eDNA profiling of mammals, birds, and fish of surface waters by mitochondrial metagenomics: application for source tracking of fecal contamination in surface waters.

Knowing the composition of animals present in aquatic ecosystems can tell us about the anthropic pressures on these environments. One of these pressures is the occurrence of fecal contamination. However, this contamination can originate from more than one animal species in areas where urban and agricultural activities overlap. Mitochondrial DNA (mtDNA) has become the standard barcoding tool to identify the presence of animal species in environment. Amplicon-sequencing metagenomics is a powerful approach to derive the animal profile in an environment. However, PCR primers targeting mtDNA of a broad range of animals are highly degenerate or generate short DNA fragments that could cause ambiguous affiliation. Here we report the development of a new set of primers targeting the mitochondrial 16S ribosomal RNA genes of a broad range of terrestrial and aquatic animals, which include mammals, birds, and fishes. These primers successfully amplified mtDNA from environmental DNA (eDNA) extracted from surface waters. Sequencing the resulting amplicons revealed the presence of mammals and birds that may contribute in fecal contamination of surface water. In one of the river samples high in fecal indicator bacteria, human and bovine mtDNA accounted for 40.5% and 4.1% of the sequences, respectively, suggesting fecal contamination by these two animals. These findings indicate that our PCR primers coupled with amplicon-sequencing metagenomics contribute in profiling the animal diversity in the surface waters and its surrounding. This approach could be a valuable tool to identify simultaneously the potential contribution of various animals as sources of fecal contamination in surface waters.