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 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; post-hoc K2P distances among these groups ranged from 4.78 to 8.20% (mean pairwise distances). Florida specimens formed a distinct, strongly supported clade (Group 1; mean K2P 7.19–8.20% from other groups) corresponding to T. sanguisuga var. ambigua Neiva, 1911. Linear discriminant analysis discriminated Florida and non-Florida morphotypes (100% resubstitution accuracy; 80–100% accuracy on small held-out random-forest test subsets), with synthlipsis width providing the strongest diagnostic character (AUC > 0.96). Based on concordant mitochondrial and nuclear evidence and geographically congruent morphological evidence, we revalidate Triatoma ambigua Neiva, 1911 stat. rev. as a distinct species within a newly defined T. sanguisuga complex and designate a neotype from the Instituto Oswaldo Cruz collection. Additional triatomine sampling in Florida and adjacent southeastern states is needed to characterize geographic boundaries. These findings have implications for Chagas disease vector surveillance in the southeastern United States.