A reliable DNA barcode reference library for the identification of the North European shelf fish fauna THOMAS KNEBELSBERGER,* MONICA LANDI, † HERMANN NEUMANN, ‡ MATTHIAS KLOPPMANN,§ ANNE F. SELL,§ PATRICK D. CAMPBELL,¶ SILKE LAAKMANN,* MICHAEL J. RAUPACH,* GARY R. CARVALHO** and FILIPE O. COSTA † *Senckenberg am Meer, German Center for Marine Biodiversity Research (DZMB), S€ udstrand 44, 26382 Wilhelmshaven, Germany, †Department of Biology, Center of Molecular and Environmental Biology (CBMA), University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal, ‡Senckenberg am Meer, Marine Research, S€ udstrand 40, 26382 Wilhelmshaven, Germany, §Th€ unen Institute of Sea Fisheries, Palmaille 9, 22767 Hamburg, Germany, ¶Natural History Museum, Cromwell Road, South Kensington, London SW7 5BD UK, **School of Biological Sciences, University of Wales, Deiniol Road, Bangor, Gwynedd LL57 2UW, UK Abstract Valid fish species identification is an essential step both for fundamental science and fisheries management. The tra- ditional identification is mainly based on external morphological diagnostic characters, leading to inconsistent results in many cases. Here, we provide a sequence reference library based on mitochondrial cytochrome c oxidase subunit I (COI) for a valid identification of 93 North Atlantic fish species originating from the North Sea and adja- cent waters, including many commercially exploited species. Neighbour-joining analysis based on K2P genetic dis- tances formed nonoverlapping clusters for all species with a ≥99% bootstrap support each. Identification was successful for 100% of the species as the minimum genetic distance to the nearest neighbour always exceeded the maximum intraspecific distance. A barcoding gap was apparent for the whole data set. Within-species distances ran- ged from 0 to 2.35%, while interspecific distances varied between 3.15 and 28.09%. Distances between congeners were on average 51-fold higher than those within species. The validation of the sequence library by applying BOLDs bar- code index number (BIN) analysis tool and a ranking system demonstrated high taxonomic reliability of the DNA barcodes for 85% of the investigated fish species. Thus, the sequence library presented here can be confidently used as a benchmark for identification of at least two-thirds of the typical fish species recorded for the North Sea. Keywords: COI, DNA barcoding, marine fishes, mitochondrial DNA, reference libraries, species identification Received 22 November 2013; revision received 29 January 2014; accepted 29 January 2014 Introduction The accurate identification of species is a pivotal compo- nent in many fields of biological research and conserva- tion efforts. In this context, the application of molecular methods is essential, especially in cases where traditional species identification based on diagnostic morphological characters fails. For fish, several DNA-based discrimina- tion/identification methods have been developed over the last decades (for an overview see Teletchea 2009) among which DNA barcoding constitutes one of the most promising techniques by using partial cytochrome c oxidase subunit I (COI) sequences for a standardized and routine species identification (Hebert et al. 2003a). So far, many studies have demonstrated the ability of this approach as a powerful tool for the identification of ray- finned fishes (Actinopterygii) from freshwater habitats (Hubert et al. 2008; April et al. 2011) as well as marine ray-finned and cartilaginous fishes (Chondrichthyes) from different geographic regions (e.g. Ward et al. 2005, 2008a; Steinke et al. 2009a; Mabraga~ na et al. 2011; Cerutti-Pereyra et al. 2012; Costa et al. 2012; Zhang & Hanner 2012; Keskın & Atar 2013; McCusker et al. 2013). Besides the performance of DNA barcoding surveys of regional ichthyofaunas, DNA barcodes have already been used to identify a great variety of source material such as fish larvae (Pegg et al. 2006; Victor et al. 2009; Hubert et al. 2010), fins (Holmes et al. 2009)and globally traded fish products (Vi ~ nas & Tudela 2009; Wong et al. 2011). At last, DNA barcoding has been applied to identify ornamental fish (Steinke et al. 2009b). Correspondence: Thomas Knebelsberger, Fax: +4944219475111; E-mail: tknebelsberger@senckenberg.de © 2014 John Wiley & Sons Ltd Molecular Ecology Resources (2014) doi: 10.1111/1755-0998.12238