Bulk DNA samples detect more macrobenthos species than eDNA from the ethanol preservative and are not affected by morphological traits of the species
The characterization of macrobenthos communities by DNA metabarcoding the ethanol preservative instead of bulk specimens would greatly speed up processing time of samples in the laboratory since the decantation and sorting step can be avoided. The use of the ethanol preservative to reliably characterize macroinvertebrate diversity and composition has been demonstrated for fresh water samples (Martins et al., 2019). Our results show that the number of morphological species detected in the ethanol preservative of each sample is lower compared to the bulk DNA samples (ESM Fig 4), which has also been observed for insect communities (Zenker, Specht, & Fonseca, 2020) and for freshwater benthic communities (Hajibabaei et al., 2019). Substantial differences between both sample types have been observed with almost no shared species between bulk DNA and eDNA (Marquina, Esparza-Salas, et al., 2019). Our results also show significant differences between communities from the bulk and ethanol preservative (Table 2), but many species were detected in both sample types. The 15 species that were exclusively detected in the ethanol preservative were mainly pelagic species. In view of the low read numbers allocated to these species (at most 153 reads) these DNA molecules are most likely “contaminant” DNA molecules that were absorbed to the organic debris. Our results show that morphological traits of macrobenthos species can explain their detection in the eDNA from the ethanol preservative. Species with soft body skeleton and large body size showed higher probability of being detected in the eDNA fraction, while these traits did not strongly impact the detection in bulk DNA samples (Fig 5). The latter illustrates that mixing or grinding the samples before DNA extraction greatly increases the detection of a variety of species in bulk DNA samples.