Identifying the unidentifiable: A PCR multiplex protocol for the diagnosis of invasive pheretimoid earthworm species, verified by morphological and barcode identification

Identification of taxa that are morphologically very similar is a standing problem in ecology and is particularly important for invasive species that may differ in their dispersal rates and environmental and economic costs. Three similar pheretimoid earthworms (genera of Amynthas, Metaphire) are important invasive species in North American forests. They often lack the diagnostic morphological characters and their hatchlings, juveniles, and cocoons are impossible to identify to species. We present a multiplex PCR protocol that accurately scores the three species and is inexpensive compared to other molecular methods. Multiplex PCR identification was as accurate as mitochondrial COI barcoding and better than morphological scoring. The method uses unique PCR fragments of different lengths for each species from the COI gene. The multiplex PCR correctly identified embryos within cocoons, juveniles, and adults of Amynthas agrestis, Amynthas tokioensis and Metaphire hilgendorfi, with 100% accuracy. Comparisons of COI sequences (GenBank) with other populations of the same species (including specimens from their native range in Japan), and many other species of earthworms showed the primers always amplify only the three target species. The multiplex PCR method is rapid and costs a fraction of standard COI barcoding. Also, tiny scratches of cells from living specimens can be entered directly into the PCR for easy identification, reducing costs even further by avoiding DNA extraction, and allows the earthworms to be conserved for ecological experiments. To show the utility of the method we present the hatching phenology of the three species which could not be done by morphology. The three species begin hatching at the same time with A. tokioensis producing the most abundant juveniles early in the season. The method facilitates studies on biogeography, phenology, life histories, and resource partitioning among the three co-occurring earthworm species. [Display omitted] •A multiplex PCR method was developed to identify morphologically similar pheretimoids.•The method was verified with identification by morphological characters and COI gene barcoding.•All life stages of pheretimoids were identified accurately using species-specific primers.•Considerable cost savings were achieved with the method allowing extensive ecological studies.•Hatchings of the three pheretimoid species began to emerge at the same time in spring.