Evaluation of Filtration and DNA Extraction Methods for Environmental DNA Biodiversity Assessments across Multiple Trophic Levels

DNA extraction from environmental samples (environmental DNA; eDNA) for metabarcoding‐based biodiversity studies is gaining popularity as a noninvasive, time‐efficient, and cost‐effective monitoring tool. The potential benefits are promising for marine conservation, as the marine biome is frequently under‐surveyed due to its inaccessibility and the consequent high costs involved. With increasing numbers of eDNA‐related publications have come a wide array of capture and extraction methods. Without visual species confirmation, inconsistent use of laboratory protocols hinders comparability between studies because the efficiency of target DNA isolation may vary. We determined an optimal protocol (capture and extraction) for marine eDNA research based on total DNA yield measurements by comparing commonly employed methods of seawater filtering and DNA isolation. We compared metabarcoding results of both targeted (small taxonomic group with species‐level assignment) and universal (broad taxonomic group with genus/family‐level assignment) approaches obtained from replicates treated with the optimal and a low‐performance capture and extraction protocol to determine the impact of protocol choice and DNA yield on biodiversity detection. Filtration through cellulose‐nitrate membranes and extraction with Qiagen's DNeasy Blood & Tissue Kit outperformed other combinations of capture and extraction methods, showing a ninefold improvement in DNA yield over the poorest performing methods. Use of optimized protocols resulted in a significant increase in OTU and species richness for targeted metabarcoding assays. However, changing protocols made little difference to the OTU and taxon richness obtained using universal metabarcoding assays. Our results demonstrate an increased risk of false‐negative species detection for targeted eDNA approaches when protocols with poor DNA isolation efficacy are employed. Appropriate optimization is therefore essential for eDNA monitoring to remain a powerful, efficient, and relatively cheap method for biodiversity assessments. For seawater, we advocate filtration through cellulose‐nitrate membranes and extraction with Qiagen's DNeasy Blood & Tissue Kit or phenol‐chloroform‐isoamyl for successful implementation of eDNA multi‐marker metabarcoding surveys.

Section: Discussionsupporting

confidence: 92%

Section: Discussionsupporting

confidence: 71%

Species‐level biodiversity assessment using marine environmental DNA metabarcoding requires protocol optimization and standardization

Jeunen

Knapp

Spencer

et al. 2019

“…Since the majority of eDNA is found in the 1–10 µm size fraction, a 0.22 µm filter effectively captures both single‐celled organisms and particulate organic matter left behind by multicellular individuals (Sassoubre, Yamahara, Gardner, Block, & Boehm, ; Turner et al, ). Therefore, metabarcoding of eDNA captured on a 0.22 µm filter from seawater enables high resolution examination of ecosystem biodiversity across multiple trophic levels (Biggs et al, ; Djurhuus et al, ; Jane et al, ; Kelly et al, ; Port et al, ; Stat et al, ). Additionally, since DNA degradation in the water column occurs within a few days to weeks, species recovered with eDNA are expected to have recently been present near the site of sample collection (Andruszkiewicz, Sassoubre, & Boehm, ; Thomsen et al, )…”

Section: Introductionmentioning

confidence: 99%

“…Since the majority of eDNA is found in the 1-10 µm size fraction, a 0.22 µm filter effectively captures both single-celled organisms and particulate organic matter left behind by multicellular individuals (Sassoubre, Yamahara, Gardner, Block, & Boehm, 2016;Turner et al, 2014). Therefore, metabarcoding of eDNA captured on a 0.22 µm filter from seawater enables high resolution examination of ecosystem biodiversity across multiple trophic levels (Biggs et al, 2015;Djurhuus et al, 2017;Jane et al, 2015;Kelly et al, 2017;Port et al, 2016;Stat et al, 2017). Additionally, since DNA degradation in the water column occurs within a few days to weeks, species recovered with eDNA are expected to have recently been present near the site of sample collection (Andruszkiewicz, Sassoubre, & Boehm, 2017;Thomsen et al, 2012) As a part of the Marine Biodiversity Observation Network (MBON), which aims to monitor biodiversity across multiple trophic levels, we are testing the applicability of eDNA metabarcoding to examine eukaryotic communities by using routinely monitored and tightly regulated marine sanctuaries as sentinel sites that will act as indicators for the status of nearby marine ecosystems.…”

Section: Introductionmentioning

confidence: 99%

Assessing eukaryotic biodiversity in the Florida Keys National Marine Sanctuary through environmental DNA metabarcoding

Sawaya

Djurhuus

Closek

et al. 2019

Self Cite

Environmental DNA (eDNA) is the DNA suspended in the environment (e.g., water column), which includes cells, gametes, and other material derived from but not limited to shedding of tissue, scales, mucus, and fecal matter. Amplifying and sequencing marker genes (i.e., metabarcoding) from eDNA can reveal the wide range of taxa present in an ecosystem through analysis of a single water sample. Metabarcoding of eDNA provides higher resolution data than visual surveys, aiding in assessments of ecosystem health. This study conducted eDNA metabarcoding of two molecular markers (cytochrome c oxidase I (COI) and 18S ribosomal RNA (rRNA) genes) to survey eukaryotic diversity across multiple trophic levels in surface water samples collected at three sites along the coral reef tract within the Florida Keys National Marine Sanctuary (FKNMS) during four research cruises in 2015. The 18S rRNA gene sequences recovered 785 genera while the COI gene sequences recovered 115 genera, with only 33 genera shared between the two datasets, emphasizing the complementarity of these marker genes. Community composition for both genetic markers clustered by month of sample collection, suggesting that temporal variation has a larger effect on biodiversity than spatial variability in the FKNMS surface waters. Sequences from both marker genes were dominated by copepods, but each marker recovered distinct phytoplankton groups, with 18S rRNA gene sequences dominated by dinoflagellates and COI sequences dominated by coccolithophores. Although eDNA samples were collected from surface waters, many benthic species such as sponges, crustaceans, and corals were identified. These results show the utility of eDNA metabarcoding for cataloging biodiversity to establish an ecosystem baseline against which future samples can be compared in order to monitor community changes.

“…There are many potential benefits that whole‐community metabarcoding of eukaryotic marine eDNA, using multiple or even single‐assay approaches, could bring to biodiversity assessment and monitoring, such as using direct measurements of biodiversity, instead of relying on biodiversity indicators (Aylagas, Borja, Irigoien, & Rodríguez‐Ezpeleta, ; Djurhuus et al, ; Lindenmayer & Likens, ; Rees, Maddison, Middleditch, Patmore, & Gough, ). Environmental DNA analysis can also be used for the detection of “hidden diversity,” without a priori knowledge of the composition of species assemblages in a particular water body (Boussarie et al, ; Lindeque, Parry, Harmer, Somerfield, & Atkinson, ).…”

Section: Introductionmentioning

confidence: 99%

Biodiversity assessment of tropical shelf eukaryotic communities via pelagic eDNA metabarcoding

Bakker

Wangensteen

Baillie

et al. 2019

Our understanding of marine communities and their functions in an ecosystem relies on the ability to detect and monitor species distributions and abundances. Currently, the use of environmental DNA (eDNA) metabarcoding is increasingly being applied for the rapid assessment and monitoring of aquatic species. Most eDNA metabarcoding studies have either focussed on the simultaneous identification of a few specific taxa/groups or have been limited in geographical scope. Here, we employed eDNA metabarcoding to compare beta diversity patterns of complex pelagic marine communities in tropical coastal shelf habitats spanning the whole Caribbean Sea. We screened 68 water samples using a universal eukaryotic COI barcode region and detected highly diverse communities, which varied significantly among locations, and proved good descriptors of habitat type and environmental conditions. Less than 15% of eukaryotic taxa were assigned to metazoans, most DNA sequences belonged to a variety of planktonic “protists,” with over 50% of taxa unassigned at the phylum level, suggesting that the sampled communities host an astonishing amount of micro‐eukaryotic diversity yet undescribed or absent from COI reference databases. Although such a predominance of micro‐eukaryotes severely reduces the efficiency of universal COI markers to investigate vertebrate and other metazoans from aqueous eDNA, the study contributes to the advancement of rapid biomonitoring methods and brings us closer to a full inventory of extant marine biodiversity.