DNA barcoding. For birds, parapatric species are defined as species with contiguous or narrow overlap zones, excluding each other geographically; these species may or may not hybridise, and may or may not represent sister species, but phylogenetic data are incomplete thus far [15]. Only few DNA barcoding studies have focused on potentially hybridising bird species thus far [12,16], which probably in part is due to the lack of cox1 sequences for crucial taxa. Especially the parapatric species pairs from the Palearctic have remained largely unstudied in this respect. Data are available for 39 pairs of proven sister species of North American birds, most of which hybridise; these have K2P (Kimura two-parameter) mtDNA distances of 0.07% to 8.2%, with an average of 1.9% [12]. For 29 out of these 39 species pairs the sequence divergences was equal or lower than the suggested cox1 threshold (2.7%) [9]. Building upon this previous work, [9], Kerr et al. (2007) [16], working with a larger number of species and samples, found significantly smaller amounts of interspecific variation between closely related allopatric bird taxa (that often are known to hybridise), potentially compromising the universal applicability of cox1 DNA barcoding. Besides the cox1 gene, other mitochondrial markers also have been widely sequenced across vertebrates for their utility in phylogenetics or to complement cox1 in DNA barcoding. In amphibians the 16S ribosomal RNA gene (16S) has been suggested as a complementary DNA barcoding marker [17]. Another protein coding gene, cytochrome b (cob), has also been suggested as a marker to determine species boundaries [18–20]. Birds are among the most intensively sequenced taxa for cox1 and cob, and there is a reasonable dataset available for 16S. Taken together, sequences of these three genes are available for a significant proportion of worldwide species diversity of birds. Furthermore, birds are taxonomically one of the best studied animal groups which indicates that a relatively low proportion of unknown, cryptic species is to be expected, and that taxonomic misidenti- fications are relatively rare, giving a reasonable degree of confidence in the specific identity of published DNA sequences. We here make use of the availability of cob and 16S data to combine these with cox1 sequences into the largest taxon set that so far has been assessed for mitochondrial divergences at different taxonomic levels. We specifically aimed to provide novel data on (a) a possible dependence of the barcoding gap between intra- and interspecific divergences from the number of sequences per species, (b) a comparison of levels of pairwise divergences among species in the same genus vs. species in different genera, and, especially, (c) the utility of DNA barcoding to discern among mainly Palearctic hybridising parapatric species. Results For none of the three genes was mean divergence within species significantly related to the sample sizes per species, as revealed by regression analysis (cox1: R2 = 0.001, p = 0.465, 16S: R2 = 0.001, p = 0.465, cob: R2 = 0.001, p = 0.338) (Figure S1). In general, intraspecific K2P distances for the three genes ranged from zero to 17.9% (cox1: 0–7.3%, 16S: 0–6.2%, and cob: 0–17.9) and intrageneric K2P distances ranged from zero to 20.1% (cox1: 0– 18.9%, 16S: 0–13.3%, and cob: 0–20.1). The lower range of values may be an effect of misnamed or misidentified taxa in GenBank, or may be real (as in the case of several taxa that form a so-called ring-species: [21–23]. Similarly, we strongly suspect that many of the the highest intraspecific distances are due to wrongly determined samples, or pseudogene sequences, recovered from Genbank. In total, only 134 out of 31,773 cob intraspecific K2P values were above 7.4% (and thus 10-fold higher than average DNA Barcoding in Birds intraspecific divergence, see below), indicating that these possibly wrong comparisons (affecting 63 species) will have a very limited effect on subsequent calculations. Cox1 gene Intraspecific K2P distances for species with $2 sequences (mean = 4.51, range = 2–122, n = 566 species) averaged 0.24% (SD = 0.59%). Intrageneric K2P distances are some 24-fold higher (5.9763.55) than the mean intraspecific K2P distances (Figure 1a, Table S1). Mean divergences within families and orders were 11.46% (SD = 3.06%) and 15.80% (SD = 3.35%) respectively (Figure S2a). K2P distances within 64 parapatric species (comparing species occurring parapatrically with at least one other related bird species) with .2 sequences (mean = 3.1, range = 2–8) averaged 0.4960.87% (Figure 2a). K2P distances between species in parapatric species pairs averaged 3.6463.29% with significant differences between those species that do hybridise (3.3563.35%) and those that do not hybridise (5.9964.24%) (p.0.001 Table 1, 2). K2P distances between species in hybridising species pairs were significantly larger than intraspecific distances and smaller than intrageneric K2P distances for all species (p,0.001, Table 2). Cob gene Intraspecific K2P distances for species with $2 sequences (mean = 4.64, range = 2–127, n = 656 species) averaged 0.74% (SD = 1.21%). Intrageneric K2P distances were some 11-fold higher (8.1163.80) than the mean intraspecific K2P distances (Figure 1b, Table S1). Mean divergences within families and orders were 13.97% (S = 3.13%) and 19.50% (SD = 3.64%) respectively (Figure S2b). K2P distances within 60 parapatric species with $2 sequences (mean = 3.51, range = 2–20) averaged 1.0761.74% (Figure 2b). K2P distances between species in parapatric species pairs averaged 3.0862.50, with a significant difference between those species that do hybridise and those that do not hybridise (p,0.001, Table 1, 2). Intrageneric K2P distances between species in hybrid species pairs were significantly different from intraspecific and intrageneric K2P distances for all species (p,0.001, Table 2). 16S gene Intraspecific K2P distances for species with $2 sequences (mean = 2.67, range = 2–12, n = 125 species) averaged 0.48% (SD = 1.06%). Intrageneric K2P distances were some seven fold higher (3.1361.92) than the mean intraspecific K2P distances (Figure 1c, Table S1). Mean divergences within families and orders were 6.51% (SD = 2.45%) and 10.69% (SD = 2.37%) respectively (Figure S2c). K2P distancess within 46 parapatric species with $2 sequences (mean = 2.22, range = 2–12) averaged 0.3360.82% (Figure 2c). K2P distances between species in parapatric species pairs averaged 1.3061.17, with no significant difference between those species that do hybridise and those that do not hybridise (p = 0.078 Table 1, 2). K2P distances between species in hybridising species pairs were significantly different from intraspecific and intragene- ric K2P distances for all species (p,0.001, Table 2). Discussion DNA barcoding efficiency of cox1 vs. cob and 16S The accuracy of distance-based DNA barcoding depends particularly on the extent of the separation between intra- and interspecific divergence in the selected marker. The ideal world for barcoding lacks any overlap between these two values. By PLoS ONE | www.plosone.org 2 January 2009 | Volume 4 | Issue 1 | e4119