| Abstract |
Environmental DNA (eDNA) metabarcoding is a relatively new, molecular tool for the detection of organisms in an environmental sample. It’s a very promising technique with emerging applications in the fields of ecology, conservation, invasive biology, biomonitoring and more. More and more studies are using eDNA nowadays, yet in the Mediterranean ecosystems, its use is limited so far and focuses mostly on planktonic communities and less on the assessment of ichthyofauna.
The main goal of this study is to develop a reliable eDNA metabarcoding protocol to study and monitor fish biodiversity in the oligotrophic ecosystems of the Eastern Mediterranean. This research has two parts: a) standardization of the method by testing two different sets of primers in aquaria with known fish species assemblage and b) estimation of the heterogeneity of fish eDNA distribution in different habitats within an area, in order to determine the most efficient sampling strategy.
To standardize the method, we conducted sampling in two tanks of the Cretaquarium and we tested different sets of primers (for genes 16S rRNA and 12S rRNA) to see their efficiency in retrieving species' composition both qualitatively and semi-quantitatively To estimate the eDNA heterogeneity in natural ecosystems, we sampled a coastal ecosystem with three different types of habitats (hard substrate, soft substrate, Posidonia meadows, mid water-column). In both cases, we analysed our samples through NGS (Next Generation Sequencing).
In regard to the aquarium samples, both primers seemed to work quite well. Most taxa in both tanks were detected up to species level and marker 16S seemed to have higher resolution. The marker 12S, has never been used in fish eDNA metabarcoding surveys in the Mediterranean sea. Building a reliable reference database of the Mediterranean fishes is of high priority in order to be able to reliably identify fishes of the Mediterranean ecosystems at species level. A rather weak correlation was also detected between actual fish biomass and relative abundance as estimated by eDNA metabarcoding.
For the coastal ecosystem sampling, we only got results with the 16S marker gene. A total of 69 taxa were detected, with 55 of them distinguished at the species level while in each sample the number of taxa detected ranged from 13 to 27. Posidonia meadows and the water column samples showed the greatest heterogeneity, in contrast to the hard and soft substrate samples that showed little differentiation within each habitat as well as between the two habitats. Based on these results, an improved protocol should include a larger volume of water filtered per sample (but some technical difficulties must be overcome), an increase in technical repetitions per sample (at least 3 PCRs) , and at least one habitat sampled per area. In conclusion, the method seems to work well, and with some small improvements, it can be used as a reliable tool for the study of biodiversity and biomonitoring of fish communities of the oligotrophic ecosystems of the Eastern Mediterranean Sea.
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Ανάπτυξη και βελτιστοποίηση μεθοδολογίας περιβαλλοντικού DNA (eDNA metabarcoding) για τη μελέτη και βιοπαρακολούθηση της ιχθυοπανίδας στα ολιγοτροφικά θαλάσσια οικοσυστήματα της Μεσογείου
