How many replicates to accurately estimate fish biodiversity using environmental DNA on coral reefs?

Abstract

Quantifying fish species diversity in rich tropical marine environments remains challenging. Environmental DNA (eDNA) metabarcoding is a promising tool to face this challenge through the filtering, amplification, and sequencing of DNA traces from water samples. However, because eDNA concentration is low in marine environments, the reliability of eDNA to detect species diversity can be limited. Using an eDNA metabarcoding approach to identify fish Molecular Taxonomic Units (MOTUs) with a single 12S marker, we aimed to assess how the number of sampling replicates and filtered water volume affect biodiversity estimates. We used a paired sampling design of 30 L per replicate on 68 reef transects from 8 sites in 3 tropical regions. We quantified local and regional sampling variability by comparing MOTU richness, compositional turnover, and compositional nestedness. We found strong turnover of MOTUs between replicated pairs of samples undertaken in the same location, time, and conditions. Paired samples contained non‐overlapping assemblages rather than subsets of one another. As a result, non‐saturated localized diversity accumulation curves suggest that even 6 replicates (180 L) in the same location can underestimate local diversity (for an area <1 km). However, sampling regional diversity using ~25 replicates in variable locations (often covering 10 s of km) often saturated biodiversity accumulation curves. Our results demonstrate variability of diversity estimates possibly arising from heterogeneous distribution of eDNA in seawater, highly skewed frequencies of eDNA traces per MOTU, in addition to variability in eDNA processing. This high compositional variability has consequences for using eDNA to monitor temporal and spatial biodiversity changes in local assemblages. Avoiding false‐negative detections in future biomonitoring efforts requires increasing replicates or sampled water volume to better inform management of marine biodiversity using eDNA.

Cross-ocean patterns and processes in fish biodiversity on coral reefs through the lens of eDNA metabarcoding

“…However, our site-based and station-based accumulation curves do not reach plateaus suggesting that our sampling effort was not sufficient to exhaustively estimate fish biodiversity for each site (electronic supplementary material, analyses, figure S5) and station (electronic supplementary material, analyses, figure S6). Twentyfive replicates (so, 12 stations in case of field duplicates) could accurately estimate biodiversity regionally due to high local turnover [53]. A higher number of eDNA samples would be necessary here to reach MOTU accumulation per site and station.…”

Disentangling the components of coastal fish biodiversity in southern Brittany by applying an environmental DNA approach

“…However, replication quality and sampling effort may vary according to the studied environment because biotic and abiotic conditions can influence degradation, deposition and detection of eDNA (Stewart, 2019). For example in highly diverse tropical marine ecosystems where eDNA shedding and decay rates tend to be higher, partially due to higher water temperature (Jo et al, 2019), a study by Stauffer et al (2021) highlighted strong dissimilarity in species composition between filtration replicates (e.g., dissimilarity β jac mean = 0.729 ± 0.102 in the West Indian Ocean; β jac mean = 0.528 ± 0.146 in the Caribbean Sea, with a major contribution of the turnover component for both sampling areas) and required additional sampling efforts to ensure robust biodiversity estimates.…”

Effects of sampling strategies and DNA extraction methods on eDNA metabarcoding: A case study of estuarine fish diversity monitoring

“…Adequate water-sampling procedures for aquatic ecosystems are important for eDNA studies. Previous research has demonstrated that a large volume of filtration water is needed to represent real fauna in habitats ( Cantera et al, 2019 ; Polanco et al, 2021 ; Pont et al, 2018 ; Stauffer et al, 2021 ; Valentini et al, 2016 ). The three filtration water volumes tested in this study are commonly used in eDNA metabarcoding studies, with high success rates for species detection ( Sales et al, 2021 ; Valdivia-Carrillo et al, 2021 ).…”

“…Notably, different extraction techniques can result in differences in DNA quantity and quality, which can subsequently impact biodiversity assessment ( Coutant et al, 2021 ; Deiner et al, 2015 ; Jeunen et al, 2019 ; Piggott, 2016 ; Wittwer et al, 2018 ). At present, studies show considerable variation in the protocols and steps involved in aquatic eDNA metabarcoding, which can impact the probability of detecting species as the protocols are not specific for fish and quantitative standards for estuarine ecosystems are lacking ( Kumar et al, 2020 ; Lear et al, 2018 ; Stauffer et al, 2021 ). Thus, understanding the differences in DNA yields generated by different protocol combinations is essential for the successful application of eDNA metabarcoding in estuarine ecosystems.…”