Pheretimoid earthworms (Amynthas, Metaphire) are aggressive invasive species with ecosystem wide effects in North American hardwood forests. Three morphologically similar annual species (Amynthas agrestis, Amynthas tokioensis, Metaphire hilgendorfi) co-occur sympatrically in invaded forest. One of the factors that contributes to the scarcity of ecological data is the difficulty of distinguishing these earthworms morphologically especially when they are in their juvenile and cocoon life stages. Identification with DNA barcoding using the mitochondrial cytochrome c oxidase subunit I (COI) gene becomes expensive when studying their spatial distribution, species assembly, or phenology. To facilitate ecological studies, we developed an inexpensive multiplex PCR method that reliably identifies the three morphologically similar species. The method is based on amplifying unique sequences of different base pair lengths within the COI gene. This allowed identification based on the position of PCR products on agarose gels. The method was verified using both morphology and full COI sequences for hundreds of earthworms from several sites. We then applied it to finding the variation in relative abundance of juveniles belonging to the three species through time.
Results/Conclusions
Sixty-two adult pheretimoids were identified both by morphology and by COI barcoding. By morphology there were 28 A. tokioensis, 12 A. agrestis, 6 M. hilgendorfi, and 16 lacked the characters needed for identification. Morphological and barcode identifications matched where morphological identification was possible. Of the sixteen unidentifiable earthworms 9 were A. tokioensis and 7 were A. agrestis. The identification for a 310 adult earthworms identifiable by morphology and PCR multiplex also agreed.
We collected 193 juveniles from a single forested site on four sampling dates from April to July 2018. Probing the time course of juvenile abundance with multiplex PCR showed that the three species began hatching at the same time in early April when A. tokioensis and A. agrestis were equally abundant and only a small number of M. hilgendorfi hatchlings was present. There after A. tokioensis juveniles were dominant and M. hilgendorfi were the least abundant within this pheretimoid community.
The multiplex PCR is not only useful for obtaining the species composition of pheretimoid communities but also for ecological studies in phenology and potential inter-specific competition. By the present method, the three earthworm species could all be identified at the same time more easily, accurately and at one tenth of the cost than the usual methods.