eDNA in a bottleneck: obstacles to fish metabarcoding studies in megadiverse freshwater systems

Abstract

The current capacity of environmental DNA (eDNA) to provide accurate insights into the biodiversity of megadiverse regions (e.g., the Neotropics) requires further evaluation to ensure its reliability for long-term monitoring. In this study, we first evaluated the taxonomic resolution capabilities of a short fragment from the 12S rRNA gene widely used in fish eDNA metabarcoding studies, and then compared eDNA metabarcoding data from water samples with traditional sampling using nets. For the taxonomic discriminatory power analysis, we used a specifically curated reference dataset consisting of 373 sequences from 264 neotropical fish species (including 47 newly generated sequences) to perform a genetic distance-based analysis of the amplicons targeted by the MiFish primer set. We obtained an optimum delimitation threshold value of 0.5% due to lowest cumulative errors. The barcoding gap analysis revealed only a 50.38% success rate in species recovery (133/264), highlighting a poor taxonomic resolution from the targeted amplicon. To evaluate the empirical performance of this amplicon for biomonitoring, we assessed fish biodiversity using eDNA metabarcoding from water samples collected from the Amazon (Adolpho Ducke Forest Reserve and two additional locations outside the Reserve). From a total of 84 identified Molecular Operational Taxonomic Units (MOTUs), only four could be assigned to species level using a fixed threshold. Measures of α-diversity analyses within the Reserve showed similar patterns in each site between the number of MOTUs (eDNA dataset) and species (netting data) found. However, β-diversity revealed contrasting patterns between the methods. We therefore suggest that a new approach is needed, underpinned by sound taxonomic knowledge, and a more thorough evaluation of better molecular identification procedures such as multi-marker metabarcoding approaches and tailor-made (i.e., order-specific) taxonomic delimitation thresholds.

The critical role of natural history museums in advancing eDNA for biodiversity studies: a case study with Amazonian fishes

“…Likewise 37 , detected and correctly assigned all 67 species with 12S previously designed primers and reference library in the São Francisco River, Brazil. In contrast 32 , assigned only 4 of 84 MOTUs to species, demonstrating problems of taxonomic resolution in the target gene and general threshold used for species assignment.…”

“…Thus, it is likely that, in the near future, DNA barcoding and eDNA metabarcoding methods will converge to use large portions of the mitochondrial genome. Regardless of the fragment or the threshold used to delimit species (e.g., 32 ), it is essential that studies involving eDNA for assessing fish diversity move towards building robust mitochondrial DNA reference libraries based on vouchered specimens.…”

“…Most metabarcoding inventories of freshwater fishes have been conducted in temperate habitats with well-characterized species diversity 28 . There have been only a few attempts to use eDNA metabarcoding in ichthyological surveys in the Neotropical region 29 – 32 , an area where understanding species-level diversity is more complex. For example 30 , built 12S eDNA metabarcoding primers based on a reference library for over 130 species known to occur in the rivers and streams of the French Guiana, and the eDNA results were compared with capture-based sampling methodologies.…”

“…They recovered a similar number of species, with a partial match to species identification, using both capture-based and eDNA approaches. Conversely 32 , used MiFish primers 3 in three localities in the central Amazon and suggested that a new approach would be necessary to evaluate the Neotropical fish fauna using eDNA metabarcoding.…”

Monitoring fish communities through environmental DNA metabarcoding in the fish pass system of the second largest hydropower plant in the world

“…Although eDNA metabarcoding is a powerful tool for biodiversity, as it has been widely used for different purposes and different taxonomic groups, including identification and quantification of Neotropical ichthyofauna 16,76,77 , many issues can hamper the metabarcoding results 7,10,78,79 . Shaw et al 10 drew attention to methodological considerations related to the eDNA sampling process for freshwater fishes.…”