Mitochondrial introgression hampers the DNA barcoding of cryptic yellow fever vectors Haemagogus capricornii Lutz and Hg. janthinomys in the Atlantic Forest, Brazil

BACKGROUND: Yellow fever is a major public health concern in Brazil, transmitted in sylvatic cycles by Haemagogus and Sabethes mosquitoes. Among them, Haemagogus janthinomys and Hg. capricornii occur in sympatry in the Atlantic Forest and females are morphologically indistinguishable, complicating vector identification during outbreaks. Here, we aimed to investigate their taxonomic status and evolutionary history using an integrative approach including morphological and phylogenetic analyses. METHODS: Mosquitoes were collected in 17 municipalities across nine Brazilian states, including simultaneous captures of both species in sympatric areas. Males were identified by genitalia morphology and molecular analyses were performed using three mitochondrial and two nuclear genes. Diversity analyses and neutrality tests were performed, and phylogenies were reconstructed with Maximum Likelihood and Bayesian inferences. Divergence times were estimated using strict molecular clock, and population history was assessed through mismatch distribution analysis and Bayesian Skyline Plots. RESULTS: A total of 79 specimens were morphologically identified, with Hg. janthinomys showing a broader geographic and altitudinal distribution than Hg. capricornii, which was usually restricted to higher elevations. Phylogenetic analyses based on mitochondrial markers revealed two clades, but did not recover clear reciprocal monophyly, thus evidencing that these markers alone cannot separate the two species. The inclusion of nuclear markers evidenced introgression events of Hg. janthinomys mitochondria in Hg. capricornii specimens in the Paraíba River Valley and Espírito Santo State, and successive breeding of Hg. capricornii on few samples morphologically identified as Hg. janthinomys in São Paulo State. Molecular clock and population history analyses evidenced that these species have probably speciated in peripatry or parapatry during the Pleistocene era at approximately 1.2 million years ago, and a recent sudden expansion of Hg. capricornii in the last 10 thousand years ago has tripled its population and likely led to a secondary contact between the two species. CONCLUSIONS: Haemagogus janthinomys and Hg. capricornii are valid and closely related species with evolutionary histories shaped by divergence during the Pleistocene era and subsequent introgression events. The use of cytochrome c oxidase subunit I gene (COI) DNA barcoding alone could not reliably distinguish them, and integrating morphology with multiple molecular markers is essential for accurate identification. Future work is needed for a finer resolution of hybridization patterns to help clarify if the observed mito-nuclear discordance reflects historical introgression or active genetic exchange between species.