Influence of Preservation Methods, Sample Medium and Sampling Time on eDNA Recovery in a Neotropical River

Abstract

Environmental DNA (eDNA) has rapidly emerged as a promising biodiversity monitoring technique, proving to be a sensitive and cost-effective method for species detection. Despite the increasing popularity of eDNA, several questions regarding its limitations remain to be addressed. We investigated the effect of sampling medium and time, and preservation methods, on fish detection performance based on eDNA metabarcoding of neotropical freshwater samples. Water and sediment samples were collected from 11 sites along the Jequitinhonha River, Southeastern Brazil; sediment samples were stored in ethanol, while the same amounts of water per sample (3L) were stored in a cool box with ice, as well as by adding the cationic surfactant Benzalkonium chloride (BAC). Sediment and water samples yielded a similar amount of fish MOTUs (237 vs 239 in the first sampling event, and 153 vs 142 in the second sampling event). Water stored in ice provided better results than those preserved in BAC (239 and 142 vs 194 and 71 MOTUs). While documenting the effectiveness of eDNA surveys as practical tools for fish biodiversity monitoring in poorly accessible areas, we showed that keeping water samples cooled results in greater eDNA recovery and taxon detection than by adding cationic surfactants as sample preservatives. Furthermore, by comparing two sets of samples collected from the same locations at a three-week interval, we highlight the importance of conducting multiple sampling events when attempting to recover a realistic picture of fish assemblages in lotic systems.

Environmental DNA metabarcoding as an effective and rapid tool for fish monitoring in canals

“…Owing to the nature of the sediment, it was not always possible to collect 10 g free of macroremains. Amplification of a fragment of the mitochondrial 12S rRNA gene was conducted using the MiFish 12S primer set (Miya et al, 2015) and library preparation were conducted according to the protocol described in Sales et al (2018). A total of 29 samples (including collection blanks and laboratory negative controls) were sequenced in a single multiplexed Illumina MiSeq (www.illuima.com) run along with samples from a non-related project (Supporting Information Methods in File S1).…”

Suppression of environmental DNA degradation in water samples associated with different storage temperature and period using benzalkonium chloride

“…Thus, BAC could be utilized for a number of species in a variety of environments. Recent investigations using eDNA metabarcoding also reported the evaluation of fish detection performance in water samples preserved by BAC (Sales et al 2019).…”

Blind assessment of vertebrate taxonomic diversity across spatial scales by clustering environmental DNA metabarcoding sequences

“…Accurately delineating 'true' biological sequences from PCR and sequencing noise has been an ongoing challenge since the emergence of next generation sequencing (NGS) technologies. Clustering sequences into molecular operational taxonomic units (MOTUs) or defining exact sequence variants (ESVs) as proxies for species is a common practice in the prokaryote microbial field but also to study unicellular eukaryotes or fungi (Huse et al 2010, Schmidt et al 2013, Zimmermann et al 2015, Callahan et al 2017) and more recently eDNA of vertebrates (Closek et al 2019, Sales et al 2019.…”

“…However, these approaches focus mainly on fungi or unicellular organisms where the concept of species remains challenging (Pawlowski et al 2018, Lladó Fernández et al 2019. Even if clustering-based analyses are increasingly used in eDNA studies targeting vertebrates (Andruszkiewicz et al 2017, Bakker et al 2017, Closek et al 2019, Sales et al 2019, using the diversity of MOTUs as a reliable proxy for species diversity has yet not been evaluated. For instance, Closek et al (2019) reported a large overestimation with more than 1300 MOTUs for 92 fish taxa only in the Californian Current upwelling ecosystem.…”