Genetic variability within Triatoma sanguisuga complex in North America and the re- description of Triatoma ambigua (Neiva 1911), with specimens from Florida, USA

Triatoma sanguisuga is the most widely distributed triatomine vector in North America, occurring in at least 23 U.S. states across and capable of transmitting Trypanosoma cruzi to humans. Historical morphological variability led to the description of several subspecies, but their taxonomic validity has remained unresolved. We integrated molecular (cytochrome b, n=58; ITS-2, n=23) and morphometric (18 characters, n=82 specimens) data to reassess species boundaries within T. sanguisuga sensu lato. Four species-delimitation methods (GMYC, ABGD, mPTP, hierBAPS) consistently identified four divergent mitochondrial lineages with K2P distances of 4.5–8.8%. Florida specimens formed a distinct, strongly supported clade (Group 1; K2P >7.5% from other groups) corresponding to T. sanguisuga var. ambigua (Neiva, 1911). Linear discriminant analysis achieved 100% classification accuracy between Florida and non-Florida morphotypes, with synthlipsis width providing the strongest diagnostic character (AUC >0.96). Based on concordant genetic and morphological evidence, we revalidate Triatoma ambigua (Neiva, 1911) stat. nov. as a distinct species within a newly defined T. sanguisuga complex and designate a lectotype from the Instituto Oswaldo Cruz collection. Sampling focused primarily on Florida; additional sampling in adjacent southeastern states is needed to characterize geographic boundaries. These findings have implications for Chagas disease vector surveillance in the southeastern United States.