<scp>DNA</scp> metabarcoding challenge: Uncovering the cryptic dominance of the genus <scp><i>Lyrella</i></scp> in tidal flat diatom communities through full‐length <scp>18S rRNA</scp> sequencing

SUMMARY DNA metabarcoding is a powerful tool for analyzing microbial diversity; however, its effectiveness is limited by the incompleteness of reference databases. In this study, we aimed to assess its utility in characterizing diatom communities and identify the limitations associated with reference coverage. We examined benthic diatom communities from two tidal flats using DNA metabarcoding targeting the V4 region of the 18S rRNA gene, which was sequenced on an Illumina MiSeq platform. Although sequences from diatom genera such as Amphora and Navicula commonly observed in tidal flats were frequently detected, the most abundant amplicon sequence variants (ASVs) at both sites could not be identified, even at the genus level. To determine their identities, we performed DNA walking and obtained nearly full‐length 18S rRNA gene sequences corresponding to the most dominant ASVs from each site. Phylogenetic analyses based on these extended sequences revealed that the most dominant unidentified ASVs at each site are distinct species belonging to the genus Lyrella , which has not previously been reported as dominant taxa in tidal flat environments, and is poorly represented in reference databases used for metabarcoding. This finding underscores a major limitation of DNA metabarcoding, that is, even the most abundant taxa may remain unidentified because of insufficient reference data. Moreover, our results highlight the value of combining metabarcoding with full‐length sequencing to uncover cryptic but potentially ecologically significant taxa. The unexpected dominance of Lyrella calls for further research focusing on its ecological characteristics and greater efforts to enrich diatom reference libraries for environmental DNA studies.