DNA Barcoding Case Studies

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Scope

Barcode all 2,000+ species of parasitoids (Tachinidae, Ichneumonidae, Braconidae, etc.) of non-leaf-mining Lepidoptera in the dry forest, rain forest and cloud forests of the 1,150 km2 Area de Conservacion Guanacaste (ACG), as reared by the inventory of these caterpillars (begin: 1978, continuing indefinitely). The current 6,000-specimen backlog (about 1000 species) is being barcoded, and 2,000/yr will be barcoded until "done" (20,000+ specimens). 30% of the morphological species will be several sympatric or parapatric species, and another 3-5% will be indistinguishable with COI barcodes (see Smith et al 2006). Vouchers are databased when the specimens reach DNA sequencing, all are sorted to species before and during barcoding, and barcoding results are correlated with classical information (e.g., as in Hebert et al 2004, Hajibabaei et al 2006, Janzen et al 2005 and Smith et al 2006). Data is available through BoLD, GenBank, the project web site, and publications, and all vouchers deposited in the NMNH/SI. Undescribed species will be described and taxonomically revised, some relying heavily on barcoding. Funding is $50,000 from the Wege Foundation, Michigan, NSF, enormous sweat-equity from the taxasphere, and in-kind contributions from the Biodiversity Institute of Ontario at the University of Guelph. Funds are for salaries, taxonomy of specimens, targeted collecting, taxonomic sorting, barcoding/sequencing, and voucher/database curating and storage; it builds on the NSF-funded inventory all of the caterpillars of the ACG (http://janzen.sas.upenn.edu).

Purpose

Proof-of-concept for new species discovery and species discrimination with barcoding for parasitoid Diptera and Hymenoptera, and as a stimulus to evolve the management of barcode-relevant information and stimulate barcode capture from older museum specimens (as well as those freshly caught in this project). These taxa are 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 a model example for all of entomological taxonomy and ecology in complex tropical habitats. It will improve the world-level knowledge of Diptera and Hymenoptera taxonomy by emphasizing the importance of going this next step in species discovery and discrimination beyond classical morphology, 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 will provide the base point for examining geographic within-species variation in barcodes (later, separate project). These species have enormous geographic ranges, so the study is multi-national.

Background

The study is an outgrowth of the thorough NSF/BS&I-supported caterpillar inventory of the Area de Conservacion Guanacaste (ACG), in motion since 1978 (and funded through 2010), and the INBio national biodiversity inventory collections. This multi-million dollar and multi-decade effort has spun off conservation, methodological, taxonomic and ecological initiatives, and adopted barcoding upon encountering it (March 2003) as a way of both increasing the quality of the inventory and contributing to a world-wide initiative with huge relevance to society in general, and the taxasphere specifically (both to museums as enormous DNA banks and baselines for indentification, and to taxonomists as to what/how they do). The ongoing ACG parasitoid inventory has already collected and partly identified more than 1000 species of caterpillar parasitoids, and this is the first serious probe of these two very species-rich Orders in one complex and species-rich place. The classical inventory and its taxonomic processing to date have established a platform for this study, for comparatively little add-on cost. 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.

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) or alcohol-preserved, and databased (and photographed) voucher specimen deposited in the CNC, TNHM, 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, pursuing which leads. Manuscripts result and sequence/collateral deposition in GenBank. The current stumbling block is getting good sequences from older specimens (Hajibabaei, Smith, 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; Alex Smith and Paul Hebert, Department of Zoology, University of Guelph, Guelph, Ontario, N1G 2W1, Canada salex@uoguelph.ca and phebert@uoguelph.ca

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