Environmental DNA of preservative ethanol performed better than water samples in detecting macroinvertebrate diversity using metabarcoding

Abstract

Aim High‐throughput pipelines supported by eDNA metabarcoding have been applied in various freshwater ecosystems. Both eDNA in ethanol (EtOH) samples (ES‐eDNA) and in water samples (WS‐eDNA) can provide comprehensive classification lists with good taxonomic resolution and coverage for determining freshwater biodiversity and biomonitoring. But, the advantages of ES‐eDNA metabarcoding over WS‐eDNA metabarcoding remain unclear for routine assessments of diversity of benthic macroinvertebrates in streams. Location Qiantang River Basin, China. Methods Here, we compared ES‐eDNA and WS‐eDNA metabarcoding to evaluate the performance of two eDNA workflows in determining biodiversity and recovery of damaged macroinvertebrate communities. All eDNA samples from the environment and bulk specimen of macroinvertebrates were processed into available molecular operational taxonomic units (MOTUs) and identified to the level of genus. Results WS‐eDNA detected more exact sequence variants (ESVs) (formerly referred to as operational taxonomic units; OTUs), than did ES‐eDNA (2,866 vs. 2,406), but fewer macroinvertebrate ESVs (381 vs. 481). Among sampling sites, the two eDNA workflows exhibited relatively large dissimilarity on inferred community composition (p < .001). Furthermore, ES‐eDNA metabarcoding exhibited more consistent with morphological identification approaches than did WS‐eDNA metabarcoding (24.24% vs. 17.63%, p = .002), especially for species identified by traditional morphology (morphotaxa). Main conclusions Based on the attributes of ES‐eDNA and WS‐eDNA, it is suggested that ES‐eDNA metabarcoding performs better than does WS‐eDNA metabarcoding in detecting local biodiversity and was consistent with morphological results, while WS‐eDNA was more suitable for exploring biodiversity patterns on a broad scale, as it is the easiest and most convenient way to collect samples. Results of this study suggest ES‐eDNA metabarcoding could be an option in building molecular measurement biomonitoring programme based on EtOH sample used for preserving biological samples.

Optimised DNA isolation from marine sponges for natural sampler DNA (nsDNA) metabarcoding

“…Beta diversity analyses also indicated that different taxa were detected using different ethanol treatments for the Horniman invasive sponge. This reaffirms findings of studies on bulk invertebrate samples (Zizka et al, 2018; Derycke et al, 2021; Persaud et al, 2021; Wang et al, 2021) and marine sponges (Jeunen et al, 2019) where eDNA from ethanol preservative detected different taxa to DNA from tissue, although eDNA from ethanol preservative generally detected more taxa than bulk invertebrate tissue but less taxa than marine sponge tissue.…”

“…Residual ethanol in tissue can retain unique DNA that may leech from tissue into ethanol (Zizka et al, 2018; Marquina et al, 2019; Jeunen et al, 2019; Derycke et al, 2021; Persaud et al, 2021; Wang et al, 2021), but also react with buffers used for DNA extraction and/or inhibit PCR amplification downstream (Schrader et al, 2012). Our DNA concentration results seem to indicate that residual ethanol contained DNA unique and/or additional to that present in sponge tissue (at least for the Horniman invasive sponge) as higher total DNA yields and post-PCR DNA concentrations were observed with the wet ethanol treatment.…”

DNA Metabarcoding of Preservative Ethanol Reveals Changes in Invertebrate Community Composition Following Rotenone Treatment

“…DNA metabarcoding of eDNA from water samples often register more macroinvertebrate taxa than approaches using morphological identification (Deiner et al, 2016;Wang et al, 2021). Several studies have also mapped biodiversity by a combination of DNA metabarcoding of aquatic eDNA and DNA from tissues of bulk samples.…”

“…DNA metabarcoding of DNA from preservative fluids, first shown by Shokralla et al (2010), is one such method as it avoids specimen destruction. Although barcoding of preservative ethanol has shown some promising results compared to barcoded specimens of the corresponding bulk samples (Hajibabaei et al, 2012), and to eDNA from water samples (Wang et al, 2021), a recent study showed few shared taxa compared to bulk tissue samples (Persaud et al, 2021).…”

Towards environmental DNA-based bioassessment of freshwater reservoirs with small volumes of water: robust molecular protocols

“…Furthermore, there is no guarantee that a protocol yielding promising results for one system would also perform well for other water bodies. Regardless of these challenges, many water eDNA metabarcoding workflows are now undergoing calibration and refinement (Cristescu & Hebert, 2018;Kumar et al, 2020;McGee et al, 2019;Ruppert et al, 2019;Y. Wang et al, 2021;Zinger et al, 2019).…”