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A paper that myself and 28 co-authors have been working on for the past several years, “Wolbachia and DNA Barcoding Insects: Patterns, Potential, and Problems” came out late last week in PLoSONE (7(5): e36514).  In it, we examine the extent to which COI sequences from Wolbachia can be detected in routine insect DNA barcoding. We further assessed the impact of Wolbachia upon the insect sequence divergence and identification accuracy.  We concluded that the presence of the Wolbachia DNA in total genomic extracts made from insects is unlikely to compromise the accuracy of the DNA barcode library; in fact, the ability to query this library for endosymbionts is one of the ancillary benefits of this large-scale endeavor. We hope that our analyses and conclusions will be of interest to a wide array of researchers as it clearly demonstrates the opportunities that DNA barcoding initiatives present for the study and understanding of the biology of perhaps the most widely distributed bacterial endosymbiont. 

Wolbachia is a genus of bacterial endosymbionts that impacts the breeding systems of their hosts. Wolbachia can confuse the patterns of mitochondrial variation, including DNA barcodes, because it influences the pathways through which mitochondria are inherited


The first portion of the paper demonstrated the rates at which bacterial COI is generated and from which insect groups in large-scale insect DNA barcoding surveys.  We examined more than 2 million insect trace files archived on the BOLD Data System (Ratnasingham, Hebert, 2007) for evidence of Wolbachia.  In only 0.16% of cases we found evidence of the bacterial endosymbiont – however, the bacterial COI was never mistaken for the insect host barcode due to the large number of differences between the two (on average 167 bp in the barcode region).  We also screened nearly 13,000 specimens specifically for Wolbachia using the wsp assay (Braig et al., 1998).


We considered the bacterial presence in the total-genomic DNA extract, and the inadvertent amplification of bacterial COI as a novel stream of metadata associated with each insect species and specimen. In the second part of the paper, we tested whether this ancillary information can be useful for the secondary testing of other hypotheses. For instance, what phylogenetic information regarding Wolbachia can be cleaned from these serendipitous amplifications?  Can we use these to learn more regarding host/symbiont codiversification? As an example of this later case, see how within one ant genus from Madagascar (Nylanderia) we generated both insect COI sequences and Wolbachia wsp sequences (Figure 1). This allows us to examine the congruence of the two tree topologies (Nye et al., 2006), and the potential for co-diversification (Merkle et al., 2010).  In this case, we observed a close correspondence between the ant and bacterial variation suggesting that if indeed these ants are judged to be different species, the bacteria may have an important part to play in telling the story of how these species arose, or maintain their distinctness. None of this would be possible without the insect COI library, nor the archive of DNA extracts.


We concluded that that a regularizing an assay for Wolbachia presence would be a useful addition to large scale insect barcoding initiatives. In addition, while COI is already one of the five multi-locus sequence typing (MLST) genes used for categorizing Wolbachia ( - Baldo et al., 2006), there is limited overlap with the eukaryotic DNA barcode region. It would be interesting to see what further benefits of aligning the biodiversity informatics libraries would be achieved by extending the current MLST into the eukaryotic DNA barcoding region. Likely, it would permit a more thorough assessment of endosymbiont diversity and create a better link between these two growing libraries of biodiversity.


Figure 1: An example of using the Wolbachia generated in DNA barcoding an insect fauna to help contextualise discoveries. Neighbor-joining trees of genetic variation (uncorrected p-dist) for insect COI and Wolbachia wsp for multiple provisional species of Nylanderia ants from three sites in eastern Madagascar. Black lines are insect COI, and red lines are Wolbachia wsp. The two trees are 72 % topologically similar and show signs of significant co-diversification (p=0.01).  (Nylanderia image from AntWeb (



Baldo L, Dunning Hotopp JC, Jolley KA, et al. (2006) Multilocus Sequence Typing System for the Endosymbiont Wolbachia pipientis. Appl Environ Microbiol 72, 7098 - 7110.

Braig HR, Zhou W, Dobson SL, O'Neill SL (1998) Cloning and characterization of a gene encoding the major surface protein of the bacterial endosymbiont Wolbachia pipientis. J Bacteriol 180, 2373 - 2378.

Merkle D, Middendorf M, Wieseke N (2010) A Parameter-Adaptive Dynamic Programming Approach for Inferring Cophylogenies. BMC Bioinformatics 11(Suppl 1), S60.

Nye TM, Liò P, Gilks WR (2006) A novel algorithm and web-based tool for comparing two alternative phylogenetic trees. Bioinformatics 22, 117-119.

Ratnasingham S, Hebert PDN (2007) BOLD: The Barcode of Life Data System Mol Ecol Notes 7, 355 - 364.


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