Environmental DNA (eDNA) is generally defined as DNA present in the environment, including the DNA inside single-celled organisms. High-throughput DNA-based community analysis methods and their resulting datasets have unveiled previously unimagined biodiversity (Sogin et al. 2006). However, questions remain on how to optimise the “depth of the investigation” through sampling schemes and strategies, particularly concerning the number of samples, the number of sequences analysed per sample, and the sample volume. The significant reduction in sequencing costs has eased constraints on the first two aspects and made DNA-based analyses more feasible for inclusion in marine monitoring programmes. This transition to incorporating eDNA-based analyses in monitoring efforts has been facilitated by the research community, which has developed laboratory protocols and adopted genetic markers with relative consistency. However, minimum reliable standards for various steps in the protocol, such as the sample volume, have not yet been established.
Current phytoplankton monitoring relies on standardised methods, such as those outlined in the HELCOM COMBINE manual (HELCOM 2017) and OSPAR CEMP guidelines (OSPAR 2016), to ensure comparability between samples and laboratories. Species identification in these methods is based on morphological differences observed using a light microscope. However, species that are small (<20 µm), exhibit variable morphology, or lack distinctive characteristics are challenging to identify reliably with this approach. Often, phytoplankton analysis also includes some consumers that are important for understanding energy transfer in the marine food webs. Those are primarily microzooplankton species, e.g. a ciliate species Mesodinium rubrum. However, similarly to the phytoplankton, there are the same difficulties in reliable species identification for microzooplankton. eDNA offers a potential solution by enabling identification of such species, as it is not dependent on the cell size or morphology. However, curated reference databases are needed to reliably identify the species based on DNA sequences.
Internationally agreed standards are needed to adopt eDNA-based methodologies in routine monitoring and facilitate comparison between samples. Currently, no such standards exist for detecting marine species and only best practices, guidelines, and protocols are available (Yarimizu et al. 2020; Salomaso et al. 2021a, 2021b; Andersson et al. 2022; Jerney et al. 2022, 2023; Andersson et al. 2024). This gap is due to the ongoing rapid development of methods and differences in infrastructures, budgets, and expertise across countries and institutes (Jacobsen et al. 2023).
The focus of this project is on the sample volume used for eDNA-based detection, as the volume of water filtered determines the sampling effort. In principle, filtering higher volumes of water (up to several litres), increases the likelihood of detecting rare species, including harmful algal bloom (HAB) species. However, the absence of established upper and lower limits for sample volume can lead to unnecessary time spent on filtration.
A prerequisite for establishing an eDNA-based monitoring standard is determining a sufficient sampling effort. To address this, an experiment was conducted in which eDNA metabarcoding was applied to samples of varying water volumes collected from different locations with varying biomass levels. By keeping all other variables constant, this approach allowed to determine the minimum reliable sample volume required for assessing phytoplankton (including microzooplankton) biodiversity and detecting HAB species. The findings of this study not only increase the cost-effectiveness of eDNA-based monitoring approaches but also ensure comparability between samples, contributing to the standardisation of eDNA methodologies.