Mapping the Genetic Relatedness of Outdoor-Biting <em>Anopheles </em>Mosquitoes in Zambia

The zoophilic and exophilic traits of outdoor-biting Anopheles has led to this group largely being overlooked for their role in malaria transmission, despite several species now recognized as locally important in regions of sub-Saharan Africa. Given the current limitations with identification of these understudied species, it is crucial to accurately correlate morphological features to molecular data. Here, we produced high quality reference sequence data for representative understudied anopheline species to better understand the phylogenetic relationships between under- and well-studied vectors of malaria. For mitochondrial genome assembly, shallow shotgun sequencing was implemented on single mosquito specimens and phylogenetic analyses were performed on the concatenated protein coding genes of the mitogenomes using a Bayesian approach. This study generated 10 complete mitogenomes focusing on less-studied taxa with an average length 15,383 bp and A-T content of 77.1% consistent with other anophelines containing 37 genes. Bayesian inference analysis yielded four main clades with molecular dating indicating that well-studied malaria vectors diverged from outdoor-biting species more than 65 million years ago. These findings support the taxonomic grouping of mosquitoes belonging to the Anopheles genus based on morphological characteristics and can provide molecular diagnostics for species identification enabling more precise and adept interventions for malaria control.