In Situ Harvesting and Molecular Identification for the Germinating Species Diversity of Dinoflagellate Resting Cysts in Jiaozhou Bay, China

Dinoflagellate resting cysts are critical to dinoflagellate ecology, acting as a key seed source for initiating harmful algal blooms (HABs) through their germination. However, the in situ germination dynamics of these cysts remain poorly understood due to technical challenges. To overcome this, we utilized the Germlings Harvester (GEHA), an in situ germination device we designed, to collect water samples containing dinoflagellate cysts germinated from marine sediments in Jiaozhou Bay, China, after 5 and 20 days of incubation. By combining the GEHA with metabarcoding analysis targeting 28S rDNA-specific primers for dinoflagellates, we identified 44 dinoflagellate species spanning 31 genera, 18 families, and 7 orders. Of these, 12 species were linked to HABs or recognized as toxic, including <i>Azadinium poporum</i>, <i>Alexandrium leei</i>, <i>Alexandrium pacificum</i>, <i>Akashiwo sanguinea</i>, <i>Karlodinium veneficum</i>, <i>Stoeckeria algicida</i>, and <i>Luciella masanensis</i>. Additionally, five species were newly identified as cyst producers, and one symbiotic dinoflagellate, <i>Effrenium voratum</i>, was detected. Our results also found that germinated dinoflagellate species increased from 23 to 34 with extended incubation, and the ratio of mixotrophic to heterotrophic species was approximately 2:1 in the samples of in situ sediments and seawater outside GEHA, as well as across germination durations (Sg-5 d vs. Sg-20 d). These findings provide essential field evidence for the role of resting cysts in driving HAB formation in this region and highlight the efficacy of the GEHA-based approach for studying in situ cyst germination dynamics, offering a robust tool for monitoring, early warning, prevention, and forecasting of HABs.