A Multilocus Nanopore eDNA Workflow for Red Algal Diversity Assessment in an Algal Reef System

Environmental DNA (eDNA) metabarcoding is a powerful tool for rapid biodiversity assessment through high-throughput sequencing of taxonomically informative genetic markers. However, the use of multiple high-resolution genetic markers in red algae (Rhodophyta) is often constrained by the short-read capacity of widely used platforms. To bridge this gap, we developed and evaluated a multilocus eDNA workflow optimized for red algal diversity using Oxford Nanopore Technologies (ONT) long-read sequencing. We targeted four genetic regions (rbcL, rpoC1, psbA, and COI-5P) and applied the workflow to one composite water sample and one composite sediment sample per site from two contrasting coastal habitats in northwestern Taiwan: a high-reef algal site (Yongan) primarily concreted by crustose coralline algae (CCA) and a low-reef sand-based site (Potou). The ONT workflow enabled commonly used red algal markers with different amplicon lengths, ranging from approximately 350 to 850 bp, to be sequenced within a single pooled run. This approach simultaneously recovered informative red algal assemblages. Marker comparison showed that although rpoC1 yielded the highest OTU (Operational Taxonomic Unit) richness, broad taxonomic and phylogenetic coverage required a multilocus strategy using rbcL, rpoC1, and psbA, whereas COI-5P was not informative for red algal recovery in our pilot workflow. Rarefaction analysis indicated that sequencing depth was sufficient to recover most of the detectable red algal ASVs within most libraries, although low red algal read numbers limited psbA recovery in both Potou libraries, especially in sediment. Our results also highlight a methodological caveat where turf and frondose algae, rather than CCA, dominated the recovered red algal reads. This result may reflect differences in eDNA shedding among different algal growth forms. Overall, our study supports the use of long-read multilocus ONT strategies for future red algal biodiversity surveys, while highlighting the need for marker-specific interpretation, improved reference databases, and calibration against traditional surveys.