New DNA markers reveal presence of Aphthona species (Coleoptera: Chrysomelidae) believed to have failed to establish after release into leafy spurge

Six species of Aphthona flea beetles from Europe have been introduced in North America for the purpose of controlling a noxious weed, leafy spurge (Euphorbia esula). In the years following the releases, five of the species have been recorded as being established at various locations. There is no evidence that the sixth species ever became established. A molecular marker key that can identify the DNA of the five established species is described. The key relies on restriction site differences found in PCR amplicons of a portion of the mitochondrial cytochrome oxidase I gene. Three restriction enzymes are required to separate the immature specimens which are not visually separable. Adults which can be quickly separated into the two black species and three brown species require only two restriction enzymes to resolve the species. Many of the original releases and relocations of the flea beetles used populations containing mixed species that were often not thoroughly characterized as to species. The markers showed the presence of two Aphthona species in North Dakota that were believed to have been absent from the state for the past decade. Without the marker assay these populations would probably have been overlooked.