Species of the dinoflagellate genus Tripos plays a crucial role as primary producers in marine ecosystems, while some Tripos species can cause harmful algal blooms (HABs). Morphology-based identification is often inconclusive because most Tripos species show remarkable morphological plasticity, leading to overestimation of Tripos biodiversity with some literature reporting as many as 800 Tripos species. Molecular marker-based analysis has been demonstrated to be effective for identifying Tripos species. However, reference sequences for Tripos species are currently very limited with only 25 Tripos species listed in the PR² database, leading to an underestimation of Tripos biodiversity. Here, we expanded the Tripos database to ascertain Tripos biodiversity in the South China Sea (SCS).
Through searching NCBI NT and phylogenetic analysis, we obtained 18S rDNA V4 reference sequences for 64 Tripos species, representing most of the 81 Tripos species listed in AlgaeBase. We then carried out the single-cell analysis, which yielded 706 ASVs across the 57 Tripos single cells, revealing 19 dominant ASVs, with a substantial number of non-dominant 18S rDNA V4 sequences identified in each cell, indicating substantial intragenomic variations (IGVs). Of the 57 single cells analyzed, 19 Tripos species were identified, including 10 known Tripos species, two putative unidentified species, and seven closely related species with identical dominant sequences. The number of Tripos species identified in the SCS was substantially more than that identified in the coastal regions of Shandong Province, which was five.
We further investigated the Tripos biodiversity by carrying out metabarcoding analysis. We identified a total of 29 Tripos species, including 12 known species, four putative unidentified species, and 13 closely related species with identical dominant sequences. Significant differences in Trips biodiversity between SCS and the coastal regions of Shandong was found.
This research highlighted the strengths and limitations of the 18S rDNA V4-based metabarcoding analysis of Tripos species. Its resolution for Tripos species is limited as many Tripos species share identical 18S rDNA V4 sequences. Other molecular markers such as ITS1, ITS2, and 28S rDNA D1-2 may be evaluated and considered. In addition, reference sequences need to be expanded to include all Tripos species to ensure effective identification. Furthermore, intensive IGVs exist in all Tripos species, which need to be evaluated for the correct interpretation of metabarcoding results. This study provides insights into the species diversity of Tripos in the South China Sea and beyond, laying a crucial foundation for future monitoring of HABs and marine ecological conservation efforts.

metabarcoding analysis, single-cell analysis, Tripos biodiversity, intragenomic variations, South China Sea