Assessing vertebrate biodiversity in a kelp forest ecosystem using environmental DNA

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.

Given the rapid rise of environmental DNA (eDNA) surveys in ecology and environmental science, it is important to be able to compare the results of these surveys to traditional methods of measuring biodiversity. Here we compare samples from a traditional method (a manual tow-net) to companion eDNA samples sequenced at three different genetic loci. We find only partial taxonomic overlap among the resulting datasets, with each reflecting a portion of the larger suite of taxa present in the sampled nearshore marine environment. In the larger context of eDNA sequencing surveys, our results suggest that primer amplification bias drives much of the taxonomic bias in eDNA detection, and that the baseline probability of detecting any given taxon with a broad-spectrum primer set is likely to be low. Whether catching fish with different nets or using different PCR primer sets, multiple data types can provide complementary views of a common ecosystem. However, it remains difficult to cross-validate eDNA sequencing techniques in the field, either for presence/absence or for abundance, particularly for primer sets that target very wide taxonomic ranges. Finally, our results highlight the breadth of diversity in a single habitat, and although eDNA does capture a richer sample of the community than traditional methods of sampling, a large number of eDNA primer sets focusing on different subsets of the biota would be necessary to survey any ecological community in a reasonably comprehensive way.

Many ocean policies mandate integrated, ecosystem-based approaches to marine monitoring, driving a global need for efficient, low-cost bioindicators of marine ecological quality. Most traditional methods to assess biological quality rely on specialized expertise to provide visual identification of a limited set of specific taxonomic groups, a time-consuming process that can provide a narrow view of ecological status. In addition, microbial assemblages drive food webs but are not amenable to visual inspection and thus are largely excluded from detailed inventory. Molecular-based assessments of biodiversity and ecosystem function offer advantages over traditional methods and are increasingly being generated for a suite of taxa using a "microbes to mammals" or "barcodes to biomes" approach. Progress in these efforts coupled with continued improvements in high-throughput sequencing and bioinformatics pave the way for sequence data to be employed in formal integrated ecosystem evaluation, including food web assessments, as called for in the European Union Marine Strategy Framework Directive. DNA sequencing of bioindicators, both traditional (e.g., benthic macroinvertebrates, ichthyoplankton) and emerging (e.g., microbial assemblages, fish via eDNA), promises to improve assessment of marine biological quality by increasing the breadth, depth, and throughput of information and by reducing costs and reliance on specialized taxonomic expertise.