Optimizing environmental DNA sampling effort for fish inventories in tropical streams and rivers

Abstract

Environmental DNA (eDNA) metabarcoding is a promising tool to estimate aquatic biodiversity. It is based on the capture of DNA from a water sample. The sampled water volume, a crucial aspect for efficient species detection, has been empirically variable (ranging from few centiliters to tens of liters). This results in a high variability of sampling effort across studies, making comparisons difficult and raising uncertainties about the completeness of eDNA inventories. Our aim was to determine the sampling effort (filtered water volume) needed to get optimal inventories of fish assemblages in species-rich tropical streams and rivers using eDNA. Ten DNA replicates were collected in six Guianese sites (3 streams and 3 rivers), resulting in sampling efforts ranging from 17 to 340 liters of water. We show that sampling 34 liters of water detected more than 64% of the expected fish fauna and permitted to distinguish the fauna between sites and between ecosystem types (stream versus rivers). Above 68 liters, the number of detected species per site increased slightly, with a detection rate higher than 71%. Increasing sampling effort up to 340 liters provided little additional information, testifying that filtering 34 to 68 liters is sufficient to inventory most of the fauna in highly diverse tropical aquatic ecosystems.

Detecting Native Freshwater Fishes Using Novel Non-invasive Methods

“…The detection probabilities of redfins and Cape Kurper using eDNA were the lowest among the three gear types, but it detected both species in all four tributaries. While slightly lower than the visual observation methods, detection probability is within the range of other eDNA studies of rare freshwater species in lotic habitats (e.g., Schmidt et al, 2013;de Souza et al, 2016;Lopes et al, 2017;Bylemans et al, 2019;Sutter and Kinziger, 2019), but was highly dependent upon sampling strategy including volume of water filtered (Wilcox et al, 2016;Cantera et al, 2019;Hunter et al, 2019), number of PCR technical replication effort (Piggott, 2016), size, material, and pore size of the filter (Majaneva et al, 2018), the seasonal hydrology of the system, and rarity of the target (Buxton et al, 2017). Some of these factors, such as rarity, cannot be controlled and are specific to the species under investigation.…”

“…Turbidity, a proxy of organic solids containing PCR inhibitors, such as tannic and humic acids, has severe negative effects on the detection of species using eDNA, and increases susceptibility of the survey to false negatives (Harper et al, 2019). PCR inhibitors may also have unpredictable effects on increasing the volume of water sampled (Cantera et al, 2019). The impact of inhibitory substances can be alleviated by diluting samples prior to amplification; however, this practice will also decrease concentrations of target DNA.…”

“…In this study, including the detections and non-detections of each eDNA replicate of each pool, instead of using the combined-replicate eDNA detection and non-detections, into the occupancy models reduced the eDNA detection probability by about 20%, revealing that there is a lot of variation in detection across replicates taken from a pool. Increasing the number of physical replicates and the total volume of water sampled should improve detection probability of both species, assuming inhibition of the PCR reaction can be overcome (Cantera et al, 2019). In addition, for these remote locations, filtering on site is also more appropriate to limit degradation of eDNA and reduce effort required to transport samples.…”

“…Nevertheless, even if the appropriate facilities for processing eDNA samples are available, methods need to be optimized for the target species, ensuring a minimum acceptable standard for deployment (Klymus et al, 2020), analogous to the MIQE standards in clinical PCR applications (Bustin et al, 2009). Once optimized, these approaches are widely applicable across broad geographic scales, taking into account environmental factors that may influence their efficacy (Cantera et al, 2019). The standardized nature of these studies has made it easier to study wide-spread invasive (Amberg et al, 2015) and commercially valuable fishes (Thomsen et al, 2016), and make detection studies more reproducible (Mauvisseau et al, 2019).…”

“…However, the effectiveness of eDNA studies may depend on the hydrological and physical aspects of the habitat (Jane et al, 2015;Balasingham et al, 2017;Harper et al, 2018), and species-specific differences in shedding, behavior, and abundance (Thomsen and Willerslev, 2015). In addition, eDNA studies have predominantly focused on temperate species, and the utility of these studies in warmer tropical and subtropical conditions may differ (Eichmiller et al, 2016;Cantera et al, 2019;Doble et al, 2019).…”