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The implementation of DNA metabarcoding and environmental DNA (eDNA) to the biodiversity assessment and biomonitoring of aquatic ecosystems has great potential worldwide. However, DNA metabarcoding and eDNA are highly reliant on the coverage of the DNA barcode reference libraries that are currently hindered by the substantial lack of reference sequences. The main objective of this study was to analyze the current coverage of DNA barcode reference libraries for phytoplankton species of the aquatic Mediterranean ecoregion in the southeast of Italy (Apulia Region) in order to assess the applicability of DNA metabarcoding and eDNA in this area. To do so, we investigated three main DNA barcode reference libraries, BOLD Systems, GenBank and SILVA, for the availability of DNA barcodes of the examined phytoplankton species. The gap analysis was conducted for three molecular gene markers, 18S, 16S and COI. The results showed a considerable lack of barcodes for all three markers. However, among the three markers, 18S had a greater coverage in the reference libraries. For the 18S gene marker, the barcode coverage gap across the three types of ecosystems examined was 32.21-39.68%, 60.12-65.19% for the 16S marker gene, and 72.44-80.61 for the COI marker gene. Afterwards, the interspecific genetic distance examined on the most represented molecular marker, 18S, was able to distinguish 80% of the species mined for lakes and 70% for both marine and transitional waters. Conclusively, this work highlights the importance of filling the gaps in the reference libraries, and constitutes the basis towards the advancement of DNA metabarcoding and eDNA application for biodiversity assessment and biomonitoring.

DNA Barcode Gap Analysis for Multiple Marker Genes for Phytoplankton Species Biodiversity in Mediterranean Aquatic Ecosystems