DNA Barcoding in Birds Figure 2. K2P Pairwise comparisons among parapatric bird species in (a) cox1, (b) cob, and (c) 16S genes. White bars are comparisons between pairs of parapatric species and grey bars are comparisons within each of these species. doi:10.1371/journal.pone.0004119.g002 including cob and 16S in our analysis, besides cox1, we have been able to test the overlap between inter- and intraspecific mitochondrial distances in a much wider array of taxa than previous analysis [9,16]. Furthermore, by specifically targeting parapatric and hybridising bird taxa which potentially are particularly problematic in DNA barcoding [14], we here provide a more stable basis to test the performance of mitochondrial DNA barcoding in these species. As apparent from Figure 1 (and Figure S2abc, Supporting information for comparisons within higher taxonomy levels for each gene) a wide gap exist between intra- and interspecies divergences for both cox1 and cob genes if all taxa within genera are compared, whereas this gap is less apparent for 16S. This indicates that mitochondrial rRNA genes may be less suitable for bird species identification despite their many other advantages like universal primer applicability [17]. In the following we thus do not use the 16S data further to discuss the distribution of sequence divergences among birds, but report the cob data as a complement to cox1 in order to base our comparisons on the maximum available number of taxa. PLoS ONE | www.plosone.org 4 January 2009 | Volume 4 | Issue 1 | e4119