The genus Coolia comprises a group of widely distributed marine benthic dinoflagellates, with several species recognized for their production of bioactive compounds associated with benthic harmful algal blooms (BHABs). In this study, the thecal plate morphology, molecular phylogenetic relationships, and toxicity of Coolia species isolated from Perhentian Islands Marine Park (Malaysia), including C. malayensis, C. tropicalis, C. canariensis, and a novel lineage within the C. palmyrensis complex, are presented. Morphological characterization was conducted using light and scanning electron microscopy, while molecular phylogenetic relationships were inferred from the nuclear encoded ribosomal RNA gene (rDNA). Cells of C. malayensis, C. canariensis, and C. tropicalis observed in this study shared similar morphology with those reported from geographically distant regions, and clustered consistently in the phylogenetic trees inferred from the 18S–ITS–28S rDNA sequences obtained through long-read Nanopore sequencing, as well as 28S and ITS rDNA sequences generated from cultured strains. For cells that resembled C. palmyrensis, our phylogenetic analyses further revealed cryptic diversity within C. palmyrensis, with the Malaysian strains forming a distinct sister lineage within C. palmyrensis clade. The genetic divergence among the strains further supported cryptic diversity within this complex. Additionally, toxicity potential of Coolia strains obtained in this study was screened using Artemia bioassay. These findings revealed previously unrecognized diversity within the genus Coolia and highlighted the importance of integrating morphological, molecular, and toxicological approaches for accurate species identification and assessment of benthic harmful dinoflagellate diversity.

Keywords: Benthic harmful algal blooms (BHABs), Coolia palmyrensis, Coolia malayensis, Coolia canariensis, long-read sequencing