Fast and efficient discrimination of the Isotoma viridis group (Insecta: Collembola) by PCR-RFLP

Abstract

Summary Identification of collembolan species is generally based on specific morphological characters, such as chaetotaxy and pigmentation pattern. However, some specimens do not match to described characters because these refer to adult specimens, often of one specific sex, or the characters are highly variable in adults (e.g. pigmentation, setae or furcal teeth). Isozymes have frequently assisted species discrimination, and also these may vary with developmental stage or environmental conditions. For identification of single species of the Isotoma viridis group, we present both direct sequencing of the cytochrome oxidase subunit II (COII) gene and a simple DNA-based molecular method. Five PCR primers amplifying the COII region (717 bp) of the mitochondrial DNA were used. The sequences clearly separated the species I. viridis , I. riparia and I. anglicana , irrespective of colour varieties within the first species. DNA amplification products of different species can also be distinguished by digestion with restriction endonucleases, followed by gel electrophoresis for separation of fragments. This restriction fragment length polymorphism (RFLP), obtained after digestion with the endonucleases Taq I, Vsp I, Mva I and Bsp 143I, revealed specific fragments that separated the three species from each other. Since restriction enzymes are sensitive to single base mutations, we suggest to use a combination of enzymes with at least two species-specific restriction sites when using the RFLP technique. For the I. viridis complex, Vsp I and Bsp 143I appear to be an appropriate combination.