Cryptic taxa in the morphospecies Bichromomyia flaviscutellata uncovered by expanded geographic sampling and COI DNA barcodes.
The Neotropical sand fly species Bichromomyia flaviscutellata is widely distributed and the main vector of Leishmania amazonensis. Previous molecular studies suggested that it comprises at least two morphologically cryptic taxa, but sampling has remained geographically limited. In this study, we expanded specimen collection across 12 Brazilian municipalities spanning the Amazon, Cerrado, and Atlantic Forest biomes, generating 62 new sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene and combining them with publicly available data. A phylogenetic gene tree, and distance- and coalescent-based delimitation methods, were used to evaluate lineage structure and assess potential species boundaries. Our results consistently revealed two deeply divergent and well‑supported mitochondrial lineages within B. flaviscutellata. One lineage (PS1) occurs across the Amazon basin, Cerrado, and an inland fragment of the western region of the Atlantic Forest, while the other (PS2) is restricted to the coastal Atlantic Forest. Additional internal structure was recovered within both lineages. Pairwise genetic distances were high between PS1 and PS2 (14%-15.7%) but remained comparatively low (< 5%) within each lineage. Other species included in the analysis, such as B. bicolor and B. olmeca, each formed a single, cohesive molecular cluster. The pronounced COI divergence observed here indicates long‑term isolation, but confirmation of these candidate species will require multilocus nuclear data. This study provides the most comprehensive molecular assessment of the B. flaviscutellata complex to date and highlights the need for integrative taxonomic approaches to formally evaluate species boundaries.