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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Review DNA barcoding for ecologists Alice Valentini1,2, Franc¸ois Pompanon1 and Pierre Taberlet1 1 Laboratoire d’Ecologie Alpine, CNRS UMR 5553, Universite´ Joseph Fourier, BP 53, 38041 Grenoble Cedex 9, France 2 Dipartimento di Ecologia e Sviluppo Economico Sostenibile, Universita` della Tuscia, Via S. Giovanni Decollato 1, 01100 Viterbo, Italy DNA barcoding – taxon identification using a standar- Here our purpose is to review the main applications of dized DNA region – has received much attention DNA barcoding in ecology, with an emphasis on the new recently, and is being further developed through an opportunities linked to the recent availability of next- international initiative. We anticipate that DNA barcod- generation DNA sequencers. First, we will present the ing techniques will be increasingly used by ecologists. new tools that make the barcoding approach for ecologists They will be able to not only identify a single species easier. We will then focus on single-species identification, from a specimen or an organism’s remains but also which is the historical fundament of DNA barcoding. determine the species composition of environmental Finally, we will discuss the unprecedented potential of samples. Short DNA fragments persist in the environ- DNA barcoding for simultaneous multiple-species identi- ment and might allow an assessment of local biodiver- fication from a single environmental sample, for biodiver- sity from soil or water. Even DNA-based diet sity assessment and for diet analysis from feces. composition can be estimated using fecal samples. Here we review the new avenues offered to ecologists by DNA New tools for new prospects barcoding, particularly in the context of new sequencing In the past 20 years the technology of DNA sequencing has technologies. greatly improved, from manual sequencing to automated sequencers. A single automated 96-capillary sequencer can A new name for an old concept provide more than 1000 sequences of 1000 base pairs (bp) The term ‘DNA barcoding’ is of recent use in the literature per day. Even non-geneticists now have easy access to [1,2]. It relies on the use of a standardized DNA region as a sequencing via companies that offer this service at a tag for rapid and accurate species identification [3]. Never- competitive price. Clearly, the development of DNA bar- theless, DNA barcoding is not a new concept. The term coding is linked to these improvements. ‘DNA barcodes’ was first used in 1993 [4], in a paper that When using the classical sequencing approach via capil- did not receive very much attention from the scientific lary electrophoresis, environmental samples (Box 2) community. The concept of species identification using require an additional step of cloning the different amplified molecular tools is older still [5]. However, the golden age DNA fragments into bacteria, followed by sequencing hun- of DNA barcoding began in 2003 [2]. The now well-estab- dreds or thousands of clones to reveal the full complexity of lished Consortium for the Barcode of Life (CBOL, http:// those samples. Such a cloning step is both expensive and barcoding.si.edu), an international initiative supporting time consuming, thus limiting large-scale applications. the development of DNA barcoding, aims to both promote New DNA sequencing technologies bypassing the cloning global standards and coordinate research in DNA barcod- step have recently been developed (Box 3), opening the way ing. For animals, the gene region proposed for the standard to applying large-scale DNA barcoding studies to environ- barcode is a 658 base pair region in the gene encoding the mental samples (Figure 1). mitochondrial cytochrome c oxidase 1 (COI) [2]. For plants More and more sequence data for the accepted barcod- the situation is controversial, and many strategies have ing markers are becoming available in public databases been proposed, either based on a single chloroplast region (GenBank, http://www.ncbi.nlm.nih.gov; EMBL, http:// [6,7] or on a combination of different regions [8,9]. www.ebi.ac.uk/embl; DDBJ, http://www.ddbj.nig.ac.jp)as Taxonomists are not the only potential users of DNA sequencing facilities improve. This greatly stimulates the barcoding, as the technique can be useful for scientists development of species identification via DNA barcoding, from other fields (e.g. forensic science, the biotechnology and enhances the design of standardized methods by and food industries and animal diet) [10]. Taxonomists are allowing a better design of ‘universal’ primers. However, concerned with DNA barcoding sensu stricto. Other scien- the quality of the sequence data in GenBank, EMBL or tists will be more interested with DNA barcoding sensu DDBJ is not always perfect [11], either as a result of lato, that is by DNA-based taxon identification using sequencing errors, contaminations, sample misidentifica- diverse techniques that can lie outside the CBOL approach tions or taxonomic problems. CBOL’s recent initiative to (Box 1; see Table S1 in the online supplementary material). build a new database specially dedicated to DNA barcoding The difference between the two approaches derives mainly will change this situation, and will provide an efficient and from different priorities given to the criteria used for accurate tool for species identification (Barcode of Life designing the molecular markers (Box 1). Data Systems, BOLD, http://www.barcodinglife.org). BOLD has been designed to record not only DNA sequences Corresponding author: Taberlet, P. ([email protected]). from several individuals per species (including primer sets, 110 0169-5347/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tree.2008.09.011 Available online 25 December 2008 Author's personal copy Review Trends in Ecology and Evolution Vol.24 No.2 Box 1. DNA barcoding: definitions and ideal properties Box 2. Environmental samples DNA barcoding sensu stricto corresponds to the identification of the An environmental sample is a mix of organic and inorganic species level using a single standardized DNA fragment. This materials taken from the environment (e.g. soil or feces). It can definition fits with the CBOL view [2]. contain live individuals (i.e. microorganisms or small macroorgan- The definition of DNA barcoding sensu lato is much less isms such as nematodes or springtails) and remains of macro- restrictive. It corresponds to the identification of any taxonomical organisms present around the sampling site. Until now, level using any DNA fragment. The identification of genera or environmental samples have been used mainly for studying families, from an environmental sample using a very short DNA microbial communities, using 16S or the internal transcribed spacer fragment that has not been recognized as the standardized barcode, of rDNA as the barcode [43 45,84]. In this case, DNA sequences of can nevertheless be considered as DNA barcoding sensu lato. Taxon – several hundreds of base pairs can be retrieved because DNA of identification with diagnostic single-nucleotide polymorphisms good quality is extracted from live microorganisms. Environmental (SNPs) or with insertions/deletions (indels) can also be considered samples should also be useful for characterizing the diversity of as DNA barcoding sensu lato (see Table S1 in the online macro-organic species (such as plant or animals) in an ecosystem. supplementary material). The identification of populations within a Here the DNA comes from dead macroorganisms, is in most cases given species, based either on diagnostic markers or on the highly degraded, and only short sequences can be amplified; the differences in marker frequencies among populations, refers to new sequencing techniques (Box 3), coupled with universal primers assignment tests [83], and should not be considered as DNA that amplify short fragments, now enable the identification of barcoding. various macro-organic taxa. A few studies have already been carried The ideal DNA barcoding system should meet the following out using different types of environmental samples: criteria [69]. water for microbial biodiversity [33] and for identifying an (i) The gene region sequenced should be nearly identical among invasive species [40]; individuals of the same species, but different between species. modern soil for comparative analyses of bacterial communities (ii) It should be standardized, with the same DNA region used for [43 45]; different taxonomic groups. – permafrost for assessing past animal and plant communities [51]; (iii) The target DNA region should contain enough phylogenetic silty ice at the base of an ice cap for assessing past animal and information to easily assign unknown or not yet ‘barcoded’ plant communities at the onset of glacial conditions [52]; species to their taxonomic group