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

Abstract

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.