DNA Barcoding in Birds Table 2. K2P distances of species in parapatric species pairs and among non-parapatric species, comparing parapatric species pairs that do hybridise with those that do not, and with intrageneric (excluding parapatric species), intraspecific (excluding parapatric species), intraspecific in hybridising parapatric species, and intraspecific in non-hybridising parapatric species (** = 0.01, t-test). Comparisons Between hybridising parapatric species Between non-hybridising parapatric species Within all genera (excl. parapatric species) Within all species (excl. parapatric species) Within hybridising parapatric species Within non-hybridising parapatric species cox1 K2P Mean6std (N) 3.3563.05 (191) 5.9964.24 (23)** 5.9963.54 (27162)** 0.2360.57 (12924)** 0.4960.87 (496)** 0.3760.29 (24)** cob K2P Mean6std (N) 2.7662.28 (662) 6.1762.36 (69)** 8.263.75 (40555)** 0.7261.15 (29191)** 1.0161.67 (2488)** 2.2262.56 (105) 16S K2P Mean6std (N) 1.3661.23 (119) 1.0160.79 (25) 3.2661.91 (1731)** 0.5761.18 (246)** 0.2860.68 (128)** 0.6361.37 (22)** Presented are mean6standard deviation (number of comparisons). doi:10.1371/journal.pone.0004119.t002 Effects of sample size on DNA barcoding gap The intrageneric distances of cox1, on average, were 24-fold higher that the sequence divergences within species (0.24 vs 5.97%). In cob the intrageneric distances were on average 11-fold higher than the sequence divergences within species (0.74 vs 8.11%). The slight differences between the two data sets may be due to a different composition of the data (intraspecific comparisons in cob possibly based on samples from more distant localities). These values are not too different from those obtained in an initial effort to test cox1 DNA barcoding in birds [9], based on two or more individuals for 130 species (0.27% vs. 7.93%). It has been argued that this alleged gap will considerably lower down if more individuals per species are sampled and when a large proportion of closely related taxa are included [14,24]. This effect was however not observed in a subsequent study [16] that analysed an average 4.1 individuals per species. Our study confirms this trend. The barcoding gap was apparent in the cox1 dataset with, on average, 4.51 sequences per species, and also in the independent cob dataset with 4.64 sequences per species. Furthermore, our regression analyses found no dependence of intraspecific divergences from the number of individuals per species included in the analysis, neither in cox1 nor in cob. The present study compared the divergence of intraspecific sequences from specimens that in a high proportion originated from widely separated geographic regions, and confirmed that cox1 sequence variation was able to identify more than 98% of the pairwise sequence divergences correctly as corresponding to variation within species. In contrast, 20% of the pairwise comparisons within genera (intrageneric sequences distances) were lower than 0.24%. Intraspecific variation identified in this study was similar to that in North American breeding birds: 0.24% vs. 0.23% and 0.27% [9,16]. These values are lower than in most other animal groups: e.g., 0.60% in Guyanese bats [25], 0.46% in Lepidoptera [26], 0.39% in marine fishes [27], 3–4% in Aneides salamanders and mantellid frogs [17]. DNA barcoding in parapatric bird species While the barcoding gap appears to hold for overall comparisons among birds even if larger numbers of individuals are included, a more critical issue is that of distinguishing related combinations of species [14]. In such species complexes, the barcoding gap may not exist, and this effect may be diluted in overall comparisons of large numbers of taxa. Numerous DNA barcoding studies conducted thus far revealed that more than 90% of species under study could be identified by this method. For example, 93% of studied species of Guyanan bats and 95% of North American bird species could be allocated correctly [25,16]. The cases where barcodes failed to separate species involved either closely allied allopatric taxa whose status, as distinct species, is uncertain, or sister taxa that hybridise. However, coalescent and character-based approaches are effective in closely related species, non-hybridising species of birds [10]. Our study showed that a high proportion of hybridising parapatric species cannot be distinguished by the suggested distance-based threshold value in DNA barcoding. The proportion was 48% (14/29) in the cox1 dataset and 78% (25/32) in the cob dataset. These different values probably were not due to different properties of the analysed genes but to different taxa included, and possibly to a higher degree of misidentified taxa (as taken from Genbank) in the cob dataset. Of the parapatric species pairs that do not hybridise, 14% (1/7) and 73% (19/26) did not meet the threshold for cox1 and cob genes respectively. Based on published [e.g., 28] and unpublished estimates (own data) the percentage of parapatric species that hybridise in the Palearctic Region is 60%, which corresponds to 10–18% of all species: [29,30] Using these values, plus the global number of parapatric species [15,28] and the proportion of parapatric species that show only a small amount of genetic divergence, we can estimate that between 250 (based on cox1) and 650 (cob) parapatric species of birds are not distinguishable by the barcoding gap. This represents some 2.5–6.0% of the total number of species. If DNA barcoding would be used as a tool for species discovery, it would fail to identify these species. However, especially in a well-known group such as birds, DNA barcoding is usually used for assigning individuals to known species. In these cases, most of the parapatric species could probably be still correctly identified, depending on the origin of the low divergences: (a) mitochondrial introgression due to hybridisa- tion, or incomplete lineage sorting, which would cause some individuals in one species being closer to individuals of another species than to conspecifics; or (b) an origin of parapatric species pairs by recent speciation, and therefore overall low genetic divergences between them. Our data set does not allow distinguishing between these two causes, but further research into this question would be useful to understand the processes influencing the perspectives and reliability of DNA barcoding in birds. Furthermore, we should keep in mind that the named taxa PLoS ONE | www.plosone.org 6 January 2009 | Volume 4 | Issue 1 | e4119