Detection of a diverse marine fish fauna using environmental DNA from seawater samples

Abstract

Marine ecosystems worldwide are under threat with many fish species and populations suffering from human over-exploitation. This is greatly impacting global biodiversity, economy and human health. Intriguingly, marine fish are largely surveyed using selective and invasive methods, which are mostly limited to commercial species, and restricted to particular areas with favourable conditions. 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 account for marine fish biodiversity. This eDNA approach has recently been used successfully in freshwater environments, but never in marine settings. We isolate eDNA from ½-litre seawater samples collected in a temperate marine ecosystem in Denmark. Using next-generation DNA sequencing of PCR amplicons, we obtain eDNA from 15 different fish species, including both important consumption species, as well as species rarely or never recorded by conventional monitoring. We also detect eDNA from a rare vagrant species in the area; European pilchard (Sardina pilchardus). Additionally, we detect four bird species. Records in national databases confirmed the occurrence of all detected species. To investigate the efficiency of the eDNA approach, we compared its performance with 9 methods conventionally used in marine fish surveys. Promisingly, eDNA covered the fish diversity better than or equal to any of the applied conventional methods. Our study demonstrates that even small samples of seawater contain eDNA from a wide range of local fish species. Finally, in order to examine the potential dispersal of eDNA in oceans, we performed an experiment addressing eDNA degradation in seawater, which shows that even small (100-bp) eDNA fragments degrades beyond detectability within days. Although further studies are needed to validate the eDNA approach in varying environmental conditions, our findings provide a strong proof-of-concept with great perspectives for future monitoring of marine biodiversity and resources.

Figure 1. Summary of results showing sampling site and panel of fish species recovered by eDNA.

Sampling locality (The Sound, Elsinore, Denmark) for this study with the three sampling sites; 1) open beach, 2) outer pier, 3) inner pier. The 15 different fish species obtained by eDNA in this study are shown with colour codes explaining in which of the three sampling sites they were found. All fish drawings by Susanne Weitemeyer ©.

Figure 2. Number of fish species recorded by 9 different conventional survey methods and eDNA at The Sound of Elsinore, Denmark.

Bars show mean number of fish species caught across surveys in 2009, 2010 and 2011 and error bars represent the standard deviation (see also Table S1). The eDNA bar represents the total amount of fish species recorded by this method in 2011. *) Depend heavily on competent experts in fish identification. **) Only possible where seabed conditions allow it.

Figure 3. Results from eDNA degradation experiment. eDNA concentration in seawater as a function of time for the two fish species; Platichthys flesus (circles) and Gasterosteus aculeatus (triangles), investigated in a 50 l aquarium.

Time points with no detection of eDNA signals are shown in red. The lines show simple exponential decay models, p<0.001 (Platichthys flesus) and p<0.05 (Gasterosteus aculeatus). Dashed line shows the suggested detection threshold of 25 DNA molecules pr 400 ml seawater. Estimated time for eDNA to degrade beyond the detection threshold was estimated to be 0.9 days for Gasterosteus aculeatus and 6.7 days for Platichthys flesus. See also Materials and Methods section.