DNA Barcoding Case Studies

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Scope

Barcode all species of Sphingidae - 93+ of an estimated total 114+ species - in the dry forest, rain forest and cloud forests of Area de Conservacion Guanacaste (ACG; 1,150 km2) in northwestern Costa Rica (30,000 rearings to date: http://janzen.sas.upenn.edu). All reared species have been barcoded, and all specimens databased. The study will COI barcode 2,500+ specimens. As many as 15% of the "species" will prove to be several sympatric or parapatric species (clumps of barcodes in the NJ tree, correlating with microgeography, food plants, and morphology), and but all have proven to be indistinguishable by their barcodes. ALL collateral data (including images) is available by the time the specimen reaches the sequencer. All species/specimens treated are sorted to morphology-based species before and during barcoding, and all barcoding results are correlated with the classical information (e.g., as in Hebert et al 2004, Hajibabaei et al 2006, Janzen et al 2005). All information is publically available through the project web site, BoLD, GenBank, publications, and deposition of the specimens as barcode vouchers in the NMNH/SI. All undescribed species will be described, with some descriptions and diagnoses relying more heavily on barcoding than others. Approximately $20,000 have been raised from the Wege Foundation for the study to date (June 2006), and the study depends heavily on in-kind contributions from the Ontario Institute of Biodiversity at the University of Guelph, NSF and ACG.

Purpose

This is a proof-of-concept for new species discovery and species discrimination with barcoding, and explores the management of barcode-relevant information, and stimulate barcode capture from older museum specimens (as well as those in this project). These particular species are primarily of importance because of the massive amount of databased collateral information (ecological and taxonomic) available for each barcoded specimen with which to verify/callibrate barcoding results, because of the ability of the ACG inventory project to obtain yet more specimens as required, and because of the availability of an ecologist (Janzen & Hallwachs) and taxonomists (Kitching, Cadiou, Haxaire) with drive and enthusiasm to carry the project to completion. The study is 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 beyond classical morphology in complex tropical systems. 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. Finally, these species range across many tens of degrees of latitude and countries, so the project will also provide a starting point for examining geographic within-species variation in barcodes (later, a separate project).

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 covering to date about a third of the estimated 9,600 species of ACG caterpillars. This multi-million dollar and multi-decade effort has spun off conservation, methodological, taxonomic and ecological initiatives, and it adopted barcoding immediately upon encountering it in 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 ACG inventory will largely generate the specimens, databasing, classical taxonomy at no additional cost, and simultaneously, integrate with the project to barcocde all the Lepidoptera of the ACG (see other case study). The Sphingidae (along with Saturniidae and Hesperiidae) are the first serious probe of a very species-rich group (just as Hebert et al 2004 was the first serious probe at a single species complex with barcoding per se - although using sequencing of various genes from a few individuals to puzzle out extra-tropical sibling species has occurred in entomology on and off for more than a decade). This is the first large barcoding cross-species project with Sphingidae, is a companion with barcoding the Saturniidae and Hesperiidae of the ACG (see other case studies), and is the first barcoding project in Costa Rica and Latin America.

Logistics

Exploratory barcoding of 5-20 specimens/species is done to the total available so as to represent the diverses ACG ecosystems and food plants. 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 and couriered to the Hebert lab at the Biodiversity Center at the University of Guelph. It is sequenced and the collateral information+sequence deposited in the Barcodes of Life Database (BoLD). I and Burns get the results by downloading a 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 is 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; the recently deceased Claude Lemaire lives on through his recent publications, and Paul Hebert and Mehrdad Hajibabaei, Department of Zoology, University of Guelph, Guelph, Ontario, N1G 2W1, Canada phebert@uoguelph.ca & mhajibab@uoguelph.ca

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