Like a map that is regularly updated, the reliability of DNA barcode databases will improve over time. To enable improvement, researchers have agreed to standardize on a particular region, to analyze multiple individuals from each species, and to revise DNA sequences and taxonomic labels as new information becomes available. By using specimens archived in museums, taxonomic identifications and DNA sequences can be re-checked. In March 2007 Med Vet Entomol 21:44, researchers from University of Wollongong, Australia, apply DNA barcoding to the identification of 9 species of forensically and medically important blowflies in family Calliphoridae. Calliphoridae blowflires cause disease in humans and domestic animals, and, in cases of murder or suspicious death, identification of blowfly species is a first step in determining the post-mortem interval. Identifications of adult flies requires specialized taxonomic knowledge and even experts have difficulty identifying egg and larval stages and the fragments of decomposed insects that may be all that is available in forensics. Nelson et al sequenced COI barcode region from legs of 52 adult flies representing 9 species in genus Chrysoma. The specimens were deposited in the Diptera collection at the School of Biological Sciences, University of Wollongong. NJ and Bayesisan analyses recovered each species as a distinct cluster, ie a well-supported reciprocally monophyletic group.
Early in the study, two complications were encountered. First, four specimens preliminarily identified as Ch. latifrons grouped with Ch. semimetallica. Second, a specimen identified as Ch. saffranea grouped with its closest relative Ch. megacephala. The adult voucher specimens were re-examined and the nuclear ITS gene was sequenced for these individuals. This confirmed that the first four specimens had been misidentified, in retrospect unsurprising given the close morphological similarity of the two species. The fifth anomalous individual was diagnosed as a hybrid based on comparison of nuclear and mitochondrial sequences. The authors conclude “the need for re-examination of misplaced specimens…highlights the importance of a voucher collection for all members of a barcode database.” I would add that the researchers’ willingness to re-examine taxonomic identifications and sequence data is just as important as the availability of voucher specimens.
Two other recent papers on blowfly identification with mitochondrial DNA showed incomplete resolution at species level, but in these the authors did not close the taxonomy-DNA circle, either by re-examining specimens or repeating sequence analysis. In Int J Legal Med 2007, Wells et al examined Lucilia sp blowflies, using published GenBank sequences and newly sequenced adult flies, and found overlap between all sister species of Lucilia for which 2 or more specimens were examined. It is unclear from this short note how many specimens were examined, their geographic origin, and whether they are stored as vouchers (online supplementary material is not available on publisher’s website at the time of this writing). There is no mention of re-examining their own specimens or analyzing other loci and of course it is not possible in most cases to confirm that taxonomic identifications and sequences in GenBank data are correct.
In July 2007 Proc R Soc B Whitworth et al examine 31 Protocalliphora individuals belonging to 12 species. Protocalliphora are Holoarctic species whose larva parasitize newly-hatched nestling birds. Blowfly larvae or pupae were collected from nests, and emergent flies were identified based on fly and pupal case morphology. As “the lower half of the abdomen of each fly was used for DNA sequencing” I assume this would not leave enough tissue for voucher specimens. They first attempted to construct a phylogeny using nuclear ITS, but found a very low level of substitutions between species and those found were all autoapomorphies, both of which suggest this is a very recently derived species complex. They were able to construct a phylogeny using amplified fragment length polymorphism (AFLP) mapping, with each species forming a reciprocally monophyletic group. This was then compared to mitochondrial sequence data.
Given that the title of the paper is “DNA barcoding cannot reliably identify….” it is inexplicable and also scientically inaccurate that they did NOT analyze the standard 648 bp COI barcode region, instead using a 374 bp fragment of COI and a 579 fragment of COII! It is likely that the results would be similar in any case, but their mitochondrial data cannot be combined with or directly compared to results with the growing DNA barcode libraries, which now contain about 260,000 barcode records from about 29,000 species. The mitochondrial sequences showed distinct clusters for 6 of the 12 species, and there were 2 other clusters comprising 2 and 4 species respectively. A separate analysis suggests these multi-species clusters reflect horizontal transfer of mitochondrial DNA among closely-related species as a result of Wolbachia infection, and the authors speculate that, since Wolbachia are found in “15-75% of insect species”, there may difficulty using DNA barcoding to resolve many insect species. To my reading, their data suggest this is a very recently derived species complex and hybridization among species is common. One of the utilities of DNA barcoding is to highlight exceptional groups, such as this one appears to be, deserving of further study. For the next studies on DNA barcoding in Lucilia and Protocalliphora, I hope the researchers retain voucher specimens and sequence the standard barcode fragment!