How Fragmented Is eDNA? A Case Study on Shark DNA in Tropical Reef Seawater

Abstract

Environmental DNA (eDNA) is broadly assumed to be highly fragmented (< 600 bp) in seawater. However, several marine eDNA studies that have successfully amplified longer fragments (from 600 up to 16,000 bp) are challenging this notion. We hypothesized that a small, yet amplifiable, proportion of eDNA templates contain fragment lengths exceeding 600 bp. To test this, we designed primers to target a series of mitochondrial fragment lengths between 119 and 15,727 bp for the tiger shark (Galeocerdo cuvier) and performed qPCR on seawater eDNA samples collected from the offshore, tropical Kimberley and Roebuck Marine Parks in Western Australia. We observed a steep decrease in eDNA copy number with increasing fragment size between 119 and 1518 bp, beyond which amplification was not successful. Importantly, we demonstrate that fragment sizes larger than conventionally targeted (e.g., 636, 840, and 1518 bp) can still be successfully amplified from seawater eDNA samples. Estimated mean nucleotide damage in seawater eDNA samples was found to be 3.9 breaks per 1000 bp; this equates to a mean undamaged fragment size of 256 bp and is less than damage observed in modern fecal DNA and ancient DNA. Characterizing the extent of eDNA fragmentation in various environmental samples will improve understanding of the genetic material available and enable practitioners to target standard length barcodes and longer hypervariable gene regions. Through the recovery of more informative data, eDNA applications will extend to finer-scale taxonomic resolution, including complex species and sub-species discrimination, as well as population analyses.