127 W. John Kress and David L. Erickson (eds.), DNA Barcodes: Methods and Protocols, Methods in Molecular Biology, vol. 858, DOI 10.1007/978-1-61779-591-6_7, © Springer Science+Business Media, LLC 2012 Chapter 7 DNA Barcoding Birds: From Field Collection to Data Analysis Darío A. Lijtmaer, Kevin C.R. Kerr, Mark Y. Stoeckle, and Pablo L. Tubaro Abstract As of February 2011, COI DNA barcode sequences (a 648-bp segment of the 5 ¢ end of the mitochondrial gene cytochrome c oxidase I, the standard DNA barcode for animals) have been collected from over 23,000 avian specimens representing 3,800 species, more than one-third of the world’s avifauna. Here, we detail the methodology for obtaining DNA barcodes from birds, covering the entire process from field collection to data analysis. We emphasize key aspects of the process and describe in more detail those that are particularly relevant in the case of birds. We provide elemental information about collection of speci- mens, detailed protocols for DNA extraction and PCR, and basic aspects of sequencing methodology. In particular, we highlight the primer pairs and thermal cycling profiles associated with successful amplifica- tion and sequencing from a broad range of avian species. Finally, we succinctly review the methodology for data analysis, including the detection of errors (such as contamination, misidentifications, or amplification of pseudogenes), assessment of species resolution, detection of divergent intraspecific lineages, and identi- fication of unknown specimens. Key words: Birds, Cytochrome c oxidase I, DNA barcodes, Collection, DNA extraction, Neighbor joining, Polymerase chain reaction, Pectoral muscle, Sequencing, Toe pad Taxonomy and phylogenetic affinities are better understood in birds than in any other large group of organisms. Additionally, they are probably the best represented group of vertebrates in frozen tissue collections, with more than 300,000 tissue samples covering nearly 75% of known bird species (1). These characteristics make birds an ideal group to analyze the effectiveness of a standardized genetic method for species identification—i.e., DNA barcoding. Consequently, they were the first taxonomic group for which a large- scale barcoding study was performed (2) and were the focus of one 1. Introduction