DOI: 10.5061/dryad.j6q573ncq
title: eDNA in a bottleneck: obstacles to fish metabarcoding studies in megadiverse freshwater systems

description: 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.
description: The eDNA component of the study was conducted in six different
 sites located in the Brazilian Amazon, with four sites inserted inside the
 Adolpho Ducke Forest Reserve and two outside.  For the
 eDNA metabarcoding, from each of the four streams within Ducke Reserve,
 three water replicates were taken from three positions (from the left
 bank, middle and right bank) along a transect at 0m, 25m, and 50m using
 500 ml water bottles resulting in a total of nine replicates per stream (9
 × 500 ml). Two water‐sample replicates were collected at site A and three
 at site C (no netting was performed at these sites). At the start of each
 sampling period, a field blank was collected, totalling four field blanks
 overall. Samples were collected prior to the start of the netting sampling
 and disposable collection bottles and syringes were used to avoid the risk
 of contamination. Water samples were manually filtered using a syringe and
 Sterivex enclosed filters (0.45μm, Merck Millipore) and kept cool until
 transport to the UK. In total, 51 samples were analyzed
 (including 41 water samples, four field blanks, two extraction blanks,
 four PCR negative controls). The eDNA extraction, amplification of the 12S
 rRNA fragment using the MiFish primer set, and library preparation
 (Illumina MiSeq ‐ V2 2 × 150 bp kit) were conducted following the
 procedure described in Sales et al. (2021) and details are provided in the referred manuscript.
description: This dataset contains the raw data generated in one Illumina
 MiSeq run (V2,  2 × 150 bp kit), following the procedures described in the
 methods section.