Multidimensional comparisons of the V4 and V9 regions of the 18S rRNA gene in characterizing microeukaryotic community across complex environmental gradients of coastal waters

The V4 and V9 regions of the 18S rRNA gene are widely employed to target microeukaryotes, serving as promising bioindicators of ecological responses or consequences to environmental stresses in coastal marine ecosystem. However, choosing 18S regions is tricky for investigations at regional scale or beyond, because between-18S-region differences in estimating microeukaryotic diversity, composition, and assembly mechanism across environmental gradients have not been comprehensively assessed. Here, we compared microeukaryotic communities using the two 18S regions across complex environmental gradients in the coastal waters of northern Zhejiang, East China Sea. Given the overall superior performance and data trend stability across total community/major supergroups or coastal zones, the V9 region based on operational taxonomic units with 97 % sequence similarity (OTU₉₇s), rather than zero-radius OTUs (ZOTUs), is recommended for estimating alpha diversity across the area. Although the beta diversity patterns of microeukaryotes based on both 18S regions were similar regardless of metrics or OTU resolutions, the inference of community assembly processes based on the null models was sensitive to the choice of 18S region, particularly at the finer OTU resolution. Remarkably, we confirmed the widespread environmental applicability of 18S regions in characterizing taxonomic distribution, often driven by bay-to-offshore gradients in salinity and nutrients (corresponding to different levels of eutrophication). This suggests that terrestrial and anthropogenic disturbances influence the trade-off between the two 18S regions in targeting microeukaryotic groups along the coastal continuum. Our findings facilitate customized strategy for estimating microeukaryotic diversities and targeting specific groups in the practice of large-scale coastal environmental monitoring under varying environmental conditions.