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DNA barcoding uses a short fragment of a DNA sequence to identify a taxon. After obtaining the target sequence it is compared to reference sequences stored in a database to assign an organism name to it. The quality of data in the reference database is the key to the success of the analysis. In the here presented study, multiple types of data have been combined and critically examined in order to create best practice guidelines for taxonomic reference libraries for environmental barcoding. 70 unialgal diatom strains from Berlin waters have been established and cultured to obtain morphological and molecular data. The strains were sequenced for 18S V4 rDNA (the pre-Barcode for protists) as well as rbcL data, and identified by microscopy. LM and for some strains also SEM pictures were taken and physical vouchers deposited at the BGBM. 37 freshwater taxa from 15 naviculoid diatom genera were identified. Four taxa from the genera Amphora, Mayamaea, Planothidium and Stauroneis are described here as new. Names, molecular, morphological and habitat data as well as additional images of living cells are also available electronically in the AlgaTerra Information System. All reference sequences (or reference barcodes) presented here are linked to voucher specimens in order to provide a complete chain of evidence back to the formal taxonomic literature.