Single-cyst morpho-molecular identification detected an unexpected high species diversity of dinoflagellate resting cysts from the coastal seas of China.

Over the past several decades, harmful algal blooms (HABs) caused by dinoflagellates frequently occurred along the coastal waters of China, with an increasing number of emerging HAB species. Due to the vital roles played by the resting cyst in the ecology of HABs, the investigation of resting cyst diversity and distribution of dinoflagellates, those causing HABs in particular, in marine sediment is of great significance. However, it has been difficult to unambiguously identify cyst species via obtaining both morphological and molecular evidence due to a variety of technological limitations (e.g. extremely simple morphology and/or small sizes of many cyst species). Although the application of high-throughput metabarcoding analysis has greatly improved the efficiency (high throughput) and accuracy (molecular identification) of cyst identification, lacking morphological evidence makes it less convincing because the sequences obtained with this approach may be doubted to be from fragmental vegetative cells or relics of eDNA. Furthermore, insufficient sequencing depths commonly adopted in studies using this technique together with the extremely large and widely-varying genome sizes of dinoflagellates have also led to the potential oversight of those species having small cell sizes and/or relatively low abundances. In this study, we employed a single-cyst morpho-molecular method (ScPCR sequencing) to identify dinoflagellate cysts from 23 sediments collected from all four seas of China. From 702 individually picked-up, micrographed, and sequenced cysts, we identified 127 species of dinoflagellates, with 63 (49.6%) fully identified to well-described species, and 64 (50.4%) that could not be determined for their species identity due to the unavailability of reference sequences. Notably, among the 63 fully-identified species, 6 had not been reported from China before, 19 are well-documented HABs-causing species (e.g. 8 Alexandrium spp., Gymnodinium catenatum, Karenia mikimotoi), and 22 were identified for the first time from one of the four seas of China. In addition, from 44 sediment samples that were collected from the East China Sea (ECS, a "hotspot" of HABs in China) and pre-processed with the sodium polytungstate protocol to concentrate their cyst assemblages, we fully identified 61 species of dinoflagellate cysts via metabarcoding analysis, including 27 species causing HABs, 10 as new records in Chinese waters, 13 as new records in the ECS, and 10 previously unreported as cyst producers. It is noteworthy that 7 (35%) of the 20 cyst species identified via ScPCR sequencing from the ECS were not detected by the metabarcoding analysis. Contrasting to that 64 species of dinoflagellate cysts had been unequivocally identified from China by 2021, the total number of cyst species identified in this study using ScPCR sequencing demonstrated the robustness of the detection technique. This study also suggests that the species diversity of dinoflagellate cysts in Chinese marine sediments is still largely underestimated, which calls for significant advancements both in the taxonomy of dinoflagellates and cyst detection technology and effort.