To aid in identifying key predators of Proconiini sharpshooter species present in California, we developed and tested molecular diagnostic markers for the glassy‐winged sharpshooter, Homalodisca coagulata (Say), and smoke‐tree sharpshooter, Homalodisca liturata (Ball) (Homoptera: Cicadellidae). Two different types of markers were compared, those targeting single‐copy sequence characterized amplified regions (SCAR) and mitochondrial markers targeting the multicopy cytochrome oxidase subunit genes I (COI) and II (COII). A total of six markers were developed, two SCAR and four mitochondrial COI or COII markers. Specificity assays demonstrated that SCAR marker HcF5/HcR7 was H. coagulata specific and HcF6/HcR9 was H. coagulata/H. liturata specific. COI (HcCOI‐F/R) and COII (HcCOII‐F4/R4) markers were H. coagulata specific, COII (G/S‐COII‐F/R) marker was H. coagulata/H. liturata specific, and lastly, COII marker (Hl‐COII‐F/R) was H. liturata specific. Sensitivity assays using genomic DNA showed the COI marker to be the most sensitive marker with a detection limit of 6 pg of DNA. This marker was 66‐fold more sensitive than marker Hl‐COII‐F/R that showed a detection limit of 400 pg of DNA. In addition, the COI marker was 4.2‐fold more sensitive than the COII marker. In predator gut assays, the COI and COII markers demonstrated significantly higher detection efficiency than the SCAR markers. Furthermore, the COI marker demonstrated slightly higher detection efficiency over the COII marker. Lastly, we describe the inclusion of an internal control (28S amplification) for predation studies performing predator gut analyses utilizing the polymerase chain reaction (PCR). This control was critical in order to monitor reactions for PCR failures, PCR inhibitors, and for the presence of DNA.

A phylogeographic analysis inferred from the partial mitochondrial cytochrome oxidase subunit I gene (433 bp) was performed with 22 populations of Diaphorina citri Kuwayama collected in the Americas and one in the Pacific. Eight populations from four countries in South America, 14 from four countries in North America, and one from Hawaii were analyzed. Twenty-three haplotypes (hp) were identified and they fell into two groups: hp1–8 were identified in South America (group 1) and hp9–23 were identified in North America and Hawaii (group 2). Hp1 and nine were present in the highest frequencies within each population and within their group, 81 and 85% for group 1 and group 2, respectively. A diagnostic nucleotide at position 48 was identified that allowed for the discrimination of the two groups; in addition, no haplotypes were shared between the two groups. An analysis of molecular variance uncovered significant genetic structure (φCT = 0.733; P < 0.001) between the two groups of the Americas. Two haplotype networks (ParsimonySplits and Statistical Parsimony) discriminated the two groups and both networks identified hp1 and nine as the predicted ancestral or founding haplotypes within their respective group. The data suggest that two separate introductions or founding events of D. citri occurred in the Americas, one in South America and one in North America. Furthermore, North America and Hawaii appear to share a similar source of invasion. These data may be important to the development of biological control programs against D. citri in the Americas.

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