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Have the cake and eat it: Optimizing nondestructive DNA metabarcoding of macroinvertebrate samples for freshwater biomonitoring

ABSTRACT: DNA metabarcoding can contribute to improving cost?effectiveness and accuracy of biological assessments of aquatic ecosystems, but significant optimization and standardization efforts are still required to mainstream its application into biomonitoring programmes. In assessments based on freshwater macroinvertebrates, a key challenge is that DNA is often extracted from cleaned, sorted and homogenized bulk samples, which is time?consuming and may be incompatible with sample preservation requirements of regulatory agencies. Here, we optimize and evaluate metabarcoding procedures based on DNA recovered from 96% ethanol used to preserve field samples and thus including potential PCR inhibitors and nontarget organisms. We sampled macroinvertebrates at five sites and subsampled the preservative ethanol at 1 to 14 days thereafter. DNA was extracted using column?based enzymatic (TISSUE) or mechanic (SOIL) protocols, or with a new magnetic?based enzymatic protocol (BEAD), and a 313?bp COI fragment was amplified. Metabarcoding detected at least 200 macroinvertebrate taxa, including most taxa detected through morphology and for which there was a reference barcode. Better results were obtained with BEAD than SOIL or TISSUE, and with subsamples taken 7–14 than 1–7 days after sampling, in terms of DNA concentration and integrity, taxa diversity and matching between metabarcoding and morphology. Most variation in community composition was explained by differences among sites, with small but significant contributions of subsampling day and extraction method, and negligible contributions of extraction and PCR replication. Our methods enhance reliability of preservative ethanol as a potential source of DNA for macroinvertebrate metabarcoding, with a strong potential application in freshwater biomonitoring.

ORGANISM(S): benthic macroinvertebrates

SUBMITTER: Filipa Matos Silva Martins

PROVIDER: S-BSST241 | biostudies-other |

SECONDARY ACCESSION(S): https://www.ebi.ac.uk/ena/data/view/PRJEB31133

REPOSITORIES: biostudies-other

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Project description:Environmental DNA (eDNA) metabarcoding has been increasingly used to monitor the community assemblages of a wide variety of organisms. Here, we test the efficacy of eDNA metabarcoding to assess the composition of Iberian freshwater fishes, one of the most endangered groups of vertebrates in Spain. For this purpose, we sampled 12 sampling sites throughout one of Spain's largest basins, the Duero, which is home to approximately 70% of the genera and 30% of the primary freshwater fish in Spain. We sampled these sampling sites in the summer by using electrofishing, a traditional sampling method, and eDNA metabarcoding of river water samples using the mitochondrial 12S rRNA gene (12S) as a marker. We also resampled four of these sampling sites in autumn by eDNA. We compared the results obtained through eDNA metabarcoding with those of electrofishing surveys (ones conducted for the present study and past ones) and assessed the suitability of 12S as an eDNA metabarcoding marker for this group of freshwater fishes. We found that the 12S fragment, analysed for 25 Iberian species, showed sufficient taxonomic resolution to be useful for eDNA approaches, and even showed population-level differences in the studied populations across the tissue samples for Achondrostoma arcasii. In most cases, a greater number of species was detected through eDNA metabarcoding than through electrofishing. Based on our results, eDNA metabarcoding is a powerful tool to study the freshwater fish composition in the Iberian Peninsula and to unmask cryptic diversity. However, we highlight the need to generate a local genetic database for 12S gene for such studies and to interpret the results with caution when studying only mitochondrial DNA. Finally, our survey shows that the high detection sensitivity of eDNA metabarcoding and the non-invasiveness of this method allows it to act as a detection system for species of low abundance, such as early invasive species or species in population decline, two key aspects of conservation management of Spanish freshwater fishes.

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Have the cake and eat it: Optimizing nondestructive DNA metabarcoding of macroinvertebrate samples for freshwater biomonitoring

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