The dinoflagellate genus Alexandrium comprises multiple toxigenic harmful algal bloom (HAB) species capable of synthesizing paralytic shellfish toxins, posing severe threats to ecosystem stability, and public health. Morphological identification lacks species-level resolution, while metabarcoding can help, but marker selection and reference datasets for Alexandrium remain unevaluated. In Shandong coastal waters, where HABs are frequent, Alexandrium diversity and dynamics are poorly known.

We refined the reference sequence datasets for the universal markers 18S rDNA V4 and 28S rDNA D1–2 and evaluated their resolving power for Alexandrium. (1) Through mining and comparative analysis of the NCBI NT database, we revised the Alexandrium reference sequences in the PR² database, identifying two erroneous sequences that do not belong to Alexandrium and discovering the 18S rDNA V4 sequences of two species (A. limii and A. ogatae); these findings have been submitted to PR². (2) Using single‑cell high‑throughput sequencing, we elucidated that numerous sequences annotated to the same Alexandrium species in public databases show high intragenomic variation. Co‑occurrence of dominant and non‑dominant variants within the same strain yields a greater number of molecular operational taxonomic units or amplicon sequence variants than actual species numbers in metabarcoding analyses. We classified non‑dominant variants into two categories: one highly similar to the dominant variant, and the other highly similar to dominant variants of other species. Thus, inclusion of only dominant variants in reference datasets can accurately represent species diversity. (3) We compared the resolution of 28S rDNA D1–2 fragments of different lengths and found that the amplicon targeted by primers DinoF/DinoR exhibits higher species discrimination. We further constructed the first dedicated 28S rDNA D1–2 dataset for Alexandrium. (4) Phylogenetic analyses based on these markers confirmed that 28S rDNA D1–2 provides significantly higher species‑level resolution than 18S rDNA V4.

Employing a 28S rDNA D1–2‑based metabarcoding approach, we resolved the composition and spatiotemporal distribution of Alexandrium across four seasons in the coastal waters of Shandong. This method identified 11 Alexandrium species, outperforming the 7 species detected via 18S rDNA V4 metabarcoding and further corroborating the superior resolution of 28S rDNA D1–2. Our results reveal pronounced seasonal dynamics, with most species exhibiting higher relative abundances in summer and autumn, whereas A. catenella was more abundant in winter and spring. Temperature emerged as the primary environmental factor associated with these seasonal variations. Overall, by establishing robust reference datasets, this study demonstrates that 28S rDNA D1–2 possesses high resolving power for Alexandrium and can be applied for species detection and spatiotemporal dynamic studies of this genus.

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