Integrative single-cell sequencing and environmental metabarcoding reveals genetic complexity and hidden diversity of...

The dinoflagellate genus Tripos is species-rich, containing many species that can cause harmful algal blooms (HABs). Although 800 species and infraspecific taxa have been described worldwide for this genus, only ∼10% are characterized and listed in AlgaeBase, due primarily to their pronounced morphological plasticity, highlighting challenges associated with Tripos taxonomy. Furthermore, the 18S rDNA V4 sequences of over 50% of Tripos species listed in AlgaeBase are not included in the reference database PR², indicating that only a small portion of Tripos is currently molecularly characterized. Here, we investigated Tripos species diversity by integrating single-cell morphological analysis, single-cell 18S rDNA V4 high-throughput sequencing, and eDNA metabarcoding, taking advantage of high Tripos diversity reported in the South China Sea (SCS). Morphologies of 89 Tripos cells isolated from SCS, together with 24 Tripos cells from the Shandong coastal region (SCR) were analysed. Altogether 33 Tripos species were identified morphologically, with 32 identified in SCS and five in SCR, confirming higher diversity in SCS than in SCR. High-throughput sequencing of the 18S rDNA V4 of these 117 Tripos single cells showed that each cell harboured one dominant amplicon sequence variant (dASV) accompanied by multiple low-abundance non-dominant variants (ndASVs), revealing pervasive intragenomic rDNA variation (IGV) and providing empirical support for prioritizing dASVs as biologically meaningful units in metabarcoding analyses. A total of 29 dASVs was identified, exhibiting one-to-one, one-to-multiple, and multiple-to-multiple relationships with morphologically defined Tripos species. Through integrative analysis of morphological taxonomic annotation and single-cell sequencing results, the 18S rDNA V4 sequences were identified, for the first time, for 13 Tripos species, including T. belone, T. muelleri var. atlanticus, T. pacificus, T. pennatus, T. scapiformis, T. geniculatus, T. lanceolatus, T. subcontortus, T. karstenii, T. teres, T. axialis, Tripos sp. ASV17, Tripos sp. ASV18, substantially expanding and refining existing reference databases. Environmental metabarcoding of 1578 field samples further corroborated these patterns, recovering 11 Tripos species and 12 Tripos ribogroups in SCS compared with six Tripos species and five Tripos ribogroups in SCR, corroborating higher Tripos diversity in SCS. Together, this study provided a comprehensive molecular-morphological reference framework for Tripos and demonstrated the value of integrating single-cell and environmental metabarcoding with morphology to improve species-level resolution in taxonomically complex dinoflagellates.