Damon P. Little

A DNA barcode for land plants

DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. However, there has been no agreement on which region(s) should be used for barcoding land plants. To provide a community recommendation on a standard plant barcode, we have compared the performance of 7 leading candidate plastid DNA regions (atpF–atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK–psbI spacer, and trnH–psbA spacer). Based on assessments of recoverability, sequence quality, and levels of species discrimination, we recommend the 2-locus combination of rbcL+matK as the plant barcode. This core 2-locus barcode will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of land plants.

Choosing and using a plant DNA barcode.

The main aim of DNA barcoding is to establish a shared community resource of DNA sequences that can be used for organismal identification and taxonomic clarification. This approach was successfully pioneered in animals using a portion of the cytochrome oxidase 1 (CO1) mitochondrial gene. In plants, establishing a standardized DNA barcoding system has been more challenging. In this paper, we review the process of selecting and refining a plant barcode; evaluate the factors which influence the discriminatory power of the approach; describe some early applications of plant barcoding and summarise major emerging projects; and outline tool development that will be necessary for plant DNA barcoding to advance.

A comparison of algorithms for the identification of specimens using DNA barcodes: examples from gymnosperms

In order to use DNA sequences for specimen identification (e.g., barcoding, fingerprinting) an algorithm to compare query sequences with a reference database is needed. Precision and accuracy of query sequence identification was estimated for hierarchical clustering (parsimony and neighbor joining), similarity methods (BLAST, BLAT and megaBLAST), combined clustering/similarity methods (BLAST/parsimony and BLAST/neighbor joining), diagnostic methods (DNA–BAR and DOME ID), and a new method (ATIM). We offer two novel alignment‐free algorithmic solutions (DOME ID and ATIM) to identify query sequences for the purposes of DNA barcoding. Publicly available gymnosperm nrITS 2 and plastid matK sequences were used as test data sets. On the test data sets, almost all of the methods were able to accurately identify sequences to genus; however, no method was able to accurately identify query sequences to species at a frequency that would be considered useful for routine specimen identification (42–71% unambiguously correct). Clustering methods performed the worst (perhaps due to alignment issues). Similarity methods, ATIM, DNA–BAR, and DOME ID all performed at approximately the same level. Given the relative precision of the algorithms (median = 67% unambiguous), the low accuracy of species‐level identification observed could be ascribed to the lack of correspondence between patterns of allelic similarity and species delimitations. Application of DNA barcoding to sequences of CITES listed cycads (Cycadopsida) provides an example of the potential application of DNA barcoding to enforcement of conservation laws.

DNA barcoding in the cycadales: testing the potential of proposed barcoding markers for species identification of cycads.

Barcodes are short segments of DNA that can be used to uniquely identify an unknown specimen to species, particularly when diagnostic morphological features are absent. These sequences could offer a new forensic tool in plant and animal conservation—especially for endangered species such as members of the Cycadales. Ideally, barcodes could be used to positively identify illegally obtained material even in cases where diagnostic features have been purposefully removed or to release confiscated organisms into the proper breeding population. In order to be useful, a DNA barcode sequence must not only easily PCR amplify with universal or near-universal reaction conditions and primers, but also contain enough variation to generate unique identifiers at either the species or population levels. Chloroplast regions suggested by the Plant Working Group of the Consortium for the Barcode of Life (CBoL), and two alternatives, the chloroplast psbA-trnH intergenic spacer and the nuclear ribosomal internal transcribed spacer (nrITS), were tested for their utility in generating unique identifiers for members of the Cycadales. Ease of amplification and sequence generation with universal primers and reaction conditions was determined for each of the seven proposed markers. While none of the proposed markers provided unique identifiers for all species tested, nrITS showed the most promise in terms of variability, although sequencing difficulties remain a drawback. We suggest a workflow for DNA barcoding, including database generation and management, which will ultimately be necessary if we are to succeed in establishing a universal DNA barcode for plants.

Commercial Teas Highlight Plant DNA Barcode Identification Successes and Obstacles

Appearance does not easily identify the dried plant fragments used to prepare teas to species. Here we test recovery of standard DNA barcodes for land plants from a large array of commercial tea products and analyze their performance in identifying tea constituents using existing databases. Most (90%) of 146 tea products yielded rbcL or matK barcodes using a standard protocol. Matching DNA identifications to listed ingredients was limited by incomplete databases for the two markers, shared or nearly identical barcodes among some species, and lack of standard common names for plant species. About 1/3 of herbal teas generated DNA identifications not found on labels. 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. Demonstrating the importance of accessible plant barcoding, our findings indicate unlisted ingredients are common in herbal teas.

A phylogenetic evaluation of Leandra (Miconieae, Melastomataceae): a polyphyletic genus where the seeds tell the story, not the petals

Melastomataceae is a tropical family of 4500–5000 species divided into nine tribes. The largest tribe, Miconieae, is composed of approximately 2200 species in 30 genera and is found exclusively in the Neotropics. Previous phylogenetic analyses of the Miconieae have suggested that many of the genera are derived from a paraphyletic Miconia. However, these analyses only included six species of the large genus Leandra, so its phylogenetic affinities remained unclear. As currently defined Leandra is characterized by acute petals and terminal inflorescences, but some species of Miconia, Clidemia and Ossaea also have these characters. In this study, we present an analysis of nrITS sequence data for a sample of 63 species of Leandra. The genus is clearly resolved as polyphyletic, but some distinct and well‐supported clades exist. Some of these partially correspond to sections recognized in the nineteenth century by Cogniaux, or to geographic distribution. The distribution of seed structure characters is better correlated with the phylogeny than traditional characters, such as petal morphology. Seed appendages in Leandra have evolved independently at least four times.

Phylogeographic relationships among Asian eggplants and new perspectives on eggplant domestication.

The domestication history of eggplant (Solanum melongena L.) has long been debated, with studies unable to narrow down where domestication occurred within a broad region of tropical Asia. The most commonly hypothesized region is India, however China has an equally old written record of eggplant use dating ca. 2000 years before present. Both regions have a high diversity of landraces and populations of putatively wild eggplant: Solanum incanum L. in India and Solanum undatum Lam. in SE Asia. An additional complication is that there is taxonomic confusion regarding the two candidate progenitors. Here, we synthesize historic, morphologic, and molecular data (nrITS sequence and AFLP) to interpret the phylogeographic relationships among candidate progenitors and Asian eggplant landraces in order to test theories of domestication. A minimum of two domestication events is supported: one in India and one in southern China/SE Asia. Results also support separate domestication of S. melongena subsp. ovigerum, a group of morphologically distinct eggplants found in SE Asia, and suggest Asian S. incanum and S. undatum may not be genetically distinct. Routes of the spread of eggplant cultivation throughout Asia are proposed, and evolutionary relationships among allied species are discussed.

DNA Barcode Sequence Identification Incorporating Taxonomic Hierarchy and within Taxon Variability

For DNA barcoding to succeed as a scientific endeavor an accurate and expeditious query sequence identification method is needed. Although a global multiple–sequence alignment can be generated for some barcoding markers (e.g. COI, rbcL), not all barcoding markers are as structurally conserved (e.g. matK). Thus, algorithms that depend on global multiple–sequence alignments are not universally applicable. Some sequence identification methods that use local pairwise alignments (e.g. BLAST) are unable to accurately differentiate between highly similar sequences and are not designed to cope with hierarchic phylogenetic relationships or within taxon variability. Here, I present a novel alignment–free sequence identification algorithm–BRONX–that accounts for observed within taxon variability and hierarchic relationships among taxa. BRONX identifies short variable segments and corresponding invariant flanking regions in reference sequences. These flanking regions are used to score variable regions in the query sequence without the production of a global multiple–sequence alignment. By incorporating observed within taxon variability into the scoring procedure, misidentifications arising from shared alleles/haplotypes are minimized. An explicit treatment of more inclusive terminals allows for separate identifications to be made for each taxonomic level and/or for user–defined terminals. BRONX performs better than all other methods when there is imperfect overlap between query and reference sequences (e.g. mini–barcode queries against a full–length barcode database). BRONX consistently produced better identifications at the genus–level for all query types.

A DNA mini-barcode for land plants.

Small portions of the barcode region – mini‐barcodes – may be used in place of full‐length barcodes to overcome DNA degradation for samples with poor DNA preservation. 591,491,286 rbcL mini‐barcode primer combinations were electronically evaluated for PCR universality, and two novel highly universal sets of priming sites were identified. Novel and published rbcL mini‐barcode primers were evaluated for PCR amplification [determined with a validated electronic simulation (n = 2765) and empirically (n = 188)], Sanger sequence quality [determined empirically (n = 188)], and taxonomic discrimination [determined empirically (n = 30 472)]. PCR amplification for all mini‐barcodes, as estimated by validated electronic simulation, was successful for 90.2–99.8% of species. Overall Sanger sequence quality for mini‐barcodes was very low – the best mini‐barcode tested produced sequences of adequate quality (B20 ≥ 0.5) for 74.5% of samples. The majority of mini‐barcodes provide correct identifications of families in excess of 70.1% of the time. Discriminatory power noticeably decreased at lower taxonomic levels. At the species level, the discriminatory power of the best mini‐barcode was less than 38.2%. For samples believed to contain DNA from only one species, an investigator should attempt to sequence, in decreasing order of utility and probability of success, mini‐barcodes F (rbcL1/rbcLB), D (F52/R193) and K (F517/R604). For samples believed to contain DNA from more than one species, an investigator should amplify and sequence mini‐barcode D (F52/R193).

DNA barcode authentication of saw palmetto herbal dietary supplements.

Herbal dietary supplements made from saw palmetto (Serenoa repens; Arecaceae) fruit are commonly consumed to ameliorate benign prostate hyperplasia. A novel DNA mini–barcode assay to accurately identify [specificity = 1.00 (95% confidence interval = 0.74–1.00); sensitivity = 1.00 (95% confidence interval = 0.66–1.00); n = 31] saw palmetto dietary supplements was designed from a DNA barcode reference library created for this purpose. The mini–barcodes were used to estimate the frequency of mislabeled saw palmetto herbal dietary supplements on the market in the United States of America. Of the 37 supplements examined, amplifiable DNA could be extracted from 34 (92%). Mini–barcode analysis of these supplements demonstrated that 29 (85%) contain saw palmetto and that 2 (6%) supplements contain related species that cannot be legally sold as herbal dietary supplements in the United States of America. The identity of 3 (9%) supplements could not be conclusively determined.