Surveying biodiverse coral reef ecosystems across the tree of life with environmental DNA

A central challenge in evaluating biodiversity using eDNA metabarcoding is that each primer set has biases resulting in selective amplification of a unique composition of taxonomic diversity. Here we test eleven primer pairs (Amaral-Zettler 18S V9, Govinarajan 18S V9, Guardiola 18S V7, Stat 18S V4, Berry 16S, MiFish E 12S, MiFish U 12S, Riaz 12S, Leray CO1, Martineau 28S-S, and Sherwood 23S) to assess Central Pacific coral reef diversity in the five kingdoms of marine eukaryotes: Chromista, Fungi, Metazoa, Plantae, and Protozoa. Zero-width OTUs (ZOTUs) were compared to public sequence databases and resolved to the finest taxonomic rank possible. The highest number of unique taxa was recovered by Govinarajan 18S V9 primers (127) largely comprised of Chromista and Metazoa, followed by Martineau 28S-S primers (100) with similar taxonomic composition. All primers except the algae-focused Sherwood 23S recovered Metazoa, while all markers except Riaz 12S recovered taxa in the kingdom Plantae. None of these primers performed particularly well for Fungi, Cercozoa or Protozoa. At the phylum level, Riaz 12S, Berry 16S, MiFish 12S U and MiFish 12S E recovered the most taxa of Chordata. Marineau 28S, Govinarajan 18S V9, and Amaral Zettler 18S V9 recovered the most Porifera taxa, with highest counts coming from the most diverse class Demospongiae. At the class level, the "fish" primers (Riaz 12S, MiFish 12S U, Berry 16S, and MiFish 12S E) preferentially recovered Actinopteri (ray-finned fishes). While each primer set proved uniquely informative for some taxa, the taxon recovery profiles provided here should aid researchers in matching the taxonomic specificity of environmental metabarcoding primers with research questions of interest.