Evaluation of two short overlapping rbcL markers for diatom metabarcoding of environmental samples: Effects on biomonitoring assessment and species resolution.

Two short diatom rbcL barcodes, 331 bp and 263 bp in length, have frequently been used in diatom metabarcoding studies. They overlap in a common 263-bp region but differ in the presence or absence of a 68-bp tail at the 5' end. Though the effectiveness of both has been demonstrated in separate biomonitoring and diversity studies, the impact of the 68-bp non-shared region has not been evaluated. Here we compare the two barcodes in terms of the values of a biotic index (IPS) and the ecological status classes derived from their application to an extensive metabarcoding dataset from United Kingdom rivers; this comprised 1703 samples and was produced using the 331-bp primers. In addition, we assess the effectiveness of each barcode for discrimination of genetic variants around and below the species level. The strong correlation found in IPS values between barcodes (Pearson's R = 0.98) indicates that the choice of the barcode does not have major implications for current WFD ecological assessments, although a very few sites (55: 3.23% of those analysed) were downgraded from an acceptable WFD class ("Good") to an unacceptable one ("Moderate"). Analyses of the taxonomic resolution of the two barcodes indicate that for many ASVs, the use of either marker - 263-bp and 331-bp - gives unambiguous assignations at species level though with differences in bootstrap confidence values. Such differences are caused by the stochasticity involved in the naïve Bayesian classifier used and by the fact that genetic distance, regarding closely related species, is increased when using the 331-bp barcode. However, in three cases, species differentiation fails with the shorter marker, leading to underestimates of species diversity. Finally, two ASVs from Nitzschia species evidenced that the use of the shorter marker can sometimes lead to false positives when the extent and nature of infraspecific variation are poorly known.