Morphogenetic characterisation, date of divergence, and evolutionary relationships of malaria vectors Anopheles cruzii and Anopheles homunculus

The mosquito species Anopheles cruzii and Anopheles homunculus are co-occurring vectors for etiological agents of malaria in southeastern Brazil, a region known to be a major epidemic spot for malaria outside Amazon region. We sought to better understand the biology of these species in order to contribute to future control efforts by (1) improving species identification, which is complicated by the fact that the females are very similar, (2) investigating genetic composition and morphological differences between the species, (3) inferring their phylogenetic histories in comparison with those of other Anophelinae, and (4) dating the evolutionary divergence of the two species. To characterise the species we used wing geometry and mitochondrial cytochrome oxidase subunit I (COI) gene as morphological and genetic markers, respectively. We also used the genes white, 28S, ITS2, Cytb, and COI in our phylogenetic and dating analyses. A comparative analysis of wing thin-plate splines revealed species-specific wing venation patterns, and the species An. cruzii showed greater morphological diversity (8.74) than An. homunculus (5.58). Concerning the COI gene, An. cruzii was more polymorphic and also showed higher haplotype diversity than An. homunculus, with many rare haplotypes that were displayed by only a few specimens. Phylogenetic analyses revealed that all tree topologies converged and showed [Anopheles bellator + An. homunculus] and [Anopheles laneanus + An. cruzii] as sister clades. Diversification within the subgenus Kerteszia occurred 2-14.2 million years ago. The landmark data associated with wing shape were consistent with the molecular phylogeny, indicating that this character can distinguish higher level phylogenetic relationships within the Anopheles group. Despite their morphological similarities and co-occurrence, An. cruzii and An. homunculus show consistent differences. Phylogenetic analysis revealed that the species are not sister-groups but species that recently diverged within the Kerteszia group, perhaps concomitantly with the radiation of bromeliads in South America or during the Pleistocene climate oscillations.