Experimental evaluation of species and genetic variability based on DNA metabarcoding from the aquatic environment

The intraspecific genetic variation is an important basis for the assessment of organisms resistance to changing environmens and anthropogenic pressures. The aquatic DNA metabarcoding provides a non-invasive method of biodiversity research, including within-species level. In this study, we aimed to assess the intraspecific variation in different taxa based on the COI barcode (~650bp) reduced to Leray region (~313bp), as well as experimentally evaluate the identification of the Operational Taxonomic Units (OTUs) and Amplicon Sequence Variants (ASVs) in marine eDNA among abundant species of the Zostera sp. community in the northern Sea of Japan: Hexagrammos octogrammus, Pholidapus dybowskii (Teleostei: Perciformes), and Pandalus latirostris (Arthropoda: Decapoda). These species were collected in two distant locations to make the mock communities and gather the aquatic eDNA both on community and individual level. The resulting COI reads of eDNA samples when blasting on the local reference produce additional OTUs which we consider to be nuclear copies of mitochondrial sequences (NUMTS). The presence of NUMTS in the eDNA samples in turn reduces the detection of ASVs, which may be related to both the low sequencing coverage and competition of NUMTS for primer binding sites during PCR.