4.3 Universality and resolution of primers
Our analysis did not identify one single outperforming metabarcode. The
universality of primers was variable among taxa, with Euka02 showing the
highest performance for some phyla (platyhelminthes, molluscs, annelids
and even some arthropods), and Inse01 showing a generally good
performance for insects. However, each of these metabarcodes has some
drawbacks. For instance, Euka02 amplifies very long sequences for some
taxa of crustaceans (Isopoda and Amphipoda; Guardiola et al.2015; Taberlet et al. 2018) thus their eDNA metabarcoding with
this marker is problematic. Conversely, Inse01 is a metabarcode
developed specifically for insects, and fails to amplify key freshwater
taxa such as Turbellaria and molluscs (Fig. 1). In our in vitroanalysis, BF1_BR2-COI showed a moderate amplification rate, still for
insects a relevant proportion of specimens were not amplified (Fig. 1).
This is in contrast with previous analyses, that successfully amplified
100% of tested insects using BF1_BR2-COI (Elbrecht & Leese 2017).
Differences might be due to DNA quality, as this primer amplifies
relatively long metabarcodes (>300 bp). Some of our
>1500 specimens were old, and this can cause DNA
degradation, while the starting material of Elbrecht and Leese (2017)
was probably of better quality. Furthermore, in several cases
BF1_BR2-COI did not amplify the DNA of our target organisms, but
amplified the DNA of contaminants, i.e. other organisms for which small
body fragments were probably present in the tube, and that perhaps
showed excellent match with the primers. Unfortunately, these conditions
(degraded DNA, and contemporaneous presence of many organisms) are
typical of eDNA metabarcoding studies, stressing the complexity of
finding appropriate primers.
Differences in performance were also strong when considering the
resolution of the metabarcodes. BF1_BR2-COI clearly showed the best
resolution while Euka02 showed a very poor performance, as in many cases
it failed even at the family level (Fig. 3; see also Tang et al.2012). COI is a highly variable region, and this has promoted its use as
standard barcode for animals (Hebert et al. 2003; Hebert et
al. 2004; Andújar et al. 2018). The excellent performance of
BF1_BR2-COI can also be explained by the relatively long amplified
region. Inse01 showed an intermediate performance, as its resolution was
insufficient for species-level identification, while genus level
identification was good for most of taxa (Fig. 3). It must be remarked
that these are optimistic values of resolution, given that our database
was far from complete, particularly at the genus-level and for some
taxa, therefore a more complete database could yield poorer resolution
values.