Environmental DNA for Detection of Endangered Grouper Species (Epinephelus spp.)

Abstract

Marine ecosystems nestle species or populations known to be threatened due to human
 overexploitation. Reliable detection and monitoring of threatened organisms is crucial for
 data-driven conservation actions. Furthermore, misidentification of species represents a major
 problem. Here, we investigate the potential of using metabarcoding of environmental DNA
 (eDNA) obtained directly from seawater samples to detect endangered grouper species
 (Epinephelus spp.). Cytochrome c oxidase subunit I (COI) fragment of mtDNA was used to
 detect groupers species in the Mediterranean Coasts. We conducted eDNA sampling at sites
 by underwater diving across the range of the Grouper species habitats in Northeastern
 Mediterranean (Antalya-Kas Region and Iskenderun Bay). eDNA was isolated from 2 liter
 seawater samples which were vacuum-filtered onto 0.45-mm membrane filters. Filters were
 then folded inwards, placed in 2 ml tubes and stored at -20 oC until DNA extraction, which
 took place within 24 hours. DNA was extracted from the water sample filters using the
 DNeasy Blood and Tissue Kit (Qiagen, USA). Manufacturer’s protocols were used during all
 steps. PCR amplification of eDNA samples were done using selective primers of COI region
 of mitochondrial DNA, and next-generation DNA sequencing of PCR application was
 conducted. For the successfully obtained COI sequences, maximum matching rates were
 revealed as 80% for Epinephelus marginatus, 78,95% for Epinephelus aeneus, 73,48% for
 Epinephelus costae, 63,45% for Epinephelus caninus, 60,12% for Mycteroperca rubra and
 57,12% for Hyporthodus haifensis. Despite the methodological challenges inherent in eDNA
 analysis, the results demonstrated that eDNA method may be proved to step towards a new
 beginning to detect and monitor endangered grouper species.