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.

The use of molecular tools (DNA barcoding and metabarcoding) for the identification of species and ecosystem biomonitoring is a promising innovative approach. The effectiveness of these tools is, however, highly dependent on the reliability and coverage of the DNA sequence reference libraries and it also depends on the identification of primer sets that work on the broadest range of taxa. In this study, a gap analysis of available DNA barcodes in the international libraries was conducted using the aquatic macroinvertebrate species checklist of the Apulia region in the southeast of Italy. Our analyses show that 42% of the 1546 examined species do not have representative DNA barcodes in the reference libraries, indicating the importance of working toward their completeness and addressing this effort toward specific taxonomic groups. We also analyzed the DNA barcode reference libraries for the primer set used to barcode species. Only for 52% of the examined barcoded species were the primers reported, indicating the importance of uploading this information in the databases for a more effective DNA barcode implementation effort and extensive use of the metabarcoding method. In this paper, a new combination of primers has revealed its experimental effectiveness at least on the species belonging to the three most represented taxa in the aquatic ecosystems of the Apulia region, highlighting the opportunity to develop combinations of primers useful at the regional level and the importance of studying DNA barcode gaps at the local/regional level. The DNA barcode coverage also varies among different taxonomic groups and aquatic ecosystem types in which a large number of species are rare. We tested the application of the DNA barcoding single species to a lagoon ecosystem (the lagoon named “Acquatina di Frigole” in the Apulia region) and we sampled two macroinvertebrate species lacking DNA barcodes from “Aquatina di Frigole” NATURA 2000 Site IT9150003, Fabulina fabula and Tritia nitida, generated two new CO1 barcodes and added them to a DNA barcode reference library.

The loss of aquatic biodiversity is increasing at a rapid rate globally. There is a worldwide effort to protect, preserve and restore aquatic ecosystems. For efficient biodiversity monitoring and reliable management tools, comprehensive biodiversity data are required. The abundance and species diversity of benthic macroinvertebrates are commonly used as indicators of the aquatic ecosystem condition. Currently, macroinvertebrate species biodiversity assessment is based on morpho-taxonomy, which could be enhanced by recent advances in DNA-based tools for species identification. In particular, DNA metabarcoding has the potential to identify simultaneously many different taxa in a pool of species and to improve aquatic biomonitoring significantly, especially for indicator species. This review is focused on the current state of DNA-based aquatic biomonitoring using benthic macroinvertebrates in the Mediterranean region.

The biodiversity of the Mediterranean Sea is currently threatened by the introduction of Non-Indigenous Species (NIS). Therefore, monitoring the distribution of NIS is of utmost importance to preserve the ecosystems. A promising approach for the identification of species and the assessment of biodiversity is the use of DNA barcoding, as well as DNA and eDNA metabarcoding. Currently, the main limitation in the use of genomic data for species identification is the incompleteness of the DNA barcode databases. In this research, we assessed the availability of DNA barcodes in the main reference libraries for the most updated inventory of 665 confirmed NIS in the Mediterranean Sea, with a special focus on the cytochrome oxidase I (COI) barcode and primers. The results of this study show that there are no barcodes for 33.18% of the species in question, and that 45.30% of the 382 species with COI barcode, have no primers publicly available. This highlights the importance of directing scientific efforts to fill the barcode gap of specific taxonomic groups in order to help in the effective application of the eDNA technique for investigating the occurrence and the distribution of NIS in the Mediterranean Sea.

Coastal lagoons are considered important habitats both for ecological functions and biodiversity worldwide. Thus, they provide relevant ecosystem services and valuable natural resources. However, coastal lagoons are highly susceptible to anthropogenic pressures that can cause biodiversity losses and require specific biomonitoring programs as well as management measures. In this research, we applied environmental DNA (eDNA) metabarcoding to investigate the biodiversity of a poorly known Mediterranean lagoon included in the European Natura 2000 Network. We used the cytochrome oxidase I (COI) gene marker to capture the entire biodiversity of this highly diversified aquatic coastal environment. With a low sampling effort and rapid laboratory practices, a large amount of valuable biodiversity data was generated and analyzed. Interestingly, this straightforward and broad molecular surveying of biodiversity unveiled a wide variety of taxonomic groups, such as benthic macroinvertebrates, zooplankton, phytoplankton, and macroalgae, which are frequently used as ecological indicators. We were able to detect species that were previously morphologically identified, as well as species never identified before. This research underlines the validity of eDNA metabarcoding in assessing the biodiversity in a poorly known and protected Mediterranean lagoon ecosystem, as well as in identifying the early warnings of environmental stressors. Finally, the research highlights the need to investigate multiple target genes and primers set for a larger analysis of specific species.