Passive eDNA collection enhances aquatic biodiversity analysis.

Environmental DNA (eDNA) metabarcoding is a sensitive and widely used approach for species detection and biodiversity assessment. The most common eDNA collection method in aquatic systems is actively filtering water through a membrane, which is time consuming and requires specialized equipment. Ecological studies investigating species abundance or distribution often require more samples than can be practically collected with current filtration methods. Here we demonstrate how eDNA can be passively collected in both tropical and temperate marine systems by directly submerging filter membranes (positively charged nylon and non-charged cellulose ester) in the water column. Using a universal fish metabarcoding assay, we show that passive eDNA collection can detect fish as effectively as active eDNA filtration methods in temperate systems and can also provide similar estimates of total fish biodiversity. Furthermore, passive eDNA collection enables greater levels of biological sampling, which increases the range of ecological questions that eDNA metabarcoding can address.


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Details of our nested study design are included in the manuscript. Briefly, to evaluate the viability of passively collecting eDNA, we submerged filter membranes approximately one meter below the surface in a pearl frame (n=150). To evaluate if submersion time affected collection of eDNA, we retrieved the filter membranes after different time intervals. We also investigated two different membrane materials. All treatments were done in triplicate over four days (June 17 to 20, 2019) at a tropical marine site (n=72 membranes) and five days (Jan 29 to Feb 2, 2019) at a temperate marine site (n=78 membranes). We then compared the species diversity obtained from passive eDNA collection to that achieved by the conventional method of actively filtering water samples. We collected nine 1 L surface water samples in sterile containers at each site and filtered the water over nine non-charged cellulose membranes using a peristaltic pump.
We chose to analyse fish eDNA because this is one of the most common assessments made in aquatic eDNA metabarcoding studies with a substantial reference database for taxonomic assignment.
We compared the species diversity obtained from passive eDNA collection to that achieved by the conventional method of actively filtering nine 1L surface water samples at each site. We chose nine 1 L actively filtered water samples for comparison because this level of replication is rarely achieved by the majority of aquatic eDNA metabarcoding fish studies.
Filter membranes were retrieved using gloved hands and sterile tweezers by Cindy Bessey at Daw Island, and by Cindy Bessey and Todd Stewart at Ashmore Reef.
Data collection at Ashmore Reef occurred over June 17 to 20, 2019. Triplicate membranes were retrieved after four, eight, 12, and 24 hours of deployment. Data collection at Daw Island occured over January 29 to February 2, 2019. Triplicate membranes were retrieved after four, eight, 12, 24 hours of deployment, and on the last day we also deployed triplicate membranes for 34 hours.
All positive controls amplified multiple reads identifying dhufish with 100% identity. However, 36 reads of the positive control showed up in two Ashmore Reef samples. Therefore, to ensure a conservative approach to detection efficiency, we required a minimum of 40 reads to count a fish species as present.
The experiment was repeated twice, once in tropical water and once in temperate waters. Both attempts were successful.
Filter membranes were placed in the deployment frame to ensure all treatments were completely submerged and had equal exposure to the marine environment.
All laboratory samples were labeled as a number and processed in the same fashion.
Sampling was conducted in the tropical waters of Ashmore Reef (June 17 to 20, 2019; average water temperature for June is~25C) and the temperate waters surrounding Daw Island (Jan 29 to Feb 2, 2019; average water temperature for June is~18C).
At Ashmore Reef, sampling took place in surface waters at the vessel mooring site (122°58.99" E, 12°14.27" S), where the depth was less than 10 meters. Sampling at Daw Island took place in the surface waters at the vessel mooring site (124°07.86" E, 33°51.01" S), where the depth was less than 20 meters.
All samples were taken from the boat at the mooring site.
Our experiments were non-invasive and caused no disturbance.