Molecular Identification of Birds: Performance of Distance-Based DNA Barcoding in Three Genes to Delimit Parapatric Species Mansour Aliabadian1,2*, Mohammad Kaboli3, Vincent Nijman 4 , Miguel Vences5 1 Institute for Biodiversity and Ecosystem Dynamics and Zoological Museum, University of Amsterdam, Amsterdam, The Netherlands, 2 Department of Biology, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran, 3 Department of Fishery and Environment, Faculty of Natural Resources, University of Tehran, Tehran, Iran, 4 School of Social Sciences and Law, Oxford Brookes University, Oxford, United Kingdom, 5 Division of Evolutionary Biology, Zoological Institute, Technical University of Braunschweig, Braunschweig, Germany Abstract Background: DNA barcoding based on the mitochondrial cytochrome oxidase subunit I gene (cox1 or COI) has been successful in species identification across a wide array of taxa but in some cases failed to delimit the species boundaries of closely allied allopatric species or of hybridising sister species. Methodology/Principal Findings: In this study we extend the sample size of prior studies in birds for cox1 (2776 sequences, 756 species) and target especially species that are known to occur parapatrically, and/or are known to hybridise, on a Holarctic scale. In order to obtain a larger set of taxa (altogether 2719 species), we include also DNA sequences of two other mitochondrial genes: cytochrome b (cob) (4614 sequences, 2087 species) and 16S (708 sequences, 498 species). Our results confirm the existence of a wide gap between intra- and interspecies divergences for both cox1 and cob, and indicate that distance-based DNA barcoding provides sufficient information to identify and delineate bird species in 98% of all possible pairwise comparisons. This DNA barcoding gap was not statistically influenced by the number of individuals sequenced per species. However, most of the hybridising parapatric species pairs have average divergences intermediate between intraspecific and interspecific distances for both cox1 and cob. Conclusions/Significance: DNA barcoding, if used as a tool for species discovery, would thus fail to identify hybridising parapatric species pairs. However, most of them can probably still assigned to known species by character-based approaches, although development of complementary nuclear markers will be necessary to account for mitochondrial introgression in hybridising species. Citation: Aliabadian M, Kaboli M, Nijman V, Vences M (2009) Molecular Identification of Birds: Performance of Distance-Based DNA Barcoding in Three Genes to Delimit Parapatric Species. PLoS ONE 4(1): e4119. doi:10.1371/journal.pone.0004119 Editor: Neil John Gemmell, University of Otago, New Zealand Received August 22, 2008; Accepted November 27, 2008; Published January 7, 2009 Copyright: 2009 Aliabadian 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: This work was partially funded by the Ministry of Science, Research and Technology of the Islamic Republic of Iran to MA. 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: M.aliabadian@uva.nl Introduction Mitochondrial DNA (mtDNA) markers have been widely applied in molecular phylogenetic studies, but deciding which mtDNA genes to use for the identification of species remains an important issue [1–3] because different parts of the mtDNA genome evolve at different mutation rates [4,5]. The choice of a suitable gene with high phylogenetic resolution will be more crucial when evaluating species delimitation of recently diverged species. MtDNA, with rapid pace of sequence changes, regularly shows pronounced divergences between closely related species [1,6] but concern has been expressed that mtDNA sequence differences among such closely related species will often be too small to allow their discrimination, and the problem will be further accentuated by phenomena of ancient sharing of haplotype polymorphisms and by introgression (e.g. [7]). Recent studies suggest that sequences of the mitochondrial cytochrome oxidase subunit I (cox1 or COI) could serve as a fast and accurate marker for the identification of animal species, and for the discovery of new species across the tree of life [8], a procedure for which the term DNA barcoding has been coined. A first major study investigated sequence variation of 25% of the species of North American breeding birds (260 species) [9]. Variation of cox1 sequences within species was an average of 20 times smaller than between species, and there was a clear gap between intra- and interspecific variation. Utilizing this barcoding gap, a standard sequence threshold was proposed to define species boundaries of around 10 times the mean intraspecific variation for the group under study [9]. DNA barcoding based on cox1 has been successful in species identification across a wide array of taxa [9–12]. For invertebrates, it has however been argued [13] that the barcoding gap may be an artifact of insufficient sampling across taxa. In general, it is especially with pairs or complexes of closely related and potentially hybridising species where DNA barcoding can be expected to encounter problems [14]. In particular the existence of so-called parapatric species may pose a challenge for PLoS ONE | www.plosone.org 1 January 2009 | Volume 4 | Issue 1 | e4119