How reliable is the humeral pale spot for identification of cryptic species of the Minimus Complex?

The Anopheles minimus Complex Theobald (Diptera: Culicidae) is composed of the 3 sibling species A, C, and E. The malaria vectors An. minimus A and C are distributed over the Southeast Asian region, whereas species E is restricted to the Ryukyu Japanese Islands. Because species A and C can be sympatric and present specific behaviors and have a role in malaria transmission, it is important to differentiate them. The literature mentioned the presence of a presector pale spot on the wing costa of An. minimus A, whereas species C may exhibit both presector and humeral pale spots. However, the reliability of their diagnostic power has not been established over large temporal and geographic surveys. From the analyses of 9 populations throughout Southeast Asia, including published data and field populations from 2 sites in Thailand, we showed that the wing patterns present spatial and temporal variations that make these two morphological characters unreliable for the precise identification of An. minimus A and C. Therefore, molecular identification remains the most efficient method to obtain an unambiguous differentiation of these 2 species. Correct species identification is essential and mandatory for any relevant study on the Minimus Complex and for the application of successful control strategies.