Comparative Morphological, Ultrastructural, and Molecular Studies of Four Cicadinae Species Using Exuvial Legs

Previous studies have suggested that exuviae can be used for the identification of cicada species, but the precise characteristics that differ among species have not been determined. Thus, we performed the first comparative analyses of the leg morphology, ultrastructure, and mitochondrial DNA sequences of exuviae of four dominant cicada species in Korea, Hyalessa maculaticollis (Motschulsky, 1866), Meimuna opalifera (Walker, 1850), Platypleura kaempferi (Fabricius, 1794) and Cryptotympana atrata (Fabricius, 1775), the source of Cicadidae Periostracum, a well-known traditional medicine. A morphological analysis revealed that the profemur length, femoral tooth angle, and distance between the intermediate and last tooth of the femoral comb are useful characteristics for identification. We also evaluated the usefulness of the size, degree of reflex, and number of spines on the mid-legs and hind legs as diagnostic features. An ultrastructural study showed that Meimuna opalifera has a unique surface pattern on the legs. The sequences obtained using exuviae were identical to previously obtained sequences for adult tissues. Moreover, in a phylogenetic analysis using CO1 sequences, each species formed a monophyletic cluster with high bootstrap support. Accordingly, multiple methodological approaches using exuviae might provide highly reliable identification tools. The integrative data provide useful characteristics for the exuviae-based identification of closely related species and for further taxonomic and systematic studies of Cicadinae.