Our data add to the growing number of successful interspecific paternal leakage studies. We suggest the need for more surveys of natural populations of hybrid individuals and for more experimental crosses between GDC-0879 Raf inhibitor species and between divergent haplotypes within species to look for paternal leakage. Such studies are important for clarifying potential problems with analyses that rely on exclusively maternal mtDNA inheritance. In addition, such studies might help clarify the reasons why mitochondrial inheritance is ever uniparental. During the emergences of Brood IX and X of 17- year periodical cicadas, we collected unmated cicadas from various locations and performed purebred and cross-species matings by enclosing males and females in small cages. Mating cages contained either males and females of the same species or males of one species with females of another species so individuals were not free to choose the species with which they mated. Natural hybridization is rare, partly because females are unresponsive to the songs of heterospecifics. We facilitated hybrid matings by placing heterospecific mating cages near homospecific cages. This arrangement allowed females to hear males of their own species and to signal sexual receptivity, increasing the odds that a heterospecific male in her own cage would mate her. After mating, females were isolated in individually marked cages surrounding live tree branches suitable for oviposition and feeding. The cages were monitored for oviposition, and at four time periods after laying, approximately 3 eggnests were collected from each female��s cage. After the eggnests were cut from the branches, the eggs were removed and stored in 100% ethanol. We found that one of the eggnests dissected from the 1-day age group was empty, so we cut and dissected another eggnest from the same Doxorubicin Topoisomerase inhibitor female; by the time we did this, the eggnest was 4 days old. All remaining eggnests were clipped from the trees just prior to hatching and the hatching nymphs were allowed to burrow into the ground in marked 1 m2 plots in a second-growth Oak-Hickory forest in Connecticut. After approximately 16 months, cicada nymphs from one control and one hybrid cross were excavated and stored in 100% ethanol. All females in this experiment were permitted to mate only once, ruling out mixed paternity among the eggs of an eggnest. DNA was extracted from legs of adult cicadas belonging to the three different Magicicada species groups using the Nucleospin Tissue kit following instructions provided by the manufacturer. Extractions were PCR amplified using primers C1-J-2195 and TL2-N-3014 for 30 cycles. PCR product was cleaned and sequenced using BigDye terminator chemistry and an ABI Prism 3100 capillary sequencer. On the basis of these sequences, we developed internal 25-mer COI primers with 39 ends that anneal to polymorphisms unique to each species group.