Improving the COI DNA barcoding library for Neotropical phlebotomine sand flies (Diptera: Psychodidae)

Abstract

Sand fly species are traditionally identified using morphological traits, though this method is hampered by the presence of cryptic species. DNA barcoding is a widely used tool in the case of insects of medical importance, where it is necessary to know quickly which species are present in a transmission area. Here, we assess the usefulness of mitochondrial cytochrome c oxidase subunit I (COI) DNA barcoding as a practical tool for species identification, correct assignment of isomorphic females, and to evaluate the detection of cryptic diversity that occurs in the same species. A fragment of the COI gene was used to generate 156 new barcode sequences for sand flies from different countries of the Neotropical region, mainly Colombia, which had been identified morphologically as 43 species. The sequencing of the COI gene allowed the detection of cryptic diversity within species and correctly associated isomorphic females with males identified by morphology. The maximum intraspecific genetic distances ranged from 0 to 8.32% and 0 to 8.92% using uncorrected p distances and the Kimura 2-parameter (K2P) model, respectively. The minimum interspecific distance (nearest neighbor) for each species ranged from 1.5 to 14.14% and 1.51 to 15.7% using p and K2P distances, respectively. Three species had more than 3% maximum intraspecific distance: Psychodopygus panamensis, Micropygomyia cayennensis cayennensis, and Pintomyia evansi. They also were split into at least two molecular operational taxonomic units (MOTUs) each, using different species delimitation algorithms. Regarding interspecific genetic distances, the species of the genera Nyssomyia and Trichophoromyia generated values lower than 3% (except Nyssomyia ylephiletor and Ny. trapidoi). However, the maximum intraspecific distances did not exceed these values, indicating the presence of a barcode gap despite their proximity. Also, nine sand fly species were DNA barcoded for the first time: Evandromyia georgii, Lutzomyia sherlocki, Ny. ylephiletor, Ny. yuilli pajoti, Psathyromyia punctigeniculata, Sciopemyia preclara, Trichopygomyia triramula, Trichophoromyia howardi, and Th. velezbernali. The COI DNA barcode analysis enabled the correct delimitation of several Neotropical sand fly species from South and Central America and raised questions about the presence of cryptic species for some taxa, which should be further assessed.

Keywords: Barcoding; Molecular systematics; Molecular taxonomy; Phlebotominae; Single-locus; Species delimitation.

Fig. 1

Map showing the sampling sites of sand flies species used in this study. Colombia: (1) Leticia (Amazonas), (2) Puerto Nariño (Amazonas), (3) Apartadó (Antioquia), (4) Remedios (Antioquia), (5) Norcasia (Caldas), (6) Samaná (Caldas), (7) Victoria (Caldas), (8) Santa Marta (Magdalena), (9) Ovejas (Sucre); Nicaragua: (10) León/Rota; Costa Rica: (11) Limón/San Vicente, (12) Limón/Sibuju; Panama: (13) Panama Oeste/Capira-Ollas Arriba; Honduras: (14) Valle/Amapala-El Caracol

Fig. 2

Neighbor-joining phenogram of COI sequences of sand fly species from the Neotropical Region. Numbers near nodes indicate bootstrap values above 70. Lateral black bars indicate the MOTU species delimitation partitions made by the algorithms ABGD, RESL, TCS, and PTP

Fig. 3

A Neighbor-joining phenogram of COI sequences of the sand fly genera Nyssomyia and Trichophoromyia. Numbers near nodes indicate bootstrap values above 70. Clusters are colored according to the nominal species. B TCS haplotype network analysis of COI sequences of the sand fly genera Nyssomyia and Trichophoromyia. Unconnected networks are delimited MOTUs and are colored according to nominal species and NJ patterns. Each circle represents a unique haplotype, with its size proportional to the number of individuals, and the small white circles represent inferred unobserved haplotypes