MetaBARFcoding: DNA-barcoding of regurgitated prey yields insights into Christmas Shearwater (Puffinus nativitatis) foraging ecology at Hōlanikū (Kure Atoll), Hawaiʻi

Morphological identification of digested prey remains from a generalist predator can be challenging, especially when attempting to match degraded remains to taxonomic keys. DNA techniques, whereby prey is sequenced and matched to large public nucleotide sequence databases, are increasingly being used to augment morphological identification. We used “metaBARFcoding” (DNA metabarcoding) to target a region of the cytochrome c oxidase subunit I mitochondrial gene to identify prey in highly-digested regurgitations from Christmas Shearwaters Puffinus nativitatis at Hōlanikū (Kure Atoll). Metabarcoding was used to bulk-process 92 water samples from regurgitations collected from 2009-2017, providing an overview of the seabird’s diet. We additionally Sanger sequenced 100 prey items from 50 randomly chosen regurgitations to verify that metabarcoding characterized key components of the diet. The metabarcoding technique identified 87 unique taxa from 29 families of fish and squid, spanning diverse taxa, including reef-associated, pelagic-oceanic, and mesopelagic species. Rare prey (frequency of occurrence < 5% of samples) constituted 66% of the species richness, demonstrating the highly diverse diet of this generalist predator. Overall, 81% of the families detected in the contemporary diet were previously documented in Christmas Shearwater diets from the Northwestern Hawaiian Islands. Our results indicate that metabarcoding the cytochrome c oxidase subunit I (COI) region is useful in identifying a wide range of taxa from highly digested regurgitations, thus facilitating this approach to study seabird diets.

Methods
These are dDNA metabarcoded (Illumina MiSeq) and Sanger sequenced individual specimens from regurgitations of Christmas Shearwaters (Puffinus nativitatis) onto the runway of Kure Atoll. They are byproducts of a tagging program - the birds often regurgitate during the tagging process. All information on DNA extraction, PCR amplification, and sequencing can be found in the Supplementary Information of the manuscript.