Long-range PCR allows sequencing of mitochondrial genomes from environmental DNA

1. As environmental DNA (eDNA) from macro-organisms is often assumed to be highly degraded, current eDNA assays target small DNA fragments to estimate species richness by metabarcoding. A limitation of this approach is the inherent lack of unique species-specific single-nucleotide polymorphisms available for unequivocal species identification. 2. We designed a novel primer pair capable of amplifying whole mitochondrial genomes and evaluated it in silico for a wide range of ray-finned fishes (Class: Actinopterygii). We tested the primer pair using long-range PCR and Illumina sequencing in vitro on a mock community of fish species assembled from pooling genomic DNA extracted from tissues. In situ we utilized long-range PCR and Illumina sequencing to generate fragments between 16 and 17kb from eDNA extracted from filtered water samples. Water samples were sourced from a mesocosm experiment and from a natural stream. 3. We validated our method in silico for 61 orders of Actinopterygii; we successfully sequenced mitogenomes in vitro from all six species in our mock community. In situ we recovered mitogenomes for all species present in our mesocosms. We additionally recovered mitogenomes from 10 of 12 species caught at the time of water sampling and two species previously only detected from eDNA metabarcoding of short DNA fragments from a natural stream. 4. Successful amplification of large fragments (>16kb) from eDNA demonstrates that not all eDNA is highly degraded. Sequencing whole mitogenomes from filtered water samples will alleviate many problems associated with identification of species from short-fragment PCR amplicon-based methods.