Reviving and characterizing three species of dinoflagellate cysts dormant for about 70 years in the East China Sea: Biecheleria brevisulcata, Biecheleriopsis adriatica , and Scrippsiella donghaienis

Many marine dinoflagellates can form resting cysts as a part of their life cycle, and the cysts could be buried in sediment and remained viable for as long as over 150 years. However, only a very limited number of cyst species have been revived from long-buried sediments and investigated in regard to a possible shift in the intra-specific genetic structure of a species detected from the historical record at a particular location. Here, we report a successful germination of three species of resting cysts that were sampled from the depth dated back to 1941±18 AD from a 44-cm sediment core from the East China Sea. Seven isolates were established from germination of single cyst isolation or multi-cyst germinations. LSU rRNA gene or ITS sequences of these strains were obtained, then they were identified to be Biecheleria brevisulcata (five strains), Biecheleriopsis adriatica (one strain), and Scrippsiella donghaienis (one strain) in terms of morphology and rRNA gene sequence. Biecheleria brevisulcata strain 1, Bps. adriatica strain 21, and S. donghaienis strain 23 were examined in detail with light microscope (LM) and scanning electron microscope (SEM), and analyzed with high performance liquid chromatography (HPLC) for their pigment compositions, and genetic diversity. We also confirmed the presence of a resting cyst of Bps. adriatica in the field for the first time. The LSU rRNA gene-based genetic distances of Bps. adriatica from that obtained from water sample, single-cell PCR sequencing for the cysts isolated from the surface sediment of the same sea area and that reported from other regions during the recent years, and ITS-based genetic distances of S. donghaienis from that obtained from cysts isolated from the surface sediment of the same location and that reported from other regions during the recent years indicated that the intra-specific genetic structure of each species in the sampling area may have shifted during the last 70 years. Our work confirms that B. brevisulcata, Bps. adriatica , and S. donghaienis , all described as new species around 2010, have inhabited the East China Sea for about 70 years. The present work reports for the first time the revival of dinoflagellate resting cysts long-buried in the coastal sediments of China, which facilitates further study on the historical occurrences of other harmful dinoflagellates and their relevance to the regional climate and environmental changes in China.