DNA BARCODING Re-integrating earthworm juveniles into soil biodiversity studies: species identification through DNA barcoding B. RICHARD,* T. DECAE ¨ NS,* R. ROUGERIE,† S. W. JAMES,‡ D. PORCO† and P. D. N. HEBERT† *Laboratoire d’Ecologie, EA 1293 ECODIV, FED SCALE, Ba ˆtiment IRESE A, UFR Sciences et Techniques, Universite ´ de Rouen, F-76821 Mont Saint Aignan cedex, France, †Canadian Centre for DNA Barcoding, Biodiversity Institute of Ontario, 50 Stone Road East, Guelph, ON, N1G 2W1 Canada, ‡Natural History Museum and Biodiversity Institute, Kansas University, 1345 Jayhawk Blvd., Lawrence, KS 66045, USA Abstract Species identification of earthworms is usually achieved by careful observation of morpho- logical features, often sexual characters only present in adult specimens. Consequently, juve- niles or cocoons are often impossible to identify, creating a possible bias in studies that aim to document species richness and abundance. DNA barcoding, the use of a short standardized DNA fragment for species identification, is a promising approach for species discrimination. When a reference library is available, DNA-based identification is possible for all life stages. In this study, we show that DNA barcoding is an unrivaled tool for high volume identifica- tion of juvenile earthworms. To illustrate this advance, we generated DNA barcodes for speci- mens of Lumbricus collected from three temperate grasslands in western France. The analysis of genetic distances between individuals shows that juvenile sequences unequivocally match DNA barcode clusters of previously identified adult specimens, demonstrating the potential of DNA barcoding to provide exhaustive specimen identification for soil ecological research. Keywords: DNA barcoding, earthworms, juveniles, species identifications Received 13 September 2009; revision received 15 November 2009; accepted 23 November 2009 Introduction Studies on the biology and ecology of earthworms often depend on species diagnoses. Species identifications in this group require minute examinations of external and ⁄ or internal morphology of adults, necessitating the involvement of expert taxonomists. In many cases, identi- fications are complicated by the lack of stable, easily scored diagnostic characters or by environmentally induced variability in morphological features. In addition, many characters important in species diagnosis involve the position and structure of the clitellum and the associ- ated tubercular pubertatis (Bouche ´ 1972; Sims & Gerard 1999), characters, which are only observable in sexually mature specimens. As a result of their lack of diagnostic characters, the identification of juveniles of closely related species (e.g. members of the genus Lumbricus) is impos- sible in most cases. As a result, taxonomists can only provide generic identifications for juveniles, hampering soil studies for species richness evaluation. Attempts to characterize earthworm species based on electrophoresis analysis – as developed for instance by Bogh (1992) – revealed higher resolution for species identification in samples containing many immature stages or incomplete body fragments, but the method remained largely ignored by soil biologists and has never been developed as a rou- tine, which could be used to process the high number of individuals that an ecological study often comprises. More recently, DNA barcoding has emerged as a promising standardized approach for rapid species iden- tifications in taxonomically complex groups. It uses a 658 bp fragment of the mitochondrial gene cytochrome-c oxi- dase I (COI) as a standard DNA tag for species discrimi- nation and identification in the animal kingdom (Hebert et al. 2003). The effectiveness of DNA barcodes in species identification has been shown in varied taxonomic groups (Hebert et al. 2004a,b; Ward et al. 2005; Smith et al. 2007; Borisenko et al. 2008) and they are increasingly being used in species descriptions as well (Decae ¨ns & Rougerie 2008; Martinez et al. 2008; Vaglia et al. 2008; Correspondence: Thibaud Decae ¨ns, Tel: 33 + (0)2 3276 9442; Fax: 33 + (0)2 3514 6655; E-mail: thibaud.decaens@univ-rouen.fr Ó 2009 Blackwell Publishing Ltd Molecular Ecology Resources (2010) 10, 606–614 doi: 10.1111/j.1755-0998.2009.02822.x