4.4 No Holy Grail for macrobenthos metabarcodes?
The heterogeneous performance of the different metabarcodes highlights the complexity of the identification of the best markers. No primer showed the best performance for all the considered metrics, as the most ”universal” marker (Euka02) showed a generally poor resolution, while the marker with the highest resolution (BF1_BR2-COI) did not successfully amplify many taxa. The selection of metabarcodes for biomonitoring is therefore a trade-off, depending on the aims of studies. Euka02 can allow a good assessment of overall biodiversity, but it is unable to tease apart closely related taxa, thus it might be not enough to define the ecological status of environments. Furthermore, the poor resolution would hamper the comparison with historical data for most of taxa. Conversely, the excellent resolution of BF1_BR2-COI could allow species-level identification, and might have more power to distinguish different communities. However, this comes at a cost. Many taxa did not amplify either because the level of DNA degradation compromises the amplification of a relatively long metabarcode, or because the poor match of the primer(s) with their target. In fact, the relatively long amplified metabarcode could limit its usefulness for application with environmental DNA extracted from water. Finally, Inse01 showed a generally good performance, but it is not appropriate for many non-insect taxa.
Given these limitations, it is unlikely that one single metabarcode will be able to fully replace the traditional biomonitoring using macrobenthic invertebrates. Nevertheless, the data obtained through multiple metabarcodes can be integrated for a more comprehensive and accurate understanding of ecological impacts on freshwater biodiversity. For instance, highly universal markers, providing a complete but coarse picture of animal biodiversity (e.g. Euka02) can be combined with markers providing a specific focus on key taxa (e.g. Inse01) or a high-resolution level (e.g. BF1_BR2-COI). The integration of multiple metabarcodes certainly increases the cost and complexity of studies, still it has the potential to provide an unprecedented amount of data, thus opening unexplored avenues to biodiversity assessment.