DNA Barcoding Case Studies

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Scope

Barcode the 9,600 species of non-leaf-mining Lepidoptera in the dry forest, rain forest and cloud forests of the 1,150 km2 Area de Conservacion Guanacaste (ACG) (http://janzen.sas.upenn). By the end of 3 years (begun 1 April 2006), nearly all ACG species will have been wild-caught and/or reared and have series of pinned adults fully databased (about 400,000 specimens). About 90,000+ will be sequenced. About 20% of the species will be several sympatric or parapatric species (clumps of barcodes in the NJ tree), and another 3-5% of morphologically-defined species will be indistinguishable with COI barcodes. All data for the barcoded specimens will be databased, all specimens will be taxonomically sorted before and during barcoding, and all barcoding results will be correlated with classical information (e.g., as in Hebert et al 2004, Hajibabaei et al 2006, Janzen et al 2005). All will be available through the web site, INBio, BoLD, Genebank, publications and the barcode (and taxonomic) vouchers in NMNH/SI. All species will eventually be described or taxonomically revised, some relying more heavily on barcoding than others. $950,000 have been raised from the Wege Foundation, Michigan, NSF, sweat-equity from the taxasphere, and in-kind contributions from the Biodiversity Institute of Ontario. Funds are used for salaries, taxonomy, targeted collecting, taxonomic sorting, barcoding/sequencing, and voucher/database curating and storage. This is part of the NSF-funded effort to inventory all of the caterpillars of the ACG (http://janzen.sas.upenn.edu).

Purpose

A proof-of-concept for new species discovery and species discrimination with barcoding for all higher taxa of Lepidoptera, a stimulus to management of barcode-relevant information, and stimulation of barcode capture from older museum specimens (as well as those freshly caught in this project). The ACG Lepidoptera per se are primarily of importance because of the massive amount of databased collateral information (ecological and taxonomic) for each specimen with which to verify/callibrate barcoding results, because of the ability of the inventory project to obtain yet more specimens as required, and because of the availability of an ecologist (Janzen PI), taxonomists and parataxonomists, and institutions (ACG, INBio and SI) with drive and enthusiasm to carry the project to completion. The study will be an example for all of entomological taxonomy and ecology in complex tropical habitats. It will improve the world-level knowledge of Lepidoptera taxonomy by emphasizing the importance of going this next step beyond classical morphology in species discovery and discrimination, no matter what taxon or in what place. Because of the enthusiasm of PI Janzen for the subject, it will also be raw material, stimulus, and concept for the evolution of the hand-held, field-friendly barcorder. The project provides a starting point for examining geographic within-species variation in barcodes (later, separate project). These species have enormous geographic ranges and are multi-national.

Background

This is an outgrowth of the NSF/BS&I-supported caterpillar inventory of the ACG, in motion since 1978 (and funded through 2010). This effort has spun off conservation, methodological, taxonomic and ecological initiatives, and adopted barcoding upon encountering it (March 2003) to increase the quality of the inventory and contribute to a world-wide initiative of relevance to society in general, and the taxasphere specifically (both to museums as DNA banks and baselines for identification, and to taxonomists as to what/how they do). The ongoing ACG and INBio inventories have already collected and partly identified as much as half of what is needed, and this is the first serious probe of an entire very species-rich order in one complex and species-rich place. The classical inventory and its taxonomic processing to date have established a platform for this study, which has comparatively little add-on cost. This is the first barcoding of an entire order in one place, and will build on the preliminary projects in the ACG (Saturniidae, Sphingidae and Hesperiidae). It will require a major pro-bono involvement of classical taxonomy, and this guild is on board for the biota of the ACG as a whole. The experienced parataxonomists and curators at INBio and in the ACG have all the background and experience to carry this to completion. The project is heavily integrated with the on-going barcode inventory of the Great Smoky Mountains National Park, the Lepidoptera of North America, and the Lepidoptera of Australia (P. Hebert, PI) (ALL-Lep).

Logistics

Exploratory barcoding of 5-20 specimens/species is done to the total available so as to represent microgeography, ecosystems and morpho-variation. Intensive exploration (20-100 individuals) of suggestive variation then reveals (or refutes) multiple species complexes and provides the baseline for unambiguous use of barcodes for identification (or lack thereof) of all species. A leg is taken from a single pinned (oven-dried) and databased (and photographed) voucher specimen deposited in the NMNH/SI or INBio (Costa Rica) and couriered to the Biodiversity Institute of Ontario at the University of Guelph. It is sequenced and the collateral information+sequence deposited in the Barcodes of Life Database (BoLD). The results are a downloaded NJ tree from BoLD, and all of us discuss, and plot the next array of legs to be collected, and pursue which leads. Manuscripts result and sequence/collateral deposition in GenBank. The current stumbling block is getting good sequences from older specimens (Hajibabaei and Hebert are working on this) and the meaning of low-level variation (all of us are working on it). Daniel Janzen, Department of Biology, University of Pennsylvania, Philadelphia, PA 19104 at djanzen@sas.upenn.edu (tel 215-898-5636, fax 215-898-8780). CO-PIs INBio, USNM/SI and the taxasphere; Paul Hebert Mehrdad Hajibabaei and John Wilson, Department of Zoology, University of Guelph, Guelph, Ontario, N1G 2W1, Canada phebert@uoguelph.ca, mhajibab@uoguelph.ca, and jwilso04@uoguelph.ca

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