We demonstrated that the protocol for molecular analysis of samples fixed in Carnoy’s solution is straightforward and
successful. The procedure should be particularly useful for molecular study of black flies, not only for DNA barcoding, but also for population genetics, phylogeographic analysis, and phylogenetics of species complexes requiring cytological
identification before molecular study. Our results also indicate the usefulness of DNA barcoding for taxonomy.
Although the success of using DNA barcoding in the subgenus Gomphostilbia was rather low, it can facilitate traditional
taxonomy. The cryptic biodiversity discovered in our study is consistent with previous reports of high levels of
hidden diversity of black flies in Thailand (Jitklang et al. 2008; Tangkawanit et al. 2009).