DNA barcoding: a new diagnostic tool for rapid species recognition, identification, and discovery James Hanken Museum of Comparative Zoology Harvard University, USA

What is DNA barcoding? Definition: Derivation of a short DNA sequence(s) that enables species identification or recognition in a particular domain of life (eucaryotes). Focus to date—in animals—has been on a 658 base-pair (bp) fragment of the mitochondrial gene, cytochrome oxidase subunit I (COI). The Barcode of Life Initiative (BOLI) would resolve barcodes for named species and use a barcoding approach to assess undescribed biological diversity. Very controversial!

What isn’t DNA barcoding? It is not intended to, in any way, supplant or invalidate existing taxonomic practice. It is not DNA-taxonomy; it does not equate species identity, formally or informally, with a particular DNA sequence. It is not intended to duplicate or compete with efforts to resolve deep phylogeny, e.g., Assembling the Tree of Life (ATOL).

“the role of any molecular diagnostic is to aid research, not to serve as an end in itself. Barcoding … is independent of questions as to whether individual taxa are species, what species are (or should be), and where they fit in a unified tree of life…. Barcoding is not an end in itself, but will boost the rate of discovery. The unique contribution of DNA barcoding to … taxonomy and systematics is a compressed timeline for the exploration and analysis of biodiversity.”

Potential applications 1) Facilitating identification and recognition of named (described) species: linking life history stages, genders; differentiating cryptic species; identifying gut contents; human disease vectors; agricultural pests; biosecurity (?). 2) Surveying and inventorying biodiversity; e.g., flagging potentially new (undescribed) species.

Differentiating cryptic species Astraptes fulgerator, skipper butterfly. Wide-ranging; southern U.S. to northern Argentina. In northwestern Costa Rica, comprises complex of 10 sympatric species that are distinct in DNA sequence (COI), larval coloration, food plants, and subtle morphological traits. D. Janzen, et al., submitted

Sympatric larvae of Astraptes fulgerator Food plant: Celtis iguanaea Food plant: Trigonia (2 species); larvae will starve if reared on plants used by other larval types.

Strengths Offers alternative taxonomic identification tool for situations in which morphology is inconclusive. Focus on one or a small number of genes provides greater efficiency of effort. Cost of DNA sequencing is dropping rapidly due to technical advances. Potential capacity for high throughput and processing large numbers of samples. Once reference database is established, can be applied by non-specialist.

Weaknesses Assumes intraspecific variation is negligible, or at least lower than interspecific values. No single gene will work for all taxa (e.g., COI is not appropriate for vascular plants, or even for some animals). Single-gene approach is less precise than using multiple genes; may introduce unacceptable error. Some of the most attractive aspects rely on future technology, e.g., handheld sequencer.

% Proportion of species pairs % n = 13,320 n = 17 Annelida, Arthropoda, Chordata, Mollusca, Echinodermata, Nematoda, Platyhelminthes Cnidaria (corals, anemones, jellyfish, sea pens, etc.) Between-species sequence divergence in COI (%)

COI sequence divergence among North American birds K2P (%)

Sequence data Barcoding must adhere to standards for specimen and data management Voucher specimens and electronic databases Digital images

Pilot projects 2) Possible targets: “All taxa”—primates, turtles, mosquitoes, tephritid fruitflies, birds, sphinx moths, salamanders, etc. Regional faunas, e.g., Gulf of Maine megafauna. Existing inventories, e.g., INBio and ACG (Costa Rica). 1) Goals and objectives: Validate barcoding approach, in general, and the use of COI, in particular (for animals); i.e., proof of principle. Assess feasibility of large-scale effort, e.g., identify bottlenecks, cost, logistic issues. 3) Would rely principally on museum specimens.

Barcoding must validate existing taxonomy before it can be offered as an identification tool, and especially before it can be used to discover new species. Pilot projects will utilize museum specimens. New inventory efforts will yield large numbers of vouchers, which must be properly accessioned, databased, and stored. Results will flag many new species requiring formal description. Additional burden on museums, herbaria, etc., but may also offer new sources of support. Role of museums (and impact) 

DNA barcoding is under way… See also Nature 426: 514 (4 Dec 2003)

and appeals to the biotech sector

Anticipated next steps (2004) January: Working group submits a grant proposal to found a coordinating Secretariat to develop a Barcode of Life Initiative (BOLI) based in Washington, DC; decision expected late March. May: Founding meeting of the BOLI Consortium (museums and herbaria) at U.S. Smithsonian Institution (NMNH). Late fall: First International Barcode of Life Conference; venue TBD.