The taxonomic status and phylogenetic relationships of Toxorhynchites (Diptera: Culicidae) species from Panama

Abstract Mosquitoes of the genus Toxorhynchites are specialized predators of container-breeding Aedes mosquito larvae. Despite their biocontrol potential, species boundaries and phylogenetic relationships remain poorly understood. Utilizing country-wide sampling across Panama, we evaluated the taxonomic status and diversity of local Toxorhynchites species. We collected 147 specimens from artificial and natural containers, yielding 97 cytochrome c oxidase subunit I (COI) barcodes from 101 sequenced individuals. A regional Neighbor-Joining (NJ) analysis revealed 4 distinct, well-supported clusters (I to IV) exhibiting high mean inter-cluster genetic divergence (4.8% to 20.2%), alongside clear morphological and ecological segregation. Subsequent global NJ, Maximum Likelihood (ML), and Bayesian analyses incorporating BOLD and GenBank sequences recovered Toxorhynchites as a strongly supported monophyletic group (posterior probability = 1.0). These analyses revealed a deep phylogenetic divergence separating Old World Tx. (Toxorhynchites; Clades A and B) from New World Tx. (Megarhina; Clades C to G) lineages, placing the split during the early Miocene at 22.29 million years ago (95% Highest Posterior Density [HPD]: 16.24 to 31.08 Ma). Panamanian Clusters I and II formed 2 distinct groupings within Clade G, nesting as sisters to Subclade G1, which is characterized by severe taxonomic discordance involving sequences labeled as Tx. moctezuma, Tx. theobaldi, and Tx. rutilus. Cluster III nested within Tx. hypoptes (1.5% divergence) in Clade E, whereas Cluster IV associated with Tx. haemorrhoidalis s.l. (>5.0% divergence) in Clade C. The high degree of taxonomic uncertainty and cryptic diversity uncovered within Clades C, D, and G underscores an urgent need for a formal taxonomic revision to accurately delimit species boundaries within the genus.