Phlebotomine DNA barcodes and new records for Argentina

MOYA, S.L.; MARÍA GABRIELA QUINTANA; PECH-MAY, A.; MANTECA ACOSTA, M.; LAMATTINA, DANIELA; GIULIANI, M.G.; SALOMÓN, O.D.; LIOTTA, D.J.

San Cristobal, Galapagos Islands

Simposio; 10th International Symposium on Phlebotomine Sandflies; 2019

Universidad de San Francisco de Quito

Correct species identification is critical for any taxonomic group of interest, and particularly for those presenting challenges related to the existence of intraspecific phenotypic variation, cryptic species, and morphologically indistinguishable females of closely related species. Therefore, besides the still required traditional taxonomist expertise, molecular markers have been used to perform evolutionary systematics and more recently to contribute in the field of alpha taxonomy as is the proposal of the International Barcode of Life initiative. Despite the criticized choice of the mitochondrial marker, the performance of the cytochrome C oxidase subunit I (COI) gene fragment was evaluated for phlebotomine fauna from different Latin American countries showing high efficiency with few exceptions generally related to recently divergent species. Therefore, our aim is to initiate a DNA barcode library and to evaluate its performance as a complementary tool for the morphological-based identification method for the Argentinian phlebotomine fauna, currently counting 13 genera and 38 species, including 6 proven vectors of visceral and cutaneous leishmaniasis agents. Once identified, sixty-one phlebotomine were chosen for DNA extraction with commercial kits from wings and legs previously dissected, the rest of the body was clarified and stored as a voucher. In addition, sixty-two archived samples were also included derived from projects in which DNA was extracted from thorax and abdomen so no voucher was kept and, in some cases, the identification was checked again post-analysis. A 527 bp COI sequence was obtained for 123 specimen belonging to 19 species with the primers LCO1490 and HCO2198. Sequences alignment was performed using Clustal-W in MEGA v.7, as well as the estimation of K2p genetic distances, intra and inter-species, which we analyzed in order to evaluate the presence of a barcode gap. The resulting Neighbor Joining dendrogram identified 18 molecular taxonomic units (MOTUs) with high bootstrap support values (≥99%) consistent with the species morphologically identified, except for Nyssomyia whitmani and Ny. neivai given their low inter-specific genetic distance (2.8%). The last two species belong to a group with evidence of introgression and low congener divergence showing a limit to the effectiveness of the barcode tool. Nevertheless, barcoding helped to solve particular misidentifications and allowed the association between females and males among morphologically similar species of some genera, e.g. Brumptomyia and Psathyromyia.In conclusion, barcoding made possible discrimination among these phlebotomine species with 95% efficiency. This study also reported new species for Argentina: Br. cunhai, Br. nitzulescui and Br. ortizi, and generated the first barcodes for Martinsmyia alphabetica, Ny. neivai, Pa. lanei, Pintomyia pessoai, Cortelezzii complex and Shannoni complex. The availability of DNA barcodes from different geographical regions allows comparisons within and between taxonomic groups that may resolve the status of some species or reveal hiding diversity of others, in both cases setting the direction of further studies towards an integrative taxonomic approach.