Correction: Authentication of Herbal Supplements Using Next-Generation Sequencing.

[This corrects the article DOI: 10.1371/journal.pone.0156426.]

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

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

<div><p>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 (<i>rbcL</i>, <i>matK</i>, 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 <i>rbcL</i> to 60% for ITS2 and 39% for <i>matK</i>. 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 <i>rbcL</i> to 97% for ITS2 and 98% for <i>matK</i>. However, <i>matK</i> delivered the highest species discrimination (~81%) followed by ITS2 (~72%) and <i>rbcL</i> (~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 <i>rbcL</i> to 69% with <i>matK</i>. Species resolution was higher in the other settings, peaking in the Woodland region at 52% for <i>rbcL</i> and 87% for <i>matK</i>. 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.</p></div

Primers used for Sanger sequencing, NGS and in IAC.

<p>Primers used for Sanger sequencing, NGS and in IAC.</p

Level of taxonomic resolution provided by <i>rbcL</i>, <i>matK</i> or ITS2 for 25 families.

<p>Level of taxonomic resolution provided by <i>rbcL</i>, <i>matK</i> or ITS2 for 25 families of vascular plant that are species-rich in Canada. The three colours show the proportion of species identified to a family (blue), genus (orange) or species (green) level.</p

Percentage of taxonomic resolution by BLAST to family, genus and species.

<p>Taxonomic resolution for <i>rbcL</i>, <i>matK</i>, and ITS2 for 25 species-rich families.</p

La composición dietaria de los Guacamayos Rojos reintroducidos refleja una adaptación gradual a la vida silvestre

Over the last two centuries, the Red-and-Green Macaw (Ara chloropterus) has become locally extinct in Argentina. In an attempt to restore its key ecosystem functions as both disperser and regulator of large-seeded plants, a reintroduction project was initiated at the Iberá National Park in northeastern Argentina. The ability of released individuals to find food is crucial, especially when working with captive-bred animals, as long-term establishment of a self-sustaining population depends on their short-term ability to exploit wild food sources. Monitoring of feeding habits is usually conducted through behavioral observation, but in recent years DNA metabarcoding has emerged as an alternative for obtaining highly resolved data on diet composition. In this study, we use a combination of both techniques to characterize the breadth and composition of the reintroduced macaws' diet. In addition, we compare the efficiency of both observational and molecular techniques to assess diet composition in a frugivorous bird. Individuals fed on a variety of plant species (n = 49) belonging to a broad phylogenetic spectrum (28 families). Dietary richness estimated by direct observation and DNA metabarcoding was similar, though smaller than the combination of the two datasets as both techniques detected at least 15 species not recorded by the other method. While the total number of detected species was higher for observational data, the rate of species-detection per sampling day was higher for DNA metabarcoding. These results suggest that a combination of both methods is required in order to obtain the most accurate account of the total diversity of the diet of a bird species. The ability of reintroduced macaws to successfully exploit local food resources throughout the year indicates a good level of adjustment to the release site, an important step towards the creation of a stable, self-sustaining population of Red-and-Green Macaws in Northern Argentina.En el transcurso de los últimos dos siglos, el Guacamayo Rojo (Ara chloropterus) se ha extinguido en la Argentina. Buscando recuperar su rol ecológico tanto de dispersor como de depredador de semillas de gran tamaño, se comenzó un proyecto de reintroducción de la especie en el Parque Nacional Iberá, en la región noreste del país. La capacidad para encontrar alimento por parte de los individuos liberados es crucial, particularmente cuando se trabaja con animales provenientes de condiciones de cautiverio, ya que el establecimiento de una población autosuficiente a largo plazo dependerá de la habilidad de éstos para explotar fuentes de alimento silvestre a corto plazo. El monitoreo de hábitos alimenticios se realiza usualmente a través de observaciones comportamentales. Sin embargo, en los últimos años la técnica del meta-código de barras de ADN ha surgido como una alternativa para la obtención de datos de composición dietaria con alto nivel de resolución. En este estudio, utilizamos una combinación de ambas técnicas para caracterizar la amplitud y composición de la dieta de los guacamayos reintroducidos. A su vez, evaluamos de forma comparativa la eficiencia de la observación directa y el código de barras genético de heces como técnicas para determinar la composición de la dieta en un ave frugívora. Los individuos se alimentaron de una amplia variedad de especies (n = 49), abarcando un amplio espectro filogenético (28 familias). La riqueza dietaria estimada por observación directa y por meta-código de barras genético fue similar, aunque menor a la resultante de la combinación de todos los datos ya que ambas técnicas detectaron al menos 15 especies no registradas por el otro método. Mientras que el número total de especies detectadas fue mayor para los métodos observacionales, la tasa de detección de especies por día de muestreo fue mayor para el análisis genético. Estos resultados sugieren que una combinación de ambos métodos es necesaria para obtener la descripción más precisa posible de la diversidad dietaria total de una especie. La capacidad de los guacamayos reintroducidos para explotar recursos alimenticios locales a lo largo del año estaría indicando un buen nivel de adaptación al sitio de liberación, un paso muy importante hacia el establecimiento de una población de Guacamayo Rojo estable y autosuficiente en el norte de Argentina.Fil: Volpe, Noelia Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Centro de Ecología Aplicada del Litoral. Universidad Nacional del Nordeste. Centro de Ecología Aplicada del Litoral; ArgentinaFil: Thalinger, Bettina. University of Guelph; CanadáFil: Vilacoba, Elisabet. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia"; ArgentinaFil: Braukmann, Thomas W. A. University of Guelph; Canadá. University of Stanford; Estados UnidosFil: Di Giacomo, Adrian Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Centro de Ecología Aplicada del Litoral. Universidad Nacional del Nordeste. Centro de Ecología Aplicada del Litoral; ArgentinaFil: Berkunsky, Igor. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil; Argentina. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Exactas. Instituto Multidisciplinario de Ecosistemas y Desarrollo Sustentable; ArgentinaFil: Lijtmaer, Dario Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia"; ArgentinaFil: Steinke, Dirk. University of Guelph; CanadáFil: Kopuchian, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Centro de Ecología Aplicada del Litoral. Universidad Nacional del Nordeste. Centro de Ecología Aplicada del Litoral; Argentin

Boxplots of MPD and MNTD for <i>rbcL</i>, <i>matK</i>, and ITS2.

<p>Boxplots comparing MPD and MNTD for the vascular plant families of Canada for <i>rbcL</i>, <i>matK</i>, and ITS2. Significance (p–adjusted < 0.005) is indicated with an asterisk(s).</p