Frontiers in Zoology BioMed Central Research Open Access Comparative performance of the 16S rRNA gene in DNA barcoding of amphibians Miguel Vences*1, Meike Thomas2, Arie van der Meijden3, Ylenia Chiari3 and David R Vieites4 Address: 1Institute for Biodiversity and Ecosystem Dynamics, Zoological Museum, University of Amsterdam, Mauritskade 61, 1092 AD Amsterdam, The Netherlands, 2Institute for Genetics, Evolutionary Genetics, University of Cologne, Weyertal 121, 50931 Köln, Germany, 3Department of Biology (Evolutionary Biology), University of Konstanz, 78457 Konstanz, Germany and 4Department of Integrative Biology, Museum of Vertebrate Zoology, 3101 Valley Life Sciences Bldg., University of California, Berkeley, CA 94720-3160, USA Email: Miguel Vences* - [email protected]; Meike Thomas - [email protected]; Arie van der Meijden - [email protected]; Ylenia Chiari - [email protected]; David R Vieites - [email protected] * Corresponding author Published: 16 March 2005 Received: 25 October 2004 Accepted: 16 March 2005 Frontiers in Zoology 2005, 2:5 doi:10.1186/1742-9994-2-5 This article is available from: http://www.frontiersinzoology.com/content/2/1/5 © 2005 Vences et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Identifying species of organisms by short sequences of DNA has been in the center of ongoing discussions under the terms DNA barcoding or DNA taxonomy. A C-terminal fragment of the mitochondrial gene for cytochrome oxidase subunit I (COI) has been proposed as universal marker for this purpose among animals. Results: Herein we present experimental evidence that the mitochondrial 16S rRNA gene fulfills the requirements for a universal DNA barcoding marker in amphibians. In terms of universality of priming sites and identification of major vertebrate clades the studied 16S fragment is superior to COI. Amplification success was 100% for 16S in a subset of fresh and well-preserved samples of Madagascan frogs, while various combination of COI primers had lower success rates.COI priming sites showed high variability among amphibians both at the level of groups and closely related species, whereas 16S priming sites were highly conserved among vertebrates. Interspecific pairwise 16S divergences in a test group of Madagascan frogs were at a level suitable for assignment of larval stages to species (1–17%), with low degrees of pairwise haplotype divergence within populations (0–1%). Conclusion: We strongly advocate the use of 16S rRNA as standard DNA barcoding marker for vertebrates to complement COI, especially if samples a priori could belong to various phylogenetically distant taxa and false negatives would constitute a major problem. Background organisms [4,5], the large-scale identification of organ- The use of short DNA sequences for the standardized isms in ecological or genomic studies [1,6] and, most con- identification of organisms has recently gained attention troversially, explorative studies to discover potentially under the terms DNA barcoding or DNA taxonomy [1-3]. undescribed "candidate" species [4,7,8]. Although it is not Among the promising applications of this method are the a fundamentally new technique [9], DNA barcoding is assignments of unknown life-history stages to adult promising because technical progress has made its large- Page 1 of 12 (page number not for citation purposes) Frontiers in Zoology 2005, 2:5 http://www.frontiersinzoology.com/content/2/1/5 scale, automated application feasible [3,6] which may were concordant in yielding more reliable and universal accelerate taxonomic progress [10]. amplifications for 16S than COI. In a set of fresh and well- preserved samples from relatively closely related mantel- Although not necessarily under the specific concepts of lid frogs from Madagascar (Table 1, Additional file 1), the DNA barcoding and DNA taxonomy, the diagnosis and 16S amplification success was complete, whereas the even definition of taxa by their DNA sequences are reali- three sets of COI primers yielded success rates of only 50– ties in many fields and organism groups, such as prokary- 70%. Considering all three primer combinations, there otes, fungi, and soil invertebrates [1,6]. To use this were two species of frogs (10%) that did not amplify for approach on a large and formalized scale, consensus of COI at all (Boophis septentrionalis and B. tephraeomystax). the scientific community is essential with respect to the most suitable genes that allow robust and repeatable Priming sites amplification and sequencing, and that provide unequiv- The variability of priming sites was surveyed using nine ocal resolution to identify a broad spectrum of organisms. complete amphibian mitochondrial sequences from Gen- While D. Tautz and co-workers [3] proposed the nuclear bank (Fig. 1), and 59 mt genomes of fishes, reptiles, birds ribosomal RNA genes for this purpose, P. D. N. Hebert and mammals (Fig. 2). A high variability was encountered and colleagues have strongly argued in favor of a 5' frag- for COI. The sequences of some species were largely con- ment of the mitochondrial gene for cytochrome oxidase, sistent with the primers: Xenopus had two mutations only subunit I (COI or COXI) [2,11]. This gene fragment has at each of the priming regions. However, other sequences been shown to provide a sufficient resolution and robust- were strongly different, with up to seven mutations, all at ness in some groups of organisms, such as arthropods third codon positions. No particular pattern was recogniz- and, more recently, birds [2,4,7,11]. able for any major group that would facilitate designing COI primers specific for frogs, salamanders or caecilians. A genetic marker suitable for DNA barcoding needs to Interestingly the variability among the amphibian meet a number of criteria [2]. First, in the study group, it sequences available was as large as or larger than among needs to be sufficiently variable to discriminate among the complete set of vertebrates at many nucleotide posi- most species, but sufficiently conserved to be less variable tions of COI priming sites (Fig. 2), indicating a possible within than between species. Second, priming sites need higher than average variability of this gene in amphibians. to be sufficiently conserved to permit a reliable amplifica- tion without the risk of false negatives when the goal is the In contrast, the 16S priming sites were remarkably con- analysis of pooled samples, e.g. when the total of inverte- stant both among amphibians and among other verte- brates from a soil sample is to be studied without separat- brates (Fig. 1, 2). A wider survey of priming sites, i.e., the ing individuals, or of environmental DNA such as alternative reverse priming sites used in arthropod and subfossil DNA remains from the soil [12,13]. Third, the bird studies [2,7], confirmed the high variability of COI in gene should convey sufficient phylogenetic information amphibians, and in vertebrates in general (Fig. 2). A to assign species to major taxa using simple phenetic screening of the first 800 bp of the C-terminal part of the approaches. Fourth, its amplification and sequencing gene in nine amphibians of which complete mitochon- should be as robust as possible, also under variable lab drial genes were available did not reveal a single fragment conditions and protocols. Fifth, sequence alignment of 20 bp where all nine species would agree in 80% or should be possible also among distantly related taxa. more of their nucleotides. Here we explore the performance of a fragment of the 16S Recovery of major groups ribosomal RNA gene (16S) in DNA barcoding of amphib- The phenetic neighbor-joining analysis using the 16S frag- ians. As a contribution to the discussion about suitable ment produced a tree that contained eight major group- standard markers we provide experimental data on com- ings that conform to or are congruent with the current parative amplification success of 16S and COI in amphib- classification and phylogeny (Fig. 3): cartilaginous fishes, ians, empirical data on conservedness of priming sites, salamanders, frogs, turtles, eutherian mammals, mam- and an example from the 16S-based identification of mals, squamates, birds. Of these, the COI tree (Fig. 4) amphibian larval stages. recovered only the lineages of cartilaginuous fishes and birds. The COI analysis did not recover any additional Results major lineage. Amplification experiments We performed independent amplification experiments 16S rDNA barcoding of tadpoles with one set of 16S primers and three published sets of From an ongoing project involving the large-scale identi- COI primers [2,7] focusing on representatives of different fication of tadpoles of Madagascan frogs [5] we here pro- frog, salamander and caecilian genera. The experiments vide data from larval and adult frog species from two sites Page 2 of 12 (page number not for citation purposes) Frontiers in Zoology 2005, 2:5 http://www.frontiersinzoology.com/content/2/1/5 VariabilityFigure 1 of priming sites in amphibians Variability of priming sites in amphibians. Variability of priming sites for 16S rRNA and COI in amphibians. Page 3 of 12 (page number not for citation purposes) Frontiers in Zoology 2005, 2:5 http://www.frontiersinzoology.com/content/2/1/5