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What is the evidence that DNA barcoding is a reliable method for species identification?

For this commentary, “DNA barcoding” refers to nucleotide sequencing of PCR-amplified DNA corresponding to an approved barcode region, namely 5′ portion of COI for animals or rbcL + matK for land plants; and “species identification” refers to assigning the name of a known species to a specimen of unknown identity.

Acceptance by scientific community. For identification of known species, I think it is fair to say that DNA testing in general and DNA barcoding in particular are generally accepted in the scientific community as reliable methods. For example, the Canadian Centre for DNA Barcoding website has a compilation of peer-reviewed publications, which includes over 500 articles published since 2003.  The primary limitation to identification is whether the relevant species and close relatives have yet been documented in the databases at the time they are queried. The BOLD database is strongest for multicellular animals (> 1,000,000 records as of May 2010; see chart), particularly arthropods and chordates. For plants, the general principles are the same, but so far there is much less documentation, as plant barcodes were not agreed-upon until last year (Hollingsworth et al PNAS May 2009), and there was not a large set of pre-existing data to work with. Nonetheless, DNA barcoding of plants is ready for practical application and is providing immediately useful information (e.g. “DNA barcoding exposes a case of mistaken identity in the fern horticultural trade” Prior et al, Mol Ecol Resources April 2010) . For fungi, from perusing database it appears that ITS (internal transcribed spacer) and COI are informally accepted as barcodes. For protists and other domains of life, results so far suggest COI will serve as a primary barcode.

Most articles focus on DNA barcoding in a particular group and assess the accuracy of identification in that group. For example, in “DNA barcoding of commercially important salmon and trout species (Oncorhynchus and Salmo) from North America” (J Agricultural Food Chem 57:8379, 2009) Rasmussen and colleagues analyzed more than 1000 samples representing the 7 commercially important salmonid species from 143 sites  across western North America including Alaska and Canada, (to capture possible variation within species) The authors found 100% separation of these species by DNA barcoding, i.e., distances among species were always greater than within species.

Forensic application. DNA barcoding for species identification has been used in legal cases (e.g. Cohen et al J Food Protection 72: 810, 2009). More general evidence is presented by Dawnay et al in “Validation of the barcoding gene COI for use in forensic genetic species identification” (Forensic Sci International 173:1, 2007). The authors conclude “this study demonstrates that the cytochrome c oxidase I gene enables accurate animal species identification where adequate reference sequence data exists.” As with any laboratory method, quality control and quality assurance (QA/QC) measures are essential (e.g. Morin et al J Heredity 101:1, 2010).

DNA barcode identification was designed to be a simple, straightforward method appropriate for wide use, and the results so far amply bear this out, including its use by high school students (e.g., “FDA pressured to combat rising ‘food fraud’,” Lyndsey Layton, Washington Post March 30, 2010). One aspect that needs work in my opinion are better explanations of the algorithms used for matching sequences to the databases and what the results mean. It still takes an expert to make sense of the data. Although the results are often obvious (e.g., 100% sequence identity to 10 barcode records of “Bos taurus (cow)”, interpretation is context dependent–a 100% match has a different meaning if a “neighboring” species differs by, say 1%, or if a congeneric species is not documented or is represented by a single record, for example. In my experience, identifications are usually straightforward, including recognizing ambiguous identifications. Nonetheless, for DNA barcoding to have the widest use, including in legal settings, it will be helpful to have better documentation of how we arrive at species diagnoses through DNA barcodes.

Biting insects transmit human and animal diseases, including protozoan (e.g., malaria, leishmania, trypanosoma (sleeping sickness, Chagas disease)), filiarial (e.g., onchocerciasis, Guinea worm), and viral (e.g., yellow fever, West Nile, dengue) diseases. Control measures rely on identifying the insects, which generally requires expert training.

There are 174 mosquito species and subspecies in North America (“Identification and Geographical Distribution of the Mosquitos of North America, North of Mexico,” Richard F. Darsie, Jr. and Ronald A. Ward, University Press of Florida, 2005). Many species bite humans, but only a handful are important disease vectors. It takes an expert to identify Culex pipiens (panel A), which is the major vector for West Nile virus in eastern U.S., and to distinguish this from other species, for example, Culiseta incidens (panel B), which does not transmit human disease. Even experts are challenged by larvae (C), and eggs (D), and the latter are small and easily overlooked (egg raft size shown in inset). Planning and/or applying control measures is best done before adults hatch, but the early stages are what is most difficult.

The reference work cited above includes morphologic keys for identification of adult females and fourth-instar larvae. However, only an expert could make use of these (e.g. “lower mesepimeral setae absent, pale basal band on abdominal tergum II narrowed, or completely interrupted, medially). If mosquito identification is important for society, then reference DNA barcodes are what is needed, as these enable many more persons to name specimens, regardless of life stage. It does not make sense to rely on reference works for the world’s mosquitos that are incomprehensible to anyone who is not already a mosquito specialist.

Leishmaniasis is a chronic parasitic infection caused by various Leishmania species, kinetoplast protozoans related to Trypanosoma (the latter includes agents of African sleeping sickness and Chagas disease, suggested as a cause of Charles Darwin’s ill health in late life).  Depending on the species involved, leishmaniasis manifests as illness ranging from non-healing cutaneous or mouth ulcers (CL) to sometimes fatal visceral infection (VL). In the Neotropics, 12 species infecting humans have been identified, all associated with CL.  Neotropical leishmaniasis is mostly zoonotic  (ie originates from animal reservoirs as opposed to human-to-human transmission), and the vectors are tiny phlebotomine sand flies, particularly Lutzomyia sp.

In March 2010 PloS Neglected Trop Diseases investigators from Smithsonian Tropical Research Institute (STRI) and Instituto Conmemorativo Gorgas de Estúdios para la Salud, Panamá, apply DNA testing to Lutzomyia sandflies collected on Barro Colorado Island, STRI’s island home in the Panama Canal. Aiming to analyze as many species as possible, Azpurua and colleagues selected 435 individuals, which they morphologically identified as representing 16 Lutzomyia and 2 Brumptomyia sandfly species, for further analysis. Over 95% of specimens in the original collection were from one species, L. panamensis, so this was not a completely representative sample; nonetheless, “the relative abundances of species collected in this study were significantly correlated to those found in a previous intensive study of sand fly community composition on the [Panama] mainland…that collected over 30,000 Lutzomyia individuals in 35 species.”

To skip to the end, COI barcodes unambiguously assigned all 49 individuals to 18 distinct lineages corresponding to named species, plus highlighted 2 genetically-divergent individuals that might represent cryptic species.  Using  primers for ITS-1 (a nuclear gene) and mini-circle DNA (part of mitochondrial genome), Leishmania were detected in 2 of 5 human-biting species, Lu. trapidoi (13/30 individuals tested, 43.3%) and Lu. gomezi (5/19 individuals tested, 26.3%). By my estimate, taking into account relative abundances of Lutzomyia sp., about 1% of Barro Colorado Island sand flies carry Leishmania. Surprisingly, DNA sequencing identified the parasite as Le. naiffi, a South American species not previously reported in Panama. Finally, using the same set of DNA extracts, the researchers tested for Wolbachia, a rickettsial intracellular insect parasite and candidate biological control agent. Wolbachia were found in 3 of 18 species, including 50% of Lu. trapidoi, the main vector of CL in Panama. As an aside, I note that the presence of Wolbachia apparently did not interfere with discriminating among sand fly species; hypothesized interference from Wolbachia was one of the early worries some expressed about DNA barcoding (e.g Whitworth Proc Biol Sci 2007).

Standardized DNA testing enables many more persons to identify insects, regardless of life stage, including those that serve as vectors for human diseases. In this report by Azpurua and colleagues, the discovery of a new species of Leishmania for Panama, and possible undescribed Lutzomyia vectors, suggests that wide application of standardized DNA testing will lead to further discoveries relevant to control of human and animal infectious diseases.

It used to be standard practice to shave the area around the incision before surgery, as it was thought that hair harbored bacteria that would cause wound infection. Beginning in the 1970s, doctors found this was unnecessary, and in fact was associated with higher incidence of post-operative infection. This history comes to mind in reading March 2010 BioTechniques report by researchers from University of Guelph demonstrating that DNA suitable for PCR and sequencing can be obtained simply by leaving specimens in alcohol overnight!

Of the three steps required to get from a specimen to a DNA barcode, namely DNA isolation, PCR (polymerase chain reaction), and sequencing, the first step is the most labor intensive and hardest to automate. Numerous protocols/kits have been developed to optimize DNA isolation from various types of specimens, such as plant vs animal tissues. As described by the Guelph researchers, “these procedures force cells to release their DNA via physical pertubation and/or chemical treatment, which is then followed by a clean-up procedure in which unwanted cellular compoents are separated from the DNA.” The researchers “hypothesized that a small amount of DNA leaks from the tissue into the preservation solution (usually ethanol), and that this DNA was amplifiable using a standard PCR protocol.” To start, they analyzed Monte Alban mescal, which is sold with a “worm” (a caterpillar of the agave moth, Hypopta agavis) in each bottle. They evaporated 50 mL mescal, re-dissolved the residue in water, applied this to a Qiagen MinElute spin column, resuspended the product in 50 ?L water, and used 2 ?L of resulting solution in a standard 25 ?L PCR reaction, with successful amplification and sequencing of 130 base mini-barcode of COI. This case was presumably challenging as mescal is only 40% ethanol and contains a variety of material that might inhibit PCR. In subsequent tests, 1 mL of 95% ethanol used to preserve specimens was evaporated, resuspended in 30 ?L of water without column purification, and 2 ?L used for PCR.

By evaporating 1 mL of ethanol in which specimens had been stored overnight (out of 2 mL total ethanol volume) and re-suspending residue in water, Shokralla and colleagues amplified and sequenced 130-base and 650-base fragments of COI and 1100-base fragment of 28s RNA from 25 whole insect specimens (mayflies, caddisflies; 1 gave COI only) and rbcL from 45 plant specimens (0.5 mm leaf samples). They also obtained COI sequences by sampling 1 mL of ethanol solution from 7 insect specimens stored at room temperature for 7 to 10 years. The researchers note this approach could facilitate for “high-throughput” analyses, as it involves liquid handling which is easy to automate, avoids destructive sampling, and could be used even when “there is simply no sample left for further analysis.” They conclude with a caution about “field sampling procedures that include placing mixtures of specimens in an ethanol jar” as this “may increase the chance of cross-contamination.”

The remarkably simple procedure reported by Shokralla and colleagues offers benefits to many persons who want to get DNA barcode identifications. I look forward to applications of this method in research and commercial laboratories, classrooms, and perhaps kitchens!