Sonet, G.,  Pauly, A.,  Smitz, N.,  Virgilio, M.,  Nagy, Z.,  Jordaens, K.,  Molle, S.,  Backeljau, T. & De Meyer, M. 2015. ‘High-throughput sequencing of PCR amplicons: a test to barcode a bee species complex (Hymenoptera: Apoidea: Halictidae) and survey Wolbachia infections’. 6th International Barcode of Life Conference. Book of abstracts. 283.

Conference abstract

Background: High-throughput sequencing of PCR amplicons, also called targeted amplicon sequencing (TAS), combines the flexibility of PCR amplification with next-generation sequencing (NGS) technologies. In comparison with Sanger sequencing, NGS potentially improves the sequencing success rate and the detection of heteroplasmy, heterozygosity in nuclear markers, and endosymbionts. Here, we applied TAS to simultaneously sequence the COI barcode region, three nuclear markers (wingless, white gene, and HOG7036- 02), and a fragment of the Wolbachia surface protein (wsp) in 24 museum bee specimens of Halictus (Seladonia). This bee genus is frequently infected by Wolbachia, and one of the species, Halictus smaragdulus Vachal, 1895, is suspected to be a species complex on the basis of the morphological variation in the male genitalia. Results obtained for the DNA barcode fragment were compared to those obtained by Sanger sequencing, using the same specimens and DNA extracts. Results: Sequencing of COI was more successful with NGS (21/24 specimens) than with Sanger sequencing (18/24 specimens). COI haplotypes obtained from both approaches were identical and showed divergences that were congruent with the male genitalia differentiation. These results suggest that H. smaragdulus comprises more than one species. No signs of heteroplasmy were observed. Nuclear markers were successfully sequenced for 15-20 (62%–83%) of the specimens, and Wolbachia was detected in 50% of the individuals. Significance: By sequencing standard DNA barcodes and specific DNA markers (including DNA fragments from Wolbachia), we produced a dataset that allows a better taxonomic interpretation of the species complex.