DNA metabarcoding of orchid-derived products reveals widespread illegal orchid trade

In eastern Mediterranean countries orchids continue to be collected from the wild for the production of salep, a beverage made of dried orchid tubers. In this study we used nrITS1 and nrITS2 DNA metabarcoding to identify orchid and other plant species present in 55 commercial salep products purchased in Iran, Turkey, Greece and Germany. Thirty samples yielded a total of 161 plant taxa, and 13 products (43%) contained orchid species and these belonged to 10 terrestrial species with tuberous roots. Another 70% contained the substitute ingredient Cyamopsis tetraganoloba (Guar). DNA metabarcoding using the barcoding markers nrITS1 and nrITS2 shows the potential of these markers and approach for identification of species used in salep products. The analysis of interspecific genetic distances between sequences of these markers for the most common salep orchid genera shows that species level identifications can be made with a high level of confidence. Understanding the species diversity and provenance of salep orchid tubers will enable the chain of commercialization of endangered species to be traced back to the harvesters and their natural habitats, and thus allow for targeted efforts to protect or sustainably use wild populations of these orchids.

[1]  M. Kasparek,et al.  European trade in Turkish Salep with special reference to Germany , 1999, Economic Botany.

[2]  Alain Viari,et al.  ecoPrimers: inference of new DNA barcode markers from whole genome sequence analysis , 2011, Nucleic acids research.

[3]  N. Yoccoz,et al.  Shedding new light on the diet of Norwegian lemmings: DNA metabarcoding of stomach content , 2013, Polar Biology.

[4]  H. D. de Boer,et al.  DNA Barcoding Reveals Limited Accuracy of Identifications Based on Folk Taxonomy , 2014, PloS one.

[5]  P. Taberlet,et al.  Glacial Survival of Boreal Trees in Northern Scandinavia , 2012, Science.

[6]  P. Taberlet,et al.  Towards next‐generation biodiversity assessment using DNA metabarcoding , 2012, Molecular ecology.

[7]  Thierry Vermat,et al.  Power and limitations of the chloroplast trnL (UAA) intron for plant DNA barcoding , 2006, Nucleic acids research.

[8]  Douglas B. Sponsler,et al.  Application of ITS2 metabarcoding to determine the provenance of pollen collected by honey bees in an agroecosystem , 2015, Applications in plant sciences.

[9]  Rodney,et al.  APPLICATION OF ITS 2 METABARCODING TO DETERMINE THE PROVENANCE OF POLLEN COLLECTED BY HONEY BEES IN AN AGROECOSYSTEM 1 , 2019 .

[10]  N. Baeshen,et al.  Biological Identifications Through DNA Barcodes , 2012 .

[11]  A. Güner,et al.  Chemical composition and physicochemical properties of tubera salep produced from some Orchidaceae species , 2010 .

[12]  Jullien M. Flynn,et al.  Toward accurate molecular identification of species in complex environmental samples: testing the performance of sequence filtering and clustering methods , 2015, Ecology and evolution.

[13]  M. Hossain Therapeutic orchids: traditional uses and recent advances--an overview. , 2011, Fitoterapia.

[14]  P. Taberlet,et al.  Fifty Thousand Years of Arctic Vegetation and Megafaunal Diet 1 Reconstruction of Arctic Vegetation from Permafrost Samples 121 , 2022 .

[15]  Jörg Römbke,et al.  Illumina metabarcoding of a soil fungal community , 2013 .

[16]  Adam Godzik,et al.  Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences , 2006, Bioinform..

[17]  Steven G Newmaster,et al.  DNA barcoding detects contamination and substitution in North American herbal products , 2013, BMC Medicine.

[18]  C. Bulpitt,et al.  The use of orchids in Chinese medicine , 2007, Journal of the Royal Society of Medicine.

[19]  Ting Gao,et al.  Validation of the ITS2 Region as a Novel DNA Barcode for Identifying Medicinal Plant Species , 2010, PloS one.

[20]  Eric Coissac,et al.  Bioinformatic challenges for DNA metabarcoding of plants and animals , 2012, Molecular ecology.

[21]  Robert A. Edwards,et al.  Quality control and preprocessing of metagenomic datasets , 2011, Bioinform..

[22]  P Green,et al.  Base-calling of automated sequencer traces using phred. II. Error probabilities. , 1998, Genome research.

[23]  C. Bulpitt The uses and misuses of orchids in medicine. , 2005, QJM : monthly journal of the Association of Physicians.

[24]  Canan Ece Tamer,et al.  A Traditional Turkish Beverage: Salep , 2006 .

[25]  Ingrid M. J. Scholtens,et al.  Advances in DNA metabarcoding for food and wildlife forensic species identification , 2016, Analytical and Bioanalytical Chemistry.

[26]  B. Gravendeel,et al.  Wild orchid tuber collection in Iran: a wake-up call for conservation , 2014, Biodiversity and Conservation.

[27]  D. Skinner,et al.  Phylogenetic analysis of Sorghum and related taxa using internal transcribed spacers of nuclear ribosomal DNA , 1994, Theoretical and Applied Genetics.

[28]  B. Gravendeel,et al.  Illegal wild collection and international trade of CITES-listed terrestrial orchid tubers in Iran , 2014 .

[29]  P. Hebert,et al.  DNA barcoding: how it complements taxonomy, molecular phylogenetics and population genetics. , 2007, Trends in genetics : TIG.

[30]  S. Oldfield,et al.  Alternatives for the bulb trade from Turkey: a case study of indigenous bulb propagation , 2002, Oryx.

[31]  Daniel Sabatier,et al.  Unveiling the Diet of Elusive Rainforest Herbivores in Next Generation Sequencing Era? The Tapir as a Case Study , 2013, PloS one.

[32]  E. Pahlich,et al.  A rapid DNA isolation procedure for small quantities of fresh leaf tissue , 1980 .

[33]  E. Sezik Destruction and Conservation of Turkish Orchids , 2002 .

[34]  E. Boersma,et al.  Prevention of Catheter-Related Bacteremia with a Daily Ethanol Lock in Patients with Tunnelled Catheters: A Randomized, Placebo-Controlled Trial , 2010, PloS one.

[35]  J. Staden,et al.  The ethnobotany of South African medicinal orchids , 2011 .

[36]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[37]  J. Arditti Fundamentals of orchid biology , 1992 .

[38]  Kang Ning,et al.  Biological ingredient analysis of traditional Chinese medicine preparation based on high-throughput sequencing: the story for Liuwei Dihuang Wan , 2014, Scientific Reports.

[39]  B. Gravendeel,et al.  DNA barcoding of tuberous Orchidoideae: a resource for identification of orchids used in Salep , 2017, Molecular ecology resources.

[40]  B. Gravendeel,et al.  Harvesting of salep orchids in north-western Greece continues to threaten natural populations , 2015, Oryx.

[41]  Rutger A. Vos,et al.  The HTS barcode checker pipeline, a tool for automated detection of illegally traded species from high-throughput sequencing data , 2014, BMC Bioinformatics.

[42]  Pierre Taberlet,et al.  Analysing diet of small herbivores: the efficiency of DNA barcoding coupled with high-throughput pyrosequencing for deciphering the composition of complex plant mixtures , 2009, Frontiers in Zoology.

[43]  K. Copenhagen New environmental metabarcodes for analysing soil DNA: potential for studying past and present ecosystems , 2012 .

[44]  Andrei Mocan,et al.  Veronica officinalis Product Authentication Using DNA Metabarcoding and HPLC-MS Reveals Widespread Adulteration with Veronica chamaedrys , 2017, Front. Pharmacol..

[45]  P. Taberlet,et al.  New perspectives in diet analysis based on DNA barcoding and parallel pyrosequencing: the trnL approach , 2009, Molecular ecology resources.

[46]  N. Fierer,et al.  Climatic warming and the future of bison as grazers , 2015, Scientific Reports.

[47]  Maria L. Kuzmina,et al.  Authentication of Herbal Supplements Using Next-Generation Sequencing , 2016, PloS one.

[48]  Ingolf Steffan-Dewenter,et al.  Increased efficiency in identifying mixed pollen samples by meta-barcoding with a dual-indexing approach , 2015, BMC Ecology.

[49]  P. Taberlet,et al.  Islands in the ice: detecting past vegetation on Greenlandic nunataks using historical records and sedimentary ancient DNA Meta‐barcoding , 2012, Molecular ecology.

[50]  James Haile,et al.  Deep Sequencing of Plant and Animal DNA Contained within Traditional Chinese Medicines Reveals Legality Issues and Health Safety Concerns , 2012, PLoS genetics.

[51]  P. Taberlet,et al.  Replication levels, false presences and the estimation of the presence/absence from eDNA metabarcoding data , 2015, Molecular ecology resources.

[52]  G. Hu,et al.  Ornithine Decarboxylase Antizyme Induces Hypomethylation of Genome DNA and Histone H3 Lysine 9 Dimethylation (H3K9me2) in Human Oral Cancer Cell Line , 2010, PloS one.

[53]  Bart Lievens,et al.  Comparison and Validation of Some ITS Primer Pairs Useful for Fungal Metabarcoding Studies , 2014, PloS one.

[54]  J. Wendel,et al.  Ribosomal ITS sequences and plant phylogenetic inference. , 2003, Molecular phylogenetics and evolution.

[55]  J. Wendel,et al.  Bidirectional interlocus concerted evolution following allopolyploid speciation in cotton (Gossypium). , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[56]  T. Stoeck,et al.  Depicting more accurate pictures of protistan community complexity using pyrosequencing of hypervariable SSU rRNA gene regions. , 2011, Environmental microbiology.

[57]  B. Gravendeel,et al.  Efforts urged to tackle thriving illegal orchid trade in Tanzania and Zambia for chikanda production , 2014 .

[58]  Vasco Elbrecht,et al.  Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol , 2015, PloS one.

[59]  S. Boessenkool,et al.  Use of Ancient Sedimentary DNA as a Novel Conservation Tool for High‐Altitude Tropical Biodiversity , 2014, Conservation biology : the journal of the Society for Conservation Biology.

[60]  H. D. de Boer,et al.  Molecular Identification of Commercialized Medicinal Plants in Southern Morocco , 2012, PloS one.

[61]  Anne Krag Brysting,et al.  Comparative authentication of Hypericum perforatum herbal products using DNA metabarcoding, TLC and HPLC-MS , 2017, Scientific Reports.

[62]  Günther Gauglitz,et al.  E-Health—a topic for analytical chemists? , 2015, Analytical and Bioanalytical Chemistry.

[63]  B. Gravendeel,et al.  Added value of metabarcoding combined with microscopy for evolutionary studies of mammals , 2016 .

[64]  M. Wilkinson,et al.  Quantitative evaluation of bias in PCR amplification and next-generation sequencing derived from metabarcoding samples , 2015, Analytical and Bioanalytical Chemistry.

[65]  V. I. Gusarov,et al.  New environmental metabarcodes for analysing soil DNA: potential for studying past and present ecosystems , 2012, Molecular ecology.

[66]  L. Baillie,et al.  Using DNA Metabarcoding to Identify the Floral Composition of Honey: A New Tool for Investigating Honey Bee Foraging Preferences , 2015, PloS one.

[67]  P. Taberlet,et al.  Power and limitations of the chloroplast trn L (UAA) intron for plant DNA barcoding. , 2007, Nucleic acids research.

[68]  B. Gravendeel,et al.  High-throughput sequencing of African chikanda cake highlights conservation challenges in orchids , 2017, Biodiversity and Conservation.

[69]  Dáithí C. Murray,et al.  Combined DNA, toxicological and heavy metal analyses provides an auditing toolkit to improve pharmacovigilance of traditional Chinese medicine (TCM) , 2015, Scientific Reports.

[70]  David L. Erickson,et al.  DNA metabarcoding illuminates dietary niche partitioning by African large herbivores , 2015, Proceedings of the National Academy of Sciences.

[71]  François Pompanon,et al.  An In silico approach for the evaluation of DNA barcodes , 2010, BMC Genomics.

[72]  T. Mitchell-Olds,et al.  Multiple hybrid formation in natural populations: concerted evolution of the internal transcribed spacer of nuclear ribosomal DNA (ITS) in North American Arabis divaricarpa (Brassicaceae). , 2003, Molecular biology and evolution.

[73]  P. Green,et al.  Base-calling of automated sequencer traces using phred. I. Accuracy assessment. , 1998, Genome research.

[74]  De‐Zhu Li,et al.  Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants , 2011, Proceedings of the National Academy of Sciences.

[75]  P. Kirk,et al.  ITS1 versus ITS2 as DNA metabarcodes for fungi , 2013, Molecular ecology resources.

[76]  Jeremy R. deWaard,et al.  Biological identifications through DNA barcodes , 2003, Proceedings of the Royal Society of London. Series B: Biological Sciences.