First Large-Scale DNA Barcoding Assessment of Reptiles in the Biodiversity Hotspot of Madagascar, Based on Newly Designed COI Primers Zolta´ n T. Nagy1*, Gontran Sonet1, Frank Glaw2, Miguel Vences3 1 Joint Experimental Molecular Unit, Royal Belgian Institute of Natural Sciences, Brussels, Belgium, 2 Zoologische Staatssammlung Mu¨nchen, Mu¨nchen, Germany, 3 Zoological Institute, Technical University of Braunschweig, Braunschweig, Germany Abstract Background: DNA barcoding of non-avian reptiles based on the cytochrome oxidase subunit I (COI) gene is still in a very early stage, mainly due to technical problems. Using a newly developed set of reptile-specific primers for COI we present the first comprehensive study targeting the entire reptile fauna of the fourth-largest island in the world, the biodiversity hotspot of Madagascar. Methodology/Principal Findings: Representatives of the majority of Madagascan non-avian reptile species (including Squamata and Testudines) were sampled and successfully DNA barcoded. The new primer pair achieved a constantly high success rate (72.7–100%) for most squamates. More than 250 species of reptiles (out of the 393 described ones; representing around 64% of the known diversity of species) were barcoded. The average interspecific genetic distance within families ranged from a low of 13.4% in the Boidae to a high of 29.8% in the Gekkonidae. Using the average genetic divergence between sister species as a threshold, 41–48 new candidate (undescribed) species were identified. Simulations were used to evaluate the performance of DNA barcoding as a function of completeness of taxon sampling and fragment length. Compared with available multi-gene phylogenies, DNA barcoding correctly assigned most samples to species, genus and family with high confidence and the analysis of fewer taxa resulted in an increased number of well supported lineages. Shorter marker-lengths generally decreased the number of well supported nodes, but even mini-barcodes of 100 bp correctly assigned many samples to genus and family. Conclusions/Significance: The new protocols might help to promote DNA barcoding of reptiles and the established library of reference DNA barcodes will facilitate the molecular identification of Madagascan reptiles. Our results might be useful to easily recognize undescribed diversity (i.e. novel taxa), to resolve taxonomic problems, and to monitor the international pet trade without specialized expert knowledge. Citation: Nagy ZT, Sonet G, Glaw F, Vences M (2012) First Large-Scale DNA Barcoding Assessment of Reptiles in the Biodiversity Hotspot of Madagascar, Based on Newly Designed COI Primers. PLoS ONE 7(3): e34506. doi:10.1371/journal.pone.0034506 Editor: Jerome Chave, Centre National de la Recherche Scientifique, France Received October 11, 2011; Accepted March 2, 2012; Published March 30, 2012 Copyright: ß 2012 Nagy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The study was funded by the Belgian Science Policy Office (Belspo). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction species delimitation and description. A solution for the second of these challenges, the Linnean shortfall [14], is not in sight. Besides The elementary question how many species of eukaryotic more posts for professional taxonomists [15], fundamentally new organisms live on Earth has in the past decades led to remarkable ideas and approaches will be required, especially in hyperdiverse controversies [1–5]. Current estimates of global species numbers groups such as small insects or nematodes. On the contrary, the differ enormously, ranging from 2 to 100 million, while ca. 1.7–1.9 initial identification of new species has been much facilitated and million species have thus far been formally described. However, accelerated by DNA-based methods, and the term DNA there is a broad agreement that the highest proportion of Earth’s barcoding has been proposed [16–19]. Similar to a long standing biodiversity—expressed in species numbers—is yet to be discov- practice in microbiology [20], a short DNA sequence of a standard ered and described. Even in several of the most prominent animal marker is used for species identification—in animals typically the groups such as vertebrates and butterflies, a remarkable propor- mitochondrial gene for cytochrome oxidase subunit I (COI). This tion of undescribed diversity is being discovered (e.g. Lepidoptera: molecular survey method has been applied in a number of [6]; fish: [7]; amphibians: [8–10]). Given that just 16,000–20,000 vertebrate taxa (e.g. birds: [21,22]; fish: [23]) and invertebrates species are being described per year [11,12] and traditional (e.g. spiders: [24], Lepidoptera: [25–27], marine invertebrates: taxonomy involves high costs (estimated between $39,000– [28], and Heteroptera: [29]). DNA barcoding has the potential to $122,000/species including salaries etc. [13]), strategies are increase the rate of discovery enormously [30] and to discover required to speed up both the process of species discovery and unexpected genetic diversity such as in butterflies [31–32] or in PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e34506 DNA Barcoding of Madagascan Reptiles amphipods [33,34]. From practical point of view, DNA barcoding hotspot, exemplified by the Madagascan ‘‘microcontinent’’ with its requires a comprehensive reference database [35]. Such reference clades of reptiles of different temporal diversification background, data sets are being assembled by the barcoding campaigns and test the performance of the method depending on barcode initiated by the Consortium for the Barcode of Life (CBOL). For length and completeness of taxon sampling. vertebrates, campaigns focusing on fish, birds and mammals have been started: the ‘Fish Barcode of Life Initiative’ (FISH-BOL, Materials and Methods www.fishbol.org; [36]), the ‘Shark Barcode of Life’ project (www. sharkbol.org), the ‘All Birds Barcoding Initiative’ (ABBI, www. Ethics statement barcodingbirds.org) and the ‘Mammal Barcode of Life’ (www. No experiments were conducted using living animals. Further- mammaliabol.org) project. A new barcoding campaign called more, none of the samples were specifically collected for this ColdCode dedicated to amphibians and non-avian reptiles has project. We exclusively used museum samples which were already been announced in September 2011, and new COI primers for available and were deposited in a tissue bank. amphibians have been published [37]. Although molecular data All field researches and collecting of specimens were approved are regularly used to discover and delimit new species of reptiles, by the Madagascan Ministe`re de l’Environnement, des Eaux et des no large-scale DNA barcoding effort has so far targeted an entire Foreˆts (Direction des Eaux et Foreˆts, DEF) under the following species-rich reptile fauna of a large region. permits: 156-MEF/SG/DGEF/DADF/SCB dated 12 December To facilitate reading, we will in the following use the traditional 2002; 238-MINENVEF/SG/DGEF/DPB/SCBLF dated 14 No- term ‘reptiles’ for species included in the vertebrate orders vember 2003; 238-MINENV.EF/SG/DGEF/DPB/SCBLF/ Squamata, Testudines, Crocodylia, and Rhynchocephalia, i.e. RECH dated 22 December 2004; 272-MINENV.EF/SG/ Sauropsida excluding birds. We continue using the term ‘reptiles’ DGEF/DPB/SCBLF/RECH dated 8 November 2005; 298- for practical reasons only and without any phylogenetic relevance. MINENV.EF/SG/DGEF/DPB/SCBLF/RECH dated 22 De- Due to technical problems in the amplification and evaluation cember 2006; 036/08 MEEFT/SG/DGEF/DSAP/SSE dated 30 of COI sequences in amphibians and reptiles linked to a high January 2008; 26/09/MEEFT/SG/DGEF/DSAP/SLRSE dated variability of sequences including priming sites, authors of previous 3 February 2009; 48/09/MEEFT/SG/DGEF/DSAP/SSE dated attempts [38] have preferred the use of a fragment of the 9 March 2009; 188/09/MEEFT/SG/DGEF/DSAP/SSE dated mitochondrial 16 S rRNA gene for DNA barcoding (sensu lato) 16 September 2009; 195/09/MEEFT/SG/DGEF/DSAP/SSE despite its non-coding characteristics and resulting alignment dated 28 September 2009. Export of specimens was approved by problems. Although recent publications claim that the difficulties the DEF under permits: 063C-EA02/MG03, dated 26 February of COI amplification in amphibians can be overcome [39], various 2003; 094C-EA03/MG04, dated 1 March 2004; 103C-EA03/ teams have experienced problems. Therefore, COI as marker for MG05, dated 15 March 2005; E 1400/06, dated 1 June 2006; molecular identification and for phylogenetic and phylogeographic 055N-EA03/MG10, dated 25 March 2010. Import of species studies has been rarely used by herpetologists. Consequently, there protected by CITES into Germany was approved by the German is a serious lack of DNA barcodes for amphibians and reptiles, and authorities (Bundesamt fu¨r Naturschutz). Voucher specimens were according to the Barcode of Life Data Systems [18] DNA barcodes euthanized using proved methods (e.g. anaesthesia with ketamine, of most taxa of reptiles (even on higher taxonomic levels) followed by ketamine overdosis) that do not require approval by an inhabiting Madagascar are virtually absent. This highlights the ethics committee. need for development and testing of primers and amplification strategies for these organisms [40]. Sampling Here we start filling this gap and provide a DNA barcoding We sampled 468 specimens of Madagascan reptiles mostly assessment of the reptile fauna of Madagascar, the fourth-largest deposited in publically accessible natural history collections (for list island in the world that has been flagged as one of the most of samples, see Table S1). About 420 of these samples were important hotspots for biodiversity conservation [41] and as a determined to belonged to 251 nominal species.