Molecular analysis supports at least two putative species within Anopheles pseudopunctipennis s.l. on the American mainland.

Anopheles (Anopheles) pseudopunctipennis, involved in seasonal malaria transmission in the Andean foothills and American coastal areas, was previously proposed as a species complex based on cross-mating experiments and population genetic analyses. In this work, a fragment of the mitochondrial gene cytochrome c oxidase subunit I or COI barcode region, and the nuclear second internal transcribed spacer (ITS2) were analysed in Colombian An. pseudopunctipennis s.l. specimens; the obtained sequences were compared to publicly available data using phylogeny and distance-based species delimitation approaches. Assemble Species by Automatic Partitioning (ASAP) and coalescent-based approaches provided strong evidence of at least two putative species on the American mainland, here designated as the North-Central and Southern lineages. The North-Central lineage is primarily found in southern/southwestern United States, Central America (Mexico and Honduras) and northwestern Colombia, while the Southern lineage is mainly detected in the Colombian Pacific and Argentina; there were some co-occurrences of these lineages in the Colombian regions. The definition of these putative species is crucial for understanding their bionomy, ecology and potential role in malaria transmission. Further research, including a more comprehensive sampling and population genetic analysis, is needed to fully elucidate their evolutionary history and demographic dynamics.