DNA barcodes reveal cryptic genetic diversity within the blackfly subgenus Trichodagmia Enderlein (Diptera: Simuliidae: Simulium) and related taxa in the New World

In this paper we investigate the utility of the COI DNA barcoding region for species identification and for revealing hidden diversity within the subgenus Trichodagmia and related taxa in the New World. In total, 24 morphospecies within the current expanded taxonomic concept of Trichodagmia were analyzed. Three species in the subgenus Aspathia and 10 species in the subgenus Simulium s.str. were also included in the analysis because of their putative phylogenetic relationship with Trichodagmia. In the Neighbour Joining analysis tree (NJ) derived from the DNA barcodes most of the specimens grouped together according to species or species groups as recognized by other morphotaxonomic studies. The interspecific genetic divergence averaged 11.2% (range 2.8–19.5%), whereas intraspecific genetic divergence within morphologically distinct species averaged 0.5% (range 0–1.2%). Higher values of genetic divergence (3.2–3.7%) in species complexes suggest the presence of cryptic diversity. The existence of well defined groups within S. piperi, S. duodenicornium, S. canadense and S. rostratum indicate the possible presence of cryptic species within these taxa. Also, the suspected presence of a sibling species in S. tarsatum and S. paynei is supported. DNA barcodes also showed that specimens from species that were taxonomically difficult to delimit such as S. hippovorum, S. rubrithorax, S. paynei, and other related taxa (S. solarii), grouped together in the NJ analysis, confirming the validity of their species status. The recovery of partial barcodes from specimens in collections was time consuming and PCR success was low from specimens more than 10 years old. However, when a sequence was obtained, it provided good resolution for species identification. Larvae preserved in ‘weak’ Carnoy’s solution (9:1 ethanol:acetic acid) provided full DNA barcodes. Adding legs directly to the PCR mix from recently collected and preserved adults was an inexpensive, fast methodology to obtain full barcodes. In summary, DNA barcoding combined with a sound morphotaxonomic framework provides an effective approach for the delineation of species and for the discovery of hidden diversity in the subgenus Trichodagmia.