Large-Scale Monitoring of Plants through Environmental DNA Metabarcoding of Soil: Recovery, Resolution, and Annotation of Four DNA Markers

In a rapidly changing world we need methods to efficiently assess biodiversity in order to monitor ecosystem trends. Ecological monitoring often uses plant community composition to infer quality of sites but conventional aboveground surveys only capture a snapshot of the actively growing plant diversity. Environmental DNA (eDNA) extracted from soil samples, however, can include taxa represented by both active and dormant tissues, seeds, pollen, and detritus. Analysis of this eDNA through DNA metabarcoding provides a more comprehensive view of plant diversity at a site from a single assessment but it is not clear which DNA markers are best used to capture this diversity. Sequence recovery, annotation, and sequence resolution among taxa were evaluated for four established DNA markers (matK, rbcL, ITS2, and the trnL P6 loop) in silico using database sequences and in situ using high throughput sequencing of 35 soil samples from a remote boreal wetland. Overall, ITS2 and rbcL are recommended for DNA metabarcoding of vascular plants from eDNA when not using customized or geographically restricted reference databases. We describe a new framework for evaluating DNA metabarcodes and, contrary to existing assumptions, we found that full length DNA barcode regions could outperform shorter markers for surveying plant diversity from soil samples. By using current DNA barcoding markers rbcL and ITS2 for plant metabarcoding, we can take advantage of existing resources such as the growing DNA barcode database. Our work establishes the value of standard DNA barcodes for soil plant eDNA analysis in ecological investigations and biomonitoring programs and supports the collaborative development of DNA barcoding and metabarcoding.