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What’s in your favorite tea? The dried and sometimes cooked or fermented bits of plants used to make teas are not easily identified to species by appearance. Over the past year I have been involved in a project testing whether DNA barcoding can identify the ingredients in commercial tea products, working with three New York City high school students and plant experts from Tufts University (Selena Ahmed) and The New York Botanical Garden (Damon Little). Student investigators Katie Gamble, Rohan Kirpekar, and Grace Young collected 146 tea products from 25 NYC locations, representing 33 manufacturers, 17 countries, and 82 plant common names–73 products were regular teas (prepared from Camellia sinensis, the tea plant) and 73 were herbal products prepared from other plant species.

Our findings are published in 21 July 2011 Scientific Reports, (Nature Publishing Group’s open access journal). About 1/3 of herbal teas generated DNA identifications indicating unlisted ingredients including weeds like annual bluegrass (Poa annua) and white goosefoot (Chenopodium album) and herbal plants like chamomile (Matricaria recutita). Matching DNA ingredients to listed ingredients was sometimes challenging–we observe that “broad-scale adoption of plant DNA barcoding may require algorithms that place search results in context of standard plant names and character-based keys for distinguishing closely-related species.”

We are pleased that our investigation has attracted press coverage including New York Times print and online editions and internationally in 65 news sites and 14 countries, including India and China, world centers of tea production. Most of the DNA work was done at The New York Botanical Garden in senior author Damon Little’s laboratory. For a small subset of samples (10) we did DNA isolation and amplification in my dining room with recycled lab equipment purchased on the internet for about $5000. Samples were mailed to a commercial facility (Macrogen) for DNA sequencing, with results available by email the next day. It cost about $15 a sample including sequencing (unidirectional). More info and pictures on our TeaBOL website!

What’s next? I am excited about enabling wider use of DNA barcoding by high school students, including Cold Spring Harbor’s Urban Barcode Project competition (I am an advisor), open to teams from all New York City schools, with a focus on public institutions. I expect that soon manufacturers of teas and herbal products (and regulators) will incorporate DNA barcode testing into their quality control practices. One of the important tasks for scientists is building up the reference databases. At the time of the study, BOLD (Barcode of Life Database) and GenBank lacked rbcL or matK records for about 1/3 of plant species listed on product labels in our study. More on herbal plant identification: (Lou et al 2010. An integrated web medicinal materials DNA database. BMC Genomics 11, 402; Smillie and Kahn 2010. A comprehensive approach to identifying and authenticating botanical products. Clin Pharm Therapeutics 87, 175).

 

Veneridae, commonly known as venus clams, are the largest family of heterodont bivalves (clams and cockles), with about 500 named species, all marine, distributed in mostly shallow water areas around the globe.  In June 2011 Plos ONE, researchers from Fisheries College, Ocean University of China apply DNA barcodes to perform what they call “taxonomy distentanglement” on 315 venerid specimens representing about 60 species collected along the coast of mainland China. This qualifies as the largest analysis of DNA barcodes for marine bivalves to date. Chen and colleagues note “species boundaries of these clams are difficult or even impossible to define accurately based solely on morphologic features,” so there is a potentially a big role for DNA characters.

The clams were collected over a 6 year period from 2004-2010, stored in 95% ethanol (marine specimens are traditionally stored in formalin, which is an effective preservative but makes it difficult to recover DNA), and deposited as voucher specimens in Fisheries College. DNA was extracted from adductor muscle (some bivalves inherit mitochondrial DNA from both male and female parents, but the male type is restricted to gonadal tissue). Given that not many bivalves have been barcoded, it is of interest to learn what primer pairs were effective (BOLD taxonomy browser lists barcodes for 966 of the approximately 10,000 bivalve species).  Starting with Folmer primers, two additional published sets and 4 sets developed for this study were used if needed, with recovery of COI from all specimens.

I note that genetic differences within Family Veneridae are remarkably large–average pairwise COI K2P distance within the family (not counting conspecific and congeneric comparisons) is around 35% and maximum is over 50%. For comparison, in birds, average and maximum distances within families are about half as large, and even within birds as a whole (Class Aves, i.e., two hierarchical levels above family), average and maximum distances are only 20% and 33%, respectively (I generated bird stats by merging public projects in BOLD and running “Distance Summary.”) I wonder if what we call Families in vertebrates and invertebrates reflect different levels on the evolutionary tree.

Back to the paper. Chen and colleagues used neighbor-joining, maximum-likelihood, and MOTU analysis to examine their data with and without 310 additional venerid sequences downloaded from BOLD/GenBank. All individuals that could be morphologically identified to species possessed distinct (reciprocally monophyletic) COI sequences, with the exception of one species pair. 11/23 sequences from specimens that could not be identified morphologically formed five monophyletic clusters, likely representing species new to science or unreported in China. The remaining 12 sequences from morphologically-puzzling specimens clustered within named species, suggesting these represent morphologically variant specimens. Sorting puzzling specimens into genetic clusters led the authors to recognize previously overlooked diagnostic morphologic characters.  A number of existing records in BOLD/GenBank prior to this study clustered with different species, suggesting these specimens were misidentified by submitters or reflected outdated taxonomy.

Chen and colleagues conclude that DNA barcoding has a third purpose in addition to species identification (assigning unknown specimens to known species) and species discovery (flagging divergent clusters), namely what they call “taxonomy disentanglement,” which other authors have called iterative or integrative use of barcoding (for example Smith et al,  Extreme diversity of tropical parasitoid wasps exposed by iterative integration of natural history, DNA barcoding, morphology, and collections, 2008 PNAS). I like the term “disentanglement”–it brings to mind the many confusions in existing classifications and specimen labels, many of which can be unknotted with DNA barcodes.

You have 1 more day! Abstract deadline is 12 midnight tomorrow, June 15,  for the Fourth International Barcode of Life Conference, Adelaide, Australia, 28 November-3 December 2011. Online submission form here.

Young scientists to help document what lives on Earth! Coastal Marine BioLabs (CMB), a private, research-based scientific educational organization in Ventura, California was awarded a 3-year NSF grant to train high school teachers and students in DNA barcoding, with the goal of contributing reference sequences to Barcode of Life Database. CMB students and their teachers will be part of the International Barcode of Life project, which aims to expand BOLD (currently about 1.2M barcodes from 130K species) to 5M records from 500K species, the largest biodiversity intiative ever. For more on how students are helping build the genetic database of global species diversity, see Sacramento Bee news story and CMB web page.

 

 

Barcode of Life Connect tops 1000 members! If you haven’t already, I encourage you to visit and join the Barcode of Life Connect site, a “network to allow DNA barcoding professionals to discuss issues, share profiles, form special interest groups, and more.”The more includes webinars and links to upcoming relevant conferences. The core of the site is the chance to connect with like-minded barcoding professionals, either directly through their profiles or through discussion groups–so far there are 40 groups ranging from “Medicinal Plants” to “Madagascar” and “Portugese-Speaking Barcoders.”

 

To get an idea of how barcoding has taken hold around the world, particularly with young scientists, try perusing recent pictures posted by Connect members–I take the liberty of re-posting some images of the investigators and their specimens-enjoy!