Molecular analysis supports at least two putative species within <i>Anopheles pseudopunctipennis</i> 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.