Geometric morphometric and molecular techniques for discriminating among three cryptic species of the Anopheles barbirostris complex (Diptera: Culicidae) in Thailand

Anopheles members of the Barbirostris complex are important vectors of malaria in Thailand. However, they are morphologically indistinguishable because they are closely related species. In this study, wing geometric mor-phometrics (GM) and DNA barcoding based on the cytochrome c oxidase subunit 1 (COI) gene were applied to differentiate cryptic species of the Barbirostris complex in Thailand. Three cryptic species of the Barbirostris complex, Anopheles dissidens (19.44%), Anopheles saeungae (24.54%), and Anopheles wejchoochotei (56.02%) were initially identified using the multiplex polymerase chain reaction assay. DNA barcoding analyses showed low intraspecific distances (range, 0.27%-0.63%) and high interspecific distances (range, 1.92%-3.68%), consistent with the phylogenetic analyses that showed clear species groups. While wing size and shape analyses based on landmark-based GM indicated differences between three species (p < 0.05). The cross-validated reclassification revealed that the overall efficacy of wing size analysis for species identification of the Barbirostris complex was less than the wing shape analysis (56.43% vs. 74.29% total performance). Therefore, this study's results are guidelines for applying modern techniques to identify members within the Barbirostris complex, which are still difficult to distinguish by morphology-based identification and contribute to further appropriate malaria control.