Filling reference libraries with diatom environmental sequences: strengths and weaknesses

Diatom species identification with DNA metabarcoding is an economical, fast and reliable alternative to identification via light microscopy for river quality monitoring. Using a short DNA sequence of the rbcL gene and ‘Diat.barcode’, a reference barcode library, enables the identification of more than 90% of the environmental sequences to species level in French rivers. But the completeness of this library is much lower in other regions, such as the tropical French overseas departments. A barcode library completion method using high-throughput sequencing data combined with microscopy count data from natural samples (Rimet et al. 2018) was applied and tested in rivers of Martinique and Guadeloupe (West Indies), for which only 45% of the environmental sequences could be identified to species level using Diat.barcode v9. Assigning barcodes to the most abundant species in the islands by this method is illustrated with <i>Ulnaria goulardii</i> and two new species belonging to <i>Nupela</i> and <i>Epithemia</i>, which are also described in this paper. The more complex situation of morphologically similar species is illustrated by reference to <i>Gomphonema designatum</i> and <i>G. bourbonense</i>. Using a combination of molecular and morphological data, their conspecificity, as <i>G. bourbonense</i>, is demonstrated with their reference barcodes. However, when several morphologically similar species and several environmental sequences belonging to the same clade are present, it is not possible to relate the barcodes to corresponding morphological species. Applying this method enabled the Diat.barcode library (v.10) to be updated, with 84% of the environmental sequences from the West Indies now identifiable at the species level. However, many morphological species still lack barcodes. In these cases, more classical methods, such as cell isolation, Sanger sequencing and morphological observations of cultures, must be applied.