DNA barcoding cannot reliably identify species of the blowfly genus Protocalliphora (Diptera: Calliphoridae).

In DNA barcoding, a short standardized DNA sequence is used to assign unknown individuals to species and aid in the discovery of new species. A fragment of the mitochondrial gene cytochrome c oxidase subunit 1 is emerging as the standard barcode region for animals. However, patterns of mitochondrial variability can be confounded by the spread of maternally transmitted bacteria that cosegregate with mitochondria. Here, we investigated the performance of barcoding in a sample comprising 12 species of the blow fly genus Protocalliphora, known to be infected with the endosymbiotic bacteria Wolbachia. We found that the barcoding approach showed very limited success: assignment of unknown individuals to species is impossible for 60% of the species, while using the technique to identify new species would underestimate the species number in the genus by 75%. This very low success of the barcoding approach is due to the non-monophyly of many of the species at the mitochondrial level. We even observed individuals from four different species with identical barcodes, which is, to our knowledge, the most extensive case of mtDNA haplotype sharing yet described. The pattern of Wolbachia infection strongly suggests that the lack of within-species monophyly results from introgressive hybridization associated with Wolbachia infection. Given that Wolbachia is known to infect between 15 and 75% of insect species, we conclude that identification at the species level based on mitochondrial sequence might not be possible for many insects. However, given that Wolbachia-associated mtDNA introgression is probably limited to very closely related species, identification at the genus level should remain possible.