Comparative evaluation of 18S rDNA V4 and 28S rDNA D1-2 regions for resolving Alexandrium species diversity to...

Many Alexandrium species can develop harmful algal blooms (HABs) associated with paralytic shellfish poisoning (PSP), making their accurate identification critical. Morphological similarity among Alexandrium species limits their identification, whereas molecular marker-based methods are effective, resulting in the accumulation of vast molecular data. However, the existence of these massive molecular sequences makes it confusing to select reference sequences for taxonomical and diversity studies. To address this, we mined and curated molecular marker sequences of 18S rDNA V4 and 28S rDNA D1-2 for Alexandrium species from NCBI NT database. We built the first reference 28S rDNA D1-2 database for 27 Alexandrium species, and for the 18S rDNA V4 region, our results expanded PR² database by adding sequences for two additional Alexandrium species and proposed to eliminate two contaminated sequences. Furthermore, we characterized intra-genomic variations (IGVs) for both 18S rDNA V4 and 28S rDNA D1-2, and experimentally confirmed the existence of 18S rDNA V4 IGVs in Alexandrium species. Both single-cell sequencing and database analysis results showed that an Alexandrium cell typically harbors IGVs with the co-existence of usually one dominant and multiple non-dominant sequences, with non-dominant sequences potentially matching another species' dominant sequence. Therefore, only dominant sequences of each Alexandrium species were proposed to be included in reference databases for clarity. We also confirmed that 28S rDNA D1-2 has higher resolution for identifying Alexandrium species. Through clarifying the characteristics of these molecular markers and providing curated datasets, this work enhances metabarcoding accuracy for early detection of toxic Alexandrium populations.