DNA barcoding birds: from field collection to data analysis

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 specimens, 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 amplification 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 identification of unknown specimens.