In this study, we investigate the origin and diversification of Trypanosoma cruzi vectors within the Rhodniini tribe (Triatominae subfamily) through phylogenetic analyses based on eight genes from 17 species and 497 specimens—the largest sampling of this tribe to date. Our results predominantly support the paraphyly of the genus Rhodnius, with the three Psammolestes species forming a well-supported monophyletic clade nested within it. In two reconstructions, however, Psammolestes and Rhodnius are recovered as reciprocally monophyletic, each with strong support. In Rhodnius, we find monophyletic pallescens and pictipes groups, but a paraphyletic prolixus group, with persistent phylogenetic discordances underscoring uncertainties in species placements. Divergence estimates suggest Rhodniini originated around 5.26 million years ago, notably more recent than previously thought. Evolution within the tribe appears shaped by geography, gene flow, and incomplete lineage sorting rather than traditional taxonomy. Only four species—P. arthuri, R. ecuadoriensis, R. neivai, and R. neglectus—are consistently supported across analyses, likely diversifying during Pleistocene climate changes. Other Rhodniini species may represent a panmictic population with minor structuring influenced by the Andes uplift. This study underscores the need for integrative research combining genetic, ecological, and biogeographical data to fully understand Rhodniini speciation and diversification.
