this species into two taxa (pulchella and darlingtoni Clench, 1943), of which the latter might prove to be a separate species rather than subspecies. The taxonomic revisions presented here result in Calisto now containing 34 species and 17 subspecies. Three species endemic to islands other than Hispaniola appear to be derived lineages of various Hispaniolan clades, indicating ancient dispersal events from Hispaniola to Puerto Rico, Cuba, and Jamaica. Overall, the degree of intrageneric and intraspecific divergence within Calisto suggests a long and continuous diversification period of 4-8 Myr. The maximum divergence within the genus (ca. 13.3%) is almost equivalent to the maximum divergence of Calisto from the distant pronophiline relative Auca Hayward, 1953 from the southern Andes (14.1%) and from the presumed closest relative Eretris Thieme, 1905 (14.4%), suggesting that the genus began to diversify soon after its split from its continental sister taxon. In general, this 'barcode' divergence corresponds to the high degree of morphological and ecological variation found among major lineages within the genus.
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http://dx.doi.org/10.3897/CompCytogen.v5i3.1185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3833777PMC
November 2013

Complete DNA barcode reference library for a country's butterfly fauna reveals high performance for temperate Europe.

Proc Biol Sci 2011 Feb 11;278(1704):347-55. Epub 2010 Aug 11.

Institute of Evolutionary Biology (UPF-CSIC), Passeig Marítim de la Barceloneta 37-49, 08003 Barcelona, Spain.

DNA barcoding aims to accelerate species identification and discovery, but performance tests have shown marked differences in identification success. As a consequence, there remains a great need for comprehensive studies which objectively test the method in groups with a solid taxonomic framework. This study focuses on the 180 species of butterflies in Romania, accounting for about one third of the European butterfly fauna. This country includes five eco-regions, the highest of any in the European Union, and is a good representative for temperate areas. Morphology and DNA barcodes of more than 1300 specimens were carefully studied and compared. Our results indicate that 90 per cent of the species form barcode clusters allowing their reliable identification. The remaining cases involve nine closely related species pairs, some whose taxonomic status is controversial or that hybridize regularly. Interestingly, DNA barcoding was found to be the most effective identification tool, outperforming external morphology, and being slightly better than male genitalia. Romania is now the first country to have a comprehensive DNA barcode reference database for butterflies. Similar barcoding efforts based on comprehensive sampling of specific geographical regions can act as functional modules that will foster the early application of DNA barcoding while a global system is under development.
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http://dx.doi.org/10.1098/rspb.2010.1089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3013404PMC
February 2011

DNA barcoding Central Asian butterflies: increasing geographical dimension does not significantly reduce the success of species identification.

Mol Ecol Resour 2009 Sep 25;9(5):1302-10. Epub 2009 Feb 25.

Department of Karyosystematics, Zoological Institute of Russian Academy of Science, Universitetskaya nab. 1, 199034 St. Petersburg, Russia Department of Entomology, St. Petersburg State University, Universitetskaya nab. 7/9, 199034 St. Petersburg, Russia McGuire Center for Lepidoptera and Biodiversity, Florida Museum of Natural History, University of Florida, Gainesville, FL 32611, USA Biodiversity Institute of Ontario, University of Guelph, Guelph, ON, Canada N1G 2W1.

DNA barcoding employs short, standardized gene regions (5' segment of mitochondrial cytochrome oxidase subunit I for animals) as an internal tag to enable species identification. Prior studies have indicated that it performs this task well, because interspecific variation at cytochrome oxidase subunit I is typically much greater than intraspecific variation. However, most previous studies have focused on local faunas only, and critics have suggested two reasons why barcoding should be less effective in species identification when the geographical coverage is expanded. They suggested that many recently diverged taxa will be excluded from local analyses because they are allopatric. Second, intraspecific variation may be seriously underestimated by local studies, because geographical variation in the barcode region is not considered. In this paper, we analyse how adding a geographical dimension affects barcode resolution, examining 353 butterfly species from Central Asia. Despite predictions, we found that geographically separated and recently diverged allopatric species did not show, on average, less sequence differentiation than recently diverged sympatric taxa. Although expanded geographical coverage did substantially increase intraspecific variation reducing the barcoding gap between species, this did not decrease species identification using neighbour-joining clustering. The inclusion of additional populations increased the number of paraphyletic entities, but did not impede species-level identification, because paraphyletic species were separated from their monophyletic relatives by substantial sequence divergence. Thus, this study demonstrates that DNA barcoding remains an effective identification tool even when taxa are sampled from a large geographical area.
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http://dx.doi.org/10.1111/j.1755-0998.2009.02577.xDOI Listing
September 2009
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