Evaluating freshwater macroinvertebrates from eDNA metabarcoding: A river Nalón case study

Abstract

Rivers are a vital resource for human wellbeing. To reduce human impact on water bodies, the European Union has established an essential regulatory framework for protection and sustainable management (WFD; 2000/60/EC). In this strategy, reliable and economic bioindicators are a fundamental component. Benthic macroinvertebrates are the group most commonly used as bioindicators through all European countries. However, their conventional assessment currently entails serious cost-efficiency limitations. In this study, we have tested the reliability of metabarcoding as a tool to record river macroinvertebrates using samples from a mock community (in vitro validation) and eDNA extracted for field validation from water from six sites within a north Iberian river (River Nalón, Asturias, Spain). Two markers (V4 region within the nuclear 18S rDNA and a fragment of the mitochondrial COI gene) were amplified and sequenced using an Illumina platform. The molecular technique has proven to be more sensitive than the visual one. A cost-benefit analysis shows that the metabarcoding approach is more expensive than conventional techniques for determining macroinvertebrate communities but requires fewer sampling and identification efforts. Our results suggest metabarcoding is a useful tool for alternative assessment of freshwater quality.

How can eDNA contribute in riverine macroinvertebrate assessment? A metabarcoding approach in the Nalón River (Asturias, Northern Spain)

“…This supports the higher sensitivity of eDNA for detecting macroinvertebrate families revealed in other studies (Deiner et al, 2016;Fernández et al, 2018). This supports the higher sensitivity of eDNA for detecting macroinvertebrate families revealed in other studies (Deiner et al, 2016;Fernández et al, 2018).…”

“…Stein, Martinez, Martinez, Stiles, Miller, and Zakharov (2014) found that DNA barcoding gives a deeper ecological signal than morphology, providing higher taxonomic richness as a result of the improvement of assignations in some groups (midges, mayflies, caddis flies, and black flies), since from DNA individuals from those groups were assigned to a species level. The application of high-throughput sequencing to eDNA samples has shown to be useful for evaluating macroinvertebrate diversity in marine and freshwater ecosystems (Aylagas et al, 2014;Deiner, Fronhofer, Mächler, Walser, & Altermatt, 2016;Dowle, Pochon, Banks, Shearer, & Wood, 2016;Fernández et al, 2018;Lejzerowicz et al, 2015), but no studies have compared results obtained by eDNA metabarcoding and that of morphological assessments for water quality. The application of high-throughput sequencing to eDNA samples has shown to be useful for evaluating macroinvertebrate diversity in marine and freshwater ecosystems (Aylagas et al, 2014;Deiner, Fronhofer, Mächler, Walser, & Altermatt, 2016;Dowle, Pochon, Banks, Shearer, & Wood, 2016;Fernández et al, 2018;Lejzerowicz et al, 2015), but no studies have compared results obtained by eDNA metabarcoding and that of morphological assessments for water quality.…”

“…However, in practice the technique is still immature. In freshwater environmental samples, Fernández et al (2018) found cytochrome oxidase subunit 1 (COI) gene as better suited than ribosomal 18S DNA for this purpose, at least partially due to the fact that there are more COI sequences of freshwater macroinvertebrates in reference databases. In freshwater environmental samples, Fernández et al (2018) found cytochrome oxidase subunit 1 (COI) gene as better suited than ribosomal 18S DNA for this purpose, at least partially due to the fact that there are more COI sequences of freshwater macroinvertebrates in reference databases.…”

“…Although it has been recently demonstrated that it is more sensitive than conventional morphological approaches for identifying macroinvertebrate families (Andújar et al, 2018;Fernández et al, 2018), the results may vary considerably depending on the specific genes and assignment criteria applied within bioinformatics pipelines (Fernández et al, 2018). From the current state of the field (Andújar et al, 2018;Deiner et al, 2017;Fernández et al, 2018;Pawlowski et al, 2018), it is clear that further validation is needed for the application of eDNA metabarcoding in water quality surveys. From the current state of the field (Andújar et al, 2018;Deiner et al, 2017;Fernández et al, 2018;Pawlowski et al, 2018), it is clear that further validation is needed for the application of eDNA metabarcoding in water quality surveys.…”

“…The upstream area of River Nalón Basin (south-central Bay of Biscay, northwest of Iberian Peninsula) was considered for field validation, as in previous studies (Fernández et al, 2018), because it contains locations of very different river water quality. The results were compared between methods and with independent indices estimated from physical and chemical indicators of habitat quality.…”