Klopfstein, Seraina, Kropf, Christian & Baur, Hannes, 2016, Wolbachia endosymbionts distort DNA barcoding in the parasitoid wasp genus Diplazon (Hymenoptera: Ichneumonidae), Zoological Journal of the Linnean Society 177 (3), pp. 541-557 : 550

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https://doi.org/ 10.1111/zoj.12380

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The Wolbachia surface protein gene (wsp) sequences indicated single infections in D. deletus , D. flixi , D. tetragonus , and the outgroup species Syrphophilus asperatus and Tymmophorus obscuripes . Multiple infections, as evident from polymorphic peaks and often also length variation in the wsp sequences, were detected for D. annulatus , D. bradleyi , D. parvus , D. tibiatorius , and D. varicoxa . For the latter three, we performed molecular cloning of the wsp gene to assess how many infections were present and whether they were similar to single infections already detected in the present study ( Table 2).

Multilocus sequence typing of the singly-infected species recovered one known and three unknown Wolbachia strains ( Table 1). Diplazon deletus and D. flixi are both infected with the same strain #92 (http:// pubmlst.org/wolbachia). The sequences of the fastevolving wsp gene differs only by a single mutation in the second hypervariable region of the gene, which is further evidence for the very close relationship between those Wolbachia infections. The same strain has also been found in two lepidopteran species from the USA ( Wolbachia MLST database, http:// pubmlst.org/wolbachia; accessed 20 May 2015). Diplazon tetragonus has two previously unknown alleles for the genes FbpA and HpcA and thus harbours a new strain. Interestingly, the wsp allele found in this species has previously been found in species as diverse as the parasitic wasp Nasonia longicornis (Darling) ( Hymenoptera , Pteromalidae ), the fruit fly Rhagoletis cerasi (Linnaeus) ( Diptera , Tephritidae ), and the vinegar fly Leucophenga maculosa (Coquillett) ( Diptera , Drosophilidae ) (http://pubmlst.org/wolbachia). Diplazon tibiatorius carries multiple though closely related wsp alleles, although MLST sequence typing only recovered a single strain, which is identical to the one found in D. tetragonus . Finally, the infections of D. varicoxa and D. parvus could not be typed by the MLST approach because of multiple infections (triple or more), although one of the cloned wsp sequences corresponds to the same allele (#23) as the infections in D. tetragonus and D. tibiatorius ( Table 2). The phylogeny of the wsp sequences of the single-infected species and the successfully cloned multiple infections ( Fig. 6 View Figure 6 ) confirms the strain typing results.

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