PCR-RFLP-based method for reliable discrimination of cryptic species within Mecinus janthinus species complex (Mecinini, Curculionidae) introduced in North America for biological control of invasive toadflaxes

Several populations of the stem-mining weevil Mecinus janthinus Germar species complex (Mecinini, Curculionidae), identified based on morphological characteristics, have been introduced in North America for the biological control of invasive toadflaxes of European origin: Linaria vulgaris Miller and L. dalmatica (L.) Miller (Plantaginaceae). According to the mitochondrial cytochrome oxidase subunit II (COII) gene haplotype divergence of Mecinus janthinus species complex, a total of 20 M. janthinus s.s., 3 M. janthinus s.l. of the ‘speciosa’ genotype and 29 M. janthiniformis haplotypes have been recorded across their native range in central and southeastern Europe. A polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) diagnostic assay of COII gene using Hpy188III and MnlI enzyme-mix, was developed for fast and cost-effective discrimination of these morphologically very similar cryptic weevil species. It is shown that digestion generates unique 4-fragment restriction profile in M. janthinus s.s., 2-fragment profile in M. janthiniformis and 3-fragment profile in M. janthinus s.l. ‘speciosa’ group of haplotypes, allowing precise identification of each species or genotype. The proposed method represents a practical tool for fast and accurate identification of the target biocontrol agents and should prevent using inappropriate weevil species in redistribution programs for biological control of invasive toadflax species.