Cross mating experiments were designed to show whether sexual isolation exists between Anopheles quadrimaculatus, A. freeborni and A. aztecus.
It was found that a definite barrier to cross mating exists between males of A. quadrimaculatus and females of A. freeborni and A. aztecus, between males of A. aztecus and females of A. quadrimaculatus and A. freeborni, and between A. freeborni males and A. quadrimaculatus females. However, A. aztecus females were fertilized as readily by A. freeborni males as were A. freeborni females.
These results support conclusions drawn from other studies that A. aztecus and A. freeborni are much more closely related to one another than they are to A. quadrimaculatus. Sexual isolation could be a secondary result of long geographic separation. There is only a partial barrier between the A. freeborni and A. aztecus and although this might contribute towards a segregation of the two populations, the readiness with which A. aztecus females will mate with males of A. freeborni, even after a long period of geographic isolation, would lead one to suspect that in this case the primary dividing mechanism was not sexual isolation. This isolation would aid, however, in keeping the present-day populations distinct should their ranges overlap.