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Abstract
Cicada orni (Linnaeus, 1758) and Cicada barbara (Stal, 1866), two closely related species of cicadas both present in Portugal, are very similar and often impossible to separate on the grounds of the morphology alone. Acoustic signals produced by males during pair formation and courtship are, however, different, which is evidence for the presence of two independent species. With a view to testing the usefulness of isozyme electrophoresis for distinguishing between this pair of species, specimens of both were collected in several locations either in sympatry or in allopatry, and then assayed for 19 enzyme loci. Starch gel electrophoresis gave evidence of genetic differentiation between the groups and strong support that they constitute two independent biological species. Moreover, three loci proved to be diagnostic for the separation between C. orni and C. barbara: aspartate aminotransferase (AAT-1), creatine kinase (CK-1) and malate dehydrogenase (MDH-1).