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Comparative Study
. 2007 Jul 22;274(1619):1731-9.
doi: 10.1098/rspb.2007.0062.

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

T L Whitworth  1 , R D Dawson, H Magalon, E Baudry
Affiliations
Comparative Study

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

T L Whitworth et al. Proc Biol Sci. .

Abstract

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.

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Figures

Figure 1
Figure 1
Phylogram of the Protocalliphora genus based upon AFLP data. The tree was generated by parsimony analysis using a heuristic search with tree bisection–reconnection. Bootstrap values are shown as percentage of 1000 replicates at each node only if they are 50% or greater. The Wolbachia infection status of each individual is shown on the tree. Individuals infected with wA1, wA2 or wB Wolbachia strains are respectively represented by an open triangle, a solid triangle and a circle. Non-infected individuals are symbolized by NI.
Figure 2
Figure 2
Phylogram of the Protocalliphora genus based upon COI and COII data (total of 953 bp). The tree was generated by maximum likelihood analysis using a heuristic search with tree bisection–reconnection. Bootstrap values are shown as percentage of 1000 replicates at each node only if they are 50% or greater. The Wolbachia infection status of each individual is shown on the tree. Individuals infected with wA1, wA2 or wB Wolbachia strains are respectively represented by an open triangle, a solid triangle and a circle. Non-infected individuals are symbolized by NI. Three clusters defined using 3 or 1.8% divergence as threshold values (§3) are shown on the figure. The three ellipses indicate cases where horizontal transfer of Wolbachia between species seems probable (§3).
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