Environmental DNA (eDNA) analysis allows the simultaneous examination of organisms across multiple trophic levels and domains of life, providing critical information about the complex biotic interactions related to ecosystem change. Here we used multilocus amplicon sequencing of eDNA to survey biodiversity from an eighteen-month (2015–2016) time-series of seawater samples from Monterey Bay, California. The resulting dataset encompasses 663 taxonomic groups (at Family or higher taxonomic rank) ranging from microorganisms to mammals. We inferred changes in the composition of communities, revealing putative interactions among taxa and identifying correlations between these communities and environmental properties over time. Community network analysis provided evidence of expected predator-prey relationships, trophic linkages, and seasonal shifts across all domains of life. We conclude that eDNA-based analyses can provide detailed information about marine ecosystem dynamics and identify sensitive biological indicators that can suggest ecosystem changes and inform conservation strategies.

Metabarcoding of marine environmental DNA (eDNA), originating from tissue, cells, or extracellular DNA, offers the opportunity to survey the biological composition of communities across multiple trophic levels from a non-invasive seawater sample. Here we compare results of eDNA metabarcoding of multiple trophic levels from individual seawater samples collected from a kelp forest in Monterey Bay, California in order to establish methods for future cross-trophic level eDNA analysis. Triplicate 1 L water samples were filtered using five different 47 mm diameter membrane filters (PVDF, PES, GFF, PCTE, and NC) and DNA was extracted from triplicates of each filter-type using three widely-used extraction methods (the DNeasy Blood and Tissue kit, the MoBio PowerWater DNA Isolation kit, and standard phenol/chloroform methods) resulting in 45 individual eDNA samples prepared with 15 workflow combinations. Each DNA extract was amplified using PCR primers for the 16S rRNA gene (microorganisms; Bacteria and Archaea), 18S rRNA gene (phytoplankton), and the 12S rRNA gene (vertebrates), and PCR products were sequenced on an Illumina MiSeq platform. The richness and community composition of microbial, phytoplankton, and vertebrate OTUs were not significantly different between any of the 0.2 µm pore-size filter types extracted with the DNeasy or MoBio kits. However, phenol/chloroform extraction resulted in significantly different community structures. This study provides insight into multiple choices for extraction and filtration methods to use eDNA metabarcoding for biodiversity assessment of multiple trophic levels from a single sample. We recommend any combination of either DNeasy or MoBio with PES, PCTE, PVDF, or NC filters for a cross trophic level comparison.