A manager's guide to using eDNA metabarcoding in marine ecosystems

Environmental DNA (eDNA) metabarcoding is a powerful tool that can enhance marine ecosystem/biodiversity monitoring programs. Here we outline five important steps managers and researchers should consider when developing eDNA monitoring program: 1) select genes and primers to target focal taxa; 2) assemble or develop comprehensive barcode reference databases; 3) apply rigorous site occupancy based decontamination pipelines; 4) conduct pilot studies to define spatial and temporal variance of eDNA; and 5) archive samples, extracts, and raw sequence data. We demonstrate the importance of each of these considerations using a case study of eDNA metabarcoding in the Ports of Los Angeles and Long Beach. eDNA metabarcoding approaches detected 94.1% (16/17) of species observed in paired trawl surveys while identifying an additional 55 native fishes and provided more comprehensive biodiversity inventories. Rigorous benchmarking of eDNA metabarcoding results improved ecological interpretation and confidence in species detections while providing archived genetic resources for future analyses. Well designed and validated eDNA metabarcoding approaches are ideally suited for biomonitoring applications that rely on the detection of species, including mapping invasive species fronts and endangered species habitats as well as tracking range shifts in response to climate change. Incorporating these considerations will enhance the utility and efficacy of eDNA metabarcoding for routine biomonitoring applications.Anacapa Toolkit: https://github.com/limey-bean/Anacapa Code for Analyses in R: https://github.com/zjgold/Port-of-LA-LB-ProjectFunding provided by: Port of Long Beach and Port of Los Angeles*Crossref Funder Registry ID: Award Number:Materials & Methods The sequence of procedures followed in the eDNA sample collection, sequencing, and analysis is summarized in Figure 3. A full detailed description of the methods is provided in Appendix 1. Field sampling: environmental DNA. To compare the efficacy of eDNA and trawling for monitoring of fish communities in the Ports of Los Angeles and Long Beach, we conducted paired eDNA and trawl surveys at seven stations on 20–21 August 2018, each of which is a standard trawl station in the conventional periodic biodiversity surveys conducted by the Ports (Tables 1, S1, S2, Figures 1 & 2C; full methods are detailed in Appendix 1). We sampled three replicate 1 L seawater samples at four locations at each of the 7 sampling stations along an approximately 150 m transect aligned with the trawl track, using the R/V Yellowfin as a sampling platform. Field Sampling: Midwater Trawls. Within 15–90 minutes of the eDNA seawater sampling, we trawled the station for 5 minutes with a 7.6 m semi-balloon otter trawl with 2.5-cm side mesh and 1.3-cm mesh cod-end deployed from the R/V Early Bird II (Figure 2D), following standard protocols of the Ports Biosurvey program, as detailed in the 2018 Biosurvey report (Wood 2021). All fish were identified to species level and standard length was recorded. See Table S2 for details of trawl locations and depths. Environmental DNA extractions, amplification, and sequencing. We exacted eDNA from the Sterivex filters within one week of collection using the DNeasy Blood and Tissue Qiagen Kit (Spens et al., 2017). We amplified eDNA with four primer sets (described below) modified with Illumina Nextera XT adapters (Illumina, San Diego, CA) in triplicate PCR replicates. We prepared sequencing libraries following the methods of Curd et al., 2019 which involved a second PCR indexing step to tag libraries (See S1 Appendix 1). Resulting indexed libraries were bead cleaned to remove fragments