In addition to primer selection challenges, the availability of reference sequences for the different taxonomic groups in databases is variable and not always up-to-date for specific metabarcoding regions. This illustrates challenges with investigating diversity using amplification of a single gene region and suggests the use of  phylogenetic analyses together with sequence comparison to databases (e.g. BLAST-based approaches) to correctly assign taxonomic affiliations (Egan et al. 2018; Hart et al. 2015; Stefani et al. XX).