Unraveling the Karenia selliformis complex with the description of a non-gymnodimine producing Patagonian phylotype

Karenia selliformis is a bloom-forming toxic dinoflagellate known for production of gymnodimines (GYMs) and causing mass mortalities of marine fauna. Blooms have been reported from coastal waters of New Zealand, Mexico, Tunisia, Kuwait, Iran, China and Chile. Based on molecular phylogeny, morphology, toxin production, pigment composition and cell growth of Chilean K. selliformis isolated in 2018 (CREAN_KS01 and CREAN_KS02), this study revealed a more complex diversity within this species than previously thought. A phylogenetic reconstruction based on the large sub-unit ribosomal nucleotide (LSU rDNA) and Internal Transcriber Spacer (ITS) sequences of 12 worldwide isolates showed that within the K. selliformis clade there are at least two different phylotypes with clear phenotypic differences. Morphological differences related to the dorsal-ventral compression, shape of the hyposome and the presence of pores on the left lobe of the hyposome. A comparison of pigment signatures among worldwide isolates revealed the existence of both acyl-oxyfucoxanthin and fucoxanthin-rich strains within the phylotypes. A LC-MS/MS screening on both Chilean 2018 K. selliformis strains showed for first time no GYMs production among cultured clones of this species. However, both CREAN_KS01 and CREAN_KS02 contained two compounds with the same mass transition as brevenal, a brevetoxin related compound. A fish gill cell-based assay showed that the CREAN_KS02 strain was highly cytotoxic but pure GYM standard did not exhibit loss of cell viability, even at cell concentrations equivalent or exceeding those reported in nature. The fatty acid profile of CREAN_KS02 included high levels of saturated (14:0; 16:0) and polyunsaturated (18:3ω6+18:5ω3; 22:6ω3) fatty acids but superoxide production in this strain was low (0.86±0.53 pmol O₂⁻ cell⁻¹ h⁻¹). A factorial T-S growth experiment using the CREAN_KS02 strain showed a μ_max of 0.41±0.03 d⁻¹ at high salinity and temperature, which points to its optimal environmental niche in offshore waters during the summer season. In conclusion, the present study provides evidence for significant genetic and phenotypic variability among worldwide isolates, which points to the existence of a K. selliformis “species complex”. The massive fauna mortality during K. selliformis bloom events in the Chilean coast cannot be explained by GYMs nor brevetoxins, but can to a large extent be accounted for by the high production of long-chain PUFAs and/or still uncharacterized highly toxic compounds.