Evaluation of two overlapping rbcL amplicons for routine diatom DNA biomonitoring
Marker choice is a critical methodological factor in DNA metabarcoding, as it directly influences taxonomic composition and abundance structure of communities. In diatoms, metabarcoding is increasingly evaluated as an alternative to microscopy-based assessments, particularly for ecological status evaluation under the Water Framework Directive (WFD). Here, we compared two widely used rbc L markers (263 bp and 331 bp long) using identical benthic diatom samples to evaluate their suitability for ecological assessment. Both markers produced highly consistent community patterns and IPS-based ecological classifications. Differences between datasets were mainly restricted to low-abundance taxa, while dominant taxa—driving most of the IPS signal—were consistently detected. Decomposition of IPS differences showed that variation was primarily driven by changes in the relative abundance of shared taxa and inconsistencies in species-level assignment, whereas marker-specific taxa had negligible influence. Overall, the 331-bp marker provided higher taxonomic resolution for abundant taxa, and its lower detection of rare taxa did not compromise ecological assessment. It is therefore well suited for routine biomonitoring under the WFD. Although marker-specific calibration may further improve quantitative accuracy, our results indicate that the 331-bp marker can be reliably applied without major methodological adjustments.