Marker choice in diatom DNA metabarcoding affects ASV resolution but not ecological status assessment
Marker choice is a critical methodological variable in DNA metabarcoding, as it directly influences taxonomic resolution and the observed community composition.For diatoms, DNA metabarcoding is being intensively evaluated as a complementary approach to traditional microscopy-based assessments, with the aim of supporting the Water Framework Directive (WFD). Despite the existence of two established markers, targeting 263-bp and 331-bp fragments of the rbcL gene, there is limited empirical evidence on how their choice affects ASV structure and ecological assessments. Here, we directly compare these two rbcL markers by simultaneously analyzing identical benthic diatom samples using both primer sets We identify two marker-dependent ASV-level mechanisms. ASV collapse occurs when multiple distinct 331-bp ASVs map to a single 263-bp ASV due to reduced sequence resolution of the shorter marker, while dropout describes ASVs detected by one marker but absent from the other. These processes substantially alter ASV composition and community structure, particularly within the long tail of rare sequence variants. Collapse severity showed a weak but significant abundance dependence among low-abundance species, indicating that marker-length effects are not uniformly distributed across the community. Despite pronounced ASV- and species-level differences, dominant ASVs were strongly correlated between markers and IPS-based ecological assessment was remarkably consistent. Paired comparisons revealed no significant differences in IPS values across marker and reference configurations, with only negligible effect sizes. Our results demonstrate that marker choice affects ASV-level resolution without compromising abundance-weighted ecological assessment, supporting the robustness of metabarcoding-derived indices during ongoing method harmonisation for regulatory biomonitoring.