Molecular diversity of benthic harmful dinoflagellates on a tropical reef: Comparing natural and artificial substrate sampling methods using DNA metabarcoding and morphological analysis

Harmful algal blooms in the benthic system (BHAB) are a major environmental problem that has increased worldwide in the context of global climate change. While systematic cell-based BHAB monitoring for risk assessment and early warning systems have been recommended, implementation of a standardized sampling method is challenging owing to the benthic nature of these harmful microalgal taxa. This study investigated the molecular diversity of benthic harmful dinoflagellates in tropical reefs of Perhentian Islands, Malaysia, using artificial substrate (AS) and sampling natural substrates (NS), combined with environmental DNA (eDNA) analysis and high-throughput amplicon sequencing targeting the small subunit (SSU) and large subunit (LSU) rDNA markers. Our results revealed that the AS method effectively captured a representative subset of the benthic dinoflagellate community, with significant taxonomic overlap between AS and NS. Both markers enabled high-resolution detection of BHAB taxa, particularly of Gambierdiscus and Ostreopsis, which are challenging to identify by light microscopy. The LSU rDNA marker provided finer taxonomic resolution, capturing a broader range of dinoflagellate species. The molecular approach consistently aligned with cell quantification data, supporting AS and DNA metabarcoding as robust methods for BHAB monitoring. The findings highlight the potential of these methods for early detection, especially areas susceptible for ciguatera and BHAB-related poisoning, offering a systematic approach for routine cell-based monitoring.