DOI: 10.5061/dryad.15dv41p0d
title: Enviromental DNA datasets of the Colombian Amazon and Orinoco basins

description: The massive loss of biodiversity in recent years has driven the
 development of rapid, cost-effective, non-invasive, and efficient sampling
 alternatives, such as environmental DNA. With this method, a water sample
 can be used to evaluate a community's diversity, in addition with low
 abundance, cryptic and threatened species detection. Therefore, in this
 study, environmental DNA was used to determine the diversity of aquatic,
 semi-aquatic and terrestrial vertebrates in the Colombian Amazon and
 Orinoco basins, which included four main subregions: Bojonawi Natural
 Reserve and adjacent areas (Vichada Department), Sierra de la Macarena
 National Park and Tillavá (Meta Department), Puerto Nariño and adjacent
 areas (Amazonas Department) and the Municipality of Solano (Caquetá
 Department). A total of 709 OTUs were identified for all locations. The
 Orinoco river showed the highest number of fish genera (68) and the
 Guayabero river, the largest number of genera for tetrapods (13). New
 taxonomic records were found on all locations, mianly in Bita, Orinoco and
 Tillavá rivers, which portrayed the highest record of unknown fish
 diversity compared with traditional surveys. Likewise, two vulnerable fish
 species and three vulnerable mammal species were identified, as well as
 four threatened mammal species, including the giant otter (Pteronura
 brasiliensis), the giant anteater (Myrmecophaga tridactyla), the two
 subspecies of the Amazon river dolphin (Inia geoffrensis geoffrensis and
 Inia geoffrensis humboldtiana) and the tucuxi (Sotalia fluviatilis). It is
 essential to improve current DNA sequence databases for the neotropics and
 standardize the methodology according to the animal of interest in order
 to develop future studies that maximize environmental DNA analyses
 efficiency.
description: 30 locations were sampled
 based on the methodologies used by NatureMetrics laboratory
 (NatureMetrics, 2019) and the studies of Lozano and Caballero (2020) and
 Caballero et al. (2021a). For each location, up to seven water subsamples
 (1L each) were taken in a plastic bottle and then were poured into a
 bucket covered with a plastic bag. The bottle, as well as the plastic bags
 were previously sterilized with 90 % ethanol thoroughly. Every water
 sample was taken with sterile gloves to avoid human DNA contamination and
 all plastic bags were changed after each sampling event to prevent the
 mixing of water from different sampling locations. The water collection
 was made each 10-20 m along a linear transect, carried out by boat for the
 rivers, canoes for the lakes-lagoons and on foot for the streams, taking
 the coordinates of each sampled point with a GPS (Garmin etrex 12 channel
 GPS). Once all the subsamples of a location were taken, the process of
 filtration began using a NatureMetrics eDNA collection kit. A 60 ml
 syringe filled with the collected water was attached to a filter disk with
 a 0.8 μm pore size. Then,
 when the filter disk was clogged and no more water could go through, the
 syringe was detached and a smaller syringe with a preserving buffer was
 used to protect the filter and avoid DNA degradation. Each filter was
 stored in an envelope with their respective field information and were
 kept cool in styrofoam cooler with ice packs.
 Filters were transported to
 Nature Metrics (Egham, Surrey, England), where laboratory procedures took
 place. DNA was extracted using Qiagen DNeasy Blood & Tissue Kit
 (see manufacturer instructions), modifying some steps to obtain increased
 DNA yields. Subsequently, DNA was purified using the DNeasy PowerClean Pro
 Cleanup kit to remove PCR inhibitors. Then, DNA extracted from each filter
 was amplified using 12 replicates, with the 12S rRNA mitochondrial gene to
 target fish as part of the ​eDNA survey - Vertebrates​ pipeline (Milan et al. 2020). Tails were added
 at the 5′ end of the primers to be complementary with
 Illumina Nextera index primers. The amplification mixture for each
 replicate contained 1X DreamTaq PCR Master Mix (Thermo Scientific), 0.4
 μM of each of the tailed
 primers, 1 μL of DNA and
 PCR grade water (Thermo Scientific) up to a total reaction volume of 10
 μL. All PCRs were
 performed in the presence of both a negative control and a positive
 control sample (mock community with a known composition, not to occur in
 Colombia). PCR conditions consisted of an initial denaturation at 95°C for
 2 min, followed by 10 cycles of 20 s at 95°C, a 30 s touchdown annealing
 step (-0.5°C per cycle) starting at 60°C, and 40 s at 72°C, 35 cycles of
 20s at 95°C, 30s at 55°C, and 40s at 72°C, and a final elongation step at
 72°C for 5 min. Amplification success was determined by gel
 electrophoresis. Amplicons were pooled
 and purified with MagBind TotalPure NGS (Omega Biotek) magnetic beads with
 a ratio 0.8:1 (beads:DNA) to remove primer dimers and then quantified
 using a Qubit high sensitivity kit according to the manufacturer’s
 protocol. All purified index PCRs were pooled into a final library with
 equal concentrations. The final library was sequenced using an Illumina
 MiSeq V2 kit at 12 pM with a 10% PhiX spike in. Sequence data were
 processed using a custom bioinformatics pipeline, USEARCH v11, for quality
 filtering, dereplication and taxonomic assignment (≥ 80% agreement in the
 overlap). Forward and reverse primers were trimmed from the merged
 sequences using cutadapt 2.3 (Martin,1994; Mathon et al. 2021) and
 retained if the trimmed length was between 80 - 120 bp. These sequences
 were quality filtered to retain only those with an expected error rate per
 base of 0.01 or below and dereplicated by sample, retaining singletons.
 Unique reads from all samples were denoised in a single analysis with
 UNOISE (Dal Pont et al. 2021), requiring retained ZOTUs (zero-radius OTUs)
 to have a minimum abundance of 8. ZOTUs were clustered at 99% similarity.
 An OTU-by-sample table was generated by mapping all dereplicated reads for
 each sample to the OTU representative sequences at an identity threshold
 of 97%. Taxonomic information was added to each OTU by
 means of sequence similarity searches against the NCBI
 ​nt​ database (GenBank) and PROTAX
 (Somervuo et al. 2016; Lozano and
 Caballero, 2021). Identifications from either source were accepted and
 these were consistent at the level at which they were made. Species and
 genus level assignments were automatically retained if supported by
 unambiguous matches to reference sequences at ≥99% or ≥95%, respectively.
 Public records from GBIF were used to assess which hits were most likely
 to be present in Colombia, in cases where there were equally good matches
 to multiple species. This allowed numerous uncertain sequences to be
 resolved to species level. OTUs that were ≥99% similar and had similar
 co-occurrence patterns were combined with LULU (Frøslev et al. 2017) and
 the OTU table was then filtered to remove low abundance OTUs from each
 sample (&lt;0.05% or &lt;10 reads). Finally, human, known food
 fish and livestock sequences were removed, in addition with OTUs
 identified above order- level.