Most of the eukaryotic diversity in the soil is hidden due to taxonomic difficulties, and the amount of labor needed to sample, extract and identify soil organisms. This is true even for animals with relatively large body size, such as earthworms. This study aimed at modifying a recently developed earthworm DNA metabarcoding methodology, which targets environmental DNA (eDNA) in the soil, and applying it to archived NEON soil samples to examine earthworm diversity on a continental scale. We compared earthworm community data derived from three datasets collected from 10 forest plots at the Smithsonian Environmental Research Center (SERC), one of the relocatable NEON sites, in Maryland, USA to evaluate the effectiveness of the eDNA approach: (1) traditional earthworm community assessment using liquid extraction from three quadrats in each plot conducted in April 2016 (the 2016 dataset); (2) eDNA data from soil cores taken in 2016 alongside the traditional earthworm community assessment following NEON’s soil sampling protocol (the eDNA dataset); and (3) long-term earthworm monitoring data collected during 2008-2016 using the same protocol as in (1) (the long-term dataset).
In the long-term dataset, a total of 12 species were observed across 10 plots at SERC. The numbers of species observed at each plot ranged from four to eight (6.3±0.4, mean±SE). In the 2016 dataset, earthworm density and fresh biomass were 168±27 individuals/m2 and 106±22 g/m2 (N=10), respectively, and a total of eight species was recorded. The numbers of species observed at each plot ranged from two to seven (4.7±0.5). At the plot level, the 2016 dataset recovered only 75±7% of the species that were present in the long-term dataset. Among the 10 plots, the 2016 dataset missed four out of the twelve, or 33% of the species recorded in the long-term dataset. The modified eDNA methodology has successfully yielded two fragments, ~30 bp and ~70 bp, respectively, of the mitochondrial 16S rRNA. Sequencing and analysis of the eDNA dataset are still underway and will be reported during the meeting. We conclude that one-time sampling using the traditional earthworm community assessment approach may underestimate species richness, and when labor and time is taken into account, we expect the eDNA approach to perform equally well or better than the traditional method.